Mössbauer investigations of high-spin ferrous heme proteins. I. Cytochrome P-450.

Anaerobically reduced samples of cytochrome P-450 from Pseudomonas putida were studied by Mössbauer spectroscopy. In the presence of an applied magnetic field the high-spin ferrous heme iron showed an intricate pattern of electric and magnetic hyperfine interactions which could be parametrized successfully in terms of a spin Hamiltonian formalism. The results imply a very low (triclinic) symmetry of the heme iron. The effects of the ligand environment and of spin-orbit coupling result in a large zero-field splitting of the electronic ground state. The electronic ground state. The electric-field gradient tensor is characterized by a large asymmetry parameter, and its principal axes are rotated substantially from the frame that defines the zero-field splitting. This study shows that high-field Mössbauer spectroscopy provides a unique tool for structural investigations of high-spin ferrous compounds and can substitute, under suitable conditions, for magnetic susceptibility measurements. The present paper focuses on the methodology and data analysis; in the subsequent paper the data obtained for P-450 are compared with new results obtained for hemoglobin, chloroperoxidase, and horseradish peroxidase.


Mössbauer investigations of high-spin ferrous heme proteins. II. Chloroperoxidase, horseradish peroxidase, and hemoglobin.

Reduced samples of chloroperoxidase, horseradish peroxidase, and deoxyhemoglobin were studied by Mössbauer spectroscopy in strong magnetic fields. The intricate paramagnetic spectra of chloroperoxidase were evaluated in detail in the framework of a spin Hamiltonian pertinent to high-spin ferrous iron. The studies strongly suggest that, in their reduced states, chloroperoxidase from Caldariomyces fumago and cytochrome P-450 from Pseudomonas putida have similar, if not identical ligand structures of the heme iron. The spectral similarities of these two proteins, noted in an earlier Mössbauer investigation, are further explored and substantiated. Reduced horseradish peroxidase and deoxyhemoglobin, on the other hand, show high-field Mössbauer spectra that differ considerably from each other and, in particular, from those of the P-450 type, suggesting a different ligand arrangement of the heme iron for each case.


Magnetic circular dichroism studies of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.


Complexes of DNA with histones f2a2 and f3. Circular dichroism studies.

Two histones from calf thymus, the slightly lysine-rich histone f2a2 and the arginine-rich f3, were combined separately, with homologous DNA. The complexes were reconstituted by means of guanidine hydrochloride gradient dialysis, and their circular dichroic (CD) spectra were examined in 0.14 M NaCl. The CD spectra of f2a2-DNA complexes are characterized by a positive band at 272 nm which is blue-shifted and greatly enhanced relative to the corresponding band for native DNA. This type of CD change was noted previously with f2a1-DNA and f2b-DNA complexes. In contrast, f3 histone causes only minor distortions in the DNA CD spectrum, and their character depends upon the state of the two sulfhydryl groups in f3. When the cysteines are reduced, f3-DNA complexes have a slightly increased positive band with a small blue shift; when oxidized disulfide is the predominant form, this CD band becomes slightly smaller than native DNA value. This laboratory has now examined complexes reconstituted from DNA and all five histones of calf thymus. The sum of the CD spectra of these complexes, although very similar to the CD curve for reconstituted complexes containing whole histone, does not approximate that of chromatin; the consequence of this observation is discussed.


S1 nuclease hydrolysis of single-stranded nucleic acids with partial double-stranded configuration.

The single-strand specific nuclease S1 from Aspergillus oryzae (EC 3.1.4.21) was purified 600-fold in 16% yield from dried mycelia. Determination of the isoelectric point of S1 nuclease as 4.3-4.4 allowed adjustment of chromatographic conditions such that the enzyme was isolated free of contaminating ribonucleases T1 and T2. S1 nuclease so purified was used for removal of single-stranded portions from the RNA of the Escherichia coli phage MS2, which has a helical content of about 65% in vitro. At 23 degrees, increasing amounts of enzyme converted the RNA to mononucleotides in about equimolar base ratios. No small intermediates of chain length 2-8 were found. At 0 degrees, MS2 RNA hydrolysis was slower and reached, in exhaustive digests, a plateau where 70% of the substrate RNA remained insoluble in 66% EtOH. With [32P]MS2 RNA, strip chart counting of 6% acrylamide-6 M urea electrophoresis patterns of such digests gave recoveries of 80-91% in the form of defined oligomer bands. On 2.5% acrylamide-0.5% agarose gels, the molecular weights of the major oligomers were found to range from 25,000 to 41,000. Similar to purified tRNAArg used as a control, these oligomers were not resistant to pancreatic RNase-RNase T1 hydrolysis at 37 degrees, and were not bound on hydroxylapatite at 50 degrees in 0.14 M sodium phosphate (pH 6.8). Melting of the oligomers gave complex profiles without a clear Tm and showed an increase in A260 of 35% at 93 degrees over that at 28 degrees. Upon formaldehyde denaturation of MS2 RNA prior to S1 nuclease hydrolysis, no resistant oligomers were found.


Immobilized diol dehydrase and its use in studies of cobalamin binding and subunit interaction.

Coenzyme B12 dependent diol dehydrase from Aerobacter aerogenes was immobilized by covalent binding to CNBr-activated Sepharose 4B. The Sepharose-bound enzyme exhibited a markedly high catalytic activity, viz., 75-95% of the specific activity of the original free enzyme. The apoenzyme acquired much greater stability to heat by immobilization. No significant difference between the immobilized and free enzymes was observed in the following properties: the affinity for coenzyme B12; the sensitivity to a sulfhydryl-modifying agent; the absolute requirement for a certain monovalent cation, such as K+, for catalysis; the susceptibility toward oxygen upon incubation with coenzyme B12 in the absence of substrate. These results suggest that the structure and function of the enzyme are not significantly influenced by immobilization on Sepharose. The immobilized enzyme was found to provide a convenient method for a study of ligand interaction with the enzyme. The subunit interaction between two dissimilar subunits, components F and S, was investigated using the component S immobilized on CNBr-activited Sepharose and free component F, and it was demonstrated that the substrate (1,2-propanedoil) promotes the hybrid formation between component F and component S, but K+ alone rather retarded the subunit association to some extent. Na+ markedly weakens the forces which bind the subunits together. The relationship between cobalamin binding and subunit structure is also discussed.


Stoichiometry, ATP/2e values, and energy requirements for reactions catalyzed by nitrogenase from Azotobacter vinelandii.

The stoichiometry of the nitrogenase ATP-dependent H2 evolution and ecetylene reduction reactions using S2O4(2-) as an electron source was studied by various techniques. For each mole of S2O4(2-) oxidized to 2SO3(2-) by the enzyme-catalyzed reactions at 25 degrees and pH 8, 1 mol of H2 (1 mol of ethylene for acetylene reduction) and two protons are produced. Under these conditions, 4.5 mol of ATP was hydrolyzed to ADP and inorganic phosphate for each S2O4(2-) oxidized. ATP/S2O4(2-) (ATP/2e) values determined at 5 degree intervals from 10 to 35 degrees were found to go through a minimum at 20 degrees. This effect is explained in terms of possible enzyme structure modifications. Calorimetric measurements for the enzyme-catalyzed H2 evolution and acetylene reduction reactions gave deltaH values of -32.4 and -75.1 kcal/mol of S2O4(2-), respectively.


Sheep liver serine-threonine dehydratase. I. Purification and evidence for covalently linked alpha-ketobutyrate as its cofactor.

L-Serine-threonine dehydratase (EC 4.2.1.16) from sheep liver has been obtained as a highly purified preparation as shown by ultracentrifuge studies and analytical disc gel electrophoresis. The dehydratase has a molecular weight of 98,000 +/- 10,000 and is composed of two nonidentical subunits with molecular weights of 41,000 and 47,000. The 41,000 subunit is covalently linked to the carbonyl reagent-sensitive coenzyme which has been identified as alpha-ketobutyric acid.


Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands.

Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.


The uncatalyzed rates of enolization of dihydroxyacetone phoshate and of glyceraldehyde 3-phosphate in neutral aqueous solution. The quantitative assessment of the effectiveness of an enzyme catalyst.

By a combination of methods involving enzyme-catalyzed reactions and classical iodination techniques it has been possible to obtain all the relevant rate constants for the uncatalyzed interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate via their common enediol intermediate. These rate constants are compared with those for the individual steps of the triosephosphate isomerase catalyzed reaction, and a quantitative picture of the effectiveness of the enzyme as a catalyst has been delineated. It is apparent that the enzyme increases the enolization rate of dihydroxyacetone phosphate by a factor of more than 10(9) over that of the uncatalyzed reaction.


Magnesium-induced self-association of calf brain tubulin. II. Thermodynamics.

The thermodynamic parameters of the magnesium ion induced self-association of calf brain tubulin in pH 7.0, 0.01 M phosphate buffer containing 10(-4) M GTP, were determined from sedimentation velocity experiments. This reaction proceeds by an isodesmic mechanism terminated by the highly favored formation of a closed ring shaped polymer of degree of association 26 +/- 4. Analysis of the variation of the apparent dimerization constant in the isodesmic mechanism s,ows that this self-association is characterized by positive enthalpy, entropy, heat capacity, and molar volume changes, as well as the binding of one additional magnesium ion, which is probably not involved as a bridge between the protein molecules. The addition of the last monomeric subunit has a free energy which is about three times that of dimer formation. Under the conditions of these experiments, tubulin binds 48 +/- 5 magnesium ions with a free energy of --2.8 kcal/mol.


An investigation of the conformational and self-aggregational processes of histones using 1H and 13C nuclear magnetic resonance.

Histone self-aggregation processes have been studied by 13C and 1H nuclear magnetic resonance (NMR) as a function of ionic strength and protein concentration. Thus has led to a model involving apolar aggregation between structured regions of these molecules. This analysis supports the validity of the acquistion of conformational data on histones by the simulation of 13C NMR spectra at high concentration. Solution conformations for histones F2B and F3 are presented.


An immunological approach to the role of the low molecular weight subunits in myosin. I. Physical--chemical and immunological characterization of the light chains.

The light chains of chicken breast muscle myosin (alkali 1 and 2, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 1.c.) have been isolated in pure form and characterized with respect to amino acid composition, uv and circular dichroism (CD) spectral properties, and molecular weight. Antibodies specific for each of the light chains have been used to demonstrate the similarity of alkali 1 and 2 (mol wt 21,000 and 16,000, respectively), and the distinctness of these from DTNB 1.c. (mol wt of 18,000). The DTNB 1.c. isolated by a variety of methods were all immunologically identical. Significant cross-reactivity was observed between corresponding rabbit and chicken light chains, confirming other indications of homology between these proteins in the two species. The immunological difference between alkali 1 and 2 was largely accounted for by an N-terminal peptide, rich in proline, alanine, and lysine, which is unique to alkali 1. The presence of antibodies to this peptide in anti-alkali 1 serum suggests an immunological approach to the question of how alkali 1.c. are distributed in myosin.


A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II.

An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.


The role of bound calcium ions in thermostable, proteolytic enzymes. I. Studies on thermomycolase, the thermostable protease fron the fungus Malbranchea pulchella.

Thermomycolase, the thermostable, extracellular, serine protease of the fungus Malbranchea pulchella (G. Voordouw, G. M. Gaucher, and R. S. Roche (1974), Can J. Biochem. 52, 981-990), binds one calcium ion with an apparent binding constant of 5 X 10(5) M-1 at 25degreesC, pH 7.50, and ionic strength 0.1. There is very little change in the overall conformation of thermomycolase upon binding of this calcium ion: no change can be detected, beyond experimental error, in the sedimentation coefficient or the aromatic and peptide circular dichroism of the enzyme. However, binding of calcium changes the absorption spectrum, the ultraviolet difference spectrum being characterized by a strong band at 237 nm and smaller bands at 280 and 295 nm. The difference molar extinction coefficient at 237 nm parallels the calcium-binding isotherm. The small changes in equilibrium properties are constrasted by large calcium-dependent changes in the rates of autolytic degradation and thermal and urea denaturation. The dependence of the second-order rate constant for autolytic degradation on the free calcium ion concentration can be quantitatively accounted for by a model in which only conformers with an unoccupied calcium binding site serve as substrates in the reaction. The calcium dependence of the first-order rate constant for thermal denaturation at 70degreesC and pH 7.0 can also be accounted for quantitatively by a model in which the critically activated intermediate has a smaller calcium-binding constant than the native form of the enzyme under these conditions. The same model also accounts for the denaturation in 8 M urea at 50degreesC, pH 7.0. Rates of hydrogen-tritium exchange are shown to decrease when the calcium ion is bound. Irrespective of the occupancy of the calcium-binding site 33% of the backbone peptide hydrogens of thermomycolase do not exchange within 24 hr at 25degreesC, pH 8.0, and ionic strength 0.1.


The role of bound calcium ions in thermostable, proteolytic enzymes. II. Studies on thermolysin, the thermostable protease from Bacillus thermoproteolyticus.

The functional properties of the four calcium ions, bound by thermolysin, appear to be very similar to those of the single calcium ion bound by thermomycolase (G. Voordouw and R.S. Roche (1975), Biochemistry, preceding paper in this issue). Hence when the free calcium ion concentration is varied in the range where the calcium double-site dissociates (G. Voordouw and R.S. Roche (1974), Biochemistry 13, 5017), no changes are observed in the sedimentation coefficient or the peptide circular dichroism. Differences in molar ellipticity and molar extinction coefficient occur in the aromatic ultraviolet region, which parallel the occupancy of the calcium binding double site. The difference spectrum, characterized by a main band at 290 nm and a somewhat smaller band at 283 nm, is interpreted as due to the transfer of a partially buried tryptophan residue to the aqueous solvent upon dissociation of the two calcium ions from the double site. This is most likely Trp-186, which is in between Asp-185 and Glu-187, two chelating amino acids of this site. From the calcium dependence of the rate constant for autolytic degradation we conclude, as for thermomycolase, that only conformers devoid of bound calcium ion serve as substrates in the reaction. This rate constant increases about 1000-fold, when the double site dissociates. Hydrogen-tritium exchange studies show the presence of a large stable strcutural core, comprising about 32% of all the peptide hydrogens present. These do not exchange-in after 24 hr at 25degreesC, pH 9.0, ionic strenth 0.1. The exchange-out of 60 slow hydrogens was found to be independent of the free calcium ion concentration in the range 2.0-8.0 X 10(-4) M, where all four calcium-binding sites are saturated. The calcium dependence of the first-order rate constant for thermal denaturation at 80degreesC, pH 7.0, indicates that thermolysin is stabilized by only one calcium ion under these conditions. These observations are rationalized in terms of a calcium-binding model for thermolysin and the known three-dimensional structure of the enzyme and its calcium-binding sites.


The orientation and accessibility of substrates on the active site of triosephosphate isomerase.

Tritiated sodium borohydride was used to reduce the substrates of triosephosphate isomerase in the presence of the enzyme, and the mixture of the four possible products (D-[1(R)-3H]; D-[1(S)-3H]-; D-[2-3H]-, and L-[2-3H]glycerol 3-phosphate) was analyzed. While enzyme-bound dihydroxyacetone phosphate is reduced completely stereoselectively and at a rate eight imes faster than in free solution, D-glyceraldehyde 3-phosphate is inaccessible to reduction by borohydride when bound to the active site of the enzyme.


Fluorescence and bioluminescence of bacterial luciferase intermediates.

An intermediate in the luciferase-catalyzed bioluminescent oxidation of FMNH2, isolated and purified by chromatography at -20degrees, was postulated to be an oxygenated reduced flavin-luciferase. Maintained and studied at -20 to -30degrees, this material exhibits a relatively weak fluorescence emission peaking about 505 nm when excited at 370 nm. It may comprise more than one species. Upon continued exposure to light at 370 nm, the intensity of this fluorescence increases, often by a factor of 5 or more, and its emission spectrum is blue shifted to a maximum at about 485 nm. Upon warming its fluorescence is lost and the fluorescence of flaving mononucleotide appears. If warming is carried out in the presence of a long chain aldehyde, bioluminescence occurs, with the appearance of a similar amount of flavine fluorescence. The bioluminescence yield is about the same with irradiated and nonirradiated samples. The bioluminescence emission spectrum corresponds exactly to the fluorescence emission spectrum of the intermediate formed by irradiation, implicating the latter as being structurally close to the emitting species in bioluminescence.


Microsomal monooxygenation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Synthesis and identification of cis and trans monohydroxylated products.

Liver microsomal hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur on the cyclohexyl ring at positions 3 and 4. Four metabolites were isolated by selective solvent extraction and purifed by high-pressure liquid chromatography. cis-4-, trans-4-, cis-3-, and trans-3-OH derivatives of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea were synthesized and their chromatographic, mass spectral, and nuclear magnetic resonance characteristics matched those of the metabolites. The position of ring hydroxylation and the identity of each geometric isomer were established by nuclear magnetic resonance using a shift reagent in conjunction with spin decoupling techniques. Microsomes from rats pretreated with phenobarbital showed a sixfold increase in hydroxylation rate (19.5 vs. 3.3 nmol per mg per min). The induction was quite selective for cis-4 hydroxylation (19-fold); however, induction of trans-4 (threefold), cis-3 (threefold), and trans-3 (twofold) hydroxylation did occur. Quantitatively the cis-4-hydroxy metabolite was 67of the total product by phenobarbital-induced microsomes and 21% for normal microsomes. Microsomes from animals pretreated wit- 3-methyl-cholanthrene gave about the same rate and product distribution that normal microsomes gave. A mixture of 80% carbon monoxide-20% oxygen inhibited formation of all four hydroxy metabolites with the inhibition ranging from 55 to 78%.


Ligand-induced self-association of human luteinizing hormone. Negative cooperativity in the binding of 8-anilino-1-naphthalensulfonate.

The self-association of human luteinizing hormone (hLH) is enhanced in the presence of 8-anilino-1-naphthalenesulfonate (ANS). Sedimentation equilibrium measurements indicate that the hormone exists primarily as a dimer in the presence of excess ANS. It is shown that, for a self-associating protein system in which monomer and dimer have different affinities and/or capacities for ligand, both the shape and the position of the binding curve depend on protein concentration. Gel filtration and fluorescence measurements indicate that the hLH dimer has a single high affintiy site (K greater than 10(6) M-1) for ANS while binding to the monomer is too weak to be observed. This leads to negative cooperativity in the binding and to a shift of the binding curve to lower free ligand concentration with increasing concentration of the hormone.


Ligand-induced self-association of human chorionic gonadotropin. Positive cooperativity in the binding of 8-anilino-1-naphthalenesulfonate.

Human chorionic gonadotropin (hCG) self-associates to form higher molecular weight species in the presence of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). Sedimentation equilibrium and fluorescence titration data have been analyzed in terms of a monomer-dimer-tetramer model in which the various oligomers have different affinities and/or capacities for the ligand. The results indicate that the ligand affinities are in the order tetramer greater than dimer greater than monomer whereas the numbers of ligand binding sites per mole of hCH are in the reverse order. Consequently, addition of ANS first shifts the equilibrium from monomer to tetramer and gives rise to positive cooperativity in the titration curves. At sufficiently high ANS concentration (approximately 0.5 mM), the equilibrium shifts back to the dimer because of its greater binding capacity. This is manifested by a second phase in the titration curve and a decrease in the polarization of ANS fluorescence. The results are discussed in terms of the general problem of ligand controlled protein association and are contrasted to results reported to the previous paper for the homolgous protein, human luteinizing hormone.


Effects of estradiol on uterine ribonucleic acid metabolism. Assessment of transfer ribonucleic acid methylation.

Immature rats treated with estradiol for selected periods of time demonstrated both increased methylation of uterine transfer ribonucleic acid (tRNA) and methylase activities. Whereas the former parameter was assessed by incubating whole uteri with [methyl-14C]methionine and measuring the incorporation of isotope into the tRNA, methylase activity was obtained by measuring the rate of incorporation of methyl groups from S-adenosyl[methyl-14C]methionine into heterologous tRNA (Escherichia coli B) in the presence of uterine cytosol preparations (100,000g supernatants). Although increased methylation of tRNA during the estrogen response was demonstrated, additional studies indicated that these results were largely attributable to an increased rate of synthesis of tRNA rather than gross changes in either the type or amount of methylated constituents present. Evidence in this regard included the inability of estrogen treatment of alter significantly the (a) resulting patterns of methyl-14C-methylated constituents of uterine tRNA, (b) the extent ot which [2-14C]guanine residues, incorporated into tRNA, become methylated, (c) the extent of methylation of precursor tRNA in the absence of tRNA synthesis, and (d) the types of methylase activities expressed in vitro.


Osmotic pressure induced pores in phospholipid vesicles.

We report a comparative study of the leadage of hydrophilic molecules from vesicles of egg lecithin (EL) and of dipalmitoyllecithin (DPL). The effect of osmotic pressure differences a leakage is consistent with a model for statistical pore nucleation process. The major difference in osmotic pressure induced leakage from DPL and EL is that the number of pore creation sites is much greater in DPL. We suggest that the difference in number of these sites also accounts for other differences in the properties of DPL and EL, namely for differences in vesicle fusion and apparent rate of "flip-flop".


Radioimmunological identification of polysomes synthesizing fibrinogen polypeptide chains.

Hepatocytes of rats stimulated by turpentine into a hyperfibrinogenemic state produce sufficient quantities of fibrinogen to permit unequivocal identification of specific polysomal complexes involved in the synthesis of this molecule. Monospecific antibodies directed against intact fibrinogen and one of its subunits, the gamma-chain, have shown two size classes of polysomes. Furthermore, it seems possible that polypeptide chain assembly may occur by having completed nascent chains bind to partially completed chains that are still attached to the polysome.


Purification of murine thymus leukemia antigen (TL). A quantitative assessment of limited proteolysis.

The murine thymus leukemia antigen (TL) has been solubilized from the tumor ASL1 and from an established cell line ASL1W, by papain digestion. When a 15-min digest was chromatographed on Sephadex G-200, two peaks of TL activity were eluted with apparent molecular weights of approximately 58,000 and 31,000. Chromatography of a 30-min digest under the same conditions resulted in elution of a single peak of activity with an apparent molecular weight of 58,000. Additional purification was carried out on the 58,000 molecular weight material by absorption to, and elution from DEAE-cellulose. The combination of gel filtration and ion exchange chromatography resulted in approximately a 150-fold purification.


Subcellular redistribution of seryl-transfer RNA during estrogen-induced phosvitin synthesis and specificity of the estrogen effect.

The estrogen-induced hepatic synthesis of the serine-rich protein, phosvitin, in chickens is accompanied by an increase in the serine acceptance of total hepatic tRNA, which is limited to two sering isoacceptors. To investigate the role of the tRNA alterations in the synthesis of phosvitin, the relative amounts of various seryl-tRNA species in isolated nuclei and in free and membrane-bound ribosomes were determined. Estrogen treatment causes a shift in the subcellular distribution of hepatic seryl-tRNA. Of the four serine isoacceptors, the amount of tRNASerAGU,AGC was specifically increased in nuclei and in membrane-bound ribosomes. Changes in total hepatic tRNA occurring during physiologic estrogenization were compared with those occurring by varying steroid hormones, times after estrogen administration, estrogen doses, animal ages, and tissue types. The changes observed demonstrate that the seryl-tRNA alterations are closely correlated with the synthesis of phosvitin. The coincident change in seryl-tRNA levels and in phosvitin synthesis, together with the specific change in cellular localization, suggests that the amount and subcellular distribution of each tRNA species are separately controlled in a manner dependent upon its frequency of use in translation.


Transbilayer exchange of phosphatidylethanolamine for phosphatidylcholine and N-acetimidoylphosphatidylethanolamine in single-walled bilayer vesicles.

A preparation of small single-walled liposome vesicles containing a 9:1 mole ratio of phosphatidylcholine to phosphatidylethanolamine was subjected to reaction with the membrane-impermeable reagent, isethionyl acetimidate hydrochloride. This reagent converted 90% of the external phosphatidylethanolamine groups to the amidine derivative, leaving the mole ratio of unreacted phosphatidylethanolamine to phosphatidylcholine on the outside surface of the vesicle much lower than that on the inside surface. Equilibration of phosphatidylethanolamine across the bilayer was then measured as a function of time by monitoring the appearance of phosphatidylethanolamine on the outside surface utilizing the reaction of the amino groups with 2, 4, 6-trinitrobenzenesulfonic acid. The results show that no new phosphatidylethanolamine appeared on the external surface of the vesicles over a period of 12 days at 22 degrees. A conservative estimate of the precision of the measurements is +/- 10%. On this basis, the estimated half-time for the equilibration of phosphatidylethanolamine across the bilayer of these vesicles must be at least 80 days at 22 degrees.


Structure of the human erythrocyte blood group P1 glycosphingolipid.

A glycosphingolipid with blood group P1 activity was extracted from an acetone powder of human erythrocyte stroma with chloroform-methanol. It was purified by chromatography on columns of silicic acid and by preparative thin-layer chromatography of the fully acetylated and deacetylated glycolipid. The purified glycolipid contained galactose, N-acetylglucosamine, and glucose in a molar ratio of 3:1:1. Treatment of the P1 glycolipid with fig alpha-galactosidase released a single galactosyl residue and destroyed the blood group activity, and the alpha-galactosidase product had the same chromatographic mobility as paragloboside. Substitution sites on the neutral sugars of the P1 glycolipid and the alpha-galactosidase product were established by identification of methylated alditol acetates, and substitution on N-acetylglucosamine was determined by identification of methyl glycoside derivatives. The terminal nonreducing disaccharide of the P1 glycolipid is Gal(alpha, 1 leads to 4)Gal. N-Acetylglucosamine was identified as the next sugar in sequence by mass spectrometric analysis of the permethylated P1 glycolipid. On the assumption that the glucose residue is linked to ceramide, we propose the following structure for the P1 glycolipid: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-NAc(beta, 1 leads to 4)Glc-Cer.


An immunochemical study of the human blood group P1, P, and PK glycosphingolipid antigens.

The erythrocyte PK and P blood group antigens have been identified as ceramide trihexoside (CTH), Gal-(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, and globoside, GalN-Ac(beta, 1 leads to 3)Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, respectively, and the following structure has been proposed for the P1 antigen: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)GlcNAc(beta, 1 leads to 3)Gal(beta, 1 leads to 4)Glc-Cer. Although the P1 and PK determinants have identical terminal disaccharides, CTH did not inhibit anti-P1. The P1 glycolipid and hydatid cyst glycoprotein inhibited the agglutination of P1K erythrocytes by anti-P1 and unabsorbed anti-P1PPK sera, but neither antigen inhibited a specific anti-PK serum. The P1 and PK glycolipids were equally effective in inhibiting the hemagglutinating activity of a lectin with alpha-galactosyl specificity obtained from ova of Salmo trutta. Anti-P sera were inhibited most effectively by human erythrocyte globoside, and to a lesser extent by Forssman glycolipid and rat kidney globoside. In the latter glycolipid the linkage between the internal galactosyl residues is alpha, 1 leads to 3, rather than alpha, 1 leads to 4, as in erythrocyte globoside. No cross-reactions between P and P1 or PK antigens were detected. New hypotheses are offered to explain the genetic regulation and biosynthesis of the P1, P, and PK antigens.


Proton magnetic resonance studies of double helical oligonucleotides. The effect of base sequence on the stability of deoxydinucleotide dimers.

The concentration dependence of the proton magnetic resonance chemical shifts of a series of deoxydinucleotides and deoxydinucleoside monophosphates in neutral H2O solution has been recorded in the 1-100 mM concentration range by the use of pulsed Fourier transform techniques. The self-complementary molecules pdG-dC, dG-dC, pdC-dG, and dC-dG and the complementary mixtures pdG-dG + pdC-dC as well as pdG-dT + pdA-dC interact at low temperatures by the formation of intermolecular hydrogen bonded dimers. Noncomplementary molecules such as pdG-dT, pdT-dG, pdG-dG, pdA-dc, and pdC-dC do not self-associate by the formation of intermolecular hydrogen bonds under the present experimental conditions. The chemical shifts of the amino protons and the base protons are consistent with the interaction of two complementary dinucleotides to form a miniature double helix. An analysis of the chemical shift of the guanine amino proton resonance as a function of dinucleotide concentration has provided approximate dimerization constants. These results show that the stability of the miniature double helices is in the order (pdG-dG)-(pdC-dC) greater than or approximately (pdG-dC)-(pdG-dC) greater than (pdC-dG)-(pdC-dG) greater than (pdG-dT)-(pdA-dC) which reflects the effect of nucleotide sequence (and composition) on helix stability.


Conformation and interaction of short nucleic acid double-stranded helices. I. Proton magnetic resonance studies on the nonexchangeable protons of ribosyl ApApGpCpUpU.

1H nuclear magnetic resonance (NMR) spectra of a self-complementary ribosyl hexanucleotide, A2GCU2, are investigated as a function of temperature and ionic strength in D2O. Seventeen nonexchangeable base and ribose-H1' resonances are resolved, and unequivocally assigned by a systematic comparison with the spectra of a series of oligonucleotide fragments of the A2GCU2 sequence varying in chain length from 2 to 5. Changes in the chemical shifts of the 17 protons from the hexamer as well as the six H1'-H2' coupling constants are followed throughout a thermally induced helix-coil transition. These sigma vs. T and J vs. T (degrees C) profiles indicate that the transition is not totally cooperative and that substantial populations of partially bonded structures must exist at intermediate temperatures, with the central G-C region being most stable. Transitions in chemical shift for protons in the same base pair exhibit considerable differences in their Tm values as the data reflect both thermodynamic and local magnetic field effects in the structural transition, which are not readily separable. However, an average of the Tm values agrees well with the value predicted from studies of the thermally induced transition made by optical methods. The values of J1'-2' for all six residues become very small (less than 1.5 Hz) at low temperatures indicating that C3'-endo is the most heavily populated furanose conformation in the helix. The sigma values of protons in the duplex were compared with those calculated from the ring current magnetic anisotropies of nearest and next-nearest neighboring bases using the geometrical parameters of the A'-RNA and B-DNA models. The sigma values of the base protons in the duplex calculated assuming the A'-RNA geometry agree (+/- approximately 0.1 ppm) with the observed values much more accurately than those calculated on the basis of B-DNA geometry. The measured sigma values of the H1' are not accurately predicted from either model. The synthesis of 35 mg of A2GCU2 using primer-dependent polynucleotide phosphorylase is described in detail with extensive discussion in the microfilm edition.


Conformation and interaction of short nucleic acid double-stranded helices. II. Proton magnetic resonance studies on the hydrogen-bonded NH-N protons of ribosyl ApApGpCpUpU helix.

A self-complementary ribohexanucleotide, ApApGpCpUpU, was synthesized and its NH-N hydrogen-bonded protons were studied by proton magnetic resonance. At 1 degree C, 0.17 M Na+, pH 7.6 with 10 mM phosphate-0.1 mM EDTA in H2O, three proton resonances are found in the low-field region with the following chemical shifts and line widths at half-height: 13.2 ppm (80 Hz), 13.5 ppm (30 Hz), and 14.2 ppm (44 Hz). The existence of these resonances indicates the formation of a self-complementary, hydrogen-bonded duplex under these conditions. Upon elevation of temperature, these three resonances sequentially broaden and finally all disappear near 35 degrees C. Unambiguous assignments of these three resonances can be made to the terminal A(1)-U(6) pairs, interior A(2)-U(5) pairs, and to the middle G(3)-C(4) pairs. The assignments were based on (i) the differential sensitivities of the line widths of these resonances to thermal variation, as well as on (ii) a comparison of the computed chemical shifts with the observed chemical shifts. The quantitative aspects of the NH proton transfer between helix, coil, and water are discussed in relationship to the line widths of these resonances and the lifetime of the helix state. The computed chemical shifts of the NH-N resonances based on the A-RNA (or A'-RNA) model agree more closely with the observed chemical shifts than the computed values based on the B-DNA model. These results suggest that the helical duplex of A2GCU2 assumes a conformation similar to A-RNA (or A'-RNA) in aqueous solution. The results on both the NH-N resonances and the C-H resonances are summarized and discussed in terms of the helical conformation of (A2GCU2)2.


Terbium binding to ribosomes and ribosomal RNA.

Terbium binding to rat liver ribosomes and ribosomal RNA (rRNA) was examined by equilibrium dialysis and fluorescence spectroscopy. Upon binding to ribosomes and rRNA, the enhancement of terbium fluorescence emission at both 488 and 541 nm was dependent only upon the amount of bound terbium and independent of ionic strength. Binding profiles for ribosomes and rRNA suggested that terbium was bound to ribosomes primarily through rRNA interactions. Data suggested that terbium mimicked characteristics previously described for interactions between ribosomes and magnesium. It is proposed, therefore, that fluorescence of terbium bound to ribosomes may prove useful in studies on the nature and extent of interactions between ribosomes and magnesium.


Surface and solution properties of steroid antibiotics: 3-acetoxylfusidic acid, cephalosporin P1 and helvolic acid.

The colloid/chemical properties of the fusidane antibiotics, 3-acetoxylfusidic acid, cephalosporin P1, and helvolic acid, and their sodium salts, were investigated. The sodium salts of 3-acetoxylfusidic acid and cephalosporin P1 were found to be detergent-like molecules with micellar properties comparable to the parent compound sodium fusidate and the bile salt sodium cholate. Critical micellar temperatures (cmt) were less than 0 degrees C except for sodium helvolate which being sparingly soluble did not form micelles between 0 and 50 degrees C. Potentiometric titrations of dilute solutions gave apparent pK values (5.2-6.5) in the range expected for carboxylated steroid detergents. The apparent pK values increased significantly once the detergent concentration exceeded the critical micellar concentration (cmc). Micellar properties were determined by surface tension, titration with a water-soluble dye (Rhodamine 6G), light scattering, and solubilization of lecithin and cholesterol. Cmc's, in the range of 1.5 to 5.6 mM, were found which varied slightly depending on the method employed and in all cases fell slightly in the presence of added NaCl. The number of monomers per micelle (aggregation number) in concentrations well above the cmc was extrapolated from Debye light scattering plots in 0.15 M NaCl. The values varied from 6 for fusidate to 14 for 3-acetoxylfusidate with sodium cephalosporin P1 having an intermediate value. Each detergent readily solubilized the phospholipid lecithin.


Actinomycin D-deoxynucleotide complexes as models for the actinomycin D-DNA complex. The use of nuclear magnetic resonance to determine the stoichiometry and the geometry of the complexes.

The use of proton and carbon-13 magnetic resonance spectroscopy for the determination of the geometry and the stoichiometry of the actinomycin D-deoxyguanosine 5'-monophosphate complex is outlined. The dimerization of actinomycin D has been reexamined by recording the proton magnetic resonance spectrum of actinomycin D to much lower concentrations through the use of Fourier transform nuclear magnetic resonance techniques. The effect of the actinomycin D dimerization on the observed chemical shifts that results from the additon of nucleotides to an actinomycin D solution is directly demonstrated by comparing the actinomycin D-nucleotide titrations at both low (approximately 0.3 mM) and high (approximately 12 mM) concentrations of actinomycin D. In the presence of excess nucleotide the chemical shifts of the actinomycin D groups were essentially the same for both the low and high concentration titrations. The complexes of actinomycin D with pdG-dC, dG-dC, deoxyguanosine 3'-monophosphate, G-C, C-G, dIMP(5'), 2, 6-diaminopurine deoxyribose, and other nucleotides were also investigated by proton magnetic resonance and visible spectral titrations. These data were interpreted in terms of the molecular geometry of the complexes and in terms of the effect of the structure of the nucleotide base on the relative binding affinity of the nucleotides for the two nucleotide binding sites of actinomycin D. The carbon-13 chemical shifts of dGMP(5') were measured as a function of concentration over the concentration range of 0.5-0.025 M. The infinite dilution carbon-13 chemical shifts were graphically estimated from the dilution curves. These values were used to calculate the changes in the chemical shifts of the dGMP carbons that result from the formation of an actinomycin D-(dGMP)2 complex. It was not possible to interpret these carbon-13 chemical shift changes in terms of only ring current effects, which thus rules out the use of carbon-13 spectroscopy in the determination of the geometries of the actinomycin D complexes with the mono- and dinucleotides. The induced chemical shifts in the proton spectra may be used in the determination of the geometries of the complexes. A consideration of these data for the above nucleotide series shows that the predominant complex formed is one in which the guanine rings in the two nucleotide binding sites of actinomycin D are oriented in a manner very similar to that observed in the cocrystalline complex of actinomycin D with deoxyguanosine.


Isolation of a protease from sea urchin eggs before and after fertilization.

Upon fertilization, sea urchin eggs (Stronglyocentrotus pupuratus) release a protease into the surrounding sea water. This protease is in a particulate form which can be solubilized. The soluble form was purified by affinity chromatography on columns of immobilized soybean trypsin inhibitor. The purified enzyme is similar to bovine trypsin both in molecular weight (22500) and in susceptibility to inhibitors such as diisopropyl phosphofluoridate and soybean trypsin inhibitor. In contrast, extracts of unfertilized eggs appear to contain an inactive form of the enzyme which can be activated by dialysis at pH 4.6. The enzyme, as purified from extracts activated in this manner, was similar in its properties to that from fertilized eggs.


Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.


The molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI refined at 2.0-A resolution.

The structure of the variable portions of a K-type Bence-Jones protein REI forming a dimer has been determined by X-ray diffraction to a resolution of 2.0 A. The structure has been refined using a constrained crystallographic refinement procedure. The final R value is 0.24 for 15000 significantly measured reflections; the estimated standard deviation of atomic positions is 0.09 A. A more objective assessment of the error in the atomic positions is possible by comparing the two independently refined monomers. The mean deviation of main-chain atoms of the two chains in internal segments in 0.22 A, of main-chain dihedral angles 6.3 degrees for these segments. The unrefined molecular structure of the VREI dimer has been published (Epp, O., Colman, P., Fehlhammer, H., Bode, W., Schiffer, M., Huber, R., and Palm, W. (1974), Eur. J. Biochem. 45, 513). Now a detailed analysis is presented in terms of hydrogen bonds and conformational angles. Secondary structural elements (antiparallel beta structure, reverse turns) are defined. A more precise atomic arrangement of the amino acid residues forming the contact region and the hapten binding site is given as well as the localization of solvent molecules. Two cis-prolines (Pro-8 and Pro-95) were detected. The intrachain disulfide bridge (Cys-23-Cys-88) occurs statistically in two alternative conformations. The structure suggests reasons for strong conservation of several amino acid residues. The knowledge of the refined molecular structure enables crystal structure analyses of related molecules to be made by Patterson search techniques. The calculated phases based on the refined structure are much improved compared to isomorphous phases. Therefore the effects of hapten binding on the molecular structure can be analyzed by the difference Fourier technique with more reliability. Hapten binding studies have been started.


Solution studies on heme proteins: subunit structure, dissociation, and unfolding of Lumbricus terrestris hemoglobin by the ureas.

The subunit structure, dissociation, and unfolding of the hemoglobin of the earthworm, Lumbricus terrestris, were investigated by light scattering molecular weight methods and changes in optical rotatory dispersion (at 233 nm) and absorption in the Soret region. Urea and the alkylureas, methyl-, ethyl-, propyl-, and butylurea, were employed as the reagents to cause both dissociation and unfolding of the protein. Analysis of the light scattering data suggests that the dissociation patterns as a function of hemoglobin concentration in the various dissociating solvents can be described in quantitative terms, either as an equilibrium mixture consisting of parent duodecamers and hexamers of 3 x 10(6) and 1.5 x 10(6) molecular weight (in 1-3 M urea, 1-2 M methyl- and ethylurea, and 1 M propylurea), as a mixture of hexamers and monomers, the latter with a molecular weight of 250000 (i.e., in 4 M urea), or as a mixture of all three species of duodecamers, hexamers, and monomers, seen in 2 M propylurea. Parallel studies by optical rotation and absorption measurements indicate that there is little or no unfolding of the subunits at urea and alkylurea concentrations where complete dissociation to hexamers and extensive dissociation to monomers can be achieved. Further splitting of the monomers (A subunits) to smaller fragments of one-third to one-quarter of the molecular weight of the monomers (B subunits) is seen in the presence of 7 and 8 M urea (pH 7) and in alkaline urea to propylurea solutions. Analysis of the dissociation data of duodecamers to monomers, based on equations used in studies of the urea and amide dissociation of human hemoglobin A from our laboratory, suggests few urea and alkylurea binding sites at the areas of hexamer contacts in the associated duodecameric form of L. terrestris hemoglobin. This suggests that hydrophobic interactions are not the dominant forces that govern the state of association of L. terrestris hemoglobin relative to polar and ionic interactions. The unfolding effects of the ureas, at concentrations above the dissociation transitions, are closely similar to their effects on other globular proteins, suggesting that hydrophobic interactions play an important role in the maintenance of the folded conformation of the subunits. Use of the Peller-Flory equation, with binding constants based on free energy transfer data of hydrophobic amino acid side chains and denaturation data used in previous denaturation studies, gave a relatively good acount of the observed denaturation midpoints obtained with the various ureas supporting these conclusions.


The effects of salts on the subunit structure and dissociation of Lumbricus terrestris hemoglobin.

The effects of the neutral salts of the Hofmeister series, NaCl, NaClO4, MgCl2, NaI, and also guanidine hydrochloride (Gdn-HCl)on the subunit organization and the state of association of Lumbricus terrestris hemoglobin were examined by light scattering molecular weight measurements. The subunit dissociation of the parent duodecameric structure of 3 x 10(6) molecular weight by various salts is similar in pattern to the sequential splitting of the associated protein to half-molecules of hexamers of 1.5 x 10(6) molecular weight, followed by further dissociation at higher reagent concentration to monomers of 250000 molecular weight. Duodecamer to hexamer dissociation is observed in 0.4 M MgCl2, 1-2 M NaCl, and 1 M Gdn-HCl, while hexamer to monomer dissociation is seen in the presence of 1 M MgCl2. All three species of duodecamers, hexamers, and monomers seem to be present in 1 M NaClO4. Further splitting of the monomers of A subunits to smaller B fragments of one-third to one-quarter molecular weight is observed in 1 M NaI solutions. Optical rotation in the peptide region and absorption measurements in the Soret region indicate the salt dissociation of Lumbricus terrestris hemoglobin is not accompanied by major changes in the folding of the subunits, except in the case of the strong protein denaturant, Gdn-HCl. Relative to the dissociation effects of the urea series of compounds reported in the preceding paper (Herskovits and Harrington, 1975), the neutral salts appear to be much more effective dissociating agents for L. terrestris hemoglobin. This suggests that polar and ionic interactions are relatively more important for the maintenance of the protein than hydrophobic interactions. This conclusion is also supported by calculations of the possible effects of binding of NaClO4, based on the Setschenow constants of the literature describing the interaction of salts with the peptide and hydrophobic alkyl group of the average amino acid found in proteins, on the standard free energy of dissociation of the duodecamer to hexamer.


Effect of vitamin B-12 deficiency on the hepatic tissue concentration of acyl carnitines.

Concentrations of carnitine, acetyl carnitine, propionyl carnitine, and long chain acyl carnitines have been measured in hepatic tissue of normal and vitamin B-12 deficient rats using radiolabelled butyrobetaine to label carnitine pools. Increased levels of propionyl carnitine were seen in the livers of vitamin B-12 deprived animals when compared to those from normal animals. Methylmalonyl carnitine was not detected in the B-12 deprived animals. Free carnitine levels were no different in the livers from the B-12 deprived animals than from the normal control animals.


Synthesis of sulphoquinovosyl diacylglycerol by higher plants.

The biosynthesis of sulphoquinovosyl diacylglycerol in germinating alfalfa seeds has been examined. Some incorporation of [35S]sulphate into the lipid occurs before chlorophyll production anh this is unaffected by chloramphenicol. Cysteic acid, molybdate, sulphite and sulpholactic acid all reduce incorporation of [35S]sulphate into sulphoquinovosyl diacylgylcerol. Some comparisons are made with other seed types. The results indicate that sulphoquinovosyl diacylgylcerol synthesis in alfalfa probably proceeds by a pathway similar to that in Euglena.


Comparison of the acylation of sn-glycerol 3-phosphate and membrane-bound lipid in the microsomal fraction from rabbit brain throughout maturation.

1. Age-related changes in the specific activity of palmitoyl-CoA synthetase, sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and the esterification of [3H]palmitate into endogenous lipid in the microsomal fraction from rabbit brain have been determined throughout development. 2. The increased specific activity of sn-glycerol 3-phosphate acyltransferase at the onset of myelination (rising in parallel with other lipogenic enzymes) is consistent with a direct role of the acyltransferase in promoting the accumulation of cerebral lipid. In adult brain microsomes, although the specific activity was low, the total activity was only 20% lower than during active myelination. 3. Palmitoyl-CoA, synthesized by the palmitoyl-CoA synthetase in the microsomal membrane, was the preferred substrate for the esterification of sn-glycerol 3-phosphate. There was no evidence for a pool of palmitoyl-CoA formed from palmitate. 4. The esterification of [3H]palmitate into membrane-bound lipid remained high throughout development and may be part of an acyl-exchange cycle via lysophospholipids. [3H]palmitate was incorporated into both neutral lipids and phospholipids, while phosphatidic acid was the major product of sn-[1(3)-3H]-glycerol-3-phosphate esterification. 5. The microsomal fraction contained a pool of unesterified fatty acid, which was activated and esterified into sn-glycerol 3-phosphate.


Biosynthesis of acyl groups on molecular species of biliary phosphatidylcholines during metabolism of [2,2,2-2H3]ethanol.

Incorporation of deuterium into different positions of individual molecular species of biliary phosphatidylcholines was determined in bile fistula rats given [2,2,2-2H3]ethanol under conditions ensuring maximal rate of oxidation for 24 h. The deuterium-labelling of the glycerol moiety of the major molecular species was about 6-8 atom% at the end of ethanol administration. The deuterium excess at each of the different positions of the glycerol moiety of 1-palmitoyl-2-linoleoyl phosphatidylcholine was less than 3 atom%. From the isotopic composition of the palmitoyl residues of the phosphatidylcholines, it was calculated that [2,2,2-2H3]ethanol supplied about 35-40% of the acetyl-CoA forming the terminal methyl group and about 25-30% of the other C2 units of the palmitic acid chain. This difference in deuterium incorporation was interpreted as being due to an isotope effect, probably in the rate-limiting carboxylation step of acetyl-CoA. Most or perhaps all of the acetyl groups derived from ethanol were introduced into the terminal methyl group without loss of deuterium. This indicates that citrate is not an important carrier of acetyl-CoA in the biosynthesis of fatty acids from ethanol.


The intermicellar bile salt concentration in equilibrium with the mixed-micelles of human bile.

The intermicellar bile salt concentration in equilibrium with the bile salt-lecithin-cholesterol mixed-micelle has been studied in human bile. Equilibrium-dialysis, used to measure the biliary intermicellar bile salt concentration, has been validated as an applicable method by studying the cholate-lecithin mixed-micelle, for which intermicellar bile salt concentration values have previously been reported. The intermicellar bile salt concentration of bile was essentially independent of ionic strength in the range 0.05-0.15 M chloride. Simple dilution of bile lowered the intermicellar bile salt concentration (about 2/3 reduction for each two-fold dilution). This reduction occurred because of a simultaneous decrease in the molar ration of bile salt/phospholipid in the micelle. Dilution of micelles with micellar bile salt/phospholipid held constant did not affect the intermicellar bile salt concentration. The relationship between intermicellar bile salt concentration and micellar bile salt/phospholipid, defined in the dilution studies, was linear in the range of study. For a composite of five biles, this relationship was described by the equation: intermicellar bile salt concentration = 1.27 (bile salt/phsopholipid) + 0.538. Data obtained on an artificial bile agreed closely with the results obtained on bile suggesting that the other constituents of bile did not affect this analysis. These findings may be helpful in understanding the process of micellar cholesterol solubilization in bile.


Influence of sialic acid groups on the retention of glycosphingolipids in blood plasma.

The removal of several glycosphingolipids from the circulation and their disposal in different tissue and fluid compartments was studied in adult rats. 3H-labeled dihydro analogs of several glycosphingolipids were injected intravenously and radioactivity was measured in arterial blood samples at subsequent time intervals, to obtain half life values for the labeled compound in the plasma. Half life values of less than 1 min were obtained for neutral glycosphingolipids whereas the half lives of labeled gangliosides were much longer and ranged from 3.8 to 21 h. The prompt removal of labeled neutral glycosphingolipids but not of the gangliosides indicates that sialic acid groups play a significant role in the retention of glycosphingolipids in the circulation. The results suggest that neutral glycosphingolipids are rapidly exchanged with their counterparts in a large extraplasma pool and that a major portion of this exchange could occur between plasma and liver. The detection of only a minute fraction of the injected glycosphingolipids in the cerebrospinal fluid indicates that a blood-cerebrospinal fluid barrier exists for these compounds in the rat.


Study of amphibian lipids. I. Characterization of monoglycosylceramides from the skin of Rana nigromaculata (Japanese pond frog).

1. Monoglycosylceramide was isolated from the skin of Rana nigromaculata (Japanese pond frog), and further fractionated into three subgroups (Fraction I, Fraction II and Fraction III) by borate-impregnated Florisil column chromatography. 2. Fraction I and Fraction II contained mainly glucose as their hexose components, while Fraction III contained galactose. 3. Major long chain bases of Fraction I and Fraction III were D-erythro-1, 3-dihydroxy-2-amino-4-trans-octadecene (4-sphingenine) and D-erythro-1, 3-dihydroxy-2-aminooctadecane (sphinganine), whereas those of Fraction II were D-ribo-1, 3, 4-trihydroxy-2-aminooctadecane (4D-hydroxysphinganine) and 1, 3, 4-trihydroxy-2-aminoeicosane (C20 homologues of 4D-hydroxysphinganine). This is the first evidence of the presence of trihydroxy base-containing glycolipids in the skin of vertebrates. 4. All three subgroups of monoglycosylceramide contained both hydroxy and nonhydroxy fatty acids ranging from C14 and C26. Saturated fatty acids represented more than 90% of the total. Some differences of the fatty acid composition in the three subgroups were also observed.


Isolation and characterization of C50-carotenoid pigments and other polar isoprenoids from Halobacterium cutirubrum.

The polar acetone-soluble lipids of Halobacterium cutirubrum were found to contain (in addition to the previously reported vitamin MK-8 and retinal) neo-bacterioruberin U, bacterioruberin, monoanhydrobacterioruberin, bis-anhydrobacterioruberin, an isomer of geranylgeraniol (with one internal cis-isoprene residue), 2,3,-di-O-phytanyl-sn-glycerol and two unidentified polar isoprenoids. All compounds were isolated in pure form by column and thin-layer chromatography, quantitated and characterized by their visible, ultraviolet, infrared, proton magnetic resonance and mass spectra and the spectra of their acetyl or silyl derivatives and/or dehydrated products.


Transfer of esterified cholesterol from serum lipoproteins to the liver.

The fate of cholesteryl esters of the serum lipoproteins was studied in intact rats and in isolated perfused rat livers. The lipoproteins of fasting rat serum were labeled in vitro with [3H]cholesteryl oleate. Following intravenous injection, it was found that the majority of the radioactive ester was rapidly taken up by the liver where hydrolysis of the ester bond occurred. At 5 min, 58% of the injected material was recovered in the liver, 85% of which was still in the ester form, while at 30 min only 22% of the liver radioactivity was in cholesteryl esters. There was very little difference in the rate at which radioactivity was taken up from the different lipoprotein classes. Similar phenomena were observed in the perfused liver, but it was found that although the radioactive esters were being taken up, there was no change in the concentrations of free or esterified cholesterol in the perfusing medium, indicating that the lipoprotein cholesteryl ester was gaining access to the liver through an exchange of molecules. After uptake, cell fractionation experiments showed that the plasma membranes had the greatest relative amounts of radioactivity, suggesting that this is the site of exchange. Small amounts of radioactivity were recovered in the bile, demonstrating that serum lipoproteins can serve as precursors of at least some of the bile steroids.


Two-carrier models for mediated transport. I. Theoretical analysis of several two-carrier models.

Several possible models of two sequential and two simultaneous carriers of different affinities are theoretically analysed. Following the analysis we suggest for each model an experimental procedure capable of testing and rejecting the model.


Two-carrier models for mediated transport. II. Glucose and galactose equilibrium exchange experiments in human erythrocytes as a test for several two-carrier models.

1. Equilibrium exchange of glucose and of galactose is measured in human erythrocytes over wide concentration ranges. One-site Michaelis-Menten-type kinetics are shown for the two sugars. 2. In view of the results, two of the two-carrier models discussed in paper I (Eilam, Y. (1975) Biochim. Biophys. Acta 401, 349-363), the different sequential carriers and the antiparallel simultaneous carriers, are rejected for the sugar transfer system. The antiparallel sequential carriers model is consistent with these results.


Characterization of autoantigenic sites on isolated dog heart mitochondria.

1. Anti-heart mitochondria autoantibodies were developed in serum from dogs following experimental myocardial infarction. 2. Heart mitochondria frozen and thawed repeatedly in a sucrose/Tris-chloride buffer retained both their functional integrity as measured by the respiratory control ratio and their ability to serve as an antigen in a complement fixation test. Mitochondria frozen and thawed in a potassium chloride/Tris-chloride buffer lost both their functional integrity and their autoantigenic activity after one freeze-thaw cycle. 3. Extraction of the heart mitochondria with acetone/water mixtures to remove phospholipids from the membrane led to a complete loss of the ability of the mitochondria to react in the complement fixation test but did not affect the ability of the membranes to bind autoantibody in absorption experiments. 4. Treatment of the mitochondrial membranes with increasing concentrations of trypsin caused a loss of up to approximately 50% of the membrane protein with a gradual decrease in the autoantigenic activity of the membrane without impairment of the ability of the membrane to bind autoantibody. 5. Removal of up to 90% of the sialic acid of the mitochondrial membrane with neuraminidase resulted in a considerable increase in the complement-fixing autoantigenic activity of the membrane without changing the apparent ability of the membrane to bind autoantibody in absorption experiments. 6. Exposure of mitochondrial membranes to autoantibody and complement caused an inhibition of both an inner mitochondrial membrane enzyme, i.e. cytochrome oxidase (48%) and an outer mitochondrial membrane enzyme, i.e. NADH cytochrome c reductase (rotenone insensitive) (37%).


Accelerative exchange diffusion kinetics of glucose between blood and brain and its relation to transport during anoxia.

An earlier study showed that unidirectional glucose transport from blood to brain decreases during perfusion with anoxic blood (Betz, A. L., Gilboe, D. D. and Drewes, L. R. (1974) Brain Res. 67, 307-316). Brain glucose levels also decrease during anoxia. Therefore, the present study was designed to investigate whether the decreased transport might be the result of decreased accelerative exchange diffusion when brain glucose levels are low. The rate of undirectional transport into brain (v) of D-[6-3H]glucose was studied in 22 isolated, perfused dog brains by means of an indicator dilution technique using 22Na as the intravascular reference. The kinetics of transport were determined over a range of blood glucose concentrations (S1) at each of five different brain glucose levels (S2). The existence of accelerative exchange diffusion for glucose was indicated by a decrease in the intercept (increase of apparent V) of a double reciprocal plot (1/v versus 1/S1) as S2 increased. This phenomenon is consistent with a model for facilitated diffusion in which the mobility of the loaded carrier is greater than that of the unloaded carrier. Although the data predict a decrease in glucose transport during anoxia, the predicted decrease (5%) is less than the observed decrease (35%). It is concluded that the simple mobile-carrier model for facilitated diffusion cannot, by itself, describe all properties of blood-brain glucose transport.


Sugar transport by intestine. Escape of galactose from preloaded mucosa of hamster jejunum.

Everted hamster jejunum was loaded with D-galactose and then escape into an initially galactose-free mucosal solution was followed. Mucosal anaerobiosis greatly increased the rate of escape, an effect which might have been caused by inhibiting reuptake from the unstirred layer and/or by augmenting the ease of unidirectional efflux across the brush border membrane. The former effect was expected because of our previous results from influx studies, and the main object here was to find out if the ease of efflux is affected by anaerobiosis. With phlorizin present in the mucosal solution during escape, information about unidirectional efflux was obtainable. We estimated that 10(-4) M phlorizin inhibited the ease of efflux via the phlorizin-sensitive pathway by about 65%. Apparently the reason why mucosal phlorizin accelerates escape of sugar from loaded mucosa, an effect which has been reported previously by others, is that it inhibits unidirectional efflux less effectively than it inhibits reuptake from the unstirred layer. Residual efflux via the phlorizin-sensitive pathway was markedly increased by mucosal anaerobiosis. This increase did not require an elevation of intracellular Na+ concentration. These results, together with those of our previous study, show that mucosal anaerobiosis abolishes uphill transport of galactose across the brush border of hamster jejunum by inhibiting unidirectional influx and by increasing the ease of unidirectional efflux. Neither of these effects requires a rise in intracellular Na+ concentration.


Experimental alteration of phospholipid-protein interactions within the human erythrocyte membrane. Dependence on glycolytic metabolism.

Phosphatidylethanolamine in freshly drawn human erythrocytes is trinitrophenylated by 2,4,6-trinitrobenzene sulfonic acid only slowly and to a maximum of 32%. After different preincubation procedures at 37 degrees C in saline media in the absence of glucose (24 h without additive, 1-5 h with 8 mM hexanol or 1-4 h with the SH reagent, 5 mM tetrathionate) the rate of subsequent trinitrophenylation of phosphatidylethanolamine, in the absence of the additives, is greatly enhanced and the amount of phospholipid reacting increased. Glucose or inosine prevent these effects, inhibitors of glycosis abolish this protection. The results indicate that in fresh as well as in glycolysing incubated erythrocytes phosphatidylethanolamine in the outer layer of the membrane lipid is shielded by a protein. Conformational changes of this protein induced by metabolic starvation and perturbing agents expose the phospholipid head group to 2, 4, 6-trinitrobenzene sulfonic acid. In addition, a "flip-flop" of phosphatidylethanolamine from the inner to the outer layer may also contribute to the effects observed.


Response of transmural electrical parameters across in vitro everted sacs of hamster jejunum to variations in oxygenation rate.

1. The response of the electrical potential difference, short circuit current, and resistance across everted sacs of hamster jejunum to variations in the mucosal solution gassing rate was investigated. 2. Contrary to previous reports by others, it was found that the potential difference responds to increases in mucosal solution gassing rate by increasing in magnitude during the first 20 min of incubation. 3. The increases in potential difference were parallelled by increases in short circuit current but not by changes in resistance. 4. Increases in mucosal solution gassing rate increased epithelial cell O2 availability and this effect was determined to be due to increased stirring by gas bubbles. From the data, it was deduced that the minimum thickness of the mucosally located functional unstirred layer is between 0.08 and 0.16 cm when less than the full magnitude of electrical activity is observed across the everted sac preparation. 5. Serosal N2 or O2 had little or no effect on electrical parameters under maximum mucosal oxygenating conditions but dramatically affected these parameters when less than maximum mucosal oxygenating conditions were used. 6. Qualitative variations in the magnitude of the short circuit current across this preparation with respect to Cl- dependence were demonstrated. These variations were dependent upon the level of O2 availability, being apparent at high levels of O2 and absent at low levels. 7. It is concluded that the thickness of mucosally located unstirred layers can determine the O2 availability to the mucosa of everted sacs of hamster jejunum and thereby influence the observable magnitude and pattern of ionic dependence of the short circuit current across this preparation.


Membrane mobility agents. II. Active promoters of cell fusion.

The membrane mobility agent, A2C, actively promotes the fusion of hen erythrocytes under conditions similar to those used by Lucy et al. for glyceryl monooleate.


Molecular order in Acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids.

The first application of deuterium magentic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37 degrees C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.


Isozymes of rat AMP deaminase.

Three AMP deaminase isozymes (EC 3.5.4.6 AMP aminohydrolase) were purified from rat heart, kidney and muscle. These enzyme preparations contained only the required isozyme. Antisera to individual isozymes were prepared and immunological relationships were tested. There was no cross-reactivity as tested by precipitation experiments. The antisera precipitated only the corresponding isozyme and there was no effect on other isozymes. These isozymes were also different in Km values for AMP and in substrate specificity. From the present studies, combined with previous results, it seems clear that the heart, kidney and muscle enzymes are different basic types. It is proposed that the muscle enzyme be designated as AMP deaminase A; the enzyme in kidney and liver, AMP deaminase B; the enzyme in heart, AMP deaminase C. Brain extracts contained five isozymes; two parent isozymes (B and C) and presumably their three hybrids.


Metabolism of monofluorobenzoates by Acinetobacter calcoaceticus N.C.I.B. 8250. Formation of monofluorocatechols.

None of the monofluorobenzoates serves as sole source of carbon and energy for growth of Acinetobacter calcoaceticus but all can contribute to growth on other substrates. The monofluorobenzoates are oxidised by bacteria pre-induced for benzoate oxidation and can themselves induce the appropriate enzymes. The initial products of oxidation have been separated and identified by gas-liquid chromatography. 2-Flurobenzoate is oxidised to catechol, fluoride and 3-fluorocatechol; 3-fluorobenzoate gives 3- and 4-fluorocatechol; 4-fluorobenzoate gives 4-fluorocatechol. The fluorocatechols appear to be partially oxidised beyond the stage of 3-oxoadipate by suitably pre-induced bacteria.


[In vitro biosynthesis of iodothyronines from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (author's transl)].

A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)2-Tyr(I)2) (equimolecular in tyrosyl rings). Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction. Synthesis of thyroid hormones from Tyr(I)2-Tyr(I)2 is faster and more important than from diiodo-3,5-L-tyrosine (Tyr(I)2). A mechanism of iodothyronine formation via Tyr(I)2-Tyr(I)2 and different from the one occuring for Tyr(I)2 is suggested.


Inability of the estrogen-induced uterine protein to serve as a substrate for the endogenous phosphokinase(s).

We have studied the phosphorylation of soluble proteins from uterine extracts by an endogenous protein kinase. The analysis of phosphorylation patterns by polyacrylamide gel electrophoresis did not reveal any significant difference in this respect between the soluble proteins from control or 17-beta-estradiol stimulated uteri. In both cases, three main components with mol. wt of about 120,000, 60,000 and 45,000 appear preferentially phosphorylated. Estrogen-induced protein did not coincide with any phosphorylated component, although some migrated very closely to it. This was observed whether phosphorylation was performed on uterine extract incubated with [gamma-3 2P]ATP or on intact organs incubated in the presence of 3 2Pi. We conclude that whatever the role of estrogen-induced protein, it is unlikely to be subjected to regulation through the phosphorylation process.


Control over activation or synthesis of phenylalanine ammonia-lyase by phytochrome in mustard (Sinapis alba L.)? A contribution to eliminate some misconceptions.

1. Density labelling with 80 atom% of 2H2O has been used to examine the mode of action of phytochrome (continuous far-red light) in increasing levels of L-phenylalanine ammonia-lyase (EC 4.3.1.5) activity in cotyledons of developing mustard seedlings (Sinapis alba L.). 2. Bandwidths and density shifts of isopycnically banded enzyme show that in darkness the enzyme was synthesized de novo, continuously turning over (half-life approx. 3 h) and that maximum labelling achievable was reached at 12 h. 3. 2-fold (6h), 5-fold (12h) and 10-fold (24 h) light-mediated increases in enzyme activity were accompanied by a similar pattern of labelling as observed in darkness. 4. Experimental evidence and theoretical arguments are presented which make it unlikely that phytochrome increases enzyme activity by slowing down the rate of degradation or by activating preformed enzyme molecules. 5. The conclusion is drawn that the rate of turnover of phenylalanine ammonia-lyase in dark-grown mustard cotyledons is too rapid compared to the measured rise in enzyme activity for density labelling to reveal directly control over the rate of synthesis de novo by phytochrome. However, the elimination of other control mechanisms leads us to the conclusion that phytochrome most probably does control synthesis of this enzyme in mustard, which agrees with the previous findings for parsley cells.


Polyamines and ornithine decarboxylase activity during growth and differentiation in Blastocladiella emersonii.

The activity of ornithine decarboxylase (EC 4.1.1.17, L-ornithine carboxy-lyase) was determined during the life cycle of Blastocladiella emersonii. The specific activity of the enzyme was found to be low in the zoospores, to rise 20-fold during germination and early growth, to fall during growth and to rise again during sporulation. This rise in enzyme activity was shown to be dependent on protein synthesis. Putrescine levels, on a per mg of protein basis, paralleled the fluctuation found in ornithine decarboxylase activity. Putrescine and spermidine were the only polyamines found in extracts of B. emersonii.


Role of calcium in exocrine pancreatic secretion. I. Calcium movements in the rabbit pancreas.

1. Calcium movements in the isolated rabbit pancreas and in rabbit pancreas fragments have been studied with the aid of 4 5 Ca2+. 2. Addition of 4 5 Ca2+ to the incubation medium of the isolated rabbit pancreas results in an immediate appearance of isotope in the secreted fluid reaching a constant specific activity in 30 min. The absolute activity in the secreted fluid is 30-40% of that in the incubation medium. 3. Addition of 10(-5) M carbachol after 2 h preincubation with 4 5 Ca2+ results in enzyme secretion accompanied by calcium release. There is also an increase in 4 5 Ca2+ secretion, but this is maximal 10 min after the protein and total calcium peaks. 4. Partial removal of 4 5 Ca2+ from the bathing medium, before stimulation, reduces the increase in 4 5 Ca2+ secretion nearly proportionally. 5. [3H]Mannitol, added to the bathing medium, appears in the secreted fluid and behaves upon carbachol stimulation similarly to 4 5 Ca2+. 6. Upon repeated stimulation with 10(-5) M acetylcholine, a 4 5 Ca2+ peak appears, even in virtual absence of enzyme secretion. In this case the peak coincides with a small total calcium peak. 7. Efflux studies of rabbit pancreas fragments, preloaded with 4 5 Ca2+, show a carbachol-stimulated 4 5 Ca2+ efflux in addition to a release of amylase. 8. These studies indicate that there are three calcium movements in rabbit pancreas which can all be influenced by cholinergic agents: (a) an extracellular route for calcium and other small molecules and ions; (b) a calcium release across the apical membrane along with the enzymes, originating from a pool which does not freely exchange with 4 5 Ca2+ in the bath; (c) a calcium flux across the serosal membrane, which involves calcium exchanging freely with 4 5 Ca2+ from the bath. The third flux is thought to result from an increase in cytoplasmic calcium, which may be involved in the stimulus-secretion coupling of pancreatic enzyme secretion.


Glycoproteins from ascitic fluid of Ehrlich ascites tumor. Isolation and chemical characterization.

Three glycoprotein bands were identified by polyacrylamide disc gel electrophoresis in the perchloric acid soluble fraction of ascitic fluid of Ehrlich ascites tumor in mice. The three proteins were first separated by a new discontinuous preparative electrophoresis apparatus described previously [1]. They were further purified on Sephadex G-100 and then were subjected to chemical characterization. These glycoproteins were rich in glutamic and aspartic acids and contained the sugar moieties galactose, mannose, fucose, N-acetyl-D-glucosamine and sialic acid. The percent sugar composition ranged from 17.7-37.3% of the total weights of these glycoproteins.


Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages.

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes. When expressed as rates of hydrolysis per 10(6) cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2-4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.


Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.


X-ray structure of the curare alkaloid (+)-tubocurarine dibromide.

X-ray structure determination of the compound (C37H42N2O6)2+ .2Br-.4CH3OH, confirms that (+)-tubocurarine is a monoquaternary salt and has established that the molecule adopts different conformations in crystals of the dibromide and dichloride salts. The crystal structure is stabilised by a number of hydrogen bonds involving the two free hydroxyl groups and the tertiary nitrogen of the tubocurarine molecule, the bromide ions and the solvent molecules. The absolute configuration of the molecule, determined by X-ray anomalous scattering, confirms the configuration assigned earlier by chemical studies.


Age-related variations in glycosylation of hydroxylysine in human and rat skin collagens.

The extent of glycosylation of hydroxylysine in human skin collagen rapidly decreased during maturation and then gradually increased in proportion to the age. This decrease of glycosylation observed during maturation was also confirmed in whole, soluble and insoluble collagens from rat skin. These findings may contribute to the investigations on the functional role of glycosylation and also on the mechanism of maturational as well as senile processes.


A theoretical model for lipid monolayer phase transitions.

We present a theoretical model for the liquid-expanded to liquid-condensed phase transition observed in many phospholipid monolayer films. The total two-dimensional pressure in the model is the sum of the hydrocarbon chain pressure and the surface pressure. The hydrocarbon chain pressure is calculated in an exteded version of a model published earlier. The surface pressure results from a lowering of the surface tension in the monolayer over that of pure water, thus producing a force on a Langmuir float. When these two contributions are added, pi/A isotherms are obtained which have slope discontinuities very similar to those observed experimentally. The results indicate that a successful model for lipid phase behavior must consider the interactions between head groups and water as well as cooperative hydrocarbon chain melting.


Lectin-receptor interactions in liposomes.

The major sialoglycoprotein of mammalian erythrocytes has been incorporated into phosphatidylcholine membranes to generate a model system, glycoprotein-liposomes. Electron microscopic examination revealed these structures to be vesicles, approximately 300 A in diameter. An aqueous compartment inside the glycoprotein-liposomes has been identified by trapped volume studies with [14C]sucrose. These glycoprotein-liposomes were found to interact with the lectins, wheat germ agglutinin, and phytohemagglutinin, to form aggregates of mainly unfused vesicles. The aggregation process has been studied by electron microscopy, 90 degrees light scattering, and differential ultracentrifugation analysis. Hapten inhibitors of the lectins were found to inhibit the lectin-induced aggregation of the glycoprotein-liposomes. Binding of 125I-labeled wheat germ agglutinin to glycoprotein-liposomes was studied by differential ultracentrifugation. Hapten inhibitors of wheat germ agglutinin were also found to inhbit the binding of 125I-labled wheat germ agglutinin to the glycoprotein-liposomes. The characteristics of the lectin interactions with glycoprotein-liposomes appeared to be phenomenologically similar to lectin-cell interactions.


Fluroescence properties of chlorophyll a and b monomolecular films at the air-water interface.

The fluorescence properties of chlorophyll a and b monomolecular films at the air-water interface were measured by a high sensitivity fluorophotometer using the photon-counting method. The fluorescence intensity of chlorophyll molecules in monomolecular films in the absence of any diluents did not decrease simply with the mean distance of chlorphyll molecules.  Over the range of the mean distances from 27 to 21 A, three fluorescence components (peaks at 685, 695 and 715 nm) of chlorophyll a were observed. In the case of chlorophyll beta, two fluorescence components (peaks at 667 and 685 nm) were observed over the range of the mean distances from 34 to 24 A. When the mean distance was 18 A, the short wavelength component of chlorophyll beta disappeared, and only the long avelength component was observed.


Transport-associated phosphorylation of 2-deoxy-D-glucose in Saccharomyces fragilis.

2-Deoxy-D-glucose transport and metabolism was studied in Saccharomyces fragilis. Inside the cells four phosphorylated and three non-phosphorylated derivatives were found and identified. Accumulation of phosphorylated 2-deoxyglucose derivatives was balanced by a concomitant decrease of cellular ATP, orthophosphate and polyphosphates. The free sugar was concentrated against a concentration gradient, contradiciting facilitated diffusion. Pulse labeling experiments revealed transport-associated phosphorylation. Theoretical considerations and analysis of the effects of iodoacetate showed that an intracellular hexokinase activity was not involved in 2-deoxyglucose phosphorylation, although this sugar is a good substrate for the enzyme in in vitro experiments.


Dielectric breakdown measurements of human and bovine erythrocyte membranes using benzyl alcohol as a probe molecule.

Dielectric breakdown of intact erythrocytes and subsequent haemolysis in the presence of increasing concentrations of benzyl alcohol were investigated by means of an electrolytical discharge chamber and a hydrodynamic focusing Coulter Counter. Low concentrations of the drug stabilized human and bovine erythrocytes against haemolysis induced by dielectric breakdown of the cell membrane in isotonic solutions, while high concentrations caused lysis similar to hypotonic and mechanical haemolysis. The stabilizing effect of the drug on electrically induced haemolysis depends on the pulse length of the applied electric field. The critical dielectric breakdown voltage of the membranes of intact cells decreases progressively with increasing benzyl alcohol concentrations, at which the membrane is also more stabilized against electrical and osmotic haemolysis. Occasionally, an increase in the dielectric breakdown voltage is observed at drug concentrations at which lysis occurs. A similar depedence of the breakdown voltage on drug concentration was found for human erythrocyte ghost cells prepared by dielectric breakdown. The results are consistent with the electromechanical model suggested for the dielectric breakdwon mechanism and with the assumption of Metcalfe, using NMR and ESR techniques, that the fluidity of the membrane increases with increasing benzyl alcohol concentration.


A hydrophobic form of the small-intestinal sucrase-isomaltase complex.

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.


Influence of Ca2+ and Mg2+ on the thermotropic behaviour and permeability properties of liposomes prepared from dimyristoyl phosphatidylglycerol and mixtures of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine.

Calorimetric experiments showed a marked effect of Ca2+ and Mg2+ on the thermotropic behaviour of dimyristoyl phosphatidylglycerol. 2. Concentrations of Ca2+ and Mg2+ lower than 1 ion to 2 molecules of phosphatidylglycerol produced a shift of the phase transition to higher temperatures and an increase in the enthalpy change which is consistent with a closer packing of the lipid molecules in the liposomes. 3. Above the 1:2 ratio, freeze-fracture electron microscopy demonstrated typical "crystal" structures both in the presence of Ca2+ and Mg2+. In the presence of Mg2+ a metastable behaviour was noticed in the calorimetric experiments. 4. A Ca2+- and Mg2+-induced shift in the transition temperature and an increase in the enthalpy change was also observed in a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine. However, these mixed samples remained liposomal in structure at any concentration of the divalent ions. 5. Liposomes prepared from a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the absence of divalent cations are permeable in the range 10-50 degrees C. Bilayers of mixtures neutralized by Ca2+ or Mg2+ were demonstrated to be completely impermeable to K+, except in the vicinity of the phase transition. 6. The leak of ions from liposomes of a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the vicinity of the phase transition temperature was considerably less in the presence of Ca2+ than in the presence of Mg2+. 7. It is concluded that there is a correlation between the calorimetric data and the permeability properties of dimyristoyl phosphatidylglycerol-containing bilayers with respect to the influence of Ca2+ and Mg2+.


Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues.

An indicator dilution technique with 22Na+ as the intravascular marker was used to measure unidirectional transport of D-[6-3H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, alpha-D-glucose has higher affinity for the carrier than does beta-D-glucose. A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.


Characterization of transport systems for the transfer of 3,4-L-dihydroxyphenylalanine into slices of rat cerebral cortex.

1. Slices of rat cerebral cortex incubated aerobically at 37 degrees C in Krebs-Ringer-bicarbonate solution accumulated 3,4-L-dihydroxyphenylalanine (L-DOPA) against its concentration gradient. With 1 mM L-DOPA in the medium, tissue-water/medium concentration ratios of about 6 : 1 are reached, which are modified by the presence of other amino acids in the medium. 2. Kinetic analysis suggested that L-DOPA influx into brain cells occurred by at least two saturable processes, which show apparent Km values in the range of 10(-3) M and 10(-5) M, respectively. 3. Prior incubation of the slices in Na+-free (choline-containing) medium at 37 degrees C depressed their subsequent uptake of L-DOPA in normal Na+-containing medium; this inhibition did not appear when the preincubation was carried out at 0-4 degrees C. Besides this effect of preincubation, most of L-DOPA influx into brain slices was independent of the actual concentration of Na+ in the medium; the two saturable processes described in this article behaved similarly in this respect. 4. Most of L-DOPA uptake by the high-Km process is mediated by an agency that resembles the Na+-independent L system described in Ehrlich cells (Oxender, D. L. and Christensen, H. N. (1963) J. Biol. Chem. 238, 2686-2699), both in its specificity and in its participation in exchange phenomena. A lesser component of uptake by a type A mediation is also suggested as contributing to the high-Km process . 5. The kinetic and specificity properties of the low-Km process of L-DOPA uptake suggest a similarity between its mediation and that of the high-affinity systems for L-tyrosine and L-tryptophan found in brain tissue preparations (Belin, M. F. and Pujol, J. F. (1973) Experientia 29, 411-413; Bauman, A., Bourgoin, S., Benda, P., Glowinski, J. and Hamon, M. (1971 Brain Res. 66, 253-263).