The B-vitamins in malnutrition with alcoholism. A model of intervitamin relationships. 1. The B-vitamin status of fifty-nine patients, mainly from the lower socio-economic classes in Bombay, with a history of chronic malnutrition, and of alcoholism of 1-5-20 years' duration, was studied before and during treatment, and in relation to their clinical, especially neurological, condition. These patients were divided into two neurological categories: (I) those with peripheral neuropathy (mainly sensory and distal) alone, (2) those with mental changes (mainly confusion and disorientation) also. Both categories frequently showed pellagrous pigmentation and mucocutaneous signs of B-vitamin deficiency. 2. Thiamin and erythrocyte transketolase (EC 2.2.1.1) activity, riboflavin, nicotinic acid, pantothenic acid, total and pyridoxal fraction of vitamin B6, folate and total vitamin B12 were estimated in the blood and the cerebrospinal fluid (CSF) of these patients, and also in the blood of sixty-nine control subjects and in the CSF of some of them. The concentrations of all the vitamins, except vitamin B12, were highly significantly lower in the blood of patients of category I compared to the controls, and erythrocyte transketolase activity and pyridoxal concentration in patients of category 2 were significantly lower than those of category I patients. Blood pantothenic acid and folate concentrations were reduced less consistently. 3. Serum vitamin B12 concentration was significantly increased (though within normal range) in the patients compared to the control group, probably because of the moderate hepatic insufficiency (as assessed by liver function tests) in the former. 4. The concentrations of thiamin, riboflavin, nicotinic acid and total vitamin B6 were also highly significantly lower in the CSF in patients of category I compared with controls. Furthermore, thiamin, nicotinic acid and total vitamin B6 concentrations were significantly lower in patients of category 2 than those of category I patients, indicating that CSF levels reflect better the neurological status of these patients. 5. There was a moderate increase in the blood concentration of all the vitamins tested, after a relatively poor hospital diet alone. There was a concurrent increase in the blood levels of thiamin, riboflavin, nicotinic acid and pantothenic acid after parenteral treatment with either thiamin or nicotinic acid. The administration of pyridoxine resulted in a significant increase in the blood levels of riboflavin and the pyridoxal fraction of vitamin B6.


Inhibition of estrogen-receptor-DNA interaction by intercalating drugs. Ethidium bromide, an intercalating drug, was shown to inhibit the in vitro DNA binding of the uterine estradiol-receptor complex. The inhibition was reversible, dose dependent, complete for total saturation of DNA intercalating sites by the dye, and proportional to the extent of intercalated drug. The binding of the receptor to phosphocellulose and poly(adenylic acid)-cellulose was not decreased by this drug. Similar inhibition was also obtained with 9-hydroxyellipticine. Denatured DNA was more efficient at binding the estrogen receptor than phosphocellulose or poly(adenylic acid)-cellulose but less efficient than native DNA. We conclude that the DNA binding of the estrogen receptor cannot be simply interpreted in terms of electrostatic interactions but requires a particular double-helical structure of DNA.


Atherogenic hyperlipoproteinemia induced by cholesterol feeding the Patas monkey. Patas monkeys were studied for 2 years on three dietary regimes: (1) commercial chow (control diet); (2) semipurified diet plus lard (fat-fed); and (3) semipurified diet plus lard and cholesterol (cholesterol-fed). The control and fat-fed animals had similar lipoproteins which were equivalent to the human very low density, low density (LDL), and high density lipoproteins. An additional lipoprotein referred to as LDL-II appeared to be equivalent to the human Lp(a). The cholesterol-fed animals developed accelerated atherosclerosis associated with a hypercholesterolemia which was characterized by (1) the appearance of beta-migrating lipoproteins (B=VLDL) in the d less than 1.006, (2) an increase in the intermediate lipoproteins and LDL, and (3) the appearance of LDL-II which contained a prominence of the arginine-rich apoprotein. The arginine-rich apoprotein was also a prominent component of the B-VLDL and intermediate lipoproteins. Characterization of this apoprotein revealed that it contained 11.5 mol % arginine, had a molecular weight of approximately 34 000, and coelectrophoresed with the arginine-rich apoprotein of man, dog, swine, rat, and rabbit.


Modification of membrane lipid: physical properties in relation to fatty acid structure. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.


Thermodynamic studies of binary and ternary complexes of pig heart lactate dehydrogenase. The thermodynamics of the reaction catalyzed by pig heart muscle lactate dehydrogenase (LDH; EC1.1.1,27) have been studied in 0,2 M potassium phosphate buffer, pH 7, over the temperature range of 10 to 35 degrees C by using oxamate and oxalate to simulate the corresponding reactions of the substrates pyruvate and lactate, respectively. The various complexes formed are characterized by Gibbs free energies, enthalpies, and entropies. The Gibbs free energies were determined by equilibrium dialysis investigations, fluorescence titrations, and ultraviolet difference spectroscopy, while the reaction enthalpies stem from direct calorimetric measurements, Formulas are given for both the temperature dependence of the equilibrium constants and the variation with temperature of the enthalpies involved in the four reactions between LDH and NADH or NAD, LDH-NADH and oxamate, and LDH-NAD and oxalate. All reactions show a marked negative temperature coefficient, deltacp, of the binding enthalpies indicating partial refolding to be associated with binary and ternary complex formation. This interpretation appears very probable in view of recent x-ray crystallographic studies on lactate dehydrogenase from dogfish, which demonstrate a volume decrease to occur on binding of oxamate to the LDH-NADH complex. The validity of the thermodynamic parameters, as derived with substrate analogues, for the actual catalytic reaction, gains strong support from the agreement between the sum of the heats involved in the four intermediary reactions reported in this study and direct determinations of the overall enthalpy associated with the catalytic process published in the literature.


Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous beta polymerase. Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.


Molecular mechanism of cardiotoxin action on axonal membranes. Cardiotoxin isolated from Naja mossambica mossambica selectively deactivates the sodium-potassium activated adenosine triphosphatase of axonal membranes. Tetrodotoxin binding and acetylcholinesterase activities are unaffected by cardiotoxin treatment. The details of association of cardiotoxin with the axonal membrane were studied by following the deactivation of the sodium-potassium activated adenosine triphosphatase and by direct binding measurements with a tritiated derivative of the native cardiotoxin. The maximal binding capacity of the membrane is 42-50 nmol of cardiotoxin/mg of membrane protein. The high amount of binding suggests association of the toxin with the lipid phase of the membrane. It has been shown that cardiotoxin first associates rapidly and reversibly to membrane lipids, then, in a second step, it induces a rearrangement of the membrane structure which produces and irreversible deactivation of the sodium-potassium activated adenosine triphosphatase. Solubilization of the membrane-bound ATPase with Lubrol WX gives an active enzyme species that is resistant to cardiotoxin-induced deactivation. Cardiotoxin binding to the membrane is prevented by high concentrations of Ca 2+ and dibucaine. Although cardiotoxins and neurotoxins of cobra venom have large sequence homologies, their mode of action on membranes is very different. The cardiotoxin seems to bind to the lipid phase of the axonal membrane and inhibits the sodium-potassium activated adenosine triphosphatase, whereas the neurotoxin associates with a protein receptor in the post-synaptic membrane and blocks acetylcholine transmission.


Structure of an apolipoprotein-phospholipid complex: apoC-III induced changes in the physical properties of dimyristoylphosphatidylcholine. The effect of ApoC-III, a major apoprotein constituent of human very low density lipoproteins, on the physical properties of dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by magnetic resonance and fluorescence techniques. The sharp gel-liquid crystalline transition usually observed at 23 C in DMPC  is both broadened and elevated when ApoC-III is bound as determined (a) from measurements of microscopic viscosity by pyrene excimer fluorescence, (b) from the distribution of di-tert-butyl nitroxide between the bulk aqueous phase and the fluid lipid phase, and (c) from the motion of fatty acyl chains of spin-labeled phosphatdylcholine. Experiments involving the translocation of ascorbate and charged nitroxide ions and the movement of paramagnetic Eu 3+ ions indicate that when ApoC-III binds to DMPC vesicles, it increases their permeability or destroys their original bilayer structure. These two possibilities were distinguishable by gel filtration of the DMPC-ApoC-III complex (approximately 34 mol mol) that indicated that the product particles were significantly smaller than the original vesicles. Taken together, the data indicate that ApoC-III binding to DMPC not only decreases the acyl chain motion of individual lipid molecules, but also induces break-down of bilamellar vesicular structure to give significantly smaller complexes.


The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link. A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.


An enzyme kinetics and 19F nuclear magnetic resonance study of selectively trifluoroacetylated cytochrome c derivatives. The reaction of cytochrome c with ethyl thioltrifluoroacetate was carried out under conditions which led to the selective trifluoroacetylation of a small number of the 19 lysines. The mixture of derivatives was separated by ion-exchange chromatography and four different derivatives with well-resolved 19F nuclear magnetic resonance (NMR) spectra were obtained. Peptide mapping techniques indicated that one of these derivatives contained a single trifluoroacetyl group at lysine 22, and another derivative was singly labeled at lysine 25. The trifluoroacetylated lysine 22 derivative was fully active toward both succinate-cytochrome c reductase (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) white the trifluoroacetylated lysine 25 derivative was fully active toward the reductase, but had a threefold greater Michaelis constant in the  cytochrome oxidase reactin. This supports the hypothesis that the cytochrome oxidase binding site is located in the heme cervice region, and that Lys-25 is important in the binding. 19FNMR spectra of the cytochrome c derivatives bound to phospholipid vesicles were obtained. The reasonably narrow line widths (35-65 Hz) and good sensitivity of the trifluoroacetyl resonances indicated that they might  be useful probes for the interaction of cytochrome c with intact mitochondria.


Alterations in lipid acyl group composition and membrane structure in cells transformed by Rous sarcoma virus. The acyl group composition of the phospholipids from normal chick embryo fibroblasts and from cells transformed by Rous sarcoma virus was determined by gas-liquid chromatography. Rous-transformed cells had less arachidonate (20:4) and more oleate (18:1) in membrane lipids than normal, growing cells. Normal density-inhibited cells had the lowest ratio of 18:1/20:4. Associated with the decreased content of 20:4 in the transformed cells was a decreased motional freedom of an incorporated spin-labeled fatty acid analogue. Arrhenius plots for uptake of 2-deoxyglucose revealed an increased apparent activation energy in the transformed cells, suggesting that the hexose transport carriers were sensitive to the changes in membrane composition and structure in fully transformed cells. However, the development of the changes in fatty acid composition occurred relatively slowly in the course of transformation, indicating that the observed compositional alterations are not likely to be a primary cause of the early changes in membrane function associated with malignant transformation.


Enzyme hyperspecificity. Rejection of threonine by the valyl-tRNA synthetase by misacylation and hydrolytic editing. Valyl-tRNA synthetase from Bacillus stearothermophilus activates thereonine and forms a 1:1 complex with threonyl adenylate, but it does not catalyze the net formation of threonyl-tRNAVal at pH 7.78 and 25  degrees  C in the quenched flow apparatus it decomposes at a rate constant of 36s-1. During this process there is a transient formation of Thr-tRNAVal reaching a maximum at 25 ms and rapidly falling to zero after 150 ms. At the peak, 22% of the (14C) threonine from the complex is present as (14C) Thr-tRNA. The reaction may be quenched with phenol and the partially mischarged tRNA isolated. The enzyme catalyzes its hydrolysis with a rate constant of 40s-1. The data fit a kinetic scheme in which 62% of the threonine from the threonyl adenylate is transferred to the tRNA. This may be compared with the rate constant of 12s-1 at which 84% of the valine is transferred to tRNAVal from the enzyme-bound valyl adenylate, and the rate constant of 0.015s-1 for the subsequent hydrolysis of Val-tRNAVal. Inhibition studies indicate a distinct second site for hydrolysis. The translocation of the aminoacyl moiety between the two sites could be mediated by a transfer between the 2'-and 3'-OH groups of the terminal adenosine fo the tRNA. The hyperspecificity of the enzyme is based on discriminating between the two competing substrates twice: once against the undesired substrate in the synthetic step, and once against the desired substrate in the destructive step.


Essential arginyl residues in fructose-1,6-bisphosphatase. Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.


Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes. The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.


Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition. The existence of distinct regions of mismatch in molecular packing at the interfaces of the fluid and ordered domains during the phase transition of dimyristoylphosphatidylcholine vesicles has been demonstrated by measuring the temperature dependence of the permeability to a spin-label cation and comparing this with a statistical mechanical calculation of the fraction of interfacial lipid. The kinetics of uptake and release of the 2,2,6,6-tetramethylpiperidinyl-1-oxycholine (Tempo-choline) spin label by single-bilayer dimyristoylphosphatidylcholine vesicles were measured using electron spin resonance spectroscopy to quantitate the amount of spin label present within the vesicles after removal of the external spin-label by ascorbate at 0 degrees C. Both the uptake and release experiments show that the Tempo-choline permeability peaks to a sharp maximum at the lipid-phase transition, the vesicles being almost impermeable to Tempo-choline below the transition and having a much reduced permeability above. The temperature profile of the permeability is in reasonable quantitative agreement with calculations of the fraction of interfacial boundary lipid from the Zimm and Bragg theory of cooperative transitions, which use independent spin-label measurements of the degree of transition to determine the cooperativity parameter. The relatively high intrinsic permeability of the interfacial regions (P approximately 0.2-1.0 X 10(-8) cm/s) is attributed to the mismatch in molecular packing of the lipid molecules at the ordered-fluid boundaries, which could have important implications not only for permeability in natural membranes (e.g., in transmitter release), but also for the function of membrane-bound enzymes and transport proteins.


Arthrobacter luteus restriction endonuclease recognition sequence and its cleavage map of SV40 DNA. The nucleotide sequence at the cleavage site of the restriction endonuclease isolated from Arthrobacter luteus (Alu) has been determined. The endonuclease cleaves at the center of a palindromic tetranucleotide sequence to give even-ended duplex DNA fragments phosphorylated at the 5'-end. The endonuclease cleaves SV40 form I DNA into 32 fragments. The order and sizes of these fragments have been determined to provide an Alu cleavage map of the SV40 genome.


Methionyl-tRNA synthetase from Escherichia coli: active stoichiometry and stopped-flow analysis of methionyl adenylate formaiton. Native dimeric methionyl-tRNA synthetase and its monomeric proteolytic fragment are shown to form and to bind 1 mol of methyionyl adenylate per polypeptide chain. Moreover, at 25 degrees C, each monomer of the dimeric native enzyme behaves independently, exhibiting the same parameters for the methionine activation reaction as does the monomeric modified enzyme. These results were obtained using several independent methods including equilibrium and nonequilibrium dialysis, active site and tryptophan fluorescence titrations, and stopped-flow by fluorescence. Stopped-flow resolution of the reversible methionine activation reaction also demonstrates that methionine and ATP-Mg2+ react without coupling to form a ternary enzyme-methionine-ATP-Mg2+ complex. This complex readily converts to enzyme-methionyl approximately adenylate-PP-Mg2+ with a standard free energy close to zero. It is concluded that the uncoupled enzyme-methionine-ATP-Mg2+ complex may resemble the transition state of the reaction at the expense of the additional state of the reaction at the expense of the additional synergistic binding energy provided by reciprocal coupling, within the site, of the methionine molecule with the adenosine and PP-Mg2+ parts of the ATP-Mg2+ molecule (Blanguet, S., Fayat, G., and Waller, J. P. (1975), J. Mol. Biol. 94, 1.).


Cobalt(III) affinity-labeled aspartokinase. Formation of substrate and inhibitor adducts. The kinase active site of the aspartokinase-homoserine dehydrogenase enzyme complex of Excherichia coli has been affinity labeled both with substrates aspartate and adenosine triphosphate and feedback inhibitor threonine. Co(III) exchange-inert adducts of aspartokinase and inhibitor or substrates were produced in situ by oxidation of Co(II) with H2O2. Emzyme-Co(III)-adenosine 5'-triphosphate (ATP), enzyme-Co(III)-aspartate, and enzyme-Co(III)-threonine ternary adducts were produced in this manner. The formation of the enzyme-Co(III)-threonine adduct leads us to conclude that threonine inhibits the kinase activity of this enzyme complex by binding in the first coordination sphere of the catalytic metal ion cofactor, a conclusion which is consistent with evidence derived from previous nuclear magnetic resonance data obtained in this laboratory. The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate, and threonine comprised a mixture of both ezyme-Co(III)-ATP-aspartate and enzyme-Co(III)-ATP-threonine adducts. The formation of the quaternary aspartate-containing adduct was unexpected, since the presence of threonine was expected to prevent access of the aspartate to the active site; most significantly however, the the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one. We believe that this shows direct steric overlap between the metal-adjacent binding sites for aspartate and threonine. Aspartate or threonine can not occupy the kinase active site simultaneously; this conclusion is consistent with the direct competitive inhibition of aspartate by threonine observed in steady-state kinetic studies.


Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives. Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3',5'-monophosphate (cAMP) dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, whereas the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. Utilizing methodology developed to evaluate the affinity of protein kinase for immogilized cyclic nucleotides it was found that all of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: (1) the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; (2) the analogous spacer-arm did not compete; and (3) the effects of enzyme concentration, MgATP, and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. In addition, the immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 muM protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 muM by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 muM protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore of the immobilized ligand molecules.


Substrate Binding of avian liver prenyltransferase. Prenyltransferase (farnesyl pyrophosphate synthetase) was purified from avian liver and characterized by Sephadex and sodium dodecyl sulfate gel chromatography, peptide mapping, and end-group analysis. The enzyme is 85 800 +/- 4280 daltons and consists of two identical subunits as judged by sodium dodecyl sulfate gel electrophoresis, peptide mapping, and end-group analysis. Chemical analysis of the protein revealed no lipid or carbohydrate components. Avian prenyltransferase synthesizes farnesyl pyrophosphate from either dimethylallyl or geranyl pyrophosphate and isopentenyl pyrophosphate. A lower rate of geranylgeranyl pyrophosphate synthesis from farnesyl pyrophosphate and isopentenyl pyrophosphate was also demonstrated. Michaelis constants for farnesyl pyrophosphate synthesis are 0.5 muM for both isopentenyl pyrophosphate and geranyl pyrophosphate. The V max for the reaction is 1990 nmol min-1 mg-1 (170 mol min-1 mol-1 enzyme). Substrate inhibition by isopentenyl pyrophosphate is evident at high isopentenyl pyrophosphate and low geranyl pyrophosphate concentrations. Michaelis constants for geranylgeranyl pyrophosphate synthesis are 9 muM for farnesyl pyrophosphate and 20 muM for isopentenyl pyrophosphate. The Vmax is 16 nmol min-1 mg-1 (1.4 mol min-1 mol-1 enzyme). Two moles of each of the allylic substrates is bound per mol of enzyme. The apparent dissociation constants for dimethylallyl, geranyl, and farnesyl pyrophosphates are 1.8, 0.17, and 0.73 muM, respectively. Dimethylallyl and geranyl pyrophosphates bound competitively to prenyltransferase with one-for-one displacement. Four moles of isopentenyl pyrophosphate was bound per mole of enzyme. Citronellyl pyrophosphate, an analogue of geranyl pyrophosphate, was competitive with the binding of 2 of the 4 mol of isopentenyl pyrophosphate bound. The data are interpreted to indicate that each subunit of avian liver prenyltransferase has a single allylic binding site accommodating dimethylallyl, geranyl, and farnesyl pyrophosphates, and one binding site for isopentenyl pyrophosphate. In the absence of an allylic pyrophosphate or analogue, isopentenyl pyrophosphate also can bind to the allylic site.


Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA. Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.


Spectroelectrochemical investigations of stoichiometry and oxidation-reduction potentials of cytochrome c oxidase components in the presence of carbon monoxide: the "invisible" copper. Spectroelectrochemical studies are presented for the carbon monoxide complex of isolated, purified cytochrome c oxidase (EC 1.9.3.1) in solutions saturated with carbon monoxide. The results indicate a stoichiometry of three equivalents per oxidase-carbon monoxide complex molecule. Formal reduction potentials (Eo) of the two copper and one heme component at pH 7.0 were obtained by means of quantitative absorbance-charge titrations in the absence and presence of cytochrome c, and by means of a Nernstian "Minnaert" plot in the presence of cytochrome c. Analysis of the absorbance-charge curves from these titrations gave an indirect determination of the high potential, "invisible" copper component. The copper potentials in the carbon monoxide complex were found to be relatively unchanged with respect to those of the native enzyme. The Eo values obtained were: high potential ("invisible") copper (340 +/- 20 mV (NHE)), low potential copper (190 +/- 20 mV), and low potential heme (250 +/- 10 mV).


Assignment of a ligand in stellacyanin by a pulsed electron paramagnetic resonance method. The electron spin echo decay envelope for the blue copper protein, stellacyanin, and for a number of other Cu(II) complexes has been studied. Particular attention was given to the form of the "nuclear modulation" patterns, which show the effects of coupling between the electron spin and the neighboring nuclei. The envelopes for the hydrated cupric complex and for copper(II) glycylglycine were essentially the same and indicative of the coupling to protons. The peptide complex contains nitrogen nuclei coupled directly to Cu(II), but the coupling constant is so large for these nuclei that a modulation pattern ascribable to 14N is not seen. For copper(II) bovine serum albumin, on the other hand, a contribution due to the coupling of the remote nitrogen belonging to a histidyl imidazole ligand was observed. The modulation pattern for this complex and for stellacyanin closely resembled one another, strongly suggesting that an imidazole is ligated to the copper in this blue protein.


Study of cytochrome oxidase co-binding site using low-temperature flash photolysis. Flash photolysis has been used to study the kinetics of CO-binding to the heme alpha isolated cytochrome oxidase. Experiments performed over the range 185-295 degrees K with various CO concentrations have revealed significant deviations from the Arrhenius relationship between rate and temperature. These findings can be explained by a model in which the heme site is considered to have three regions between which CO can move; only from the innermost one can binding to the heme iron take place. The relative enthalpies and entropies of the three regions are calculated.


Characteristics of the corticosterone-receptor complex in rat liver cytosol. Using a gel filtration on Sephadex G-150 in low ionic strength, it was possible to separate a corticosterone-binding protein in rat liver cytosol from corticosteroid-binding globulin after incubation of cytosol with [3H]corticosterone. The corticosterone-protein complex ("alpha-Complex") had a sedimentation coefficient of 8-9 S in low ionic strength. In high ionic strength, the alpha-Complex rapidly dissociated with a half-life of 15 h, compared to a half-life of 31 h for the hepatic dexamethasone-receptor complex under identical conditions (0 degrees C). The alpha-Compelx was saturable with an excess of unlabelled corticosterone of dexamethasone and was sensitive to heat and protease digestion. It is stressed that quantitation of the corticosterone-receptor complex must include separation of the receptor from corticosteroid-binding globulin as this protein binds corticosterone with high affinity and with a saturable amount of binding sites.


Visible absorption spectra of quantum mixed-spin ferric heme proteins. Recently, it has been shown that the magnetic data for Chromatium ferricytochrome c' at pH 7 are consistent with quantum mechanically (as distinguished from thermally) mixed mid-spin (S = 3/2) and high-spin (S = 5/2) heme. Visible absorption spectra of the protein measured at 77 degrees K and 293 degrees K, pH 7, show peaks at 400, 490, and 632 nm. The observation of a 630 nm band in quantum mixed-spin heme spectra, and the spin state-dependence of the band intensity, are discussed in the context of the iron-ligand structure for quantum mixed-spin heme inferred from magnetic data.


Synthesis of inducible enzyme in Escherichia coli recovering from prolonged energy starvation. A marked breakdown of ribosomes and rRNA occurs in Escherichia coli cells during prolonged deprivation of a carbon source (energy starvation). In E. coli recovering from energy starvation: (a) synthesis of RNA started immediately, total protein synthesis showed a delay of 5 to 10 minutes; (b) beta-galactosidase, tryptophanase and serine deaminase could not be induced in the first 50--70 min; (c) a lag of 60 min in the synthesis of beta-galactosidase was observed in a lac constitutive mutant of E. coli; synthesis of the constitutive enzyme malate dehydrogenase did not shown any delay. RNA synthesized in the early stages of recovery contained a higher percentage of low molecular weight molecules than RNA synthesized after 70 min of recovery or during exponential growth. Messenger RNA specific for beta-galactosidase was not synthesized for the first 50--60 min of recovery even when the specific inducer was added to the cultures.


Distribution of protein kinase activities in subcellular fractions of rat brain. The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.


Studies on the interaction of propranolol with erythrocyte membranes. The interaction of propranolol with erythrocyte membranes at concentrations stabilizing intact erythrocytes against hypotonic hemolysis produced corresponding perturbations in membrane protein and particularly membrane phospholipid components as monitored by increases in the reactivity of membrane amino and sulfhydryl groups towards trinitrobenzenesulfonic acid and 5,5'-dithio-bis(2-nitro-benzoic acid), respectively. Membrane-propranolol interactions were also analyzed in terms of alterations produced in the kinetic properties of membrane enzymes. These experiments provided evidence that propranolol-induced perturbations were sufficiently generalized as to influence the activity of enzymatic processes associated with both inner and outer membrane surfaces. Configurational changes in membrane phospholipids were implicated in these effects of propranolol, which included alterations in functionally significant membrane-cation interactions, It is suggested that the findings described here may provide a basis for understanding molecular aspects of membrane stabilization in other systems.


A spin label study of rat brain membranes. Effects of temperature and divalent cations. Rat brain myelin, synaptosomal plasma membranes and synaptic vesicles were spin labelled with stearic acid nitroxide derivatives. Their electron spin resonance spectra were studied as a function of temperature and devalent ions (Ca2+ and Mg2+) concentrations. (1) Synaptosomal plasma membranes and synaptic vesicles show identical temperature variations of their order parameter (S = 0.58 at 35 degrees C and S = 0.72 AT 22 DEGREES C). Myelin appears more rigid (S = 0.66 at 35 degrees C and S = 0.76 at 22 degrees C). A discontinuity of the order parameter variation as a function of temperature, is observed between 14.5 degrees C and l9.5 degrees C with the three types of membranes. (2) The hydrophobic core of these membranes is very fluid. No transition temperature is observed. The measured values of the spin label rotation correlation times and rotational activation energies are 2.1 and 2.8 ns at 35 degrees C and 3.1 and 3.6 kcal/mol respectively for synaptosomal plasma membranes and myelin. (3) Ca2+ enhances the membrane rigidity (12+/-0.7% increase of the order parameter at 35 degrees C in the presence of 10(-3) M Ca2+) and increases the transition temperature. At a lower extend, similar effects are observed with Mg2+.


Monamine oxidase in outer membrane of skeletal muscle mitochondria. (1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay procedure for the assay of monoamine oxidase in muscle mitochondria is described. It is based on teh procedure using side-chain [2-14C]-tryptamine as substate described by Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate concentration of 0.25 mM. (3) The Km of the muscle mitochondrial enzyme at pH 8.0 is 1.34 - 10(-5) M and that of the liver enzyme under the same conditions is 2.5 - 10(-5) M. Muscle mitochondria contain only one quarter of the activity of enzyme present in liver mitochondria. (4) Monoamine oxidase is shown to be in the outer membrane of skeletal muscle mitochondria and thus to be a suitable marker enzyme for use in the fractionation of these mitochondria.


Evidence for a secretion of thyroxine by isolated hog thyroid cells. Isolated thyroid cells prepared from hog thyroid glands by tryptic dispersion were incubated with 131I- for 1--6 h. Free [131I]thyroxine was identified in the incubation medium by three chromatographic methods. Neither [131I]iodotyrosines nor [131I]triiodothyronine were detected. The [131I]thyroxine released in the medium by 100 mul of cells (packed cell volume) after a 6-h incubation period amounted to 1.16% (S.E. = +/- 0.39) of the total radioactivity. The medium [131I]thyroxine represented 15--25% of the total [131I]thyroxine synthesized during the 6 h of incubation. Thyrotropin, 1--60 munits/ml, increased the medium [131I]thyroxine content 2-4 fold. Dibutyryl cyclic AMP mimicked the effect of thyrotropin. The amount of medium [131]thyroxine was strictly related to the amount of incubated cells but was independent of the volume of the incubation medium. When prelabeled cells were incubated in the presence of methimazole the increase in medium [131I]thyroxine was quantitatively related to a decrease in the intracellular [131I]thyroxine. Addition of dinitrotyrosine, an inhibitor of the deiodinase activity, induced the release of iodotyrosines in the incubation medium. That the incubation supernatant of isolated thyroid cells did contain free thyroxine but not iodotyrosines suggests that the normal mechanisms of proteolysis of thyroglobulin and deiodination of iodotyrosines inside the cells are preserved. From these data, it was concluded that the thyroxine release by isolated cells represents a real secretion.


Production of superoxide anion during the oxidation of hemoglobin by menadione. Menadione in the presence of oxyhemoglobin will accelerate the formation of methemoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.


Kinetic studies of Rhus vernicifera laccase. Evidence for multi-electron transfer and an oxygen intermediate in the reoxidation reaction. 1. The reoxidation of reduced Rhus vernicifera laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) by molecular oxygen has been studied by optical absorption and EPR methods. 2. The reoxidation by oxygen of the type 1 Cu+ and the two-electron acceptor is characterized by a second-order rate constant of about 5-10(6) M-1-s-1. 3. The appearance of an optical intermediate (with an absorbance maximum around 360 nm) parallels the reoxidation of type 1 Cu+ and the two-electron acceptor. It disappears in a first-order reaction with a half-time of 20 s. A similar intermediate is formed during normal turnover. 4. The type 2 Cu+ appears to be reoxidized in an intramolecular reaction with a half-time of about 20 s, suggesting a correlation between the reoxidation of this site and the disappearance of the optical intermediate. 5. The results suggest that three electrons are rapidly transferred to oxygen leading to the formation of an enzyme-bound oxygen intermediate.


Protein kinases from liver mitochondria of tumour-bearing rats. The mitochondria of liver of Yoshida ascites tumour-bearing rats contained two forms of protein kinase distinguishable on the basis of their kinetic properties, substrate specificity and responses to cyclic adenosine 3',5'-monophosphate (cAMP). One of these (kinase I) was activated 2-3 fold by cAMP while the other form (kinase II) was insensitive to the action of cAMP. Kinase I which was selective towards histone F1 as substrate was obtained as a homogeneous preparation and was observed to have a molecular weight of 170 000 by Sephadex G-150 gel filtration. Protein kinase II appeared to be a smaller protein with molecular weight of 54 000 and was specific towards acidic proteins namely casein and phosvitin. Protein kinases isolated from liver mitochondria of normal rats showed variations in respect to elution profile of DEAE-cellulose and electrophoretic mobility. The preparation corresponding to kinase I did not show stimulatory responses to cAMP.


Human thyroid cyclic nucleotide phosphodiesterase. Its characterization and the effect of several hormones on the activity. Cyclic AMP and cyclic GMP phosphodiesterase activities (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were investigated in the human thyroid gland from patients with hyperthyroidism. Low substrate concentration (0.4 muM) was used. About 60% of the cyclic-AMP and 80% of the cyclic-GMP hydrolytic activities in the homogenate were obtained in the soluble fraction (105 000 X g supernatant). The thyroid gland contains two forms of cyclic-AMP phosphodiesterase, one with a Km of 1.3-10(-5) M and the second with a Km of 2-10(-6) M. Cyclic-AMP and cyclic-GMP phosphodiesterase were purified by gel filtration on a Sepharose-6B column. Cyclic-AMP phosphodiesterase activities were found in a broad area corresponding to molecular weights ranging from approx. 200 000 to 250 000 and cyclic-GMP phosphodiesterase activity was found in a single area corresponding to a molecular weight of 260 000. Cyclis-AMP phosphodiesterase activities were stimulated by the protein activator which was found in human thyroid and this stimulation was dependent on Ca2+. Stimulation of cyclic-AMP phosphodiesterase by the activator was not significant even in the presence of enough Ca2+. The effect of D,L-triiodothyronine, D,L-thyroxine, L-diiodotyrosine, L-monoiodotyrosine, L-thyronine, L-diiodothyronine, thyrotropin, hydrocortisone, adrenocorticotropin, cyclic-AMP and cyclic-GMP on the phosphodiesterase activities was studied. Cyclic-AMP, cyclic-GMP, D,L-triiosothyronine, D,L-thyroxine, adrenocorticotropin and hydrocortisone where found to inhibit the phophodiesterase. Triiodothyronine and thyroxine inhibited cyclic-AMP phosphodiesterase more effectively than cyclic-GMP phosphodiesterase. Thyroxine was a more potent inhibitor than triiodothyronine. The concentration of cyclic AMP producing a 50% inhibition of cyclic-GMP phosphodiesterase activity was 5-10(-5) M, while the concentration of cyclic GMP producing a 50% inhibition of cyclic-AMP phosphodiesterase was 3-10(-3) M. Both cyclic-AMP and cyclic-GMP phosphodiesterase activities in the homogenate of hyperthyroidism, thyroid carcinoma and adenoma were higher than in normal thyroid tissue, when assayed with a low concentration of the substrate (0.4 muM). When a higher concentration (1 mM) of cyclic nucleotides was used as the substrate, cyclic-AMP hydrolytic activity in adenoma tissue was similar to that of normal tissue, while the other activities were higher than normal.


The estimation and comparison of molecular weight of angiotensin I converting enzyme by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. The angiotensin I converting enzyme from rat lung was observed to be a glycoprotein containing 8.3% carbohydrate and consisting of a single polypeptide chain with an estimated molecular weight of 139 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 150 000 by sucrose density gradient sedimentation analysis. A comparison of the mobility of angiotensin I converting enzyme from rat lung, rabbit lung, and two hog lung sources on sodium dodecyl sulfate-polyacrylamide gels indicates that all four enzymes have very similar molecular weights and subunit structures. Some previously reported molecular weight discrepancies appear to be due to anomalous behavior of the enzyme of gel filtration.


Effect of carbamidomethylation of cysteine residues G11(104)alpha on the properties of hemoglobin A. A modified hemoglobin tetramer has been prepared containing carbamidomethylated G11(104)alpha cysteine residues. The molecule is electrophoretically identical to hemoglobin A, at pH 8.6, contains 2 titratable sulfhydryl groups per tetramer, and shows a normal oxygen affinity at half-saturation. However, the cooperative oxygen binding is significantly decreased. As the G11(104)alpha cysteine residues are located at the alpha1beta1 contact point in the hemoglobin tetramer, the results of this study indicate that modification within this portion of the molecule does not interfere with the assembly of subunits to form a tetramer or the resultant p50 but can cause a significant alteration of cooperative oxygen binding. In addition, spin-labels attached to this cysteine residue are not sensitive to changes in conformation which may take place at this contact point during oxygen binding. It is therefore possible that modification of the G11(104)alpha cysteine residue abolishes the contribution of the alpha1beta1 contact point to the cooperative oxygen binding phenomenon.


Conformational studies of corticotropin1-32 and constitutive peptides by circular dichroism. Circular dichroism spectra on corticotropin1-32 and its constitutive N-, and C-terminal peptides are determined in water and trifluoroethanol under several conditions in the aromatic and peptide spectral regions. Furthermore, the effects of pH and varied mixtures of water-trifluoroethanol are examined on the corticotropin1-32 molecule. The results show that the N- and C-terminal series have a different behaviour in both aqueous and organic media. Corticotropin and the former peptides display "random" spectra in water, and alpha-helix type spectra in trifluoroethanol, while the latter have "random" spectra in both solvents. In the holopeptide corticotropin, the side chain-side chain effects, as reflected by the titration curves obtained from variations in the aromatic region, support the idea of an helical organization of part of the backbone even in aqueous solution. When going from water to trifluoroethanol corticotropin1-32 undergoes a conformational change which leads to an alpha-helix, following a linear pathway. These results, together with other observations, indicate the possible role of the conformation of corticotropin molecules in their biological life.


Resonance raman studies of a c type algal cytochrome. Deuterium shifts and a comparison with mammalian cytochrome c. A c type cytochrome isolated from Synechococcus lividus grown on water and 2H2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment on the vibrations of the c type heme in the algal cytochrome.


A structural study of pig liver glyceraldehyde-3-phosphate dehydrogenase. A substantial portion of the primary structure of pig liver glyceraldehyde-3-phosphate dehydrogenase has been investigated and the results compared with those previously reported for the pig muscle enzyme. Liver and muscle glyceraldehyde-3-phosphate dehydrogenases show the same amino acid content, and the first N-terminal residues occur in the same sequence. No differences in N-terminal residues and amino acid composition have been evidenced by analysis of several tryptic peptides, which account for about 50% of the total amino acid sequence. From the electrophoretic mobilities of peptides T8 T9 and T25 it is concluded that residues Asp 60, Asp 67 and Glu 220 in the reported sequence of the pig muscle enzyme must be present as amides in the liver enzyme. The NAD+ content was found to be 2 mol per tetramer, while higher values have been reported for the muscle enzyme from various mammalian sources. The reactivity of lysyl side chains towards pyridoxal 5'-phosphate has been examined: the results indicate that Lys 212 is the main site reacted in fully inactivated pig liver holoenzyme. A similar result has been found for rabbit muscle apoenzyme, whereas rabbit muscle holoenzyme reacts at Lys 212 and 191.


Calcium-binding proteins in electroplax and skeletal muscle. Comparison of the parvalbumin and phosphodiesterase activator protein of Electrophorus electricus. A soluble calcium-binding protein has been isolated from the red skeletal muscle of the electric eel (Electrophorus electricus). The purification procedure involved ammonium sulfate precipitation, gel filtration on Sephadex G-75 in the presence of 45Ca2+, and chromatography on QAE-Sephadex. This procedure resulted in the isolation of a protein which was homogeneous upon polyacrylamide gel electrophoresis. The calcium-binding protein was found to be a typical parvalbumin by the following criteria: (1) molecular weight of 11 000; (2) pI of 4.7; (3) 1.9 mol Ca2+ bound per mol protein; Kd of approx. 10(-7) M; (4) no detectable phosphorus; (5) amino acid composition included nine residues of phenylalanine, single arginine, and no tyrosine or tryptophan; (6) ximax at 259 nm; (7) 260 nm:280 nm absorbance ratio of 4.78. Only one parvalbumin could be detected in muscle. Immunoprecipitation assay revealed that the parvalbumin was a major soluble component of skeletal muscle (0.10 mg/mg soluble protein), but could not be detected in liver, kidney, brain, spleen, heart or electroplax. Comparison of the parvalbumin with a calcium-binding protein previously isolated from electroplax revealed that the two proteins were different as judged by a variety of chemical criteria. These results suggest that during embryological development of electroplax the parvalbumin is lost and that it is not required for the function of electric tissue.


Platelet lipoproteins. A comparative study with serum lipoproteins. The nature of human platelet lipoproteins was studied in two series of experiments. In the first series, whole platelets were utilized for extraction of lipoproteins by three different methods: chloroform/methanol/phenol; saline; or sucrose-gradient ultracentrifugation of platelet homogenates. By polyacrylamide gel electrophoresis we were able to demonstrate the existence of lipoprotein in the extracts obtained by the last two methods. These lipoproteins were found not to share antigenic determinants with alpha and beta serum lipoproteins. The second series of experiments utilized platelets solubilized either in sodium deoxycholate or sodium dodecyl sulfate. The solubilized product was characterized by double immunodiffusion and polyacrylamide gel electrophoresis. The nonidentity between plasma and platelet lipoproteins previously demonstrated in the first series of experiments was confirmed. This nonidentity was also supported by a comparison between the apoproteins of purified serum lipoproteins and platelet proteins released after solubilization with sodium dodecyl sulfate. No identical protein fractions were found. Our results suggest that, unlike erythrocyte membrane lipoproteins, the platelet lipoproteins are structurally different from plasma lipoproteins.


Studies on pig serum lipoproteins. III. Affinity chromatography of native lipoproteins on concanavalin A-sepharose. The comparison of the binding capacities of the three major classes of pig serum lipoproteins, very low-density, low-density and high-density lipoproteins, to concanavalin A, was demonstrated by affinity chromatography on concanavalin A-Sepharose. Very low-density lipoprotein was separated into two fractions (60 to 66% of total protein was adsorbed). Each fraction had different electrophoretic mobility in pore size gradient gel. The majority of the carbohydrate was found in the adsorbed fraction. The carbohydrate content of the unadsorbed fraction was 0.14% sialic acid. 0.47% hexosamine and 0.93% neutral sugars, and of the adsorbed fraction, 2.05, 3.21 and 4.20%, respectively. The adsorbed and unadsorbed fractions contained fucose, mannose and galactose in the molar ratio of 1.0 : 3.6 +/- 0.2 : 2.2 +/- 0.4 and 1.0 : 3.1 +/- 0.2 : 2.5 +/- 0.3, respectively. Based on these results, two different molecular species were proved to be present in very low-density lipoproteins. In high-density lipoproteins, 80 to 85% of the total protein was not adsorbed on concanavalin A-Sepharose in spite of the presence of mannose in the apoprotein. In contrast to these lipoproteins, low-density lipoprotein was completely adsorbed on concanavalin A-Sepharose. However, the separation of the subfractions of low-density lipoprotein as well as the subfractions of high-density lipoprotein could not be achieved by this affinity column. The carbohydrate content of eluted fractions of low-density and high-density lipoproteins was identical with the previously reported values obtained in native lipoproteins. This difference in affinities for concanavalin A was also evidenced by gel electrophoretic profiles in urea and in sodium dodecyl sulfate which showed different glycoprotein distribution in each class of lipoproteins.


Determination of human apolipoprotein E by electroimmunoassay. 1. Apolipoprotein E ("arginine-rich" polypeptide) was isolated from delipidized human very low density lipoproteins by agarose column chromatography in the presence of 6 M guanidine-hydrochloride. 2. An electroimmunoassay ("rocket" electrophoresis) is described for quantitative determination of human serum apolipoprotein E. Purified apolipoprotein E was used for the preparation of monospecific antisera and standardization of assay. This sensitive, specific, rapid (time required for the completion of the assay is 5 h) and precise (the within- and between-assay coefficients of variation are 5 and 8%, respectively) assay is applicable to measurement of apolipoprotein E in whole serum and density classes. The results correlated well with those obtained by radial immunodiffusion (r = 0.85). 3. Serum apolipoprotein E levels of normal subjects and hyperlipoproteinemic phenotypes IIa, IIb and IV were the same (10 to 16 mg/100 ml). In contrast, patients with type III and V hyperlipoproteinemias had markedly elevated serum apolipoprotein E levels )27 and 25 mg/100 ml, respectively). The apolipoprotein E in serum of normolipidemic subjects was equally distributed among three major lipoprotein density classes: d less than 1.030 g/ml (27%), d 1.030-1.063 g/ml (36%)and d 1.063-1.21 g/ml (37%).


A major site of bicarbonate effect in system II reaction. Evidence from ESR signal IIvf, fast fluorescence yield changes and delayed light emission. In order to determine the major site of bicarbonate action in the electron transport complex of Photosystem II, the following experimental techniques were used: electron spin resonance measurements of Signal IIvf, measurements of chlorophyll a fluorescence yield rise and decay kinetics, and delayed light emission decay. From data obtained using these experimental techniques the following conclusions were made: (1) absence of bicarbonate causes a reversible inactivation of up to 40% of Photosystem II reaction center activity; (2) there is no significant effect of bicarbonate on electron flow from the charge accumulating S state to Z; (3) there is no significant effect of bicarbonate on electron flow from Z to P-680+; (4) electron flow from Q-- to the intersystem electron transport pool is inhibited by from 4- to 6-fold under bicarbonate depletion conditions.


Energy-linked regulation of glucose and pyruvate oxidation in isolated perfused rat heart. Role of pyruvate dehydrogenase. 1. The regulation of glycolysis and pyruvate oxidation under varying conditions of ATP and oxygen consumption was studied in isolated perfused rat hearts. Potassium-induced arrest was employed to inhibit the ATP consumption of the heart. 2. Under the experimental conditions, the beating heart used solely glucose as the oxidisable substrate. The glycolytic flux through the aldolase step decreased in pace with the decreasing oxygen consumption during the potassium-induced arrest of the heart. The decrease in glucose oxidation was larger than the inhibition of the oxygen consumption, suggesting that the arrested heart switches to fatty acid oxidation. The time course and percentage changes of the inhibition  of pyruvate oxidation and the decrease in the amount of the active form of pyruvate dehydrogenase suggest that the amount  of active pyruvate dehydrogenase is the main regulator of pyruvate oxidation in the perfused  heart. 3. To test the relative significance of the possible mechanisms regulating covalent interconversions of pyruvate dehydrogenase, the following parameters were measured in response to the potassium-induced cardiac arrest: concentrations of pyruvate, acetyl-CoA, CoA-SH, citrate, alpha-oxoglutarate, ATP, ADP, AMP, creatine, creatine phosphate and inorganic phosphate and the mitochondrial NADH/NAD+ ratio. In cardiac tissue the adenylate system is not a good indicator of the energy state of the mitochondrion, even when the concentrations of AMP and free cytosolic ADP are calculated from the adenylate kinase and creatine kinase equilibria. Only creatine phosphate and inorganic phosphate undergo significant changes, but evidence of the participation of the latter compounds in the regulation of the pyruvate dehydrogenase interconversions is lacking. The potassium-induced arrest of the heart resulted in a decrease in pyruvate, a slight increase in acetyl-CoA, a large increase in the concentration of citrate and an increase in the mitochondrial NADH/NAD+. The results can be interpreted as showing that in the heart, the pyruvate dehydrogenase interconversions are mainly regulated by the pyruvate concentration and the mitochondrial redox state. Concentrations of all the regulators tested shifted to directions which one would expect to result in a decrease in the amount of active pyruvate dehydrogenase, but the changes were quite small. Therefore, the energy-linked regulation of pyruvate dehydrogenase in intact tissue is possibly mediated by the equilibrium relations between the cellular redox state and the phosphorylation potential recently confirmed in cardiac tissue.


Effects of colicins K and E1 on the glucose phosphotransferase system. 1. Glycerol-grown cells of Escherichia coli and its mutant uncA, treated with colicin E1 or K, exhibited a several-fold higher level of alpha-methylglucoside uptake than untreated cells. This stimulation was independent of the carbon source present during the uptake test. In a mutant strain that has elevated levels of alpha-methylglucoside accumulation the addition of colicin E1 or carbonylcyanide m-chlorophenylhydrazone (CCCP) did not further enhance the uptake. 2. Colicins K and E1 decreased the apparent Km for alpha-methylglucoside uptake significantly and increased the V about twofold. The exit of the glucoside was severely inhibited by the colicins. 3. In the presence of colicins, alpha-methylglucoside is still accumulated via the phosphoenolpyruvate-phosphotransferase system since no accumulation or phosphorylation occurs in an enzyme I mutant. The colicins increased the relative intracellular concentration of phosphorylated alpha-methylglucoside, possibly by inhibiting the dephosphorylation reaction, and caused an excretion of this compound. 4. The results are interpreted as indicating that energization of the membrane has an inhibitory effect on the phosphotransferase system. Possible modes of action are discussed.


Respiratory components and oxidase activities in Alcaligenes eutrophus. 1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H2 oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H2 oxidase by 50% and solubilizes the bulk of the ATPase. 2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa3, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm). 3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2--3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H2 oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20-100% stimulation of oxygen uptake). 4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.


Uptake of radiolabeled galactosyl-(alpha1 goes to 4)-galactosyl-(beta1 goes to 4)-glucosylceramide by human serum lipoproteins in vitro. Human serum was exposed to various amounts of [6-3H] galactosyl-(alpha1 goes to 4)-galactosyl-(beta1 goes to 4)-glucosylceramide under standardized conditions in vitro, and the uptake of the lipid by serum lipoproteins was determined. Of the bound glycolipid, 2% was isolated with very low density, 24% with low density-, 47% with high density lipoproteins and 27% with the ultracentrifugal residue. The distribution was different from the distribution of endogenous galactosyl-galactosylglucosylceramide, indicating that the glycolipid is probably an integral part of the lipoprotein complexes in vivo.


Study of the lipid binding characteristics of the apolipoproteins from human high density lipoprotein. I. Electron microscopic and gel filtration studies with synthetic phosphatidylcholines. The characteristics of the lipid - protein complex produced by the addition of the major apolipoproteins (apo AI and apo AII) of human high-density lipoprotein to synthetic phospholipids has been studied. Under the in vitro conditions utilized, apo AI binds to 1,2-dimyristoyl-sn-glycerophosphocholine and 1,2-dipalmitoyl-sn-glycerophosphocholine liposomes, but does not alter their morphologic characteristics. This binding occurs at temperatures above or below that of the transition (Tt) of the lipid bilayer. In contrast, apo AII spontaneously generates small, homogeneous disc-shaped lipid-protein complexes (50 X 10 a) from large phospholipid globules or from liposomes prepared with these lipids. This type of complex was only formed when the lipid/apo AII mixtures were warmed above the transition temperatures. The incorporation of apo AI into this small complex with apo AII may be greatly facilitated or inhibited depending on the sequence of addition of the various components. Under optimal circumstances, a maximum of 1 molecule of apo AI is incorporated with each molecule of apo A II into complexes with these two synthetic phospholipids.


Studies of the lipid binding characteristics of the apolipoproteins from human high density lipoprotein. II. Calorimetry of the binding of apo AI and apo AII with phospholipids. The interactions of lysophosphatidylcholine and synthetic 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC) liposomes with the isolated HDL-apolipoproteins, apo AI and apo AII, has been studied by microcalorimetry. Complex formation is a highly exothermal process characterized by a maximal enthalpy of about --200 kcal/mol of apoprotein when added to DMPC at 28 degrees C in 0.05 M sodium carbonate/bicarbonate buffer, pH 9.6. For the apo AI apoprotein, the binding consists of two processes, one endothermal occurring at low phospholipid/protein ratios and one exothermal predominant at higher phospholipid levels. The endothermal process has been attributed to a lipid-induced disaggregation of the apo AI while the exothermal process is similar to the binding of apo AII or apo HDL to phospholipids. The binding of a constant AI and apo AII, demonstrates the existence of a maximal association at a 1 : 1 molar ratio of the apolipoproteins. The sequential binding of DMPC to apo AI and apo AII suggests the existence of cooperativity between the two apoproteins in phospholipid binding as apo AII promotes the incorporation of apo AI into a protein-phospholipid complex.


Phospholipid biosynthesis in sarcoplasmic reticulum membrane during development. Biosynthesis of phosphatidic acid, phosphatidylcholine and phosphatidylethanolamine in the sarcoplasmic reticulum membrane has been  investigated. The results show that sarcoplasmic reticulum, in addition to its main function, i.e. transport and accumulation of Ca2+, is able to synthetize phospholipids by the same pathways as endoplasmic reticulum of other tissues. The changes of activity of enzymes involved in phospholipid biosynthesis during muscle development have been analysed. The extent of sn-glycero-3-phosphate and lysophosphatidylcholine acylation by acyl-CoA or free fatty acids in the presence of ATP and CoA is the same at every stage of development. The specific activity of glycerolphosphate acyltransferase(s) increases progressively during development up to about the 10th day of postnatal life and then decreases to the adult level. Linoleate esterifies sn-glycero-3-phosphate to a higher extent than palmitate, especially during postnatal period. The main product of sn-glycero-3-phosphate acylation is phosphatidic acid. The specific activity of lysolecithin acyltransferase increases from the embryonic period to a maximum between the 4th and the 9th day of postnatal life followed by a decrease to the adult value. the low embryonic value to a maximum at about the 3rd day of postnatal life, followed by a decrease to the adult value. The activity of cholinephosphotransferase decreases from a high value observed during the earliest embryonic period studied until the 3rd day before birth, and then begins to increase again from about the 5th day of postnatal life. The activity of ethanolaminephosphotransferase decreases continuously with age. The main product of phosphatidylethanolamine methylation is phosphatidylmonomethylethanolamine. The specific activity of phosphatidylethanolamine methyltransferase increases from


Partial purification and properties of diglyceride kinase from Escherichia coli. Diglyceride kinase (diacylglycerol kinase, E.C. 2.7.1.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold. The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycerol or other phospholipids. Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides. The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs. The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.


Abnormal membrane phospholipid content in subcellular fractions from the Morris 7777 hepatoma. 1. Mitochondrial and microsomal fractions were prepared from normal rat liver and the Morris 7777 hepatoma and characterized by the use of the marker enzymes, succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase. 2. The phospholipid content per mg membrane protein of Morris 7777 hepatoma mitochondria was increased by 75% as compared with mitochondria from normal rat liver. Microsomes from this poorly-differentiated tumor were found to have a 45% decrease in the content of phospholipid. These abnormalities were independent of tumor size or age. 3. The percent phospholipid content of the subcellular fractions was determined, and revealed an increase in the percent sphingomyelin in both the microsomal and mitochondrial fractions of the tumor. Decreases in the percent phosphatidylcholine and phosphatidylethanolamine were noted in tumor microsomes as compared with normal liver. Diphosphatidylglycerol was not found in significant quantities in the microsomal fraction of this hepatoma line. 4. The content of the various phospholipid classes per mg protein in the respective mitochondrial and microsomal fractions was determined. Large increases in nearly all the major phospholipid classes were found in tumor mitochondria; tumor microsomes were characterized by an increased content of sphingomyelin but the content of nearly all other phospholipids was significantly decreased. These findings suggest the presence of disturbances in the regulation of phospholipid metabolism in subcellular organelle membranes of the Morris 7777 hepatoma.


Molecular weight of Yaba monkey tumor virus DNA. The molecular weight of Yaba virus DNA was determined by the isolation of intact DNA genomes from Yaba virus, which had been purified by two sucrose density gradient and one potassium tartrate gradient centrifugations, by cosedimentation with T2 DNA. The molecular weight of Yaba DNA was calculated to be 119 - 10(6).


Repair of x-ray-induced single strand breaks in toluenized Escherichia coli cells. We have used sedimentation in alkali to estimate the repair of X-ray-induced single strand breaks in the DNA of irradiated toluenized Escherichia coli cells. Extensive repair requires no exogenous cofactors except ATP although other individual NTPs (except U) or dNTPs can substitute for ATP. There is no repair in polA or resA cells and since nicotinamide mononucleotide (NMN) inhibits repair in wild type cells we interpret the results as indicating that both ligase and polymerase I are needed for repair but that the amount of any gap filling is small and extensive repair replication is not necessary.


The ATP dependence of the incision and resynthesis steps of excision repair. Escherichia coli made permeable by treatment with toluene can perform a mode of DNA synthesis that is stimulated by ultraviolet radiation and closely resembles the resynthesis step of excision repair. If ultraviolet-irradiated toulene-treated cells are incubated in an assay mixture with ATP but without the four deoxyribonucleoside triphosphates (dNTPs) or NAD, accumulations of single-strand breaks in the DNA are detected by alkaline sucrose gradient analysis. A second incubation with the dNTP'S and NAD but without ATP produces nonconservative DNA synthesis in strains with normal levels of DNA polymerase I. However, in PolA strains, ATP must be present during the second incubation in order to produce measurable amounts of ultraviolet-stimulated DNA synthesis. These results suggest that in strains deficient in DNA polymerase I there may be two ATP-dependent steps in this repair pathway, one required for incision and one associated with resynthesis.


The restriction endonucleases in Bacillus amyloliquefaciens N strain. Substrate specificities. Two species of restriction endonuclease were isolated by gel filtration and DEAE-cellulose chromatography from a cell-free extract of Bacillus amyloliquefaciens (B. subtilits) N strain; a lower molecular weight endonuclease (endonuclease R.BamNI) and a higher molecular-weight one (endonuclease R.BamNx). Both of them required only Mg2+ for their activities. Endonuclease R.BamNx introduced a larger number of site-specific scissions in Excherchia coli phage lambda DNA that endonuclease R.BamNI did. Endonuclease R.BamNx cleaved Bacillus phage phi 105C DNA at the specific sites which are classified into two groups: one type of sites is modified by B. amyloliquefaciens H strain in vivo while the other is not affected. It was also active on DNA'S OF E. coli phage T7, lambdadvl, Simian virus 40 (SV40) and colicinogenic factor ColEI and was inactive on DNAs of Bacillus phages phi 29 and M2. Endonuclease R.BamHI isolated from H strain by Wilson and Young. This endonuclease was active on DNAs of phage lambda, lambdadvl and SV40, adn was inactive on DNAs of phages phi 105C, phi 29, M2 and T7, and ColEI DNA.


Further purification and characterization of T4 endonuclease V. T4 endonuclease V, which is involved in repair of ultraviolet-damaged DNA, has been purified 3600 fold from T4D-infected Escherichia coli. The enzyme shows optimal activity at pH 7.2 and does not require added divalent ions. Endonuclease V attacks both native and heat-denatured DNA provided that the DNA has been irradiated, and the enzyme activity is dependent on the dose of ultraviolet irradiation. The rate and the extent of the reaction are greater with irradiated native DNA although the Km values for the two types of DNA are the same (2.25 - 10(-5) M). The enzyme is readily inactivated by heat and is sensitive to p-chloromercuribenzoate. Endonuclease V-treated irradiated DNA is degraded by spleen phosphodiesterase only when the DNA has been treated with alkaline phosphatase, suggesting that the enzyme produces 5'-phosphoryl termini.


Repair of DNA damaged by methyl methanesulfonate in bacteriophage T4. A highly purified preparation of T4 endonuclease V does not degrade DNA alkylated with methyl methanesulfonate, and the methyl methanesulfonate sensitivity of T4 wild type and x mutant is not affected by the v mutation. Thus, T4 endonuclease V, the v gene product, does not seem to be involved in a repair or an abortive repair of methyl methanesulfonate-damaged T4 DNA. The x and y genes of T4 and the polA and the uvrD genes of Escherichia coli are concerned with the repair of methyl methanesulfonate-induced damages in T4 DNA. Since effects of the polA and the x or y mutations are additive, it is supposed that there are at least two pathways for the repair of methyl meth-anesulfonate-damaged T4 DNA, one controlled by the x and the y genes and the other in which E. coli DNA olymerase I is involved. The partial suppression of the x gene mutation at high temerature was also demonstrated.


Lateral diffusion, order parameter and phase transition in phospholipid bilayer membranes containing tocopheryl acetate. Lateral diffusion coefficient and order parameter measurements were made with pyrene excimer optical probes and fatty acid spin label probes respectively in pure dipalmitoyl phosphatidylcholine membranes and in membranes doped with tocopheryl acetate. The investigation shows, that the lateral diffusion coefficient for pyrene in dipalmitoyl pholphatidylcholine membranes is decreased whereas the order parameter of the fatty acid chains is slightly increased in the inner part of the membranes by the addition of tocopheryl acetate. The fluid-solid equilibrium phase diagram of dipalmitoyl phosphatidylcholine/tocopheryl acetate mixed membranes has been constructed from the measurements of the partition of (2,2,6,6-tetramethylpiperdine-1-oxyl) TEMPO spin labels between lipid and aqueous regions as function of temperature. In the membranes tocopheryl acetate induces a strong broadening of the temperature range of the phase transition. At low tocopheryl acetate concentrations dipalmitoyl phosphatidylcholine and tocopheryl acetate seem to be completely miscible in the solid and in the liquid crystalline state.


Properties of spin labelled membranes of Fusarium oxysporum f. sp. lycopersici. Growth temperature-induced compositional changes in membranes of Fusarium oxysporum provided a test system for study of the relationship between physical properties and composition. Growth at 15 degrees C was characterized by a decrease in phospholipid content relative to sterol content, a shift in phospholipid composition from phosphatidylcholine to phosphatidylethanolamine and a marked enhancement in the amount of polyunsaturated fatty acids in the phospholipid and triglyceride classes. Uptake of a spin labelled analog of stearic acid during growth and subsequent solution of the probe in the membranes allowed estimation of viscosity and molecular order of the membranes of live cells and of isolated membrane preparations. Less than 1/20 of the intracellular label was accessible to sodium ascorbate while none was released by sodium dodecyl sulfate. All of the label in live cells was reduced by in vivo respiratory activity above 20 degrees C but this process could be reversed or avoided by added ferricyanide. A cholestane spin probe was also incorporated into the membranes. The probes were not reduced as readily in isolated membranes and hence fluidity of the membranes could be assessed over a wide temperature range. At low temperatures (-10 degrees C) a nonlethal, liquid-solid phase transition was indicated in isolated membrane lipids while at higher (lethal) temperatures (40-45 degrees C), discontinuities appeared in Arrhenius plots of rotational correlation time. Activation energies for isotropic rotation of the stearate probes in the membranes changed markedly in this temperature range and this effect correlated closely with loss of viability of conidial cells. Correlation times for stearate probes showed little variation with growth temperature nor were any breaks in Arrhenius plots of this parameter detected in the range 0-35 degrees C in whole cells or isolated membranes. The data indicated control of membrane physical properties within close tolerances throughout the physiological temperature range regardless of growth temperature. It was concluded that this homeostatic phenomenon was due to the counteractive effects of sterol/phospholipid ratio, phospholipid composition and fatty acid polyunsaturation since the condensing and fluidizing components of the isolated total membranes vary in a reciprocal manner.


The insulin receptor of the turkey erythrocyte. Characterization of the membrane-bound receptor. The insulin receptor of the turkey erythrocyte has previously been shown to be very similar to that of the mammalian insulin receptors. As a first step in the isolation of this receptor a highly purified plasma membrane fraction has been prepared. The binding characteristics of the purified membrane-bound receptor were identical to those found with intact erythrocytes, but the membrane preparation had very little insulin-degrading activity. Isolation of the membrane by the methods described gave a 100-fold purification of the insulin receptor with 67% yield.


Interaction of steptolysin O with sterols. A quantitative study of the specific inhibitory power of cholesterol and other sterols on the hemolytic properties of streptolysin O is reported. This streptococcal exocellular protein is a cytolytic toxin which disrupts cytoplasmic membranes of eukaryote cells. The structural characteristics, particularly the stereochemical ones required for a steroid molecule to inhibit the cytolytic activity of streptolysin O, have been investigated in detail. By immunodiffusion techniques, in agar gel plates or tubes containing sterols, the formation of hydrophobic complexes between streptolysin O and inhibitory steroids, but not non-inhibitory steroids except lanosterol, is shown. Upon interaction with inhibitory steroids streptolysin O loses its immunoreactive properties towards neutralizing and precipitating homologous antibodies. An interpretation of the mechanism of biomembrane disorganization by streptolysin O is discussed in the light of its steroid binding properties.


Fucolipid metabolism as a function of cell population density in normal and murine sarcoma virus-transformed rat cells. The incorporation of isotopically labeled fucose into the lipids of normal and murine sarcoma virus-transformed rat cells as a function of cell population density was examined. When normal cells were seeded at low cell density, the levels of the major fucolipids, i.e., fucolipids III and IV, were substantially reduced, but then they increased as the cells approached confluency. This variation in synthesis of fucolipids III and IV appeared to be primarily related to cell density and not to cell growth. Chase experiments revealed that the reduced level of fucolipids III and IV in sparse normal cells is due to decreased synthesis rather than to increased catabolism. In contrast to the observations with normal rat cells, the high level of fucolipid III and the low level of fucolipid IV in murine sarcoma virus-transformed rat cells was shown to be independent of cell population density.


Regulation of ovarian steroidogenesis. The disparity between 125I-labelled choriogonadotropin binding cyclic adenosine 3',5'-monophosphate formation and progesterone synthesis in the rat ovary. The binding of 125I-labelled human choriogonadotropin, formation of cyclic adenosine 3',5'-monophosphate (cyclic AMP), and synthesis of progesterone were examined in ovarian cells from immature rats. Collagenase dispersed ovarian cells were found to respond specifically to lutropin-like activity. The equilibrium dissociation constant (Kd) for the binding of 125I-for the binding of 125I-labelled choriogonadotropin was 1.7-10(-10) M. Progesterone synthesis was increased at least 40-fold and cyclic AMP formation 10-fold in response to maximum hormonal stimulation. The concentration of choriogonadotropin which stimulated progesterone synthesis maximally in Eagle's minimum essential medium--0.1% gelatin (2 ng/ml), resulted in minimal (less than 30% of maximum) increases in cyclic AMP accumulation and hormone binding. Similarly, binding of choriogonadotropin was not saturated at a hormone concentration (50 ng/ml) that stimulated maximal cyclic AMP formation. These results are consistent with the existence of receptor reserve in the ovarian cell. A marked shift in the dose vs. response relationship for progesterone synthesis occurred when fetal calf serum was used to supplement Eagle's minimum essential medium, however. Under these experimental conditions, progesterone synthesis reached a maximum at a hormone concentration of the same order of magnitude as did cyclic AMP formation. It is concluded that the degree of spare receptor effect observed may depend not only on an absolute amount of excess receptor, but also on the readiness of the system to respond in a given fashion.


Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells. Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polyoma virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.


Analysis of the electron paramagnetic resonance spectrum of the cobalamin intermediate in ribonucleotide reduction. EPR absorption-derivative lineshapes have been computed and least-squares fitted to the spectrum of the intermediate derived from 5'-deoxy-5'-adenosylcobalamin in the ribonucleotide reductase reaction. A Gaussian-type intrinsic lineshape was assumed and the effects of inhomogenous broadening, rotation of coordinate axes of the A-tensor relative to the g-tensor, angular dependence of transition probability and ligand hyperfine splitting have also been investigated. When the overall spectrum was computed as the sum of the lineshapes corresponding to two distinct Co(II) species, A and B, each having rhombic symmetry, the least squares procedure converged to a much better fit than with a single species, and matched almost all of the features of the experimental spectrum. The magnetic properties of A and B were compared with those of a series of other Co(II) complexes by a plot of g - g versus A - A. The results eliminate cobalt with 5-coordination to nitrogen for A and B, and suggest low-spin cobalt complexes having strongly distorted 6-fold coordination. The possibility that the sixth, symmetry-decreasing ligand is the oxygen molecule is excluded by the chemistry of the system and by the EPR properties of previously reported cob(II)alamins. It is suggested that the sixth ligand is a carbonyl, amide or sulfhydryl group of an enzyme sidechain which is inserted off-axis into the coordination position so as to exert the observed symmetry-lowering effect.


Simultaneous selection of mutants in gluconeogenesis and nucleoside catabolism in Salmonella typhimurium. Penicillin selection in minimal thymidine medium, used to select mutants in deoxynucleoside catabolism, also yields a high percentage (37%) of mutants in fructose diphosphatase. The expression of the deo regulon is retarded in the mutants defective in the glyconeogenic pathway.


On the role of the collagen carbohydrate residues in the platelet. Collagen interaction. It has been proposed that the platelet : collagen interaction is mediated in part by the collagen carbohydrate residues. To test this hypothesis we have oxidized monomeric and polymeric collagen with sodium periodate under conditions specifically designed to minimize destruction of periodate-susceptible bonds other than in the carbohydrate residues. Oxidation of the collagen significantly reduced its ability to interact with platelets. The extent of inhibition paralleled the extent of carbohydrate destruction. Oxidation with periodate also delayed the polymerization of the monomeric collagen, but even after polymerization the oxidized collagen failed to initiate the release reaction. These observations suggest that the collagen carbohydrate residues may be either near to or part of the site(s) on the collagen molecule required for platelet adhesion.


Purification, characteristics and sequence of a peptide containing an essential lysine residue. Glutamate dehydrogenase (EC 1.4.1.2-4) has been purified and crystallized from the acetone powder of tuna liver. The enzyme has a molecular weight of 333 000 +/- 15 000 as evaluated by sedimentation equilibrium and constists of six identical subunits. Unlike the bovine enzyme the molecular weight does not increase with increasing protein concentration indicating that the tuna enzyme has no tendency to polymerize. The amino acid composition and peptide maps of the tuna and bovine liver enzyme are similar, suggesting considerable homology between the two enzymes. Furthermore, from the tryptic digest a hexadecapeptide containing a lysine residue reactive to pyridoxal 5'-phosphate exhibits the same composition and sequence as the peptide containing the reactive lysine-126 in the sequence of the bovine enzyme. The molecular activity is 25 and 510 mol of substrate per mol enzyme per s, respectively, for the glutamate oxidation and the alpha-ketoglutarate reduction with NAD or NADP as coenzymes. The enzyme is regulated by pyridine nucleotides like other vertebrate enzymes, but it also exhibits some coenzyme specificity, the activity being about fifteen times higher with NAD than with NADP.


Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver. Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.


Affinity chromatography of angiotensin I-converting enzyme from rabbit lung using hippurylhistidylleucyl-OH. Approximately 50-fold purification of angiotensin I-converting enzyme (Peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was achieved by affinity chromatography using the synthetic substrate Hippuryl-His-Leu-OH. The specific activity of the enzyme was increased from 0.044 units/mg protein to 1.911 units/mg protein for Hippuryl-His-Leu-OH and from 0.33 nmol/min per mg protein to 13.8 nmol/min per mg protein for angiotensin I.