Ligand binding to the adenine analogue binding protein of the rabbit erythrocyte. Adenine analogue binding protein of rabbit erythrocytes reversibly binds [3H] adenosine with a KD of 5.3 X 10(-9) M, an association rate constant of 1.4 X 10(-12) M-1 min-1 and a dissociation rate constant of 7.5 X 10(-3) min-1, as estimated by a nonlinear curve-fitting program applied to data on the time course of the binding reaction. Independent estimates of KD by Scatchard plots and of the dissociation rate constant by dilution or adsorption of free [3H] adenosine on charcoal or by the addition of excess adenosine agreed closely with the estimates from the curve-fitting program. Inhibition of [3H] adenosine binding by a series of 77 adenosine analogues was used to define the factors determining the binding affinity of this nucleoside. These are: (1) the size and aromaticity of the purine base; (2) a glycosylic torsion angle of approximately -120 degrees; (3) the ribo configuration of the 2'-and 3'-hydroxyls and also the 5'-hydroxyl. Bulky substituents in the region of C-2' and to a lesser extent in the region of C-3' reduce binding affinity.


Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers. The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorportion of up to 50 mol percent cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers.


By-product analogues for bovine carboxypeptidase B. A series of monocarboxylic and dicarboxylic acid sulfur-containing by-product analogues of lysine and arginine has been synthesized and tested as competitive inhibitors of bovine carboxypeptidase B. The most effective derivatives were guanidinoethylmercaptosuccinic acid and aminopropylmer-captosuccinic acid with Kis of 4 and 8 X 10(-6) M, respectively. Kinetics studies established the pure competitive nature of the inhibition. Mixed studies with the alkylating reagents bromoacetyl-D-arginine and bromoacetamidobutylguanidine established their efficiency in protecting the active-center glutamic acid and tyrosine of bovine carboxypeptidase B, respectively, from irreversible alkylation. Kinetic studies with bovine carboxypeptidase A and porcine carboxypeptidase B showed a lack of efficiency for A and high degree of efficiency for B.


Amino acid sequence of the diazooxonorleucine binding site of Acinetobacter and Pseudomonas 7A glutaminase--asparaginase enzymes. Acinetobactor glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine, reduced with sodium borohydride, and cleaved with cyanogen bromide. Radioactivity was present only in a 96-residue-N-terminal peptide which eluted as the second peptide peak on Sephadex G-50. Radioactivity was released with the threonine in position 12 during automatic sequencing of this peptide. The amino acid sequence of a 60-residue tn-terminal segment and a 16-residue C-terminal segment of this peptide was determined. Pseudomonas 7 A glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine and reduced with sodium borohydride. Radioactivity was released with the threonine in residue 20 during automatic sequencing of the whole enzyme. Analysis of 26 N-terminal residues showed that an 8-residue segment containing the radioactive threonine was identical with that in Acinetobacter glutaminase-asparaginase and in Escherichia coli asparaginase. Additional identical residues were noted in the N-terminal regions of these enzymes.


Hydrolysis of proteins using dipeptidyl aminopeptidases: analysis of the N-terminal portion of spinach plastocyanin. The exopeptidases dipeptidyl aminopeptidases I and IV were used to hydrolyze the N-terminal portion of spinach plastocyanin to dipeptides. The enzymes were used individually as well as in a mixture and the dipeptides were analyzed by combined gas chromatography-mass spectrometry. Data are presented for native plastocyanin and the S-methylated protein. Of the 98 residues which make up this protein, the first 44 were released in the form of 22 dipeptides by the combined action of DAP I and DAP IV. These dipeptides were aligned by homology to other plastocyanins of known sequence. The results demonstrate the versatility of the two enzymes in hydrolyzing proteins to obtain information on their primary sequence.


Heterogeneity in the 3'-terminal sequence of ribosomal 5S RNA synthesized by isolated HeLa cell nuclei in vitro. Isolated HeLa cell nuclei synthesize ribosomal 5S RNA of very nearly correct sequence and size. The in vitro product was resolved according to size on formamide-containing polyacrylamide gels and the fractions were subsequently hybridized to recombinant DNA containing the 5S genes from Xenopus mulleri. It could be shown that the 5S RNA synthesized in vitro differed only very slightly in size from the mature species labeled in vivo and contained a few extra nucleotides in some of the molecules. Analysis of the 3'-terminal base of molecules synthesized independently with four different nucleotides showed that the chains were almost exclusively terminated with uridine. Digestion of the in vitro product with ribonuclease T1 and analysis of the oligonucleotides on DEAE-Sephadex A-25 in the presence of 7 M urea revealed that the molar yield of the internal fragments agreed well with the expected theoretical values. The 3'-terminal fragments, however, were found to be present in three different species with the sequences CUUOH, CUUUOH, CUUUUOH which occurred with a frequency of about 60%, 20%, and 20%, respectively. From these data we conclude that 5S RNA synthesis in isolated HeLa cell nuclei was correctly initiated but that termination occurred with a slight ambiguity, adding either one or two uridine residues to some of the chains.


Occurrence of an uncoupler-resistant intermediate type of phosphate-water oxygen exchange reaction catalyzed by heart submitochondrial particles. The hydrolysis of ATP catalyzed by phosphorylating vesicles prepared from bovine heart mitochondria by ultrasonic disruption was studied in H218O. Provided that an ATP-generating system was included to prevent accumulation of ADP due to hydrolysis, the addition of 20 mM arsenate or 0.5 mM 2,4-dinitrophenol to the incubation mixture either singly or together, had little or no effect on the number of oxygen atoms from H2O incorporated (on the average) into each molecule of Pi formed by hydrolysis (the O:P ratio). As the ATP concentration was reduced from 2.0 to 0.05 mM, the O:P ratio increased from about 1.4 to over 2.0 and, although dinitrophenol significantly increased the ATPase activity, it did not significantly alter the O:P ratio for a given ATP level. This implies that the uncoupler does not act directly on the terminal transphosphorylation step. Companion experiments were performed in which 18O label was placed either initially in H2O or Pi. Under conditions where extensive exchange from H218O into Pi occurred, no 18O was lost from medium Pi under identical circumstances, thus showing that the exchange was intermediate and did not involve medium Pi. Kinetic plots of v vs. v/S were nonlinear with respect to ATPase activity. The kinetic data, as well as the Pi = H218O exchange data, are consistent with enzyme models having multiple forms of catalytic sites. Several models are evaluated and attempts are made to distinguish between some of the simpler cases of these models.


Oxidation of low-spin iron(II) porphyrins by molecular oxygen. An outer sphere mechanism. Hexacoordinate low-spin iron(II) porphyrins are oxidized by molecular oxygen in amine solvents at room temperature by a process that is acid dependent. The visible and NMR spectra of solutions of the iron complexes and the influence of axial ligands upon the rate of oxidation are consistent with an outer sphere mechanism that entails the dissociation of a protonated 1:1 iron porphyrin-oxygen pi complex as the rate-limiting step.


Extracellular hydrolases of the lung.

Extracellular hydrolases of the lung. A pool of acid hydrolases exists within the acellular lining material of the alveoli and distal airways of the lungs. These extracellular hydrolases, obtained using pulmonary lavage procedures, appear to be of a selected variety insofar as some hydrolases (beta-N-acetylglucosaminidase and alpha-mannosidase) are highly active while others (beta-glucuronidase and arylsulfatase) are barely detectable. The origins of these hydrolases were investigated. Neither leakage of serum nor cell damage can account for the presence of the extracellular hydrolases in lavage effluents. Electrophoretic mobilities on acrylamide gels indicate that the extracellular hydrolases generally differ from those found in serum. Cytoplasmic soluble enzymes such as lactate dehydrogenase were used to monitor cell damage and show that the extracellular hydrolases did not originate from cell leakage during the lavage procedure. Hydrolases similar to those found extracellularly are associated with highly purified lysosome-free lamellar bodies isolated from homogenates of lung. The extracellular hydrolases are probably selected by the type 2 cells of the pulmonary alveolar epithelium during their selection of lamellar bodies.


Purification and characterization of a DNA single strand specific endonculease from human cells. An endonuclease with DNA single-strand specificity has been purified from KB cells. The enzyme has a pH optimum at 9.2, requires Mg2+ for activity, and is inhibited by mono- or divalent cations. Its sedimentation coefficient of 4.6 S is based on sucrose gradient sedimentation, and it has a molecular weight of 54 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme specifically catalyzes the endonucleolytic cleavage of denatured DNA, yielding acid-soluble oligonucleotides which contain 5'-phosphoryl termini. The rate of hydrolysis of poly(dT) is approximately eightfold greater than that observed with denatured DNA, although the Km for both substrates is 1.74 X 10(-5) M. The relative rates of hydrolysis of homopolymers by the endonuclease are: poly(dG) greater than poly(dT) greater than poly(dA) greater than poly (dC). Purified enzyme preparations also hydrolyze poly(U), releasing acid-soluble products. This activity cosediments in sucrose gradients with the DNA endonuclease activity, suggesting that both activities are contained in the same enzyme molecule.


Recombination of the biologically active peptides from a tryptic digest of bovine growth hormone. Two bovine growth hormone peptides containing residues 96 to 133 in one and a larger peptide corresponding to residues 1 to 95 and 151 to 191 linked by a disulfide bond have been recombined. Recombination was performed in 1.0 N acetic acid without and with 8 M urea and the recombined peptides were purified by gel filtration. Evidence of recombination of the peptides has been established by gel filtration, polyacrylamide gel disc electrophoresis, amino acid composition, ultraviolet absorption, circular dichroism, and intrinsic fluorescence. The molar growth promoting activity of the recombined peptides measured by tibial width assay in hypophysectomized rats was 10% that of the native hormone and greater than the sum (1%) of the growth promoting activities of the individual peptides. These studies suggest that some contribution of the larger peptide is necessary for more complete expression of the growth promoting activity of the bovine growth peptide corresponding to residues 96 to 133.


Biosynthesis of the macrolide antibiotic chlorothricin: basic building blocks. The biosynthesis of chlorothricin (I), a macrolide antibiotic isolated from Streptomyces antibioticus Tü 99, has been studied by feeding experiments with 14C- and 3H-labeled precursors. Acetate and propionate, but not methionine and mevalonate, were incorporated into the macrocylic aglycone of the antibiotic. Glucose and the various carbon atoms of tyrosine, except the carboxyl carbon, also contributed label to the aglycone. Glucose also seems to be a specific precursor of the 2-deoxyrhamnose moiety, probably via a process involving a hydrogen shift from C-4 to C-6 of the hexose. The substituted 6-methylsalicylic acid moiety seems to be derived from acetate and one O-methyl group provided by methionine; shikimic acid is not incorporated.


Primary photochemical processes in isolated reaction centers of Rhodopseudomonas viridis. Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P*) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state P+I-) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P+ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P+I- (or PF) lives approx. 15 ns. It decays, in part, into a longer lived state (PR), which appears to be a triplet state. State PR decays with an exponential time of approx. 55 microseconds. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P*. Most of P* then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P* or P+ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I- suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.


Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts. The MgCl2-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3',4'-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a pico-second time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp --At1/2, where A was found to be 0.052 ps-1/2, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl2 produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence deday law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.


Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga. The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by pico-second laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a. A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp --At1/2 transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.


Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes). The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.


On the rate-limiting step in the transfer of long-chain acyl groups across the inner membrane of brown adipose tissue mitochondria. Brown adipose tissue mitochondria predominantly oxidize fatty acids in order to generate heat for non-shivering thermogenesis, and have an unusually high capacity for net transfer of long-chain fatty acyl groups from the outer to the inner (matrix) compartment. The activities of the "outer" and "inner" carnitine long-chain acyltransferases have been estimated in isolated mitochondria of cold-acclimated guinea pits by the continuous spectrophotometric recording of the redox level of flavoproteins in the acyl-CoA dehydrogenase pathway. This redox level is determined by the intramitochondrial content of acyl-CoA under the selected experimental conditions. The apparent initial rate of the "inner" acyltransferase (palmitoyl-L-carnitine added) is three order of magnitudes higher than the "outer" acyltransferase (palmitoyl-CoA added), and this difference is not influenced by the substrate concentration, pH and reaction temperature. Thus, the "outer" acyltransferase reaction is rate limiting in the transfer of long-chain acyl groups across the inner membrane of these mitochondria and catalyzes a non-equilibrium reaction in the intact organelle. Estimates of the absolute rate of the "outer" long-chain acyltransferase indicate that it exceeds that of rat liver mitochondria by a factor of 20.


Proton electrochemical gradient and rate of controlled respiration in mitochondria. The correlation between deltamuH, the proton electrochemical potential difference, and the rate of controlled respiration is analyzed. deltamuH (the proton concentration gradient) is measured on the distribution of [3H]acetate, and deltapsi (the membrane potential) on the distribution of 86Rb+, 45Ca2+ and [3H]triphenylmethylphosphonium used either alone or simultaneously. The effects of the addition of ADP + hexokinase (state-3 ADP) and of carbonylcyanide trifluoromethoxyphenylhydrazone (state-3 uncoupler) on respiration and deltamuH are not equivalent: the uncoupler depresses deltamuH more than ADP at equivalent respiratory rates. The effects of the additions of nigericin-valinomycin and of ionophore A23187 (state-3 cation transport) and of carbonylcyanide trifluoromethoxy-phenylhydrazone (state 3-uncoupler) on respiration and deltamuH are also not equivalent: the uncoupler depresses deltamuH more than A23187 and nigericin + valinomycin at equivalent respiratory rate. A23187 is very efficient in stimulating respiration with negligible deltamuH changes.


Proton electrochemical gradient and phosphate potential in mitochondria. The paper reports an analysis of the relationship between deltamuH the proton electrochemical potential difference, and deltaGp, the phosphate potential. Depression of deltamuH and deltaGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b) nigericin (+ valinomycin); (c) KCl (+ valinomycin); and (d) rotenone. The uncoupler depresses deltamuH more than nigericin (+ valinomycin), KCl (+ valinomycin) and rotenone at equivalent deltaGp. The deltaGp/deltamuH ratio is about 3 at high values of deltamuH. When deltaGp and deltamuH are depressed by nigericin (4 valinomycin) the deltaGp/deltamuH ratio remains constant. When deltaGp and deltamuH are depressed by uncouplers, the deltaGp/deltamuH ratio increases hyperbolically tending to infinity while deltamuH tends to zero. The absence of constant proportionality between deltaGp and deltamuH indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.


Galactose transport in human erythrocytes. The transport mechanism is resolved into two simple asymmetric antiparallel carriers. The kinetic properties of the mediated transport of galactose in human erythrocytes are investigated at 20 degrees C. Different methodological procedures are used to acquire a complete kinetic description of the system. Under zero-trans conditions the uptake of galactose is mediated by two distinctly different carriers (defined as alpha and beta) having significantly different Mic;aelis parameters: alpha K = 12.7 mM and beta K = 81.5 mM, but similar maximal velocities, approx. 40 nM.min-1. The zero-trans efflux procedure reveals apparently one single carrier with K = 74.4 mM and V = 241 mM.min-1. Under equilibrium-exchange conditions the galactose transport is mediated apparently by a single site with K = 146 mM and V = 521 mM.min-1. The data for the alpha-carrier are analyzed in terms of the simple carrier model as formulated by Lieb and Stein (Biochim. Biophys. Acta (1974) 373, 178). Application of several rejection criteria for the simple carrier failed to indicate lack of fitness of the alpha-carrier to a simple asymmetric carrier. From the analysis of the kinetic data it is inferred that the transport of galactose across the human erythrocyte membrane is mediated by two simple asymmetric carriers operating in antiparallel fashion. Using this model and the data of zero-trans and equilibrium-exchange, it is shown that the predicted half-saturation constants for both uptake and efflux in infinite-cis conditions fully agree with the experimentally derived values. Further analysis of the kinetic data indicate that the translocation of the unloaded alpha-carrier is the rate-limiting step in galactose uptake. Under equilibrium-exchange conditions the unloaded carrier is asymmetrically distributed across the membrane so that its concentration is 8 times higher on the inner side of the membrane. Using the value of 3.3.10(5) hexose carriers per cell, the turnover number of galactose exchange is 6.5.10(4) molecules/carriers per min.


A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. We have worked out a simplification of the procedure described by Schmitz et al. (Biochim. Biophys. Acta (1973) 323, 98--112) for the preparation of brush border membranes from small intestine. The procedure ultimately adopted is simple, rapid, does not necessarily require scraping and can be started from fresh or frozen material. It can be scaled up easily, allowing a quick production of large amounts of brush border membrane vesicles. These vesicles prove to be excellently suited for transport studies, as suggested by our measurements of D-glucose transport. Using these vesicles, the mode of choline transport across the brush border membrane was also investigated. Choline transport was found to occur by a saturable component with a Km of 83 +/- 4 micrometer (at 20 degrees C) and by a non-saturable component. It is independent of the presence of Na+ and appears to be non-electrogenic.


Non-isothermal potential of phospholipid bilayer films. The thermal electrical potential generated in isochemical conditions by a temperature gradient at the two sides of lipid bilayer leaflets is measured. The experimental results agree rather well with the theoretical predictions. The cephaline (from sheep brain) bilayer behaves like a film with zero charge while the phosphatidylcholine (from egg yolk) film performs like a charged membrane. The results presented suggest that the measurement of electrical thermal potential is an interesting method to investigate the electrical behaviour of bilayer membranes.


Coupling polylysine to glass beads for plasma membrane isolation. Solid glass beads for use in isolating cell membranes were coated with a stable, covalently attached layer of polylysine. The optimal conditions for coating the bead surface were established and the beads were tested by measuring the attachment of human erythrocyte plasma membranes. When compared to other beads, such as those with absorbed polylysine or protamine, none retained red-cell membranes as well as glass beads with covalently linked polylysine.


Membrane isolation on polylysine-coated glass beads. Asymmetry of bound membrane. Erythrocyte membranes isolated on polylysine-coated glass beads exhibit many of the properties of the native membrane. Gel electrophoresis indicates that all major protein components of the membrane are retained during membrane isolation. The membrane integrity and accessibility of selected components was tested using non-penetrating probes. In general, membranes on beads displayed accessibility properties typical of inside-out vesicles. The accessibility of membrane acetylcholinesterase to assay reagents, as well as membrane accessibility to the actions of neuraminidase, trypsin and galactose oxidase-NaB3H4 demonstrated that the protoplasmic surface of membrane isolated on beads was exposed, while the extracellular surface was inaccessible. The differential accessibility of the membrane surfaces demonstrates the feasibility of investigating asymmetry of membranes isolated on cationic glass beads.


Interactions of acetylcholine receptor and acetylcholinesterase with lipid monolayers. The interaction of acetylcholine receptor and acetylcholinesterase with lipid monolayers was followed by measuring changes in surface pressure. When injected into the subphase of a lipid monolayer, the proteins caused increases in surface pressure from 5 to 10 dynes/cm, indicating a penetration of protein into the monolayer. At pH values below the isoelectric point of the proteins the incorporation was improved. The same was observed when Ca2+ (2mM) was added. The presence of the enzyme in the mixed film could be demonstrated by using diiso [3H] propyl fluorophosphate-labelled acetylcholinesterase as well as by measuring enzyme activity. Acetylcholine receptor was shown to be present in the mixed film by using a complex made of the receptor and alpha-[3H]neurotoxin.


13C NMR studies on [4-13C] cholesterol incorporated in sonicated phosphatidylcholine vesicles. 1. 90.5 MHz 13C NMR linewidth measurements were performed on mixed sonicated [4-13C] cholesterol/phosphatidylcholine vesicles of different fatty acid composition. 2. From the Dy3+ -induced shift of the C4 resonance of cholesterol it suggested that this part of the molecule is localized in the ester bond region of the bilayer. 3. The local motion of the cholesterol ring system is restricted and independent of fatty acid composition. 4. At cholesterol concentrations below 30 mol percent the ring system becomes more immobilised when the fatty acids of the phosphatidylcholine molecules enter the gel state.


Phase transitions in phospholipid model membranes of different curvature. 1. Nuclear magnetic resonance, light scattering and freeze fracturing electron microscopic techniques were used to characterize the size of unilamellar phospholipid vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. 2. Differential scanning calorimetric and light scattering analyses showed that very small unilamellar vesicles obtained by the sonication method exhibit a downward shifted, largely broadened phase transition with a slightly decreased enthalpy change when compared with multilayered liposomes. 3. The phase transition of vesicles with variable diameter as obtained by injection methods resembled the pattern of multilayered liposomes the more the diameter was increased. 4. Repeated cycling through the lipid phase transition was shown to have a progressive effect on a fusion process. This effect was strongly increased when the osmolarity of the medium was enhanced (e.g. by the addition of cryoprotectors). Furthermore it was shown that ice-water of the systems caused abrupt fusion of the lipid structures. 5. Controversial results in the literature on the thermotropic behavior of vesicles could be explained in terms of these fusion processes.


Use of deuterated phospholipids in Raman spectroscopic studies of membrane structure. I. Multilayers of dimyristoyl phosphatidylcholine (and its -d54 derivative) with distearoyl phosphatidylcholine. The temperature dependence of the Raman spectrum has been studied for binary phospholipid mixtures of dimyristoyl phosphatidylcholine (and its chain deuterated -d54 derivative) with distearoyl phosphatidylcholine. Two distinct melting regions are observed for the 1 : 1 mole ratio mixture. The use of deuterated phospholipid permits the identification of the lower (approximately 22 degrees C) transition with primarily the melting of the shorter chain component, and the higher (approximately 47 degrees C) transition primarily with the melting of the longer chains. The C-H stretching vibrations of the distearoyl component respond to the melting of the dimyristoyl component, an apparent consequence of alterations in the lateral interactions of the distearoyl chains. These changes in the C-H spectral region suggest that phase separation does not occur in the gel state for this system. The results are in reasonable accord with recent calorimetric studies (Mabrey, S. and Sturtevant, J.M. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3862-3866). The feasibility of using deuterated phospholipids to monitor the conformation of each component in a binary phospholipid mixture is demonstrated.


Inhibition of protein phosphorylation and induction of protein cross-linking in erythrocyte membranes by diamide. This report presents studies on the effect of diamide on protein phosphorylation in erythrocyte membranes. Diamide, a thiol-oxidizing reagent, nonspecifically inhibits cyclic Amp-dependent and -independent autophosphorylation of red cell memvranes, but not the activity of the solubilized membrane cycle AMP-independent protein kinases. Analysis of diamide-treated membranes by gel electrophoresis indicates that diamide is capable of inducing cross-linking of membrane proteins. The action of diamide, both in the inhibition of membrane autophosphorylation and in the cross-linking of membrane proteins, is very similar to that of Cu2+. o-phenanthroline complex. Our data indicate that diamide inhibits erythrocyte membrane autophosphorylation by perturbing the protein substrates.


Factors affecting the relative magnitudes of the ouabain-sensitive and the ouabain-insensitive fluxes of thallium ion in erythrocytes. A maximal rate of the ouabain-sensitive 204-Tl influx in human erythrocytes can be attained at trace concentrations of Tl+ in Mg2+ isotonic media free of K+ and Na+. The maximal influx of Tl+ from isotonic Mg(NO3)2 at 20 degrees C and pH 7.4 was 0.45 mM.l(-1).h-1 with a Km of 0.025 mM. In contrast to the active influx of Tl+, the passive Tl+ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl+ and K+ studied. The rate constants of Tl+ passive fluxes in human and cat erythrocytes can be related to pH by the equation log kin(OUT)= -A + B.pH, where A and B are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl+ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl+ is about two orders of magnitude faster than those of K+ or Rb+. An extra non-Coulombic interaction between Tl+ and membrane ligands appears to be involved providing an accumulation of Tl+ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl+ in both directions.


Transbilayer movement of cholesterol in phospholipid vesicles under equilibrium and non-equilibrium conditions. 1. The exchange of [3H] cholesterol between phospholipid: cholesterol vesicles and an excess of red cell ghosts is examined. 2. Using a number of different phophatidylcholines, only the cholesterol thought to be associated with the outer half of the bilayer (about 70 percent) is available for exchange, suggesting that at least at equilibrium the transbilayer movement of cholesterol or "flip-flop", occurs very slowly, if it occurs at all. 3. The rate of exchange of cholesterol between the vesicles and the ghosts is dependent on the nature of the fatty acid chain of the phospholipids, being a function of both the fatty acid chain length and the degree of unsaturation. 4. Under non-equilibrium conditions, when cholesterol is being both exchanged and depleted from the lipid vesicles to red cell ghosts, the previously non-exchangeable vesicle cholesterol becomes available for exchange, suggesting that under these conditions "flip-flop" can occur.


Specificity of Ca2+ and Mg2+ binding to phosphatidylserine vesicles and resultant phase changes of bilayer membrane structure. Differences in the interaction of Ca2+ and Mg2+ with phosphatidylserine vesicles were revealed by binding studies, differential scanning calorimetry and X-ray diffraction. The two cations produced structurally different complexes with phosphatidylserine as evidenced by phase transition characteristics, lamellar spacings, and hydrocarbon packing. Ca2+ effectively completes with Mg2+ for phosphatidylserine binding sites. Analysis of the binding data showed that Ca2+ had a ten-fold greater intrinsic binding constant for phosphatidylserine.


Association of glycolytic enzymes with particulate fractions from nerve endings. Several glycolytic enzymes were observed to have between 40-90% of their activities associated with the particulate fractions of lysed nerve endings. The enzymes showing high particulate activity in lysed nerve endings were hexokinase (EC 2.7.1.1), aldolase (EC 4.1.2.13), glucosephosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.27). With the exception of phosphofructokinase, 80% or more of the particle associated activity of each enzyme was solubilized by salt treatment indicating the association with particles was ionic. Sub-fractionation of lysed nerve endings showed hexokinase and fumarase (EC 4.2.1.2) had the highest specific activity in the same fractions which is consistent with observations indicating that hexokinase is associated with mitochondria. The other glycolytic zymes having high particulate activity, aldolase, glucosephosphate isomerase, phosphofructokinase, glyceraldehyde-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, showed enrichment in fractions containing synaptosomal membranes, i.e. the fractions having highest specific activity of acetylcholinesterase (EC 3.1.1.7) and (Na+ + K+)-ATPase (EC 3.6.1.3).


Purification of two forms of the associated 3-dehydroquinate hydro-lyase and shikimate:NADP+ oxidoreductase in Phaseolus mungo seedlings. Two associated enzymes, 3-dehydroquinate hydro-lyase (EC 4.2.1.10) and shikimate:NADP+ oxidoreductase (EC 1.1.1.25), have been purified from Phaseolus mungo seedlings. These enzymes were purified 6900- and 9700-fold, respectively, but they were not separable. Moreover, two activity bands of the shikimate:NADP+ oxidoreductase were detected after polyacrylamide gel electrophoresis and the two peaks also have 3-dehydroquinate hydro-lyase activity. The two forms of the associated enzymes showed only small differences in molecular weight, Km value, pH optimum and the responses to some inhibitors.


Interaction of methylchymotrypsin with Streptomyces subtilisin inhibitor. The effect of methylation of histidine-57 of alpha-chymotrypsin with Streptomyces subtilisin inhibitor was examined. Methylchymotrypsin was isolated by affinity chromatography on inhibitor-Sepharose, and the interaction of this inactive enzyme with inhibitor was quantitatively analyzed by two different methods: the spectrophotometric titration of difference spectrum resulted in the complex formation and the application of competitive enzyme assay by using substrates of large Km values. The former method gave values of 8.6 . 10(-6) M as dissociation constant (Kd) of methylchymotrypsin . inhibitor complex and 0.91 as the number of binding sites (n) per inhibitor monomer, both of which were almost equivalent to those for native enzyme . inhibitor complex. By the latter novel method, values of 7.9 . 10(-6) M and 1.08 were obtained for Kd and n, respectively, for interaction of inhibitor with alpha-chymotrypsin, and 8 . 10(-6) M as Kd for methylchymotrypsin . inhibitor complex. These results indicate that methylation of histidine-57 of active site in alpha-chymotrypsin molecule does not affect essentially the binding ability to inhibitor and the modified enzyme binds stoichiometrically to inhibitor, as the native enzyme does, with a molar ratio of 1:1 per inhibitor monomer.


Photodynamic properties of pyridoxal phosphate bound to cystathionase-gamma-lyase. The cofactor pyridoxal phosphate bound through an aldimine linkage to lysine residues of the enzyme cystathionase (L-Cystathione cysteine-lyase (deaminating), EC 4.4.1.1) is very stable to irradiation with light of 420 nm. The catalytic function of the enzyme remains unaffected indicating that the cofactor is not an efficient photosensitizer of essential amino acid residues. This unusual stability of the cofactor to irradiation can be ascribed to the presence of aldimine linkages as demonstrated by studies conducted on model compounds. The binding of a reversible inhibitor (L-allylglycine) to the catalytic site of the enzyme does not facilitate photooxidation of the cofactor. On the contrary, irradiation of the cofactor in the presence of the inhibitor results in photodestruction of the inhibitor.


Partial purification and some properties of pyruvate carboxylase from the flight muscle of the locust (Schistocerca gregaria). A procedure is described for the partial purification of pyruvate carboxylase (pyruvate:CO2 ligase (ADP-forming), EC 6.4.1.1) from the flight muscle of the locust (Schistocerca gregaria). Characterisation of the kinetic properties of this enzyme indicates that it is activated by acetyl-CoA, is insensitive to inhibition by di- and tricarboxylic acids and exhibits an apparent Km for HCO3-(16 mM) which differs by an order of magnitude from that observed for other pyruvate carboxylases. It is suggested that activation of this locust flight muscle pyruvate carboxylase during the rest leads to flight transition may result from increases in the concentrations of pyruvate and HCO3- under these conditions.


Purification and characterisation of D-amino acid aminotransferase from Rhizobium japonicum. Rhizobium japonicum has D-amino acid aminotransferase and alanine racemase activities. The D-amino-acid aminotransferase has been partially purified and characterized. This enzyme has a broad specificity and is very active with D-alpha-aminobutyrate and D-aspartate as well as D-alanine and D-glutamate. The stereospecificity of the enzyme for D-amino acids was apparently absolute with respect to product inhibition, pyridoxamine formation as well as catalytic activity. The apparent molecular weight was 58,000 and the pH optimum was 7.8-7.9. The equilibrium constant in the direction of D-glutamate formation was 1.9. Initial-velocity kinetic studies indicate the enzyme acts by a ping-pong mechanism. The dissociation constant for pyridoxal phosphate and the Michaelis constants (+/- standard errors) for D-alanine and 2-oxoglutarate were determined to be 0.51 +/- 0.06 micrometer, and 2.13 +/- 0.18 and 0.058 +/- 0.005 mM respectively. The enzyme is moderately inhibited (30%) by 4 mM p-chloromercuribenzoate.


Effects of saturated and unsaturated fats given with and without dietary cholesterol on hepatic cholesterol synthesis and hepatic lipid metabolism. Hepatic cholesterol synthesis was studied in rats after consuming diets of varying neutral lipid and cholesterol content. Cholesterol synthesis was evaluated by measuring 3-hydroxy-3-methylglutaryl-CoA reductase and by determining the rate of 3H-labeled sterol production from [3H]mevalonate. Results were correlated with sterol balance data and hepatic lipid content. Hepatic cholesterol synthesis was relatively great when cholesterol was excluded from the diet. The source of neutral dietary lipids, saturated vs. unsaturated, produced no change in hepatic sterol synthesis. Values for fecal sterol outputs and hepatic cholesterol levels were also similar in rats consuming either saturated or unsaturated fats. When 1% cholesterol was added to the diet, hepatic cholesterol synthesis was suppressed but the degree of suppression was greater in rats consuming unsaturated vs. saturated fats. This was associated with greater accumulation of cholesterol in livers from rats consuming unsaturates and a reduction in fecal neutral sterol output in this group as opposed to results from rats on saturated fats. Cholesterol consumption also altered the fatty acid composition of hepatic phospholipids producing decreases in the percentages of essential polyunsaturated fatty acids. It is concluded that dietary cholesterol alters cholesterol and fatty acid metabolism in the liver and that this effect is enhanced by dietary unsaturated fats.


Monoacylglycerol hydrolase activity of isolated rat small intestinal epithelial cells. 1. Albumin is the preferred stabilizer of the higher monoacylglycerol substrates, since the highest activity was measured with albumin rather than with Triton X-100 or other detergents tested. 2. The monoacylglycerol hydrolase activity may be strongly influenced by the amount of albumin used as the only emulsifier. Possible models for the physical states of the substrate are discussed. 3. The reaction rates with 1- and 2-monoacylglycerols are generally similar but may vary according to the physical states of the substrates. 4. The same enzyme hydrolyzes both 1- and 2-isomers since the hydrolytic activities were found to be competitive rather than additive. For both isomers identical apparent Km values less than 0.1 mM were obtained. 5. A comparison of the rates of hydrolysis of 1- and 2-monopalmitoylglycerol by the villus preparation at various temperatures confirmed that generally the reaction rates are similar and that the energy of activation is about 15 kcal/mol, so that the Q10 is about 1.8. 6. It is speculated that the microsomal level of long-chain acyl-CoA is an important determinant in the fate of the resorbed monoacylglycerol, since acylCoA is not only a substrate for the reacylation reactions but also an inhibitor of monoacylglycerol hydrolase.


The lipid composition of the non-photosynthetic diatom Nitzschia alba. The lipid composition of the non-photosynthetic marine diatom, Nitzschia alba, has been quantitatively determined. Triglycerides accounted for 20% of the cell dry weight and 87% of the total lipids. Smaller amounts of 1,2- and 1,3-diglycerides, free sterol (24-methylene cholesterol), hydrocarbons and an unknown component were the remaining neutral lipids detected. Phosphatidylsulfocholine (phosphatidyl S,S-dimethylmercaptoethanol), present in amounts of 0.8% of cell dry weight (35% of total polar lipids), was the major polar lipid component. Other phospholipids were lysophosphatidylsulfocholine, phosphatidylglycerol, phosphatidylinositol and cardiolipin, but both phosphatidylcholine and phosphatidylethanolamine were completely absent. Another novel sulfolipid, deoxyceramide sulfonic acid, as well as the sulfate ester of the free sterol, were also present. Considerable amounts of the four lipids often associated with photosynthetic organisms, mono- and di-galactosyl diglycerides, sulfoquinovosyl diglyceride and phosphatidylglycerol, were identified in N. alba. However, the fatty acid components of the glycosyl diglycerides did not show the high amounts of polyunsaturated acids (18 : 2, 18 : 3) normally found in photosynthesizing organisms. All polar lipids were found to be associated with various cell membrane fractions in N. alba.


Identification of the sulfolipids in the non-photosynthetic diatom Nitzschia alba. The four major sulfolipids in the non-photosynthetic marine diatom, Nitzschia alba, were isolated in pure form and their structures were established spectrometrically and by identification of their hydrolysis products as (a) 24-methylene cholesterol sulfate, (b) 1-deoxyceramide-1-sulfonate, (c) phosphatidyl sulfocholine (a sulfonium analogue of phosphatidylcholine) and (d) sulfoquinovosyl diglyceride. The major characteristic fatty acids of the sulfolipids were: for the deoxyceramide sulfonate, 16 : 0 (26%) and 16 : 1-delta3-trans (64%); for the sulfonium analogue, 14 : 0 (30%), 18 : 1 (12%), 18 : 2 (8%), 20 : 5 (27%) and 22 : 6 (4%); and for the sulfoquinovosyl diglyceride (two species, respectively), 14 : 0 (9%, 22%), 16 : 0 (16%, 28%), 18 : 1 (8%, 22%), 20 : 5 (42%, 23%) and 22 : 6 (14%, 2%). Traces of lyso-derivatives of sulfoquinovosyl diglyceride and phosphatidyl sulfocholine were also detected. The deoxyceramide sulfonate and the phosphatidyl sulfocholine represent novel membrane lipid components not previously detected in other organisms. They may however have a widespread distribution in marine diatoms and perhaps in marine organisms generally.


Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle. The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.


Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane. This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 . 10(5). The molecule does not contain sugars. The dissociation constant of the protein . sulfobromophthalein complex has been found to be 4. 10(-6) M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.


Fractionation of nonhistone proeins on a column of daunomycin-CH-Sepharose 4B. Daunomycin was coupled via its amino group to omega-carboxypentyl agarose (CH-Sepharose 4B). Nonhistone proteins from rat leukemia cells (DBLA-6) were fractionated on a column of the adsorbent. The adsorption of nonhistone proteins to the column was increased by high salt concentration (4 M NaCl) and was reversed by 20% glycerol (v/v), indicating a hydrophobic interaction. Complexity of the chromatographic patterns may reflect the occurrence of several species of binding protein in the tumor cells used. Thus the hydrophobic chromatography in the presence of a high concentration of salt was a useful method for fractionation of nonhistone proteins under non-rigorous conditions.


Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography. We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.


Chemical and functional homology of myotoxin a from prairie rattlesnake venom and crotamine from South American rattlesnake venom. Myonecrosis is a serious result of rattlesnake bite and constitutes a persistent clinical problem. In the current study we have isolated crotamine from the venom of Crotalus durissus terrificus to test its ability to cause structural damage to skeletal muscle, and to make direct chemical comparisons with Myotoxin a, a myotoxic polypeptide we recently isolated from prairie rattlesnake (Crotalus viridis viridis) venom. Disc gel electrophoresis, isoelectric focusing, circular dichroic spectroscopy, and amino acid analysis, all indicated a high degree of chemical similarity. Light microscope histology revealed that crotamine caused vacuolizationof skeletal muscle fibers, qualitatively the same as the vacuolization caused by Myotoxin a. The ability of these two basic snake venom polypeptides to cause structural damage to skeletal muscle fibers has significant implications toward more complete understanding of the cause of snake venom-induced myonecrosis.


Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization. Human fibrinogen was illuminated in the presence of methylene blue. The resulting photooxidized fibrinogen was devoid of polymerization activity and thrombin-induced coagulability. The initial rate of the thrombin catalysed release of fibrinopeptides from photooxidized fibrinogen was normal. It was shown that illumination of photooxidized fibrinogen and photooxidized fragment N-DSK caused the modification of histidine residues. Tryptophan residues were also modified. When fibrinogen was photooxidized immediately after the addition of thrombin, the capacity to polymerize was lost. The inhibition of polymerization was less marked when oxidation was initiated at the time when polymerization began or thereafter. Photooxidized fibrinogen acts as an inhibitor of the polymerization of fibrin monomers. Photooxidized fibrinogen has affinity for thrombin-activated fibrinogen-Sepharose and thrombin-activated fragment N-DSK-Sepharose. When the former conjugate is illuminated in the presence of methylene blue its affinity for fibrinogen is decreased. It is concluded that the fragment N-DSK domain of fibrinogen is affected by photooxidation.


A new hemoglobin variant. HB Yatsushiro alpha 2 A beta 2 60 Val replaced by Leu. This study was performed to establish the structural abnormality of a new hemoglobin variant discover-d in a Japanese patient with angina pectoris. The hybridization of the separated hemoglobin with canine hemoglobin revealed a beta-chain anomaly. Peptide betaTp-6 was found to be abnormally located on the peptide map of tryptic digests of the S-carboxymethylated beta-chain from the variant hemoglobin. A structural study on the abnormal betaTp-6 revealed that the variant hemoglobin differs from hemoglobin A by substitution of leucine for valine at residue 60 of the beta-chain. This new variant hemoglobin is designated as hemoglobin Yatsushiro after the name of the city where the propositus lived. The patient is hematologically healthy and his clinical history has nothing to do with this abnormal hemoglobin.


Multiple isoferritins in mouse liver: demonstration by polyacrylamide gel electrophoresis. Unfractionated pure ferritin isolated from the livers of female or male mice forms five narrow protein-positive bands on standard 5% polyacrylamide gel disc electrophoresis (pH 9.0). Since all of these sub-bands also contain iron, they are interpreted as being isoferritins. The multiple sub-bands are very unlikely to be artifactually generated by the analytical procedure used since they are not found in horse spleen ferritin when this is coelectrophoresed with female mouse liver ferritin. The present results provide an independnet indication that many isoferritins indeed can be found within a single organ.


Interaction of rabbit hemopexin with bilirubin. The interaction of hemopexin with bilirubin was characterized by spectrophotometric, fluorimetric and circular dichroic techniques. Hemopexin rapidly forms an equimolar complex with libirubin that has an apparent dissociation constant Kd, of 7.5.10(-7) M. The association alters the absorption band of bilirubin near 150 nm, quenches the fluorescence of tryptophan residues of hemopexin, enhances the fluorescence of bilirubin, and induces strong ellipticity extrema in bilirubin of --60 . 10(3) deg . cm2 . dmol-1 at 465 nm and +70 . 10(3) deg . cm2 . dmol-1 at 415 nm. However, the conformation-sensitive ellipticity aband at 231 nm of hemopexin is not altered. In displacement experiments using circular dichroism, heme readily replaced bound bilirubin, indicating that bilirubin and heme are bound at the same site on hemopexin. Even at molar ratios of hemopexin to albumin of 3 to 1, human serum albumin removes bilirubin from hemopexin. Hemopexin is thus unlikely to have a role in the transport of bilirubin in serum.


Effect of some alcohols on in vivo and in vitro aged collagen. 1. The effects of mixtures of ethanol or n-propanol with 0.15 M NaCl or 0.1 N HCl on the isometric melting and load/extension behaviour of rat tail tendon were examined. 2. Both intra and intermolecular effects were observed but aging of the sample had little effect upon these. 3. The extension modulus in 0.15 M NaCl begins to decrease rapidly at temperatures above the molecular melting temperature (TD). This reduction in modulus is accelerated by alcohol, although the alcohol is not effective below TD.


Studies on lectins. XXXVII. Isolation and characterization of the lectin from Jimson-weed seeds (Datura stramonium L.). The lectin of Jimson-weed seeds (Datura stramonium L.) was isolated by affinity chromatography on a polysaccharide mixture from mycelium of Aspergillus niger. The lectin yields two bands on disc electrophoresis, it has sedimentation coefficient s20,w = 3.8 S and its apparent molecular weight estimated by thin layer gel chromatography is 120,000. The lectin reduced with mercaptoethanol yields on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate three zones corresponding to subunits of molecular weight 72,000, 45,000 and 25,000. The lectin contains large amounts of cystine, glycine, 6.3% of hydroxyproline residues, 4.5% glucosamine and 28% of neutral sugar, predominantly arabinose. The lectin is nonspecific in human erythrocyte ABO system, it is not inhibited by simple sugars but is inhibited by a partial hydrolysate of chitin-containing mixture of polysaccharides from Aspergillus niger.


Studies on lectins. XXXVIII. Isolation and characterization of the lectin from black locust bark (Robinia pseudacacia L.). The lectin of black locust (Robinia pseudacacia) bark was isolated by specific adsorption on formaldehyde-fixed human erythrocytes and elution with a borate solution. The lectin is homogeneous on disc electrophoresis and ultracentrifugation (s20,w = 5.8 S) but yields three bands on isoelectric focusing. It has a molecular weight of approximately 110,000 and consists of two types of subunit (mol. wt 29,000 and 31,500). Its pI is approximately 5.9; it contains high amounts of aspartic acid, threonine and serine, no cysteine and very little methionine. Also 7.2% of covalently bound neutral sugar and 0.47% of glucosamine are present. The lectin is nonspecific in agglutination of human erythrocytes, it is inhibited by high concentrations of N-acetyl-D-galactosamine and is mitogenic in rabbit lymph node lymphocytes.


Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation. The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1.  The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.


Some phytotoxic glycopeptides from Ceratocystis ulmi, the Dutch Elm Disease pathogen. Ceratocystis ulmi, the causal agent of Dutch Elm Disease, produces phytotoxic glycopeptides in culture. A mixture of phytotoxic glycopeptides has been prepared by affinity chromatography on a concanavalin A-Sepharose column and collectively they have been termed the toxin. The polydisperse component that makes up the majority of toxin (80%) by weight has a molecular weight of about 2.7.10(5). The large molecular weight component (less than 5%) elutes at the void volume of a Bio-Gel A 50 m column. The other component (15%) appears as a trailing peak on the edge of the major component and has an approximate molecular weight of 7.10(4). The toxin is composed of 83% sugar residues, primarily rhamnose and mannose, and 7% amino acid residues. Methylation analysis coupled with mild acid hydrolysis indicates that the backbone of the polysaccharide portion of the toxin is composed of alpha -1,6-linked mannosyl residues with a 3-linked terminal rhamnosyl residue linked to C-3 of almost every mannosyl residue. The carbohydrate portion of the molecule is linked to the peptide via O-glycosidic linkages to both threonyl and seryl residues. All three components of the toxin are capable of causing wilt in stem cuttings of American elm.


The synthesis of oxylate from hydroxypyruvate by isolated perfused rat liver. The mechanism of hyperoxaluria in L-glyceric aciduria. Hydroxypyruvate and glycolate inhibited the oxidation of [U-14C]glyoxylate to [14C]oxalate in isolated perfused rat liver, but stimulated total oxalate and glycolate synthesis. [14C]Oxalate synthesis from [14C]glycine was similarly inhibited by hydroxypyruvate, but conversion of [14C1]glycolate to [14C]oxalate was increased three-fold. Pyruvate had no effect on the synthesis of [14C]-oxalate or total oxalate. The inhibition studies suggest that hydroxypyruvate is a precursor of glycolate and oxalate and that the conversion of glycolate to oxalate does not involve free glyoxylate as an intermediate. [14C35Hydroxypyruvate, but not [14C1]hydroxypyruvate, was oxidized to [14C]oxalate in isolated perfused rat liver. Isotope dilution studies indicate the major pathway involves the decarboxylation of hydroxypyruvate forming glycolaldehyde which is subsequently oxidized to oxalate via glycolate. The oxidation of serine which is subsequently oxidized to oxalate via glycolate. The oxidation of serine to oxalate appears to proceed predominantly via hydroxypyruvate rather than glycine or ethanolamine. The hyperoxaluria of L-glyceric aciduria, primary hyperoxaluria type II, is induced by the oxidation of the hydroxypyruvate, which accumulates because of the deficiency of D-glyceric dehydrogenase, to oxalate.


Effect of colchicine on estrogen action. I. Inhibition of 17 beta-estradiol-induced water and potassium uptake in the immature rat uterus. The effects of colchicine on 17 beta-estradiol-induced water and electrolyte uptake in the uterus of the immature rat have been examined 6 h after treatment with this estrogen. Estradiol stimulates an increase in total uterine Na+, K+ and water while intracellular Na+ and K+ concentrations remain relatively unchanged. Assuming the sodium space is equivalent to the extracellular space, the extracellular fluid compartment increases about 84% in response to estradiol. Similarly, the intracellular compartment increases by about 62%. The uptake of water into the cellular compartment may be a direct response to a stimulation of K+ accumulation by uterine cells. Colchicine inhibits both estradiol-induced rise in intracellular potassium and both intra- and extracellular water.


Effect of colchicine on estrogen action. II. Translocation of 17 beta-estradiol cytosol receptor complex into the nucleus. Colchicine has previously been shown in our laboratory to inhibit 17 beta-estradiol stimulation of uterine water uptake in the immature rat measured 6 h after administration of the agents. We sought to determine whether this effect was mediated through colchicine action on translocation of estradiol receptor complex into the uterine cell nucleus. The time course of estradiol effect on uterine water uptake was followed with and without concurrent colchicine administration up to 6 h after administration. At no time during this period did there appear to be any influence of colchicine on translocation of the estradiol receptor complex into the nucleus. Examination of physical chemical characteristics of the nuclear estradiol receptr complex after estradiol and estradiol plus colchicine treatments revealed no  observable differences. Thus, colchicine inhibition of estradiol-stimulated uterine water retention does not appear to be mediated through inhibition of nuclear translocation of estradiol-receptor complex nor to be due to any reduced retention time of estradiol-receptor complex in uterine nuclei.


Oxidative and phosphorylative activities of the gastric mucosa of animals and humans in relation to the mechanism of stress ulcer. In order to elucidate the mechanism of stress ulceration, which is mainly involved with the corpus mucosa of the stomach, the aerobic energy metabolism of the corpus mucosa of rats, rabbits and humans was investigated and compared with those of the antral mucosa as well as other organs such as heart, kidney and liver. The results showed that the activities of respiration and oxidative phosphorylation and the concentration of the respiratory chain components were far greater in the corpus mucosa than in the antral mucosa, the activities in the former being almost equal to that of the highly aerobic tissue, kidney cortex. Furthermore, the endogenous respiratory rate (in the absence of added substrate) was also much higher in the corpus mucosa than in the antral mucosa, indicating that oxygen demand at resting state (during the period of H+ secretion) is also higher in the corpus mucosa. We conclude that in the corpus mucosa of the stomach the aerobic energy metabolism is predominant. It is suggested that circulatory insufficiency may easily result in cell damage mainly of the corpus mucosa due to energy deficit.


Dihydropyridine precursors of elastin crosslinks. Dihydrodesmosine and dihydroisodemosine are dihydropyridines which are believed to be the immediate biosynthetic precursors of desmosine and isodesmosine, the stable pyridinium ion crosslinks of elastin. It has recently been reported that appreciably amounts of dihydrodesmosine and dihydroisodesmosine accumulate in elastin. In view of the ease with which such dihydropyridines are oxidized to the corresponding pyridinium ions by O2 and other mild oxidants, dihydrodesmosine and dihydroisodesmosine would not be expected to accumulate in elastic tissues. It therefore seemed appropriate to analyse elastin samples for dihydrodesmosine and dihydroisodesmosine using techniques different from these previously employed for this purpose. The results of these investigations indicate that dihydrodesmosine and dihydroisodesmosine do not accumulate to a measurable extent in elastin.


Sodium exchange between two sites. The binding of sodium to halotolerant bacteria. The longitudinal and transverse relaxation curves of sodium undergoing exchange between two sites are presented. When the two sites are 'bound' and 'free' sodium respectively, the relaxation curves are, in general, not exponential. It is shown that in some cases only one exponential decay could be detected experimentally though the true decay curve is much more complicated. In such cases where the population of 'free' and 'bound' sodium are equal, only 40-70% of the total intensity would be detected, depending on the lifetime of sodium in the two sites. It is also shown that the fast exchange approximation, usually applied in the interpretation of sodium relaxation curves, might lead to wrong conclusions. Measurements of sodium relaxation times in halotolerant bacteria show that T1 and T2 are different and frequency-dependent. The intensity of the sodium sugnal is 40% of the total sodium concentration. It was possible to simulate the relaxation behaviour and intensity measurements by applying the following model. There are three types of sodium, the extracellular sodium (A) which exchanges with part of the intracellular sodium (B) and a fraction (C) which is bound but does not exchange with the extracellular sodium. It was possible to estimate the physical properties of sodium at site B. The quadrupole coupling constant (e2qQ/H)B = 9.10(6) rad/s, the correlation time taucB = 5.5.10(-7) s and the lifetime of sodium at site B, tauB = 6.10(-4) s.


Studies on lectins. XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins. By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0--40%, their sedimentation coefficient shows the vales of 2.5-5.7 S; the molecular weight of an O-alpha-D-mannopyranosyl polyacrylamide copolymer (29% mannose, so20,w = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.


Microbial metabolism of aliphatic glycols. Bacterial metabolism of ethylene glycol. A species of Flavobacterium isolated from pond water by its ability to grow aerobically on ethylene glycol as the role source of carbon initially oxidised the diol to glyoxylate via glycollate. The glyoxylate was metabolised by the glycerate pathway to acetyl-CoA. The acetyl-CoA was further metabolised by the tricarboxylic acid cycle plus malate synthase acting anaplerotically.


Estrogen-induced synthesis of riboflavin-binding protein in immature chicks. Kinetics and hormonal specificity. The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay procedure. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A two-fold amplification of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulations with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered 125I-labelled protein. Simultaneous administration of progesterone did not affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively counteracted the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the antiestrogens per se were completely ineffective in substituting for estrogen in the inductive process.


Precocious differentiation of mouse parotid glands and pancreas induced by hormones. Changes of alpha-amylase activity (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in mouse parotid gland and pancreas were investigated during embryonic and postnatal development. Amylase activity in the parotid gland increased from around day 12 and reached the adult level on day 30. On the other hand, the activity in the pancreas increased during the last stage of gestation, decreased after birth, and then gradually increased from around day 15, reaching the adult level on day 35. Precocious differentiation of the parotid gland was induced by injections of hydrocortisone or thyroxine after birth, but these hormones did not have additive effects on the parotid gland. Injection of insulin had little effect when given alone, but suppressed the effects of the other two hormones on the gland. Only hydrocortisone increased the amylase activity in mouse pancreas during postnatal development, the other two hormones causing slight decrease in pancreatic amylase. Adrenalectomy and injection of hydrocortisone affected the parotid gland but not the pancreas of adult mice. These results suggest that hydrocortisone is involved in cytodifferentiations of the parotid gland and pancreas, and in maintenance of the parotid gland. Thyroxine may also be important in differentiation of the parotid gland.


Carbohydrate-binding specificity of pokeweed mitogens. The carbohydrate-binding specificity of two pokeweed (Phytolacca americana) mitogens (Pa-1 and Pa-2) was investigated by means of hemagglutination inhibition assays and the quantitative inhibition of the binding of 125I-labeled lectins to human erythrocytes using various oligosaccharides, glycopeptides and glycoproteins as hapten inhibitors. Among the inhibitors employed in this study, chitin oligosaccharides and the glycopeptides and glycoproteins which bear sugar chains of the type found in serum glycoproteins, particularly PAS-1 glycoprotein and band-3 glycoprotein of human erythrocyte membranes, exerted strong inhibitory activity. The inhibitory constants of band-3 glycoprotein toward the binding of both mitogens to human erythrocytes were found to be very close to the association constants of the mitogens to the cells. Furthermore, the results of competitive binding studies between Pa-1 and Pa-2 indicated that these mitogens share a common oligosaccharide chains on the erythrocyte surface. To isolate the membrane receptors for these two mitogens, the solubilized membranes of human erythrocytes were subjected to affinity chromatographies using Pa-1-Sepharose 4B and Pa-2-Sepharose 4B as specific adsorbents. In both cases of these two specific adsorbents, band-3 glycoprotein was found to bind most strongly. These results suggest that two pokeweed mitogens have essentially the same carbohydrate-binding specificity and they bind primarily to the sugar chains of band-3 glycoprotein, possibly to the core structure of the sugar chains containing a di-N-acetylchitobiose moiety, on human erythrocytes.


Voltage-dependent capacitance in lipid bilayers made from monolayers. Electrocompression has been measured in lipid bilayers made by apposition of two monolayers. The capacitance C(V), as a function of membrane potential, V, was found to be well described by C(V) = C(O) [1 + alpha(V + delta psi)2] where C(O) is the capacitance at V = O, alpha is the fractional increase in capacitance per square volt, and delta psi is the surface potential difference. In lipid bilayers made from monolayers alpha has a value of 0.02 V-2, which is ca. 500-fold smaller than the value found in solvent containing membranes. In asymmetric bilayers made of one neutral and one negatively charged monolayer, delta psi values were found to be those expected from independent measurements of surface charge density. If the fractional increase in capacitance found here is a good approximation to that of biological membranes, nonlinear capacitative charge displacement derived from electrostriction is expected to be less than 1% of the total gating charge displacement found in squid axons.


Inner voltage clamping. A method for studying interactions among hydrophobic ions in a lipid bilayer. Ketterer, et al. (1971) have suggested that a combination of electrostatic and chemical interactions may cause hydrophobic ions absorbed within a bilayer lipid membrane to reside in two potential wells, each close to a membrane surface. The resulting two planes of charges would define three regions of membrane dielectric: two identical outer regions each between a plane of absorbed charges and the plane of closest approach of ions in the aqueous phase; and the inner region between the two planes of adsorbed charges. The theory describing charge translocation across the inner region is based on a simple three-capacitor model. A significant theoretical conclusion is that the difference between the voltage across the inner region, V(i), and the voltage across the entire membrane, V(m), is directly proportional to the amount of charge that has flowed in a voltage clamp experiment. We demonstrate that we can construct an "inner voltage clamp" that can maintain, with positive feedback, a constant inner voltage, V(i). The manifestation of proper feedback is that the clamp current (after a voltage step) will exhibit pure (i.e., single time-constant) exponential decay, because the voltage dependent rate constants governing translocation will be independent of time. The "pureness" of the exponential is maximized when the standard deviation of the least-square fit of the appropriate exponential equation to the experimental data is minimized. The concomitant feedback is directly related to the capacitances of the inner and outer membrane regions, C(i) and C(o).Experimental results with tetraphenylborate ion adsorbed in bacterial phosphatidylethanolamine/n-decane bilayers indicate C(i) approximately 5 . 10(-7)F/cm(2) and C(o) approximately 5 . 10(-5)F/cm(2).


Electrostatic interactions among hydrophobic ions in lipid bilayer membranes. We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.


A mechanism for 1/f noise in diffusing membrane channels. The diffusion polarization effect is shown to produce 1/f (omega-1) noise in the conductance of membranes containing diffusing ion channels. The magnitude and frequency range of the effect are calculated.


Myosin subfragment-1 attachment to actin. Expected effect on equatorial reflections. The characteristic equatorial X-ray pattern from a relaxed vertebrate skeletal muscle changes when the muscle is activated. In particular, there is a simultaneous decrease in the intensity of the first reflection (I10) and increase in the intensity of the second (I11). This observed change is almost reciprocal. When compared with the predictions of computer modeling, it produces a strong argument that the intensity change is due to a redistribution of myosin heads (myosin subfragment-1 or S-1), which results from the formation and configuration changes of actin-myosin links. Computer modeling shows that different actin-S-1 configurations will give different numerical values for I10 and I11, assuming the same number of attachments. For a given configuration, the intensity changes are a nonlinear function of attachment number, so that direct scaling of force to reflection intensity may be difficult. Data from active muscle are consistent with the notion that in different states of active muscle, i.e. shortening or isometric, there are different average configurations of actin-myosin attachment and different numbers of actin-myosin links.


Agnogenic myeloid metaplasia: a clonal proliferation of hematopoietic stem cells with secondary myelofibrosis. The glucose-6-phosphate dehydrogenase (G-6-PD) types and chromosomes of hematopoietic and other tissues were determined in a woman with agnogenic myeloid metaplasia. The patient was heterozygous at the X-linked G-6-PD locus so that both B and A isoenzymes were found in nonhematopoietic cells. In contrast, only one G-6-PD type was found in granulocytes, red cells, and platelets. She also had a distinctive chromosome abnormality in blood cells but not in other tissues. These results indicate that agnogenic myeloid metaplasia is a disorder of a pluripotent stem cell and provide strong evidence that it is of clonal origin. In contrast to blood cells, the patient's cultured marrow "fibroblasts" had normal chromosomes and both B and A G-6-PD types, suggesting that the marrow fibrosis is a secondary abnormality. Thus, at least in this case of agnogenic myeloid metaplasia, the hematopoietic cell proliferation appears to be clonal, and, by inference, possibly neoplastic, whereas the marrow fibrosis is probably not clonal, and therefore appears to be secondary.


Heterogeneity of human colony-forming cells. Culture supernatants from established human fibroblast cell lines (FCM) and from phytahemagglutinin-stimulated peripheral blood leukocytes (LCM) were compared with respect to their stimulatory effects on the colony formation by human marrow leukocyte precursors in vitro using the methylcellulose culture system. The cultures were performed using specimens from children with a variety of disorders. LCM stimulation consistently produced a significantly higher proportion of macrophage colonies than did FCM stimulation, whereas FCM stimulation resulted in a significantly higher number of number of neutrophil colonies. Based on the sequential examination of colony numbers in 35 FCM-and 11 LCM-stimulated cultures, the colonies in LCM-stimulated cultures (predominantly macrophage colonies) survived much longer than those in FCM-stimulated cultures (predominantly neutrophil colonies). Cell fractionation experiments by velocity sedimentation on four different specimens revealed that the majority of FCM-responsive cells were large and formed pure neutrophil colonies, while the majority of LCM-responsive cells were smaller and formed either pure macrophage or mixed neutrophil-macrophage colonies. These observations suggest that human colony-forming cells consist of at least two distinct cell populations different in (1) cell size, (2) response to two different conditioned media, LCM and FCM, and (3) type of colonies each population forms.


Proliferative state of normal in vitro colony-forming cells during development of L5222 rat leukemia and their reaction to chemotherapy. In the experimental rat leukemia, L5222, the decrease of normal in vitro colony-forming cells (CFU-C) after chemotherapy with daunomycin is much less than in nonleukemic controls. The leukemia is therefore used here to test the hypothesis that in leukemia the CFU-C are expelled from the active cell cycle to a resting state and are thereby less sensitive to cycle-dependent chemotherapeutic agents. The L5222 leukemia has the advantage that the leukemic blast cells do not form colonies in agar culture so that normal CFU-C can be assessed under leukemic conditions. To compare the proportions of CFU-C in the S-phase in normal and leukemic rats, two S-phase-specific agents, 3H-thymidine and hydroxyurea, were used to kill proliferating bone marrow cells. Following treatment with 3H-thymidine in vitro, about 41% of the CFU-C were killed in normal and about 25% in leukemic bone marrow. Hydroxyurea administered in vivo resulted in the death of about 33% and 26%, respectively. The results indicate that fewer normal CFU-C are in S-phase in the L5222 leukemia, which might help to explain how enough normal stem cells survive chemotherapy to regenerate the bone marrow.


Regulation of megakaryopoiesis in long-term murine bone marrow cultures. Megakaryocytes and their precursor cells were sustained in mouse bone marrow suspension cultures for over 4-6 wk. Megakaryocyte precursor cells were detected by their capacity to form colonies of megakaryocytes in semisolid agar cultures. Colony formation was dependent on the presence of medium conditioned by a myelomonocytic leukemic cell line (WEHI-3CM). Megakaryocytes from the liquid and semisolid cultures were identified by cytoplasmic acetylcholine esterase and by ultrastructural analysis. The suspension medium from the bone marrow liquid cultures which sustained megakaryopoiesis was not directly acitive in stimulating megakaryocyte colony formation in the semisolid agar cultures, but potentiated the number of colonies detected when WEHI-3CM was present. Bone marrow-conditioned medium increased the sensitivity of megakaryocyte progenitor cells to the stimulus in WEHI-3CM. Addition of the activities present in the two sources produced a quantitative assay for the detection of mouse megakaryocyte progenitor cells. These studies showed: (1) that no inductive regulator of in vitro clones of megakaryocytes was present in the supernatants from the long-term marrow cultures and, (2) that at least two factors were necessary for the induction of megakaryocyte progenitors to proliferate and differentiate in semisolid cultures in vitro.


Hypoprothrombinemia: case report.

Hypoprothrombinemia: case report. A patient with a significant history of spontaneous and posttraumatic bleeding was found to have hypoprothrombinemia. His prothrombin (factor II) activity by clotting assay was 9.5% and his factor II antigen was 5%. Crossed immunoelectrophoresis and sucrose density gradient ultracentrifugation of the patient's plasma showed his prothrombin to be qualitatively indistinguishable from normal prothrombin by these techniques.


Differences in the terminal steps of complement lysis of normal and paroxysmal nocturnal hemoglobinuria red cells. The number of microscopically visible lesions produced on the membrane for a given degree of lysis on normal cells as on PNH cells. Since complement lesions were not formed until C8 or C9 was incorporated into the complement sequence, the results suggest that increased lysis of red cells in PNH is due at least in part to more efficient penetration of the PNH membrane by the terminal lytic sequence of complement. Furthermore, the efficiency of the terminal lytic sequence in the lysis of PNH cells when complement was activated by the alternative pathway and the classical pathway was analyzed. There was no significant difference (p less than 0.01) in the number of lesion present at an equivalent degree of lysis when initiated by antibody, cobra venom factor, or acidification. Thus, the efficiency of the terminal lytic sequence does not vary with different modes of activation.


Interaction between Hb Hasharon and alpha-thalassemia: an approach to the problem of the number of human alpha loci. We report the case of an Italian infant girl from Polesine (Po delta region in northern Italy) who was heterozygous for Hb Hasharon and alpha-thalassemia, did not synthesize any normal HbA, and had 3% HbH on electrophoresis. Hematologic and biosynthetic studies on Hb Hasharon carriers of the propositus' family suggest the possibility that the Hb Hasharon gene is linked to an alpha-thalassemia gene. On the other hand, in the Askenazy carriers of Hb Hasharon, Hb Harsharon is probably linked to a normal alpha gene. In comparing Hb Hasharon's behavior with that of other alpha variants, particularly HbG Philadelphia, frequent recombinations between alpha structural genes were suggested. The possible identity between the single alpha locus and the alpha2-thalassemia genotype is discussed.


Defective iron uptake and globin synthesis by erythroid cells in the anemia of the Belgrade laboratory rat. Erythroid cell iron uptake and globin synthesis were studied in the anemia of the Belgrade Laboratory rat (gene symbol, b), an autosomal recessive trait characterized by hypochromia and hyperferrinemia. Reticulocyte protein and globin synthesis, as measured in vitro by the incorporation of 3H-L-leucine, were significantly diminished in b/b animals, although no major imbalance between alpha-and beta-chain production was observed in b/b reticulocytes. The incorporation in vitro of 3H-L-methionine into marrow cell globin demonstrated no difference between b/b animals and +/? control animals in the proportion of alpha-to beta-chain production. The transfer of iron from plasma to reticulocytes, as measured in vitro by the uptake of 59Fe, was significantly decreased in b/b animals; Sephadex G 200 chromatography of b/b red cell lysates did not reveal the accumulation of 59Fe in nonhemoglobin fractions found when heme synthesis was inhibited with isoniazid. The magnitude of the reticulocyte iron-uptake defect was greater than the reticulocyte globin-synthesis defect, suggesting that the former is the primary lesion.


The effects of glycerol detubulation, of manganese ions, of dantrolene and of nitrate ions on the responses of isolated chronically denervated soleus muscles of the mouse to acetylcholine. 1 In the isolated chronically denervated soleus muscle of the mouse acetylcholine produced a biphasic contraction. 2 In muscles detubulated by glycerol treatment, the first phase of the acetylcholine contraction was almost abolished, but the second was only slightly reduced or was unaffected. 3 Manganese ions (10 mM) reduced the first phase of the acetylcholine response, but enhanced the second. 4 Dantrolene sodium (5.94 x 10(-5) M) reduced the first phase of the acetylcholine response, but not the second. 5 Nitrate ions (118 mM) augmented the first phase but not the second. 6 It was concluded that the first phase requires the excitation-contraction-coupling sequence: membrane depolarization-T-tubules-Ca2+ release from sarcoplasmic reticulum. In contrast, the second phase appears to occur independently of these processes.


Functional changes in cerebral 5-hydroxytryptamine metabolism in the mouse induced by anticonvulsant drugs. 1 Acute administration of clonazepam, diazepam, and diphenylhydantoin to mice elevated cerebral 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA); chronic administration had less effect. 2 Acute administration of clonazepam and diazepam but not diphenylhydantoin raised cerebral trytophan levels; chronic administration of clonazepam caused a smaller elevation of cerebral tryptophan but chronic administration of diazepam still caused a large rise in cerebral tryptophan. 3 Neither clonazepam nor diazepam caused induction of drug metabolizing enzymes on chronic administration but diphenylhydantoin had a marked effect. 4 These data suggest that the altered 5-HT metabolism caused by these compounds is unrelated to a common action on tryptophan levels, and that the reduced effect of clonazepam and diazepam on chronic administration cannot be attributed to increased metabolism of these compounds. 5 Clonazepam induced abnormal head movements in mice in a dose-dependent manner. Pretreatment of animals with tranylcypromine increased the intensity of movement, although pargyline was without effect. Similar effects were observed with diazepam and diphenylhydantoin, suggesting that the increase in cerebral 5-HT caused by these compounds is of functional significance in stimulating 5-HT receptors.


The effect of lithium on the increase in forebrain 5-hydroxyindoleacetic acid produced by raphe stimulation. 1 The change in forebrain 5-hydroxyindoleacetic acid (5-HIAA) concentration induced by raphe stimulation has been studied in rats treated with Li+ or 0.9% w/v NaCl solution (saline) for 10 days. 2 Raphe stimulation increased the forebrain concentration of 5-HIAA in both groups of animals. Chlorimipramine abolished this effect in the control group, but not in the Li+ group. 3 The inhibition of 5-hydroxytryptamine (5-HT) uptake by chlorimipramine was not affected by pretreatment with Li+ or by the addition of Li+ to synaptosomal suspensions in vitro. 4 It is suggested that the production of 5-HIAA following raphe stimulation in Li+-treated animals is derived from the metabolism of 5-HT which remains within the intracellular environment. The consequence of this in relation to transmitter release is discussed.


Effects of minoxidil and nitroprusside on reflex increases in myocardial contractility. 1 The effects of nitroprusside and minoxidil on increases in myocardial contractility resulting from carotid artery occlusion were investigated in anaesthetized dogs. The results were compared with those produced by intravenous influsion of noradrenaline. 2 Nitroprusside and minoxidil attenuated the pressor responses produced by carotid artery occlusion. 3 Nitroprusside, but not minoxidil, attenuated the maximal myocardial contractility resulting from carotid occlusion. 4 The pressor and contractility responses to noradrenaline infusion were unaffected by either agent. 5 Nitroprusside failed to alter the myocardial responses produced by dimethylphenylpiperazinium. 6 These results, in conjunction with those of other investigators who have demonstrated that nitroprusside does not affect the release of noradrenaline from adrenergic neurons, suggest that nitroprusside may inhibit sympathetic nervous system reflex activity via an afferent and/or central component.


Effects of delta1-trans-tetrahydrocannabinol on mechanical performance of isolated heart muscle preparations. 1 The effects of four concentrations of delta1-trans-tetrahydrocannabinol (delta1-THC) (0.1, 1, 5 and 20 microgram/ml) were compared to that of the vehicle (ethanol, 0.5 microliter/ml) on the mechanical performance of isolated cardiac muscle of the cat and rat. 2 In rat isolated papillary muscles, delta1-THC (20 microgram/ml) caused a decline in mechanical performance (-8% in developed tension and -11% in Vmax) in contrast to the apparent lack of effect of ethanol. 3 All other parameters of mechanical performance studied in both rat and cat papillary muscles were unaffected by delta1-THC when compared with ethanol. 4 It was concluded that delta1-THC in concentrations up to 20 microgram/ml had negligible effect on contractile performance, the time course of contraction or muscle elasticity in rat and cat isolated papillary muscle preparations under the conditions studied.


Characterization of the adrenoceptor in the isolated cremaster muscle of the guinea-pig. 1 Contractions of the isolated cremaster muscle of the guinea-pig obtained in response to direct electrical stimulation were augmented by salbutamol, isoprenaline, adrenaline, noradrenaline and orciprenaline (in that order of potency), and by potassium chloride. Phenylephrine and xylometazoline had no effect. 2 The augmenting effect of isoprenaline on the twitch response was not altered by yohimbine or tolazoline, but was totally prevented by butoxamine, (--)-1-(4-nitrophenyl)-2-isopropylamino-ethanol (INPEA), (+/-)-INPEA, sotalol, metalol and practolol; (+)-INPEA was much less effective. The augmenting effect of potassium was not altered by beta-adrenoceptor blockade. 3 The results are consistent with the view that the augmenting effect of the sympathomimetic agents is mediated through beta2-type of adrenoceptor in the skeletal muscle fibres.


Subcellular distribution of etorphine in rat brain and evidence for in vitro stereospecific binding. 1 Control experiments were carried out by homogenizing rat brain at 0 degrees C with sucrose containing various concentrations of [3H]-etorphine. Subcellular fractionation of this homogenate showed that the distribution of the labelled drug amongst the primary fractions was dependent on the concentration of etorphine in the homogenate. 2 Rats were injected intravenously with 0.2 and 20 microgram/kg of [3H]-etorphine. The brains were homogenized and fractionated in sucrose containing 4.2 x 10(-5) M unlabelled etorphine in order to control redistribution artifacts. Different distribution profiles in the subcellular fractions were observed at these two dose levels. 3 Concurrent administration of either cyprenorphine or naloxone with intravenous etorphine, caused a shift of the labelled drug from the P3 fraction to the supernatant fraction. 4 The subcellular distribution of intravenously administered [3H]-etorphine was also studied by homogenizing brains in etorphine-free sucrose, and sucrose containing either levorphanol or dextrorphan. From these experiments it was concluded that the P3 microsomal fraction is a major site to which in vivo etorphine is stereospecifically bound in the rat brain.


The cardiovascular effects of cyclopropane in the intact, decerebrate and pithed rabbit preparations. 1 The anaesthetic cyclopropane was given to intact, decerebrate and pithed unanaesthetized rabbit preparations to determine the relative importance in vivo of its central and peripheral cardiovascular effects. 2 Cyclopropane elevated both the heart rate and the mean arterial pressure in the intact rabbit. 3 In the decerebrate rabbit, cyclopropane elevated the heart rate and efferent cervical preganglionic nerve activity and diminished the magnitude of these components of the aortic baroreceptor reflex, the mean arterial pressure being unaffected. 4 Apart from slight myocardial depression, cyclopropane was largely without effect in the pithed rabbit. 5 It is concluded that cyclopropane produces its cardiovascular effects by supra-collicular activation eliciting an elevation of mean arterial pressure, a central sub-collicular activation producing an increase in heart rate, and that in vivo the peripheral effects of cyclopropane are of minimal importance in comparison to these central effects.