Specific modification of the alpha chain C-terminal carboxyl group of hemoglobin by trypsin-catalyzed hydrazinolysis.

In human deoxyhemoglobin a salt bridge links the alpha carboxyl of Arg-141 of each alpha chain to the epsilon-amino group of Lys-127 of the opposite alpha chain. These salt bridges are believed to contribute to the constraints in the quaternary deoxy (T) structure that lower its oxygen affinity. We have tested this hypothesis by incubating hemoglobin with 2 M hydrazine and trypsin which catalyzes specifically the reversible hydrazinolysis of the alpha carboxyl of Arg-141alpha. X-ray analysis shows the major structural difference between native deoxyhemoglobin and hydrazide deoxyhemoglobin to be the loss of the Arg-141alpha1-Lys-127alpha2 salt bridge and its Arg-141alpha2-Lys-127alpha1 counterpart. Accurate oxygen equilibrium curves of hydrazide hemoglobin show that blocking of the salt bridge has raised the oxygen affinity of the T structure while leaving that of the quaternary oxy (R) structure unchanged.


Studies of the conformation of modified dinucleoside phosphates containing 1,N6-ethenoadenosine and 2'-O-methylcytidine by 360-MHz 1H nuclear magnetic resonance spectroscopy. Investigation of the solution conformations of dinucleoside phosphates.

Seven dinucleoside monophosphates containing epsilonA (1,N6-ethenoadenosine) and 2'-O-methylcytidine were studied by 360-MHz proton magnetic resonance and compared with unmodified dimers and component monomers at 4, 20, 45, and 75 degrees C. These studies show that the dimers exhibit preference for the gg and g'g' conformations for the C-4'-C-5' and C-5'-O-5' bonds, respectively, and that dimerization induces an increase of the population and inflexibility of the 3'-endo conformations for the ribose ring. Three stacked (or stable) conformations for dimers, I, II, and III, in equilibrium with an unstacked (or open) form in solution, are suggested by dimerization shifts of ribose protons. Conformation I exhibits anti, gg, 3'-endo, phi' = 203 to approximately 211 degrees, omega' = 300 degrees, omega = 290 degrees, g'g', gg, 3'-endo, and anti conformation from the 5' end to the 3' end of the dimer. Conformation II shows anti, gg, 3'-endo, phi' = 203-211 degrees, omega = 30 degrees, omega = 100 degrees, g'g', gg, 3'-endo, and anti conformation. Conformation III is anti, gg, 2'-endo, phi' = 260 degrees, omega' = 50 degrees, omega = 220 degrees g'g', gg, 3'-endo, and anti (x approximately 100 degrees) conformation. The dimers, PupPu and PupPy, prefer conformations I and II, while PypPu and PypPy prefer conformation II. Introduction of epsilonA for the base of -pN induces an increase of conformations II and III, while the epsilonA substitution for the Np- residue induces an increase of conformation I. 2'-O-methylation of the Cp- residue of CpC decreases conformation I and increases conformation II. Based on the stable solution conformations of these dimers, a possible conformation of the anticodon loop is proposed, which is an alternative to the one observed in the crystal of tRNAPhe.


Structure of parental deoxyribonucleic acid of synchronized HeLa cells.

We have investigated the structure of parental DNA as a function of the cell cycle phase of HeLa cells. DNA was isolated from synchronized HeLa cells 0, 5, 8, and 12 h after release from a second exposure to 2 mM thymidine. These DNA preparations were characterized by CS2SO4/AgClO4 buoyant density, sensitivity to a single-strand specific nuclease, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1 nuclease. The cultures were staged according to cell cycle phase by measurements of DNA content per cell by flow microfluorometry. The cell cycle phases were G1/S (0-h culture), S (5-h culture), G2 (8-h culture), and G1 (12-h culture). There are no nuclease-sensitive sites in G2. As the cells enter G1, the number increases, with a maximum being reached in the S phase. The number of breaks in DNA with respect to cell cycle phase follows the same pattern. The amount of single strandedness, measured by buoyant density and nuclease sensitivity, is also minimal in G2, increases in G1, with a maximum achieved in the S phase. It appears that there is a chromosomal cycle, reflected as continuous structural changes in the DNA molecule, as cells traverse the cell cycle.


Differential translation of rat liver albumin messenger RNA in a wheat germ cell-free system.

Rat liver polysomal RNA directs the synthesis of albumin in a wheat germ cell-free system. The in vitro product has the same mobility in polyacrylamide-sodium dodecyl sulfate gels as purified rat liver albumin and can be immune precipitated with rabbit anti-rat liver albumin serum. The incorporation of [35S] methionine into total protein in an in vitro system increased linearly with increasing messenger RNA concentration until a saturation plateau was reached. The synthesis of albumin compared to total protein increased continually with increasing RNA concentration. Addition of the polynucleotide poly(A) to the cell-free system inhibited incorporation, presumably through interference with the initiation process, and increased the relative albumin synthesis. Translation of albumin messenger RNA was also resistant to inhibition by high levels of potassium acetate where total incorporation was cut in half. The results suggest that the messenger RNA for albumin, in addition to the mRNAs for the beta chain of globin and for the immunoglobulin polypeptides, is particularly efficient in initiating protein synthesis.


Exposure of tryptophanyl residues and protein dynamics.

The acrylamide quenching reaction is shown to be very discriminating in sensing the exposure of fluorescing tryptophanyl residues in globular proteins. The quenching rate constants for some proteins, such as aldolase and human serum albumin, are reported to be independent of the solvent viscosity, indicating that the reaction is limited by penetration of the quencher through the protein matrix. Temperature-dependent studies are performed to determine the activation energy and entropy for the penetration of acrylamide into these proteins. The tryptophanyl residues in aldolase are shown to be shielded by a large activation energy barrier, while the single residue in human serum albumin is shielded by a large activation entropy barrier. These parameters characterize the nature of the protein matrix enveloping the fluorophors.


Structural study of spectrin from human erythrocyte membranes.

Human erythrocyte spectrin prepared from fresh blood is a mixture of different association states. Depending on the manner of preparation, the two-chain dimer or the tetramer predominates. These forms are not in rapid thermodynamic equilibrium. The molecular weight of the dimer by sedimentation and diffusion and by light scattering is about 5 X 10(5). The frictional properties indicate a low or moderate asymmetry (axial ratio in the range 2-10), and from the angular dependence of light scattering intensity an upper limit of about 80 A can be set for the radius of gyration. The tetramer similarly has a moderate asymmetry. Electron microscopy reveals that the dimer is a compact, slightly elongated molecule, and that the tetramer probably consists of two parallel dimers. On increasing the concentration of solutions containing spectrin dimers, oligomers are formed, which are not rapidly dissociated on dilution. At very low protein concentrations (below about 0.05 mg/mL) there is evidence of the onset of a rapid dissociation equilibrium between dimers and single chains. Other physical properties of the spectrin have been measured. The size and shape of the spectrin molecule would seem to rule out any major physical resemblance to myosin.


Interaction of hemoglobin with red blood cell membranes as shown by a fluorescent chromophore.

Hemoglobin quenching of the fluorescence intensity of 12-(9-anthroyl)stearic acid (AS) embedded in the red blood cell membrane occurs through an energy transfer mechanism and can be used to measure the binding of hemoglobin to the membrane. The binding of hemoglobin to red cell membranes was found to be reversible and electrostatic in nature. Using a theory of energy transfer based on Förster formulation, the quantitative data for the binding were derived. The number of binding sites was found to be 1.4 +/- 0.2 X 10(6) molecules per cell and the binding constant was 0.85 X 10(8) M-1.


Separation and characterization of microtubule proteins from calf brain.

Electrophoresis of microtubule preparations purified from calf brain by repeated cycles of assembly and disassembly shows that they contain many proteins in addition to alpha- and beta-tubulin. These additional proteins constitute about 17% of the total material present after five cycles of assembly and disassembly. Both one-dimensional and two-dimensional (P.H. O'Farrell (1975), J. Biol. Chem. 250, 4007) electrophoretic techniques have been used to characterize them. They can be divided into two groups: one that contains proteins which remain in constant quantitative ratio to tubulin during the purification cycles, and one composed of proteins which are removed during purification, although inefficiently. Gel-filtration chromatography of cold-depolymerized microtubule preparations yields a polydisperse fraction of high molecular weight containing most of the non-tubulin proteins. This fraction contains flexible filaments about 100 A in diameter similar to those reported by R.A.B. Keats and R.H. Hall ((1975), Nature (London) 247, 418). It is suggested that these fibers are neurofilaments, and that they may be the major source of the group of inefficiently removed proteins.


Photoactivated heterobifunctional cross-linking reagents which demonstrate the aggregation state of phospholipase A2.

Two novel heterobifunctional cross-linking reagents, which can be used to attach photoactivatable nitroaryl azides to primary amino groups of proteins, have been synthesized. The two compounds, N-5-azido-2-nitrobenzoyloxy-succinimide and ethyl N-5-azido-2-nitrobenzoylaminoacet-imidate-HCl, as well as ethyl 4-azidobenzimidate-HCl have been attached to lysine residues of cobra venom phospholipase A2 without a loss in enzymatic activity. Subsequent illumination of the modified forms of the enzyme at appropriate wavelengths under conditions in which the native enzyme exists in an aggregated state led to the formation of covalently linked dimers and large aggregates which could be separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate.


Effect of double-bonds on bimolecular films in membrane models.

The effect of unsaturation (especially by cis-bonds) is studied on bimolecular films of saturated and unsaturated alkylammonium ions and alkanols between silicate surfaces as model systems for lipid layers in membranes. Three types of structures are observed: all-trans-blocks, kink-blocks and gauche-blocks. The knowledge of the sequence of these phases and their thermal transitions provides detailed deductions about the role of double-bonds. cis-Unsaturated chains are taken up in bimolecular films as isomers with cis-trans-gauche conformation. This conformation makes the shape of the chain similar to that of kinked chains (chains with gauche-trans-gauche (--) conformation) and enables the incorporation into the film without greater sterical hindrance. The experimental results are in good agreement with X-ray measurements on biological membranes by Engelman (Engelman, D.M., J. Mol. Biol. 47, 115--117 (1970) and 58, 153--165 (1971)). Increasing the concentration of cis-chains decreases the transition temperature of the kink-blocks into gauche-blocks. The variation of the transition temperature with concentration of cis-unsaturated chains in the model system is similar to that observed for Escherichia coli membranes. It is suggested that phase changes in biomembranes are of the same nature: transition of kink-block analogues as ordered phases into gauche-block assemblies as less ordered phases.


Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum.

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidycholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A2 of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphinogomyelin which is not hydrolysed by the former enzyme.


Properties of amphotericin B channels in a lipid bilayer.

Properties of individual ionic channels formed by polyene antibiotic Amphotericin B were studied on brain phospholipid membranes containing cholesterol. The ionic channels have a closed state and an open one (with conductance of about 6.5 pS in 2 M KCl). The conductance value of an open channel is independent of cholesterol concentration in the membrane of pH in the range from 3.5 to 8.0. The voltage-current characteristics of a single channel are superlinear. Zero current potential value in the case of different KCl concentrations in the two solutions indicates preferential but not ideal anionic selectivity of a single channel. Channel conductivity grows as the electrolyte concentration is increased and tends to a limiting value at high concentrations. A simple model having only one site for an ion was shown to represent satisfactorily an open channel behaviour under different conditions. An individual ionic channel performs a large number of transitions between the open and closed states during its life-time of several minutes. Rate constants of these transitions depend on the kind and concentration of salt in aqueous solutions. The switching system functioning is not influenced by an ion situated inside the pore.


Adaptation of biological membranes to temperature. The effect of temperature acclimation of goldfish upon the viscosity of synaptosomal membranes.

The fluidity of synaptosomal membrane preparations isolated from goldfish acclimated to 5, 15 and 25 degrees C and from rat has been estimated using the fluorescence polarisation technique with 1,6-diphenyl-1,3,5-hexatriene as probe. Membranes of cold-acclimated goldfish were more fluid than those of warm-acclimated goldfish when measured at an intermediate temperature, indicating a temperature-dependent regulation of this parameter. Similarly, membranes of warm-acclimated goldfish were more fluid than those prepared from rat brain. Liposomes prepared from the purified phospholipids of goldfish and rat synaptosomal preparations showed differences similar to those of the native membranes. Increased membrane fluidity of cold-acclimated goldfish was correlated with a decrease in the proportion of saturated fatty acids of the major phospholipid classes and an increased unsaturation index in choline phosphoglycerides. Rat membranes showed a substantial reduction in unsaturation index and an increase in the proportion of saturated fatty acids compared to the membranes of 25 degrees C-acclimated goldfish. The cholesterol content of synaptosomal membranes of goldfish was unaffected by acclimation treatment. The role of homeoviscous adaptation in the compensation of the rates of membrane processes during thermal acclimation, and upon the resistance adaptation of poikilotherms to extreme temperatures is discussed.


Translocation of hyaluronic acid in cell surface of cultured mammalian cells after x-irradiation and its recovery by added adenosine triphosphate.

To investigate the mechanism of radiation-induced decrease in cell electrophoretic mobility and its recovery by added adenosine triphosphate, specific enzymes and buffer solutions of different ionic strength were utilized. Decrease in the mobility of irradiated cells was detected only with the buffer solution of ionic strengths higher than 0.100. In this range of ionic strengths, removal of hyaluronic acid from cell surface by hyaluronidase had no effect on the electrophoretic mobility of irradiated cells, while the enzyme treatment resulted in 27% mobility reduction in non-irradiated cells. The removal of sialic acid and chondroitin sulfate by their specific enzymes resulted in the similar decrease in mobility either in irradiated and non-irradiated cells. These results suggest that the X-ray induced translocation of hyaluronic acid from the peripheral zone of O--7.5 A into the deeper zone of about 10--17 A, if we use the Debye-Hückel's thickness of ion atmosphere for an approximate estimate of effective depth of electrokinetic plane of shear. Hyaluronic acid reappeared to the peripheral zone by the subsequent incubation after small dose irradiation, or by the addition of 1 mM adenosine triphosphate with Ca2+.


The transposition of molecular classes of phosphatidylcholine across the rat erythrocyte membrane and their exchange between the red cell membrane and plasma lipoproteins.

1. The molecular composition of phosphatidylcholine is similar in the inner and the outer layer of the rat erythrocyte membrane. 2. The rate of exchange of the various molecular classes of phosphatidylcholine between rat plasma and the red cell membrane does not depend on the degree of unsaturation of the different classes. 3. The transposition of the molecular classes of phosphatidylcholine between the inner and the outer layer of the rat erythrocyte membrane is more pronounced for the more unsaturated classes.


Effect of high substrate concentrations on active transport parameters.

A general model is described to account for the observation that steady-state accumulation ratios (mainly of non-electrolytes) decrease with increasing solute concentration, frequently reaching values of less than unity. Three variants of the model are treated, all of them including the assumption that the immediate supply of the source of energy is limited and its local concentration is appreciably reduced by its interaction with the transport system. Consequences of this assumption for the kinetic parameters of the initial rate of transport are analyzed and compared with experimental data.


Comments on the quantitative interpretation of biomembrane structure by Raman spectroscopy.

The possibility of quantitation of information obtained from laser Raman spectra of aqueous lipid dispersions is discussed. It is shown that the all-trans chain order parameter ST introduced by Gaber and Peticolas ((1977) Biochim. Biophys. Acta 465, 260) for the characterization of biomembrane structure is of restricted applicability. This order parameter may give adequate information if polar head groups are not affected at all by the interaction resulting in trans-gauche isomerization. To demonstrate this, data on the effects of mono- and divalent ions on the all-trans chain order parameter are given. The lateral order parameter proved to be suitable for quantitative studies even in the case of ion-head group interaction.


The membrane volume occupied by anesthetic molecules: a reinterpretation of the erythrocyte expansion data.

Seeman and coworkers (Seeman, P. (1972) Pharmacol. Rev. 24, 583--655) calculated that anesthetic agents expand membrane volume ten times more than the van der Waals volume of the agent alone. Their calculation was based on the assumption that the thickness of the erythrocyte membrane expands at the same rate as the surface area. However, recent data on bilayer membranes demonstrate that an expansion of membrane surface area is accompanied by a decrease in membrane thickness. A reinterpretation of their erythrocyte area expansion data using an appropriate contraction of membrane thickness suggests the volume in a membrane occupied by anesthetic molecules is approximately equal to their van der Waals volume.


The effect of inorganic phosphate on sodium fluxes in dog red blood cells.

The effect of extracellular inorganic phosphate on Na+ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na+ efflux increases by 2- to 3-fold and Na+ influx increases approximately 2-fold. This enhancement of Na+ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4'-iso-thiocyantostilbene-2,2'-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na+ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na+ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.


Carnitine movement across muscle cell membranes. Studies in isolated rat muscle.

L-Carnitine uptake and exodus was studied in rat extensor digitorum longus muscle in vitro. A saturable transport process was observed, which had an apparent Km of 60 micron and V of 22 nmol/h per g tissue. Transport was inhibited by 2,4-dinitrophenol, sodium azide, anaerobiosis, ouabain, and sodium ion depletion. Analogs of L-carnitine containing a quarternary ammonium group were found to inhibit uptake (D-carnitine, Ki = 400 micron; gamma-butyrobetaine, Ki = 60 micron, choline chloride, Ki = 14 mM), while those not containing this functional group (gamma-aminobutyrate, D,L-beta-hydroxybutyrate) had no significant effect at concentrations 100 times the apparent Km of L-carnitine. Carnitine exodus from rat extensor digitorum longus muscle consisted of two phases. The rapid initial phase was attributed to leakage of L-carnitine from damaged muscle fibers, as it proceeded at nearly the same rat at 0 degrees and 37 degrees C, and then leveled off to a rate of near zero after 1 h of incubation in vitro. The quantitatively more important phase of exodus showed a latency of 1-2 h and then proceeded at a linear rate of 40-45 nmol/h per g tissue. The results of this study support the contention that L-carnitine is taken up by a carrier-mediated, active transport system in rat extensor digitorum longus muscle. Functionally, the transport system for uptake is distinct from the process by which carnitine is lost from this muscle.


Effects of cytochalasin B and colchicine on attachment of a major surface protein of fibroblasts.

We have investigated the effects of the drugs cytochalasin B and colchicine on the surface levels of the large, external, transformation-sensitive (LETS) glycoprotein. Colchicine neither removed LETS protein from the surface, nor inhibited its regeneration after removal by mild trypsinization. Cells treated with cytochalasin B, however, showed both a 2-3-fold increase in the turnover rate of their surface LETS protein and a marked inhibition in its regeneration. Inhibition of regeneration was not due to inhibition of synthesis or transport to the surface. In fact, in the presence of cytochalasin B, increased quantities of LETS protein were released into the medium. The results are consistent with the idea of an association of LETS protein with the actin-containing microfilaments. However, other possible explanations, such as effects on cellular morphology or on transport of sugar precursors cannot yet be excluded.


Calcium effects on human erythrocyte membrane proteins.

The effects of Ca2+ on human erythrocyte membrane proteins were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Ca2+ had several effects on normal human erythrocyte membrane proteins. It affected the binding of cytoplasmic proteins to the membrane, produced a non-reversible aggregation of several membrane proteins and activated apparent proteolysis of membrane proteins. The Ca2+ effect could be obtained with isolated, washed membranes when the erythrocyte cytoplasm was added. These studies indicate that the Ca2+-induced membrane proteolysis and aggregation effects are not due simply to its presence at the time of hemolysis as previously suggested (Carraway, K.L., Triplett, R.B. and Anderson, D.R. (1975) Biochim. Biophys. Acta 379, 571-581), but are the result of more complex interactions between the erythrocyte membrane and cytoplasmic factors.


Isolation of pigmented granules involved in extra-retinal photoreception in Aplysia californica neurons.

Many neurons in the ganglia of Aplysia californica contain pigmented, membrane-bound granules (lipochondria), which are thought to mediate the light response of some of the neurons, including the giant cell of the abdominal ganglion. A method of isolating the lipochondria by centrifugation of ganglia homogenates has now been developed. Electron microscopy was used to demonstrate that most of the lipochondria remain morphologically intact. As shown by X-ray microanalysis, isolated lipochondria contain the same elements, including calcium, as do lipochondria in intact giant cells. The calcium can be released into the medium by treatment of the organelles with the Ca2+ ionophore A23187. It appears that the lipochondria of Aplysia ganglia are similar in their morphology, elemental content and susceptibility to the ionophore. Two pigments were isolated from the lipochondria, and chromatography and spectrophotometric studies indicated that they are beta-carotene and a "retinol-like" compound.


Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to Mycoplasma membranes.

Growing cells of sterol-requiring Mycoplasma hominis and sterol non-requiring Acholeplasma laidlawii were used to test the ability of cholesterol-dipalmitoyl phosphatidylcholine dispersions to serve as cholesterol donors to these organisms. Dispersions with high cholesterol to phosphatidylcholine ratios were more effective than dispersions with low cholesterol to phosphatidylcholine ratios in donating cholesterol to the membranes of both mycoplasmas and in promoting growth of the sterol-requiring species. M. hominis took up almost three times as much cholesterol as did A. laidlawii. In addition, significant quantities of the phosphatidylcholine component of the dispersions were found to be associated with M. hominis membranes as against none in the A. laidlawii membrane preparations. In all cases, the percentage of cholesterol taken up by M. hominis from the dispersions exceeded that of phosphatidylcholine by a factor of 3-5. These results were interpreted to suggest that all the cholesterol taken up by A. laidlawii is transferred from the dispersion to the membranes by a process which involves only a transient contact between the organisms and the lipid dispersions, whereas a certain amount of the cholesterol taken up by M. hominis may also be derived from lipid dispersions adhering to or fusing with the cell membranes.


Localization of the amino phospholipids in sarcoplasmic reticulum membranes revealed by trinitrobenzenesulfonate and fluorodinitrobenzene.

The chemical probes for amino compounds 2,4,6-trinitrobenzensulfonate (TNBS) and 1-fluoro-2,4-dinitrobenzene (FDNB) were utilized to determine the localization of the amino phospholipids in the sarcoplasmic reticulum membranes. At low concentrations (less than 1 mM). TNBS does not penetrate the sarcoplasmic reticulum membrane, while FDNB readily penetrates it. The results show that about 70% of the total phosphatidylethanolamine is located on the external surface of the membrane, about 20% is on the internal surface and 10% is probably strongly interacting with the proteins since it is not accessible to the probes. In contrast, most of the phosphatidylserine is located on the inner surface of the membrane. This molecular distribution of the amino phospholipids supports a structural assymmetry of the sarcoplasmic reticulum membrane.


Labeling of membranes from erythrocytes and corn with fluorescamine.

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.


Studies of asymmetric membrane assembly.

The major capsid protein of M13 bacteriophage is incorporated at each stage of infection into the host plasma membrane with its amino terminus exposed on the outer surface. Purified M13 coat protein is incorporated with the same asymmetry into synthetic phosphatidylcholine vesicles formed near the Tm of the lipid by a cholate dilution technique. We now report that the lipid in the pre-dilution mixture exists as mixed micelles of uniform size. Prior to dilution, the coat protein is present in at least two states of aggregation, both of which behave similarly in the model membrane assembly reaction. No detectable lipid-protein interaction occurs prior to dilution. Upon dilution there is rapid production of small closed vesicles and coat protein is converted to a chymotrypsin-resistant form, presumably reflecting its incorporation into these vesicle bilayers. Formation of large (greater than 6000 A diameter) vesicles occurs slowly with preservation of coat protein asymmetry and internal volume. A model for this assembly reaction is proposed.


Phase diagrams and the kinetics of phospholipid exchange for vesicles of different composition and radius.

Interactions between dimyristoyl- and dipalmitoyl phosphatidylcholine vesicles have been studied by light scattering of dilute dispersions (concn less than or equal to 1 mg/ml). It was found that at 50 degrees C, the two types of vesicles exchange lipid molecules, irrespective of the initial radii of the pure vesicles. When the initial radii are different, the process leads eventually to vesicles of intermediate size. The reaction follows second order kinetics. At room temperature no reaction was observed. The phase diagrams for several lipid systems are discussed.


Voltage-induced pore formation and hemolysis of human erythrocytes.

Isotonic suspensions of human erythrocytes were exposed to single electric pulses of intensity at a few kV/cm and duration in microseconds. Upon pulsation, the cell membranes became permeable to Na+ and K+, and the erythrocytes eventually hemolysed through the colloid osmotic effect of hemoglobin. The enhanced permeability is attributed to the formation of pores in the cell membranes. These pores are formed within a fraction of a microsecond, once the transmembrane potential induced by the applied electric field reaches a critical value of 1.0 V. Increased field intensity and pulse duration, or pulsation at low ionic strengths all expand the pore size, leading to an accelerated hemolysis reaction. In contrast to this expansion process, the initial step of pore formatin is governed solely by the magnitude of the transmembrane potential: the critical value of the potential stays essentially constant in media of different ionic strengths, nor does it change appreciably with varying pulse duration. An abrupt increase in membrane permeability at a transmembrane potential adround 1 V has been observed in many cellular systems. It is suggested that a similar mechanism of pore formation may apply to these systems as well.


Allosteric cooperativity during intestinal cotransport of sodium and chloride in freshwater prawns.

Coupled influxes of sodium and chloride across the mucosal border of a freshwater prawn intestine were sigmoidal functions of luminal ion concentrations, indicative of a cooperative allosteric transport process. This process had a higher affinity for Cl (KCl = 94 mM) than for Na (KNa = 155 mM), maximally transported twice as much cation as anion (JNa max = 1.6; JCl max = 0.75 mumol-cm-2-h-1), and exhibited identical Hill interaction indices for both ions nNa = 3.4; nCl = 3.5). The suggestion is made that this cooperative carrier mechanism may be regulatory, maintaining relatively constant luminal ion concentrations which, in turn may facilitate ion-dependent absorption of non-electrolytes.


Isolation of plasma membrane from eukaryotic cells on polylysine-coated polyacrylamide beads.

The conditions for covalently binding polylysine to polyacrylamide beads used for membrane isolation have been analyzed. Larger amounts of bead bound polylysine were required for maximimizing plasma membrane purification from HeLa cells than from Dictyostelium discoideum. The least was needed for erythrocytes. The amount of polylysine bound to the bead was dependent on the carboxyl content of the bead and on the concentration of the polylysine used during the bead-polylysine coupling reaction.


Influence of incorporated cerebrosides on the interaction of liposomes with HeLa cells.

The presence of synthetic N-palmitoyl dihydrolactocerebroside in preparations of neutral multilamellar liposomes enhances their fusion interaction with cultured HeLa cells some 5-7 fold. The effect appears to be carbohydrate-specific, and is probably not attributable to a change in the physical characteristics of the liposome bilayer due to the presence of the cerebroside.


Chloride self exchange in Ehrlich ascites cells. Inhibition by furosemide and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid.

The effects of furosemide and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) on steady-state Cl- flux were studied in Ehrlich mouse ascites cells. At 10 mM, furosemide inhibited isotopically-determined Cl- flux by 86% without changing cell Cl- content, indicating that influx and efflux were depressed by the same amount. These results suggest that at least 86% of the steady-state Cl- flux may occur as a one for one exchange. Half of the inhibitory effect was not reversed by vigorous washing with albumin-Ringer. A smaller portion of steady-state Cl- flux was inhibited by SITS. The maximum effect of SITS was reached near 0.6 mM; at this concentration Cl- flux was reduced by 37% without an alteration in cell Cl- content. Possible competition of environment Cl- and SITS was investigated by replacing environment Cl- with acetate or NO3. These anions reduced the efficacy of SITS because they depressed cell Cl- turnover themselves, apparently acting on the same exchange process.


Exchange and aggregation in dispersions of dimyristoyl phosphatidylcholine vesicles containing myristic acid.

Processes occurring in dispersions of dimyristoyl phosphatidylcholine containing myristic acid have been studied by light scattering of dilute dispersions (concn. less than or equal to 1 mg/ml) at temperatures above and below the phase transition temperatures of these dispersions. The transition temperatures increase with increasing mol fraction of myristic acid. Above these temperatures, vesicles with different mol fractions of myristic acid exchange lipid molecules. The exchange process leads to vesicles having phase transition temperatures and radii, which ar both intermediate between the initial transitions and radii, respectively. In contrast with the observations above the phase transitions, it was found that when dimyristoyl phosphatidylcholine/myristic acid vesicles were cooled to a few degrees below the phase transition, larger particles were formed. These observations are consistent with a mechanism consisting of vesicle aggregation followed by fustion of the aggregated vesicles. The aggregation process is of second order in the vesicle concentration, and its rate increases with increasing mol fraction of myristic acid.


Distribution of lysophosphatidylcholine in single bilayer vesicles prepared without sonication.

The cholate method originally introduced by Kagawa et al. (J. Biol. Chem. (1973) 248, 676-684) and further developed by Brunner et al. (Biochim. Biophys. Acta (1976) 455, 322-331) has been used to prepare single bilayer vesicles containing 5 mol% lysophosphatidylcholine embedded in a matrix of phosphatidylcholine. The distribution of lysophosphatidylcholine over outer and inner monolayer was found to be highly asymmetric (ratio 9:1), as determined by lysophospholipase treatment of the vesicles. This distribution is similar to the value found in sonicated vesicles. Up to 20 mol% cholesterol could be incorporated in the vesicles by the cholate method. The method was succesfully used also for the preparation of single bilayer vesicles from total rat liver microsomal lipids, to which 5 mol% of 1-[1-14C]palmitoyl lysophosphatidylcholine had been added. Surprisingly, almost 100% of lysophosphatidylcholine in the latter vesicles was directly available for hydrolysis by sophospholipase. In contrast, only 70% of the lysophosphatidylcholine is sonicated vesicles of similar composition could be hydrolyzed by lysophospholipase.


Preferential interaction of pentagastrin with the gel state of dimyristoyl glycerophosphocholine.

Fluorescence and circular dichroism spectra indicate that pentagastrin interacts with dimyristoyl glycerophosphocholine more strongly below the phase transition temperature of the lipid than above it. Studies on the interaction of several peptides with dimyristoyl glycerophosphocholine suggest that this property may be related to the ability of these peptides to form amphipathic structures containing two hydrophobic amino acids separated by two other amino acids. Pentagastrin has a marked effect on the proton magnetic resonance spectra of dipalmitoyl glycerophosphocholine below the phase transition temperature indicating the the peptide decreases the motional freedom of the lipid.


Lateral phase separations and structural integrity of the inner membrane of rat-liver mitochondria. Effect of compression. Implications in the centrifugation of these organelles.

When maintained in the vicinity of the lower transition temperature of their membrane lipids, rat-liver mitochondria undergo lysis as shown by the release of malate dehydrogenase, (an enzyme located within the mitochondrial matrix), in the surrounding medium. Structural changes take place in the membranes of mitochondria subjected to increasing pressure at 0 degrees C, when the pressure reaches 750 kg/cm2. Freeze-fracture electron microscopy shows the appearance of smooth areas devoid of particles in fracture faces of mitochondrial membranes, together with zones, where aggregated particles can be seen. Concurrently, a suppression of the malate dehydrogenase structure-linked latency is observed. These structural changes can be prevented by increasing the temperature at which compression is performed. The freeze-etching observations suggest that lateral phase separations occur in mitochondrial membranes subjected to high pressure. This can be explained by supposing that pressure promotes the gel-phase appearance in a lipid system and raises the transition temperature since the transition liquid crystal lead to gel is accompanied by a decrease in volume. The deterioration of mitochondria subjected to high pressure is interpreted as a result of the lateral phase separation induced by compression in the membranes. These results are discussed with respect to our interpretation of the damaging effects that hydrostatic pressure, generated by centrifugation, exerts on rat-liver mitochondria.


Cytochalasin B inhibition of brain glucose transport and the influence of blood components on inhibitor concentration.

The effect of cytochalasin B on cerebral glucose transport and metabolism was investigated in 19 isolated perfused dog brain preparations. Cytochalasin B is a potent, non-competitive inhibitor of glucose transport at the blood-brain interface. Both glucose transport into (Ki = 6.6 +/- 1.9 micrometer) and out of the capillary endothelial cell are inhibited. The inhibition is readily reversible by perfusion with blood containing no cytochalasin B. After 2 min of exposure to 30 micrometer cytochalasin B, the cerebral oxygen consumption decreases by 31% probably due to decreased availability of glucose for oxidative metabolism. About one-half of the cytochalasin B that is dissolved in blood is bound to erythrocytes and other blood components while the remainder is free.


alpha-Nucleosides in biological systems. Crystal structure and conformation of alpha-cytidine.

The structure of alpha-cytidine, C9H13N3O5, monoclinic with space group C2 and cell parameters a = 20.064 (3) A, b = 7.100 (1) A, c = 7.860 (2) A, beta = 104.60 (2) degrees, Z = 4, was determined by X-ray diffraction using a combination of direct methods, Patterson and difference Fourier techniques and refined by block-diagonal least-squares to a final R of 0.033 for 1002 reflections measured on a diffractometer. The glycosidic torsional angle, chiCN = -28.4 degrees, is in the anti region; the sugar pucker is C(2')exo-C(3')endo in a nearly pure 32H twist; and the conformation of C(4')-C(5') is gauche-gauche. The molecules are bound by hydrogen bonds in the lattice with little likelihood of base-stacking interactions. The molecular features of the compound are compared and contrasted with those of its naturally occurring beta-anomer, and some biological implications of this structure, and alpha-nucleosides in general, are discussed.


Size and distribution of polyadenylic acid sequences in Drosophila polytene DNA and RNA.

[3H]Poly(U) hybridizes very rapidly to polytene DNA from Drosophila hydei. When hybridization is performed at 30 degrees C in 2 X SSC to a large excess of DNA, 95% of the poly(U) becomes ribonuclease resistant. Also, complementary RNA transcribed in vitro from polytene DNA hybridizes to poly(U). 023--0.25% of the DNA is composed of (dA)-rich sequences and 0.23--0.31% of cRNA hybridizes to [3H]poly(U). The length of the (dA)-rich sequences on the DNA and cRNA is 40 nucleotides. The Tm values of these hybrids formed between DNA or cRNA-poly(U) is 45 degrees C. The poly(A) fragments from cytoplasmic RNA ranged from 80 to 170 nucleotides in lenght, and migrated in polyacrilamide gels as a broad peak. The average sizes of the poly(A) fragments from the poly(A)-containing RNA transcribed by nuclei isolated from salivary glands in vivo or in vitro were 40, 70, 170 and 70 nucleotides, respectively. Hybridization in situ of [3H]-poly(U) to chromosome squashes indicated that the (dA)-rich sequences are randomly distributed over the whole genome.


Interaction of chromatin components with nuclear 5.0 S RNA fraction that stimulates RNA synthesis.

Chick liver nuclear 5.0 S RNA, which stimulates RNA synthesis on chromatin, binds preferentially to the deoxyribonucleoprotein in homologous chromatin. The proteins found in the isolated deoxyribonucleoprotein-5.0 S RNA complex are total amount of both H4 and H3 histone, about 20% of nonhistone protein and about 50% of both H2a and H2b histone found in the original chromatin. Cesium chloride equilibrium centrifugation of the deoxyribonucleoprotein-5.0 S RNA complex after fixation with formaldehyde shows that the 5.0 S RNA is bound to certain proteins (acid-soluble and -insoluble) in the complex. The stimulation of RNA synthesis by the nuclear RNA fraction is due to the conversion of inaccessible region of DNA to RNA polymerase into an accessible one and presumably not due to an increase in the number of binding sites for RNA polymerase in the chromatin. The release of non-histone protein from chromatin following the addition of the nuclear RNA fraction is also briefly discussed.


Effects of erythromycin on membrane-bound chloroplast ribosomes from wild-type Chlamydomonas reinhardi and erythromycin-resistant mutants.

1. Treatment of wild-type cells of Chlamydomonas reinhardi with high concentrations of erythromycin results in increased recovery of membrane-bound chloroplast ribosomes, presumably by preventing polysomal runoff during harvesting of cells. No such membrane-retention effect is detected if erythromycin is added after harvesting of cultures, before cell breakage. 2. Growth of wild-type cells is inhibited by 10 microgram/ml erythromycin, but a concentration twice as high is required to increase recovery of membrane-bound wild-type ribosomes. On the other hand, the concentrations of erythromycin which inhibit growth of mutant ery-M1b produce a membrane-retention effect. Mutant ery-U1a is resistant to high concentrations of erythromycin and no membrane-retention effect is detectable at concentrations which produce one in wild type and ery-M1b. 3. These results can be reconciled by a two-point model of the mechanism of erythromycin action on chloroplast ribosomes in Chlamydomonas.


Template specificity of rat mitochondrial DNA polymerase.

Mitochondrial DNA polymerase was purified 2300-fold over isolated mitochondria from rat liver. Template-primer specificities of this enzyme were investigated. Activated DNA was satisfactorily used as an active template-primer, but both native and denatured DNAs showed a slight activity. Synthetic polynucleotide, poly(dA) - oligo(dT)10 was found to have a high efficiency under the same condition for activated DNA. When the closed-circular, nicked and gapped Co1E1 DNAs were employed as a template-primer, the enzyme could only utilize the gapped DNA, indicating that the displacement synthesis was not catalyzed by the enzyme itself. The enzyme also copied poly(A) - oligo(dT)10 in high efficiency at pH 7.5 in the presence of MnCl2. Such RNA-directed DNA polymerase activity of the enzyme was further characterized. Cofractionated endouclease activity was completely separated from the enzyme by glycerol gradient centrifugation.


Action of intercalating agents on the activity of DNA polymerase I.

The effect of intercalating compounds such as 9-aminoacridine, quinacrine (atebrin), proflavine and daunomycin on the activity of DNA polymerase I(EC 2.7.7.7) was studied in vitro and compared with the binding of these acridines to native DNA. The enzyme kinetics were followed at various concentrations of DNA 3'-OH primer end groups and constant concentrations of deoxynucleosidetriphosphates as well as under the opposite conditions. The Km values for the DNA 3'-OH end groups were 16--38 nM and for the deoxynucleosidetriphosphates 2--5 micrometer, depending on the buffer and pH used in the enzymatic assay. All acridine derivates inhibit the DNA polymerase; at variable DNA concentrations a competitive inhibition was observed, where the Ki values ranged between 0.87 and 8.5 micrometer. At variable concentrations of deoxynucleosidetriphosphates and constant DNA concentration a non-competitive inhibition was observed. On denatured 3'-OH DNA as well as on poly(dA) - (dT)10 as substrate no inhibition by 9-aminoacridine was observed. 5'--3' exonuclease activity of DNA polymerase is inhibited by 9-aminoacridine but 3'--5' exonuclease activity on denatured DNA is not influenced by this intercalating compound. The affinity of the acridines to DNA was determined spectrophotometrically under conditions similar to those in the enzymatic assay and the computed frequency of intercalation was related to the inhibition of enzymatic activity. The mechanism of inhibition is explained by a disturbance of the structure of the double helical DNA due to the interaction of the bound acridine derivates.


Occurrence of a complex of aminoacryl-tRNA synthetases in lactating rat mammary gland.

(1) Postmicrosomal particles were isolated from the cytosol of lactating rat mammary gland. These particles contained up to 40% of the cytosol protein, and about 40% of the total aminoacyl-tRNA synthetase activity of the cytosol. (2) Sucrose density gradient centrifugation of these postmicrosomal particles showed that the activity of seventeen synthetases was distributed between one or other of two major gradient regions, corresponding to values of 20--28 S and 5--7 S. Arginyl and cysteinyl tRNA synthetases showed peaks in both major regions. (3) A complex of eight aminoacyl-tRNA synthetases (those for lysine, leucine, isoleucine, methionine, glutamine, arginine, cysteine and valine) of molecular weight 3 - 10(6) was isolated from the postmicrosomal particles by gel filtration and chromatography on DEAE-cellulose.


A cytoplasmic exoribonuclease from HeLa cells.

An exoribonuclease has been purified from the cytoplasm of HeLa cells. The enzyme produces 5'-AMP as the only product from poly(A). The degradation proceeds in a 3' to 5' direction, and a 3'-OH terminus is required. In addition to poly(A), the enzyme degrades other synthetic homopolymers as well as natural messenger, and ribosomal RNAs. The enzyme can also degrade the poly(A) tract of messenger RNA. DNA and double-stranded RNA are resistant to the enzyme.


Progesterone-regulated changes in transcriptional events in rabbit uterus.

The role of steroid receptors in the early events of progesterone action was elucidated by examining the temporal relationship between the nuclear accumulation of progestin receptor and changes in activities of RNA polymerases I and II, as well as that of chromatin template in rabbit uterus. Following a 5-day estrogen pretreatment, the animals received an intravenous injection of progesterone (10 mg), after which they were killed at timed intervals. Nuclear progestin receptor level, as measured by an exchange assay, reached the peak value 30 min after hormone administration (11 600 to 46 600 sites/nucleus) and declined to the control levels by 4 h. Changes in the activities of RNA polymerase I and II did not follow identical time courses: polymerase I rose at 30 min and remained elevated for 2 h and then declined to about 75% of the pretreatment activity, whereas RNA polymerase II activity increased more rapidly (at 15 min), and was followed by a sharp decrease to about 50% of the initial value. Thereafter, the latter enzyme activity rose slowly and reached the pretreatment level within 12 h of progesterone administration. Early changes in chromatin template activity were similar to those in RNA polymerase I with a second rise by 8--10 h. The early inhibition of transcriptional events by progesterone may result from antiestrogenic properties of this steroid. Accumulation of nuclear progestin receptor occurs at a similar time to early changes in the transcriptional events suggesting a regulatory role for the hormone receptor complexes.


Ribose 1-phosphate metabolism in Ehrlich ascites tumor cells in vitro.

Ribose 1-phosphate concentrations have been measured in tumor cells incubated with purine and pyrimidine nucleosides and with glucose. Highest concentrations (0.15 to 0.2 mumol/ml of cells) were attained in cells incubated with inosine. Although uridine was cleaved at approximately the same rate as inosine, as judged by lactate accumulation, concentrations of ribose 1-phosphate that accumulated were only approximately 0.06 mumol/ml. Ribose 1-phosphate accumulation in tumor cells incubated with inosine was dependent on the phosphate concentration of the medium up to at least 25 mM. Ribose 1-phosphate formed from inosine was readily converted both to phosphoribosyl pyrophosphate and to lactate.


The interaction of propidium diiodide with self-complementary dinucleoside monophosphates.

The interactions of a quinacrine derivative, methylated at both the aromatic and aliphatic nitrogens, and propidium diiodide with the dinucleoside monophosphates CpG, GpC, UpA and ApU have been investigated using 13C-NMR (for the quinacrine derivative prepared with [13C]methyl substituents and 1H-NMR and ultraviolet-visible spectroscopy. The quinacrine derivative displayed negligible interaction with the dinucleosides at concentrations up to 5 - 10(-4) M. Propidium did form complexes with dinucleosides even at concentrations as low as 10(-4) M. Propidium displayed a pyrimidine-purine binding preference and gave especially large changes in ultraviolet-visible and 1H-NMR spectra in the presence of CpG. This suggests that propidium forms an intercalated complex with a Watson-Crick hydrogen-bonded CpG dimer. At higher concentrations UpA and GpC gave similar spectral changes indicating that they could also form significant amounts of an intercalated complex with propidium under appropriate conditions. The changes caused by ApU were small under all conditions and were more similar to the effects caused by mononucleotides. These results indicate that, at least for phenanthridines, cationic side chains do not greatly inhibit complex formation with dinucleoside monophosphates, and suggest that the weak interaction of the quinacrine derivative with dinucleosides is due to weaker interactions of the acridine ring system with nucleoside bases relative to the phenanthridine ring system.


Differential effects of heparin on replicative DNA synthesis and unscheduled DNA synthesis.

The effect of heparin on DNA synthesis was compared between replicative DNA synthesis and unscheduled DNA synthesis. Replicative DNA synthesis in permeable cells or nuclei prepared from rapidly growing mouse ascites sarcoma cells was inhibited by heparin. Unscheduled DNA synthesis in nuclei isolated from normal rat liver or from mouse ascites sarcoma cells in stationary phase was stimulated by heparin at low concentrations but inhibited by high heparin concentrations. DNA polymerase activity assayed with activated calf thymus DNA and DNA polymerase alpha purified partially from mouse ascites sarcoma cells was inhibited with either calf thymus histones or heparin. DNA synthesis inhibited with histones was partially reactivated by heparin. Replicative DNA synthesis in permeable cells was inhibited by adding histones to the assay mixture, and the inhibited DNA synthesis was partially reactivated by low concentrations of heparin. These results indicated that the replicated sites (or replication machinery) in permeable cells or nuclei were largely unrestricted by histones and that heparin inhibition of replicative DNA synthesis was due to the direct inhibitory interaction of heparin with some essential component(s), such as DNA polymerase, of replication machinery.


Fractionation of Saprolegnia diclina (Oomycetes) satelite DNAs by AgNO3/Cs2SO4 density gradient centrifugation.

Saprolegnia diclina DNA has been fractionated using preparative AgNO3/Cs2SO4 and CsCl density gradients. In addition to the previously identified major satellite DNA, there are two minor DNA components banding at 1.682 and 1.701 g - cm(-3) in CsCl. Purified major satellite DNA bands at 1.707 g - cm(-3) giving a base composition of 48% G + C in good agreement with 47% G + C calculated from its Tm value. The nuclear DNA base composition is 58% G + C by both methods. The base composition of the major satellite DNA suggests that it may represent ribosomal DNA cistrons.


PMR-relaxation and steric computations give unequivocal nucleoside conformations.

The configuration and the conformation of alpha and beta anomers of pyrazomycin, cytidine and pseudouridine in aqueous solution have been investigated by 1H-NMR at 250 MHz. T1 proton relaxation measurements are an excellent method to determine the conformation of the base around the glycosidic linkage. Frequently, steric hindrance considerations can help to decide which conformations are possible in nucleoside anomer pairs. The proton-proton coupling constants indicate that the N conformer is largely predominant in the alpha anomers while the S conformer is particularly abundant in beta-pyrazomycin. The steric hindrance is much larger for alpha than for beta-nucleosides and change of a C-C to a C-N glycosidic bond reduces considerably the rotational possibilities of the base. The relaxation data show that alpha-cytidine adopts the anti conformation with gamma = 200 degrees in good agreement with the crystal structure and with the sterical computations. In the other case, when the syn and anti conformations are sterically accessible, the orientation of the base may be completely different from one nucleoside to the other. It can be predicted neither from the crystal structure nor from comparisons with similar compounds. For alpha-pseudo-uridine the predominant orientation of the base (gamma = 120 degrees) is in the boundary of the syn-anti regions; for beta-cytidine the syn (gamma = 65 degrees) and anti (gamma = 215 degrees) conformations are equiprobable at room temperature while beta-pseudouridine shows the syn conformation with gamma = 40 degrees, the smallest angle observed until now. There is no correlation between the N/S and syn-anti ratios.


Spectrophotometric and fluorescence polarization studies of the binding of ethidium, daunomycin and mepacrine to DNA and to poly(I-C).

The changes in the visible absorption spectrum and the degree of fluorescence polarization of daunomycin and mepacrine on binding to DNA have been compared with those shown by the archetypical intercalating agent ethidium. The changes are qualitatively similar for the three drugs, namely a bathochromic shift and hypochromicity (giving an isosbestic point) and an increase in polarization of fluorescence when irradiated with polarized light. These effects are typical of intercalation. The effect of changing the nucleic acid conformation has been investigated by repeating the studies with poly(I-C) (a nucleic acid in the A conformation). Upon interaction with poly(I-C) only ethidium shows effects typical of intercalation, neither daunomycin nor mepacrine intercalates into poly(I-C) helix.


Nucleic acid binding drugs. Part IV. The crystal structure of the anti-cancer agent daunomycin.

The crystal structure has been determined of the anti-cancer drug daunomycin, as the hydrochloride monohydrate pyridine salt. The overall structure, previously determined by X-ray analysis of an N-bromoacetyl derivative (Anguili, R., Foresti, E., Riva Di Sanserverino, L., Isaacs, N.W., Kennard, O., Motherwell, W.D.S., Wampler, D.L. and Arcamone, F. (1971) Nat. New Biol. 234, 78-80) has been confirmed, although substantial conformational differences are observed. The conformation described here is very similar to that found for the related drug carminomycin I (Wani, M.C., Taylor, H.L., Wall, M.E., McPhaill, A.T. and Onan, K.D. (1975) J. Am. Chem. Soc. 97, 5955-5956; Pettit, G.R., Einck, J.J., Herald, C.L., Ode, R.H. Von Dreele, R.B., Brown, P., Brazhnikova, M.G. and Gause, G.F. (1975) J. Am. Chem. Soc. 97, 7387-7388); it is suggested that this represents a significantly stable molecular conformation; an intramolecular C(7)...O(9) hydrogen bond is invoked to account for this. This conformation is likely to be at least close to that of daunomycin when bound to DNA.


DNA repair enzymes in ataxia telangiectasia and Bloom's syndrome fibroblasts.

Ataxia telangiectasia, Bloom's syndrome and normal fibroblasts were compared as to the capacity of their cellular extracts to enhance the priming activity of gamma-irradiated colicin E1 DNA for purified DNA polymerase. It was found that an ataxia strain had substantially lower, and a Bloom's syndrome strain had slightly lower capacity than a normal strain; while the activities of apurinic site specific endonuclease in these extracts were comparable.


Studies on the purification and characterization of malate dehydrogenase from Mycobacterium phlei.

Malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was purified from Mycobacterium phlei using (NH4)2SO4 precipitation followed by chromatography on Sephadex G-200, DEAE-cellulose on DEAE Sephadex A-50. The purified preparation homogeneous on column chromatography, polyacrylamide gel electrophoresis and sodium dodecyl sulphate gel electrophoresis had a molecular weight of 86 860. The native enzyme was composed of four subunits of equal molecular weight (21 550) and was thermostable upto 50 degrees C for 15 min. Some kinetic constants of the enzyme was determined. Tyrosine and isoleucine were identified as the N- and C-terminal amino acids respectively. The effects of various activators and inhibitors on the activity of the purified enzyme were studied. The purified enzyme exhibited maximum excitation and emission at 278 and 345 nm respectively. Amino acid composition of the enzyme was determined. Treatment of the enzyme with acid and urea resulted in dissociation of the enzyme followed by loss of catalytic activity. The dissociated enzyme could however be reconstituted by bringing the pH back to neutrality or by removal of urea from the enzyme solution.


Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves.

Ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, EC 1.6.7.1) from spinach leaves has been purified according to a new procedure. The enzyme shows the presence of five molecular forms as identified by isoelectric focusing, namely a, b, c, d and e with pI values of 6.0, 5.5, 5.2, 5.0 and 4.8, respectively. All the bands are catalytically active and are clearly identifiable after the first steps of the purification procedure. The basic pattern of the ferredoxin-NADP+ reductase forms is the same whether extracted from one or many spinach plants and is not affected by the different purification procedures used. Two distinct classes of molecular weight have been found for the isolated forms b, c and d as measured by sodium dodecyl sulphate electrophoresis, with values of 33 000-34 000 for the first and 36 000-38 000 for the later two forms. Gel electrophoresis in non-denaturing media at different gel concentrations gives the same order of molecular weight values, thus ruling out the possibility that the native enzyme is a dimer, as has been reported by Schneeman, R. and Krogmann, D.W. ((1975) J. Biol. Chem. 250, 4965-4971). No significant kinetic differences were detectable for the isolated forms of ferredoxin-NADP+ reductase.


Plant pyruvate dehydrogenase complex purification, characterization and regulation by metabolites and phosphorylation.

The pyruvate dehydrogenase complex was purified from mitochondria of cauliflower, Brassica oleracea var. botrytis floral buds to a specific activity of 5.4 mumol of NADH/min per mg of protein. The pyruvate dehydrogenase complex required CoASH, NAD+, thiamine pyrophosphate and Mg2+ for the oxidative decarboxylation of pyruvate. The kinetic analysis of the complex gave a series of parallel lines for all substrates. Product interaction patterns showed that NADH is competitive with NAD+; acetyl-CoA is competitive with CoASH; and NADH and acetyl-CoA uncompetitive with pyruvate. These kinetic patterns suggest a multisite ping-pong mechanism as described by Cleveland ((1973) J. Biol. Chem 248, 8353). The noncompetitive inhibition of NADH versus CoASH, and acetyl-CoASH versus NAD are not predicted by this mechanism. Regulation of the complex was more sensitive to the NADH/NAD+ ratio than acetyl-CoA/CoASH ratio. Hydroxypyruvate and glyoxylate inhibited the complex noncompetitively versus pyruvate. The pyruvate dehydrogenase complex was inactivated and phosphorylated by ATP. The ATP dependent inactivation is believed to be enzyme catalyzed by a pyruvate dehydrogenase complex kinase. However, no evidence was found for a plant pyruvate dehydrogenase complex phosphatase. The results suggest that the cauliflower pyruvate dehydrogenase complex is regulated by a phosphorylation-dephosphorylation mechanism.


Increased activity of rat liver nucleolar protein kinase following triiodothyronine administration.

Triiodothyronine (T3) administration to thyroidectomized rats induces a significant increase in the nucleolus-associated protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. The general properties of the protein kinase solubilized from liver nucleoli have been investigated. Mg2+ (20 mM) is essential for the reaction and an appropriate concentration of NaCl (100 mM) is required to achieve maximal phosphorylation rates. The optimal pH for casein phosphorylation is 7.6. The kinase phosphorylates casein more efficiently than phosvitin and displays an almost undetectable activity towards histones and protamine. No significant stimulation of the kinase activity by cyclic AMP has been detected. The apparent Km values for casein and ATP are 1.5 mg/ml and 1.5-10(-5) M, respectively, and are not affected by the hormone administration.


Post-proline dipeptidyl aminopeptidase (dipeptidyl aminopeptidase IV) from lamb kidney. Purification and some enzymatic properties.

Post-proline dipeptidyl aminopeptidase (dipeptidylpeptide hydrolase, EC 3.4.14.1), also known as glycylprolyl beta-naphthylamidase or dipeptidyl aminopeptidase IV, was isolated and purified in an overall yield of 20% from autolyzed extracts of lamb kidney by CM-cellulose and column chromatography on DEAE-Sephadex and Sephadex G-200. Purified enzyme was homogeneous by disc gel electrophoresis and ultracentrifugal analysis and was most active at pH 7.8 using Gly-Pro beta-napthylamide as substrate. The Km values for Gly-Pro beta-naphthylamide and Ala-Ala beta-naphthylamide were 0.63 and 0.77 mM, respectively. The proline-containing peptides were hydrolysed more than 10-fold faster. By isoelectric focusing a pI of 4.9 was determined. The enzyme was estimated to be 230 000 +/- 15 000 by the sedimentation equilibrium method and sodium dodecyl sulfate polyacrylamide gel electrophoresis indicating that the enzyme is composed of two identical subunits with molecular weights of 115 000. It was inhibited by the active-site directed, irreversible inhibitor diisopropylphosphorofluorofluoridate. Post-proline dipeptidyl aminopeptidase, in contrast to the endopeptidase post-proline cleaving enzyme [9,10] (Walter R. (1976) Biochim. Biophys. Acta 422, 138-158, and Koida, M. and Walter, R. (1976) J. Biol. Chem. 251, 7593-7599) exhibits no endopeptidase activity. Instead it is an exopeptidase with a high specificity for NH2-terminal-free peptides containing a proline residue in the penultimate position and releases the dipeptide with proline being the COOH-terminal moiety. The name "post-proline dipeptidyl aminopeptidase" is suggested.


The effect of sample viscosity on the deacylation of p-fluoro-trans-cinnamoylchymotrypsin.

The deacylation of p-fluoro-trans-cinnamoylchymotrypsin at pH 5.0 in the presence of variable amounts of polyvinylpyrrolidone has been examined. The sample viscosity at the highest polymer concentration used was over 200 times greater than that of pure buffer but no effects on the deacylation kinetics were observed.


A reinvestigation of the mechanism of Pseudomonas testosteroni delta 5-3-ketosteroid isomerase.

The mechanism of the isomerisation of delta 5-3,17-androstenedione by the isomerase (3-oxosteroid delta 4-delta 5-isomerase, EC 5.3.3.1) of Pseudomonas testosteroni has been reinvestigated with delta 5-[4-beta-2H]androstenedione as substrate in H2O and delta 5-androstenedione in 2H2O. A precise localisation of the label in delta 4-androstenendione has  revealed that the previously reported 4 beta leads to 6 beta deuterium transfer accounts for only a part of the reaction. Along with this process, removal of the 4 alpha proton is also occurring. This has already been observed with mammalian isomerases. Hence the assumed difference in mechanism between the bacterial and mammalian enzymes is very unlikely.


Xanthine oxidase in lentil (Lens esculenta) seedlings.

Until recently there were no reports regarding the presence of xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) in plants. Direct evidence for its presence in lentil seedlings is reported here. Xanthine oxidase activity increases with the period of germination, reaching a maximum at 24 h and decreasing thereafter. The pH optimum for its activity is at pH 8.0. Almost equal activity is observed against xanthine and hypoxanthine. The Km for xanthine is 1.05 mM, and considerable inhibition is observed at high substrate concentration.


Antibodies to lipoprotein lipase. Application to perfused heart.

An antibody was prepared against purified rat heart lipoprotein lipase. 1. This antibody showed marked species specificity. It inhibited almost totally the lipoprotein lipase activity from all rat tissues examined (i.e., heart, adipose, postheparin plasma, and mammary gland), while having no effect on the activity of lipoprotein lipase partially purified from rabbit, guinea pig and bovine heart and from bovine milk. The antibody also had no effect on the hepatic lipase activity of rat postheparin plasma. 2. After antibody to rat heart lipoprotein lipase was recirculated for 5 min through isolated rat hearts, little or no lipoprotein lipase activity could be detected in the perfusate during 0-20 s of a subsequent non-recirculating perfusion with buffer containing 1 unit heparin/ml. 3. Following recirculation of antibody to lipoprotein lipase for 10 min and a non-recirculating perfusion with buffer for 2 min, the hearts no longer oxidized any significant amounts of 14C-labelled palmitate chylomicron triacylglycerol fatty acid to 14CO2 during a 15-min perfusion. The data give compelling evidence that the functional fraction of lipoprotein lipase in hearts is at the endothelial cell surface accessible to lipoprotein lipase antibody.


Mitochondria from human term placenta. III. The role of respiration and energy generation in progesterone biosynthesis.

The interrelationships among the processes of progesterone biosynthesis, respiration and energy production in human term placental mitochondria were examined. All substrates (citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, glutamate, glycerol 3-phosphate, ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) previously found to stimulate oxygen uptake and ATP synthesis in placental mitochondria supported progesterone synthesis from endogenous and exogenous cholesterol. Citrate support of progesterone synthesis was stimulated by NADP+ but not NAD+. It was inhibited by fluorocitrate and trans-aconitate but not by arsenite, rotenone, antimycin, cyanide or 2,4-dinitrophenol. Ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine support of progesterone synthesis was stimulated by NAD+ and NADP+ and was inhibited by ADP, rotenone, antimycin, cyanide and 2,4-dinitrophenol. Cyanide inhibition was relieved by an exogenous ATP regenerating system and ADP inhibition was reversed by oligomycin. Progesterone synthesis supported by alpha-ketoglutarate + malonate was stimulated by NAD+ and NADP+, and was completely inhibited by arsenite. 2,4-Dinitrophenol was strongly inhibitory both in the absence and presence of rotenon or antimycin. Stimulation by ATP was enhanced by rotenon, antimycin and oligomycin and inhibited by 2,4-dinitrophenol. Thus, metabolic control of progesterone synthesis by the energy status of the mitochondrial system was demonstrated when reducing equivalents were supplied via NADH or the respiratory electron transport chain.


The specificity of 1-acyl-sn-glycerol 3-phosphate acyltransferase in microsomal fractions from lactating cow mammary gland towards short, medium and long chain acyl-CoA esters.

The 1-acylglycerolphosphate actyltransferase from a microsomal fraction of lactating cow mammary gland was active towards acyl-CoAs of chain length C8-C18, but not towards butyryl-CoA or hexanoyl-CoA. The lack of activity towards butyryl-CoA and hexanoyl-CoA explains why butyric and hexanoic acid are largely excluded from the sn-2 position of triacylglycerols from cow milk. The chain length specificity of the acyltransferase was C16 greater than C14 greater than C12 greater than C10 greater than C8, which is essentially the same as the order with which the fatty acids are found at the sn-2 position of cow milk triacylglycerols. The specificity was not affected by the nature of the fatty acid (palmitic or oleic acid) at the sn-1 position of 1-acylglycerolphosphate, as predicted by the theory of noncorrelative acylation.


Purification of lysophospholipases. Application of a continuous spectrophotometric assay using thioester substrate analogs.

Purification of lysophospholipases was monitored with four analogs of the natural lysophosphatidylcholine substrate, including two analogs with an acylthioester bond. In all chromatographic procedures employed, peaks of enzymatic activity towards each of the substrates were coincidental; moreover, the ratio of thioester to oxyester hydrolysis rates remained essentially constant over a more than 500-fold purification. These findings strongly support the conclusion that the hydrolysis of the thioester substrates truly reflects the specificity of lysophospholipases, thus allowing the use of a convenient spectrophotometric assay for these enzymes.


The structure and morphology of the abnormal serum lipoprotein-X.

The structure and morphology of an abnormal lipoprotein particle present in the serum of patients with obstructive jaundice has been investigated by gel filtration, electron microscopy and NMR spectroscopy. Lipoprotein-X is a spherical lipoprotein particle with an average Stokes diameter of approximately 40 nm and a wide size distribution ranging from 20 to 70 nm. Different from all lipoprotein structures known so far lipoprotein-X is a hollow particle (= vesicle) with a water-filled internal cavity surrounded by a continuous, single bilayer which is impermeable to cations and K3Fe(CN)6. The packing of the bilayer is tighter and the segmental motion of both the polar group and the hydrocarbon chains are significantly reduced relative to typical phosphatidylcholine bilayers. In terms of segmental motion and anisotropy of packing the lipoprotein-X bilayer closely resembles a model bilayer system consisting of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin and cholesterol mixed in the same molar ratio as in lipoprotein-X. In that model system the phospholipid distribution between the two layers of the bilayer is asymmetric with (sphingomyelin + lysophosphatidylcholine) being preferentially located on the inner layer and phosphatidylcholine preferentially on the outer layer of the bilayer. By analogy with the model system the phospholipid distribution in lipoprotein-X bilayers is proposed to be also asymmetric.


Base stimulation of phospholipid metabolism in neuroblastoma cells. I. Kinetics of incorporation of N-methylated ethanolamine bases.

The effects of nitrogen bases on the regulation of phospholipid metabolism in neuroblastoma cell cultures were investigated. An increase in the total cellular phospholipids was observed up to 24 h following plating. Addition of monomethyl- and dimethylethanolamine bases resulted in a stimulation of the synthesis of their corresponding phospholipids. The average rates of synthesis of phosphatidylmonomethyl- and phosphatidyldimethylethanolamine were 0.09 and 0.12 nmol/microgram DNA per h, respectively. The labeling patterns of the various phospholipid species from ortho[32P]phosphate have been determined. They suggest that the synthesis of the analogs proceeded entirely via a phosphate mediated pathway rather than through a base exchange mechanism. A number of distinct patterns for the incorporation of bases into acyl-, alkyl- and alkenyl-containing phosphoglyceride species were indicated. The polar head group composition appeared to be intimately related to the type of bond of the hydrocarbon residue.


Characterization of phospholipase A from pulmonary secretions of patients with alveolar proteinosis.

Phospholipase A2 (EC 3.1.1.4) from the insoluble pulmonary secretions that accumulate in the lungs of patients with alveolar proteinosis has been purified. The pure enzyme gives a single sharp band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. Amino acid analysis of the protein shows high content of cystine, aspartic acid, glutamic acid, serine, glycine, leucine and lysine. Only one N-terminal residue, alanine, can be detected. Gel filtration as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis indicate an apparent molecular weight of 75 000 for the enzyme. The enzyme activity has a pH optimum between 7.5 and 8.5 and is stimulated by sodium deoxycholate and CaCl2.


Microdetermination of dolichol in tissues.

A method is described for the rapid purification and analysis of tissue dolichol on a nanomole scale. The assay is based on a radioisotope dilution technique in which [3H]dolichyl palmitate is used as tracer and acetylation with [14C]acetic anhydride serves as the quantitating reaction. The acetylation conditions were characterized with respect to time, temperature and concentration of reagents. When this method was applied to pig liver, a value was obtained which fell within the range of previously published results based on a gravimetric assay. In chicken, dolichol levels were found to be high in oviduct and low or absent in red blood cells and plasma.


Regional distribution of water and glycosaminoglycan in immature articular cartilage.

The distribution of water and glycosaminoglycan in different functional regions of bovine immature articular cartilage were studied. There was always more water in each articulating than in the corresponding growing zone, but there was less water in both zones in the areas of maximum contact. There was more hyaluronate and much more keratan sulphate in the articulating areas of maximum contact than in the minimum contact areas. In the growing zone the distribution of these two glycosaminoglycans did not vary as significantly but there was slightly more keratan sulphate in the area of maximum contact. Regional variations in chondroitin sulphate content were also present although not as striking as those of keratan sulphate. The results suggest that some keratan sulphate may be synthesized as a reaction to load.


Colchicine increases hepatic alkaline phosphatase activity.

In vivo administration of colchicine increases the activity of alkaline phosphatase significantly in the livers of rats. Prior treatment with cycloheximide prevented the induction of the enzyme by colchicine suggesting that de novo protein synthesis was essential for the effect of colchicine on alkaline phosphatase activity. Bilateral adrenalectomy did not affect the response of alkaline phosphatase following the administration of colchicine. This indicates that the rise in the level of alkaline phosphatase in liver caused by colchicine is not secondary to the release of glucocorticoids.


Vitamin B-12 biosynthesis. A model system for isopropanolamine formation by reaction between reduced corrinoid and threonine.

When a solution of 14C-labeled L-threonine is incubated with aquocobalamin in the presence of excess of a reductant such as 2-mercaptoethanol or sodium borohydride, a labeled corrinoid adduct is formed. The corrinoid participates stoichiometrically, and not catalytically. Since 35S-labeled 2-mercaptoethanol is not part of the adduct, the reduction of the corrinoid from the Co(III) to the Co(II) form is a prerequisite for the reaction. Various independent tests show the formation of isopropanolamine. The reaction system is an attractive model for the so far unexplained decarboxylation of threonine to isopropanolamine in the course of corrinoid biosynthesis.


Heterogeneity of steroid hormone receptor in adult rat lung cytosol.

Chromatography on cellulose DEAE-52 columns revealed that the glucocorticoid receptor for rat lung cytosol consisted of a component in the 0.001 M prewash, revealed with synthetic steroids and natural mineralocorticoids, a second component eluted with 0.04 M PO4, labelled with triamcinolone, dexamethasone, and a third moiety in the 0.06 M PO4 region, evident with natural glucocorticoids (corticosterone, cortisol) as well as mineralocorticoids (aldosterone, deoxycorticosterone). The third component coeluted with rat blood serum transcortin in double labelled experiments. Rate lung was devoid of another component in the 0.02 M PO4 found in rat liver supernate and of the mineralocorticoid receptor evident only in rat kidney. Chromatography on Sephadex G-200 columns revealed a shift of radioactivity from a higher to a lower molecular weight region in the presence of 0.4 M KCl.  Collectively, these studies indicate the subunit nature of the lung receptor as evidenced in most tissues hitherto tested. Moreover, polymorphism within a given subunit component can not be revealed by competition alone, as attempted by others, but can be revealed under selected conditions of physical separation.


Effect of local anesthetics on plasma protein secretion by rat hepatocytes.

The effects of some local anesthetics on plasma protein secretion by rat liver slices have been studied and have been compared with those of colchicine. Rat liver slices were pulse-labelled with L-[14C]leucine for 9 min at 37 degrees C, collected on filter paper, washed with non-radioactive leucine and reincubated in the presence or absence of the drug to be tested. The radioactive plasma proteins produced were obtained by immunoprecipitation from either the chase medium or from the washed slices. Chlorpromazine, (3.10(-5) M), dibucaine (10(-5) M), lidocaine (10(-3) M) and procaine (5.10(-5) M) inhibited both the synthesis and secretion of plasma protein but did not affect the uptake of L-leucine into the slices nor the incorporation of phosphate into intracellular nucleotide phosphates or into phospholipids. The inhibition of secretion elicited by these drugs is probably not due to the inhibition of protein synthesis since cycloheximide, when added to the chase medium at a concentration which completely inhibits protein synthesis, did not inhibit plasma protein secretion, while cycloheximide plus procaine did inhibit secretion and also caused a retention of non-secreted plasma proteins within the slices. Unlike colchicine, however, procaine did not cause the retained plasma proteins to accumulate in Golgi-derived secretory vesicles, but showed a more general effect causing a distribution among several cell fractions.


Glycosylation of endogenous proteins through dolichol derivatives in reticulocyte plasma membranes.

It has been found that rabbit reticulocyte plasma membranes contain the enzymatic activities responsible for the synthesis of all the polyprenol-bound sugars that are intermediates in the glycosylation of proteins. The final reaction in which an oligosaccharide in transferred from a dolichol pyrophosphate derivative to an endogenous protein was detected in reticulocyte but not in erythrocyte plasma membranes.


CNDO molecular orbital calculations on porphyrins--I. Ground and excited states of porphyrin, divinylporphyrin and tetraphenylporphyrin.

Semi-empirical CNDO/2 MO calculations are reported for the ground states of porphyrin, 2,4-divinylporphyrin (DVP), and alpha, beta, gamma, delta-tetraphenylporphyrin (TPP). Results for TPP refer to the conformation with all phenyl groups perpendicular to the porphyrin ring, calculated to be 108 kJ mol-1 more stable than the coplanar conformation. The substituents withdraw electron density where they are attached to the porphyrin ring, increasing selected orbital energies. The vinyl groups also modify the electron population at nitrogen. CNDO/S calculations with extensive configuration interaction are reported for excited states. The lowest singlet states are closely similar in energy and composition for all three molecules, except for an extra state and more complex compositions in DVP above 3 eV. The lowest triplet states of porphyrin and DVP are very similar, while those of TPP are comparable in energy or composition but not both. Experimental data on the excited states are broadly consistent with the calculations, although comparisons for the excited triplets are tentative.


CNDO molecular orbital calculations on porphyrins--II. Ground states of dilithium and disodium porphyrin.

CNDO/2 calculations are reported for dilithium and disodium porphyrin. The total energy is calculated as a function of the metal-ring distance for symmetrical (D4h) structures. For dilithium porphyrin, the equilibrium metal-ring distance is 0.87 A and the metal-metal vibrational frequency is 123 cm-1. For disodium porphyrin, the distance is 1.64 A and the frequency is 77 cm -1. Little mixing of metal and porphyrin orbitals takes place; the two lowest unoccupied and the two highest occupied MOs hardly differ from those in porphyrin, but lower MOs are considerably rearranged.


Distribution of 9 trace metals in 17 anatomic sites of dog heart tissue and their classification using pattern recognition.

The concentrations (geometric means of 11 determinations) of 9 trace metals in 17 anatomic regions of dog heart tissue and blood vessels were determined. The relationships recognized within the set of trace metal profiles were comparable to those reported earlier for the cardiovascular systems of pig [J. Mol. Cell Cardiol 6, 383 (1974)] and those of beef [Bioinorg, Chem. (in press)]. These included differences in trace metal concentrations that allowed differentiation of the following tissues from each other: (1) ordinary and specialized myocardium from blood vessels and heart valves, (2) blood vessels from heart valves and (3) arterial from venous tissues.


The complexes of adenosine and organocobalamins with palladium (II).

The complexes of adenosine, methylcobalamin, adenosylcobalamin and the derivatives of adenosylcobalamin with tetrachloropalladate (II) are described. The compositions of the complexes were established by means of spectrophotometric titration. Depending on the structure of organocobalamins, three types of complexes with tetrachloropalladate (II) are formed. In the case of methylcobalamin, the palladium (II) is coordinated at the "lower" benzimidazole ligand. In adenosylcobalamin, palladium (II) coordinates the "upper" adenosyl ligand. The N-7-position in adenosine ligand and N-3-position in 5,6-dimethylbenzimidazolyribotide are the most probable coordination sites of palladium (II).


A proton magnetic resonance investigation on 1:1 cytidine and guanosine silver(I) complexes.

Solid 1:1 silver (I) complexes with cytidine and guanosine have been isolated from aqueous solutions at pH 10. PMR spectra in deuterodimethylsulfoxide-d6 solutions have been measured. Results show that binding sites for cytidine are the nitrogen atom of the NH2 group and N-3. In the Agguanosine complex, pmr data are in agreement with the formation of a chelate ring involving N-7 and O-6. Chemical shift changes of the amino protons show that NH2 is also another binding site.


Inorganic and nutritional aspects of cancer: a conference report.

The Inaugural Conference of the International Association of Bioinorganic Scientists (I.A.B.S.) was devoted entirely to inorganic and nutritional aspects of cancer. The reports of twenty-five invited speakers indicate that this area of cancer research has already produced major advances and that further significant progress may be expected to occur in the near future.


[Purification of two beta-D-glycosidases from the digestive juice of Achatina balteata].

Two beta-D-glycosidases have been purified from the digestive juice of Achatina balteata by acetone fractionation, ion exchange chromatography through DEAE-Sephadex A-50, ammonium sulphate fractionation and gel chromatography through Sephadex G-200. The preparations are homogeneous by p/lyacrylamide gel electrophoresis. Both enzymes are highly specific for the beta-D-anomeric configuration of the glycosidic linkage. They hydrolyse lactose, cellobiose and synthetic beta-D-galactosides, -glucosides and -fucosides at a pH optimum of 5,2 to 5,6 and are inactive on alpha-glycosides. The hydrolyzed substrates are recognized by the same catalytic site as shown by mutual competition studies between substrates and competitive inhibition observed with aldonolactones and glycopyranoses such as D-galactose, D-glucose and D-fucose. The different substrates are not hydrolyzed at the same rate by the two enzymes. They also differ by their electrophoretic mobility, their behaviour in gel chromatography and their stability towards pH and heat. The most salient property is the important beta-D-fucosidase activity of the two purified enzymes.


Hydrogen bonding of amino acid side chains to nucleic acid bases.

Absorption studies of hydrogen bonding association between nucleic acid bases and amino acid side chains show that association constants can be determined from difference absorption spectra in cyclohexane and chloroform. Association constants for the binding of 9-ethyladenine, 1-cyclohexyluracil and 1-cyclohexylcytosine to side chains of serine, threonine, aspartic acid, lysine, cystéine, methionine and tyrosine are reported. Results obtained in chloroform and cyclohexane are compared.


[Reversible enlargement of proteins in rat liver mitochondria: analysis by polyacrylamide gel electrophoresis].

Intramitochondrial large amplitude protein movements were studied by polyacrylamide gel electrophoresis on the intermembranal fluids of mitochondria which were incubated in presence of movement effectors. The specific and reversible property of this phenomenon was confirmed by this method. Its bilateral nature was observed. The eventuality of a partial inner membrane damage provoked by the synergic action of digitonine and movement effector, was discussed, evaluated and dismissed.


Single-case designs and clinical biofeedback experimentation.

Rigorous evaluation of the effects of biofeedback with clinical populations is necessary, but practical problems often preclude utilization of between-groups experimental designs involving large numbers of clients with clinically relevant problems. Single-case experimental designs provide a viable alternative for answering most research questions. In addition, single-case designs possess several distinct advantages for biofeedback research, including a focus on clinical significance, the use of variability as data not error, unique procedures for establishing generality of findings, and an ability to deal with ethical concerns in clinical research. Basic procedures in the use of single-case experimental designs are described and illustrations in clinical biofeedback research are provided.


Cephalic vasomotor feedback in the modification of migraine headache.

The effect of cephalic vasomotor response (CVMR) and frontalis electromyographic (EMG) feedback on control of temporal arterial vasoconstriction and frontalis muscle activity in migraine and muscle contraction headache patients was investigated. A single subject multiple baseline design (across subjects and responses) was introduced to evaluate (1) patterning in the two physiological systems and (2) the effects of CVMR and EMG feedback on headache activity. The data indicated that (a) all four patients demonstrated an ability to control CVMR activity during CVMR feedback and EMG during EMG feedback, (b) idiosyncratic patterns of physiological activity emerge during feedback training, and (c) learned control of the pain mechanism for muscle contraction and migraine headaches was related to reduced frequency and duration of these headaches.


Independent biofeedback self-regulation of EEG alpha and skin resistance.

Skin resistance and EEG alpha were recorded concurrently during alpha biofeedback, in which the participant attempted to control alpha, and during skin resistance biofeedback, in which the participant attempted to control skin resistance. Alpha production changed significantly (p less than .001) during alpha biofeedback, indicating successful self-regulation of alpha, but did not change significantly during skin resistance biofeedback. Similarly, skin resistance changed significantly (p less than .001) during skin resistance biofeedback but did not change significantly during alpha biofeedback. The results show independent control of alpha and skin resistance, and may reflect independent self-regulation of cognitive and somatic tension-relaxation systems.


Locus of control and frontal electromyographic response training.

Forty-eight normal subjects from a college population, representing extreme internal and external orientations on a locus of control scale, were provided an auditory signal in a laboratory relaxation setting. For one group (feedback) the pitch of the signal varied as a function of frontal electromyographic (EMG) levels, while for another group (control) the pitch was constant. The feedback subjects acquired lower EMG potentials than did the control subjects, and the internal subjects in the feedback condition acquired lower levels than did the external subjects. In the control condition, no consistent differences in EMG levels between internal and external subjects were obtained. These results were stable across two replications, three ethnically distinct groups, and both sexes. Additional measures designed to reflect the effects of the frontal EMG training as a generalized relaxation technique for this population, including finger temperatures and a variety of postexperiment queswtionnaire ratings of relaxation, yielded negative results. Tests of other predictions from the locus of control construct are also discussed.


The use of EMG feedback in the treatment of a severe case of blepharospasm.

In this case report, a 50-year-old female with a chronic blepharospasm (spasmodic winking) around both eyes was treated with 17 sessions of electromyographic (EMG) feedback. EMG level and spasm frequency were monitored during baseline, placebo, and feedback treatment sessions. There was a marked decrease in both EMG level and spasm frequency, which generalized to her everyday life. Improvement was maintained at 4-month follow-up.


[A new method for synthesis of some nucleoside 5'-phosphonate esters].

A new method for synthesis of nucleoside-5'-phosphonates containing a reactive halogen atom in the phosphonate residue has been developed. In order to achieve selective inhibition of individual enzymes and to elucidate the structure of their active sites, the synthesis of 5-'chloromethyl-, 5'-(beta-bromoethyl)-and 5'-(N-chloroacetylaminomethyl)-phosphonate esters of adenosine and previously unknown analogous derivatives of uridine have been carried out. The method described may also be used to synthesize 5'-phosphonate esters of nucleosides, which contain radioactive labels.


[Regeneration of rhodopsin in marine teleost fishes in the presence of exogenic of 11-cis-and all trans-retinals].

Regeneration of rhodopsin of the sea perch (Sebastodes schlegeli) and walleye pollock (Theragra chalcogramma) was studied in different preparations of eye after the addition of 11-cis- and all trans-retinals and at the expense of endogenic sources. It was established that the regeneration of rhodopsin in the eye cup, homogenate of retina and rod outer segments (ROS) at the expense of endogenic sources was extremely low and it was slightly forced by the addition of the pigment epithelium to the last two preparations. The binding degree of 11-cis retinal of walleye pollock ROS decreased with the increase of temperature of the incubation mixture and the increase of duration of storage of ROS at the temperature--10 degrees. Partial removing of lipids by hexane decreased the percentage of regeneration of the ROS preparation; on the other hand, the peroxidation of lipids practically did not affect the regenerative ability of fresh-obtained preparations. After solubilization of opsin by digitonin and sodium cholate, 40- and 50%-decreases of binding of 11-cis retinal were observed respectively. The digestion of ROS by papain, which caused no changes in the spectral properties of dark preparations, completely eliminated the regenerative ability of ROS. Regeneration of ROS at the presence of all trans-retinal proceeded at a very low speed. The specificity of the process of regeneration of rhodopsin in fishes is discussed.


[Study of the topography of the nitrogenase active center by the electron microscopy method with the use of the electron density labels].

The electron microscopy method has been used for studying the topography of the active centre of the nitrogenase in combination with the method of electron density labels. The active centre has been shown to place on the nitrogenase macromolecule asymmetrically. The non-heme iron atoms of the nitrogenase active centre have been regularly packed and located on the macromolecule as a quadrangular cluster closely to the surface of subunits. Two free sulfhydryl groups of the ATP-site of the nitrogenase active centre are in close proximity to the cluster. ATP-site of the nitrogenase active centre containing these groups is located on the Fe-enzyme, while the main part of the iron-containing cluster is located on the Mo-Fe-enzyme.


[Two simple methods for isolation of DNA from various sources using cetavlon].

Two general methods for the isolation of DNA from various sources based on the use of cetyltrimethylammonium bromide (cetavlon, CTA-Br) are described. Cetavlon is a strong cationic detergent precipitating DNA from diluted salt solutions. Cells are lysed and cellular components are dissolved in the presence of cetavlon, 5 M urea, 0.1 M EDTA and 2 M NaCl (KCl). In the first method pure DNA is precipitated in the form of CTA-salt by direct dilution of the lysate to bring the concentration of NaCl (KCl) down to 0.5 M after the removal of the main part of proteins by deproteinization with chloroform. In the first method pure DNA is precipitated in the form of CTA-salt by direct dilution of the lysate to bring the concentration of NaCl (KCl) down to 0.5 M after the removal of the main part of proteins by deproteinization with chloroform. In the second method DNA is purified on the hydroxyapatite column after cell lysis and the removal of cell debris by centrifugation. Both methods are suitable for rapid isolation of pure DNA from various sources with recovery about 80% and average molecular weight 20-10(6) and higher without use of ribonuclease, pronase and amylase.