Ribonucleoside 3'-di- and -triphosphates. Synthesis of guanosine tetraphosphate (ppGpp). A procedure has been outlined for the synthesis of ribonucleoside 3'-di- and -triphosphates. The synthetic scheme involves the conversion of a ribonucleoside 3'-monophosphate to its 2'-(5'-di)-O-(1-methoxyethyl) derivative, followed by successive treatments of the blocked ribonucleotide with 1,1'-carbonyldiimidazole and mono(tri-n-butylammonium) phosphate or pyrophosphate. The resulting ribonucleoside 3'-di- and -triphosphate derivatives are then deblocked by treatment with dilute aqueous acetic acid, pH 3.0. The use of this procedure is illustrated for adenosine 3'-monophosphate, which has been converted to its corresponding 3'-di- and -triphosphates in 61% overall yield. The decomposition of adenosine 3'-di- and -triphosphates to adenosine 2'-monophosphate, adenosine 3'-monophosphate, and adenosine cyclic 2',3'-monophosphate as a function of pH at 100 degrees has been studied as has the attempted polymerization of adenosine 3'-diphosphate with polynucleotide phosphorylase. Also prepared was guanosine 5'-diphosphate 3'-diphosphate (guanosine tetraphosphate; ppGpp), which was accessible via treatment of 2'-O-(1-methoxyethyl)guanosine 5'-monophosphate 3'-monophosphate with the phosphorimidazolidate of mono(tri-n-butyl ammonium) phosphate. The resulting blocked tetraphosphate was deblocked in dilute aqueous acetic acid to afford ppGpp in an overall yield of 18%.


Proton magnetic resonance titration curves of the three histidine residues of staphylococcal protease. Proton magnetic resonance spectra of staphylococcal protease, a serine protease from Staphylococcus aureus, strain V8, are presented. Initial proton spectra were obtained at 220 MHz, and more detailed studies of the aromatic region were carried out by correlation spectroscopy at 250 MHz. The overall spectrum bears a close resemblance to one calculated from the sum of spectra of the component amino acids. Chemical shifts of the three tyrosine, four phenylalanine, and three histidine residues appear to be equivalent at pH 3.7 and 8.5 indicating that they are all in normal chemical environments in the enzyme. The staphylococcal protease contains a large number of slowly exchanging protons. In fact, interpretable spectra of the aromatic region were obtained only after extensive exchange of N-H groups with deuterium from the D2O solvent. Proton magnetic resonance titration studies of the three histidine residues indicate that these have normal chemical shifts and pK' values. When the data are fitted to single noninteracting titration curves, the histidine pK' values are 7.19 plus or minus 0.02, 6.85 plus or minus 0.03, and 6.69 plus or minus 0.02. The titration curves of two of the histidine residues indicate negative cooperativity. A possible explanation for this is a direct electrostatic interaction between the two histidines. The titration data for these histidines give a significantly better fit to such a mutual interaction model than to noninteracting titration curves. The component microscopic dissociation constants have been calculated. Mutual interaction leads to pK' displacements of 0.31 unit; which indicates a distance of approximately 7 angstrom between the two interacting histidine rings according to the model of Tanford and Roxby. The proton resonances of the two interacting histidines are doubled in the pH region 6.7-7.0 suggesting the presence of two forms of the enzyme having lifetimes in excess of 30 msec.


Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion. The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.


Acetylation of ribosome-associated proteins in vitro by an acetyltransferanse bound to rat liver ribosomes. Incubation of rat liver ribosomes with [1-14-C]acetyl-coenzyme A results in the incorporation of [14-C]acetyl into a material insoluble in cold trichloroacetic acid. The acetyltransferase involved in the self-acetylation of ribosomes can be released by high salt washing of the ribosomes; the activity of the solubilized enzyme can be assayed using histones as acetyl acceptors. Electrophoretic analysis of acetylated risosomes or ribosomal proteins indicated that the acetyl radicals are associated with a group of relatively basic proteins, having molecular weights ranging from 10,000 to 45,000. Chromatographic analysis of the enzymatic hydrolsates of proteins extracted from acetylated ribosomes indicates that acetylation is mainly or exclusively NH2 terminal. Almost 80% of the acetyl proteins are released from the ribosomes by high salt treatment. Most of the acetyl radicals not solubilized by the high salt treatment were found in the 60S subunit, associated with a protein(s) having an apparent molecular weight of 43,000. This acetyl protein(s) was released from the 60S subunit by EDTA treatment and was found in a ribonucleoprotein complex having a bouyant density of 1.56.


Magnetic circular dichroic spectra of cobalt(II) substituted metalloenzymes. The magnetic circular dichroic (MCD) spectra of cobalt(II) sugstituted metalloenzymes have been studied and compared to a series of four-, five-, and six-coordinate cobalt(II) model complexes previously examined (T. A. Kaden et al. (1974), Inorg. Chem. 13, 2582). The MCD spectra of cobalt substituted carboxypeptidase A, procarboxypeptidase ta, and thermolysin are consistent with earlier deductions of tetrahedral coordination from absorption spectra and also with X-ray structure analysis. Inhibitors fail to alter their MCD spectra significantly. The MCD spectra of cobalt alkaline phosphatase and carbonic anhydrase are more complex and their pH dependence and alteration by inhibitors are  discussed in terms of known cobalt(II) models.


Characterization of a ribonuclease from bovine brain. An alkaline ribonuclease (pH optimum near 8) has been purified from whole beef brains and found to have a base specificity like that of bovine pancreatic ribonuclease, but in most other respects to be distinguishable from the enzymes of bovine pancreas, semen, or brain nuclei. The preparation appears homogeneous in sedimentation equilibrium and probably so in polyacrylamide gel electrophoresis under normal or dissociating conditions. Sedimentation equilibrium and SDS gel electrophoresis both indicate a molecular weight of 2.4-2.6 times 10-4, and tryptic and chymotrypic peptide patterns are consistent with a protein of this size. No dissociation into subunits has been attained. The enzyme is not precipitated by antiserum to pancreatic ribonuclease, although its activity is inhibited by this antiserum with low efficiency. In comparisons of the hydrolysis of RNA the brain enzyme was found to have a similar specificity to pancreatic RNase, but to have a loser Km for RNA and to produce significantly different oligonucleotides upon partial hydrolysis of bacteriophage RNA, suggesting differences in the mechanism of substrate recognition. In contrast, nuclease inactivation by iodoacetate at pH 5.5 is indistinguishable for pancreatic or purified brain RNase.


Synthesis and enzymic activity of 8-acyl and 8-alkyl derivatives of guanosine 3, 5-cyclic phosphate. Several new 8-alkyl and 8-acyl derivatives of quanosie 3',5'-cyclic phosphate (cGMP) and inosine 3',5'-cyclic phosphate (cGMP) were prepared by direct alkylation or acylation of the parent cyclic nucleotide via free radicals generated in situ. These compounds have been examined for their ability to stimulate a cGMP-dependent protein kinase, and several of the cGMP derivatives were as active in this regard as cGMP. These compounds proved to be quite ineffective when tested for their ability to activate an adenosine 3',5'-cyclic phosphate (cAMP) dependent protein kinase. In addition, these 8-substituted cGMP derivatives are not substrates for a phosphodiesterase preparation from rabbit kidney, but do show inhibition of the hydrolysis of cAMP by crude phosphodiesterase preparations from rabbit lung and beef heart.


Chloride flux in bilayer membranes: chloride permeability in aqueous dispersions of single-walled, bilayer vesicles. Aqueous dispersions of phosphatidylcholine vesicles were utilized to determine bilayer permeability to 36-Cl as a function of pH and temperature. These dispersions were comprised of single-walled vesicles, homogeneous in size, prepared by sonication of purified egg phosphatidylcholine under argon followed by fractionation on a molecular sieve. Permeability constants calculated from the inward flux of 36-Cl and the geometric parameters of these vesicles proved to be dependent on both pH and temperature. Analysis of these dependences leads to the conclusion that 36-Cl permeation in the presence of KCl is due principally to a carrier mediated exchange process involving a phospholipid-HCL complex. Net permeation by H-36-Cl may make a small contribution to the 36-Cl flux, however, studies carried out at very low chloride concentrations show that this flux is much smaller than the exchange flux. Thus chloride permeability for the exchange process is 1.5 times 10- minus 11 cmsec- minus 1 while the corresponding coefficient for the net flux of H-36-Cl is 1.0 times 10- minus 12 cm sec- minus 1 at pH 7. The activation energy for the 36-Cl exchange flux was found to be 19 plus or minus 2 kcal/mol. This value is similar to that obtained for the transbilayer "flip-flop" of phosphatidylcholine molecules in a similar system (Kornberg and McConnell, 1971). This correspondence together with the fact that the experimentally determined flux of 36-Cl agrees well with that calculated from the "flip-flop" parameters, strongly suggests that the flux of 36-Cl and "flip-flop" of phosphatidylcholine may be the same process.


Polyacrylamide gels copolymerized with active esters. A new medium for affinity systems. A new and versatile method for linking biologically active ligands to a polyacrylamide matrix is reported. Active esters of acrylic acid (N-succinimicyl acrylate and N-phthalimidyl acrylate) were synthesized, then copolymerized with acrylamide and N,N'-methylenebisacrylamide. Displacement of the active ester in the gel thus formed by various ligands containing aliphatic amino groups resulted in the formation of stable amid bonds between the ligands and the polyacrylamide gel. The affinity gel thus prepared has the following advantages: (i) resistance to chemical and microbiological degradation, (ii) ease of control of ligand level and higher levels of ligand possible, (iii) ease of control of porosity, and (iv) total displacement of the active ester under suitable conditions. Efficacy of this system was tested by preparation of 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside derivative polyacrylamide gel by the described method. It was found to be more effective for purification of wheat germ agglutinin than the previously published affinity chromatography systems and the wheat germ hemagglutinin was obtained in crystalline form. In addition, partial resolution of isolectins was obtained from the affinity gel witha pH gradient.


An acid induced conformational transition of denatured cytochrome c in urea and guanidine hydrochloride solutions. Previous work has shown that at neutral pH ferricytochrome c (horse heart) retains certain residual structures in concentrated solutions of urea or guanidine hydrochloride (Tsong, T. Y. (1974), J. Biol. Chem. 249, 1988). Present studies reveal that cooperative unfolding of these residual structures can be achieved by acidification of the protein to pH 4 in 9 M urea but can only be partially achieved in a 6 M guanidine hydrochloride solution. The evidence that the residual structures unfold in 9 M urea upon acidification is twofold. (1) Further uncoupling of the Trp-59-heme interaction occurs; this is reflected in the intensification of the tryptophan fluorescence from 55 to 90 percent relative to that of free tryptophan in the same solvent. (2) The intrinsic viscosity of the protein solution increases from 15.0 to 21 ml/g. The acidification also induces a spin-state transformation of the heme group at pH 5 both in urea and in guanidine hydrochloride. Acidic titration of the protein in urea and guanidine hydrochloride indicates that the unfolding involves the absorption of a single proton. However, the kinetics of the spin-state transformation are triphasic. These results suggest that the displacement of the ligand His-18 by a solvent molecule and the subsequent disintegration of the residual structures are complex processes and involve at least three kinetic steps. The ineffectiveness of guanidine hydrochloride as a denaturant for ferricytochrome c is shown to be due to the presence of the high concentration of Cl minus which can stabilize certain elements of the protein structure.


Kinetic studies on the five principal components of normal adult human hemoglobin. The five principal components of human hemoglobin (Ala, Alb, Alc, Ao, and A2) have been isolated by column chromatography and by preparative isoelectric focusing in gels. The isoelectric points and a number of kinetic parameters have been determined for each hemoglobin. The greatest kinetic differences are found in the binding of CO to the deoxy conformation. At pH 7, A0 and A2 are nearly identical in their overall reaction with CO, whereas the initial lag phase characteristic of crude hemolysate and A0 is greatly reduced in Ala and Alc and is essentially absent in Alb. The general effect of p-mercuribenzoate bindind on CO association is to magnify kinetic differences among the hemoglobins, diminish the initial lag phase, and increase the overall rate of CO binding. Hemoglobin Ala is anomalous in that the overall CO binding rate actually decreases after reaction with the mercurial. In terms of an Adair model with four association constants the rate constant for the binding of the first molecule of CO (1l') showed the greatest variation among the five hemoglobins, with A0 having the smallest constant, and Alb the largest. For the native hemoglobins, 1l' for Alb was more than twice that for A0; for the mercurated hemoglobins, the difference was greater than threefold. Raising the pH form 7 to 8 increases 1l' for all hemoglobins, but Ala is anomalous in having a slower overall rate for CO binding at the higher pH. At pH 9, the time course of CO binding is biphasic for all hemoglobins, with A0, the fastest, and Ala, the slowest, differing by nearly threefold in rate. The equilibrium constant for the tetramer-dimer equilibrium was determined by flash photolysis. The largest dissociation constant occurs for Ala and is 4.4 times that for A0, and 5.6 times that for Alc, the least dissociated of the hemoglobins. The overall oxygen dissociation reaction is biphasic for Ala and Alb, with the two phases differing by a factor of 5; the dissociation reactions for the other three hemoglobins appear essentially monophasic. The kinetics of dissociation of the first oxygen molecule from oxyhemoglobin are very similar for all five hemoglobins, as are the association kinetics for CN-minus and N3-minus binding to the five methemoglobins.


Sulfhydryl groups in hemoglobin. A new molecular probe at the alpha1 beta 1 interface studied by Fourier transform infrared spectroscopy. Infrared absorption bands due to sulfhydryl groups (VSH) of alpha-104(G11) and beta-112 (G14) cysteine residues of human carboxyhemoglobin (HbCO) have been observed near 2560 cm- minus 1 by use of Fourier transform infrared (FTIR) spectroscopy. The beta-93 cysteine SH groups absorb infrared radiation so weakly that they are not distinguished from background. Only single SH absorption bands due to the alpha-104 cysteines of pig and horse hemoglobin are observed. The SH absorption bands from human HbCO disappear in alkali, are broadened by detergent or guanidinium chloride, and show a complex titration curve, and an isotopic frequency shift (VSD/VSH equals to 0.7267) virtually identical with that reported for methanethiol. The integrated absorption coefficient (epsilonmM(area)) for 0.1 M ethanetiol increased with H-bond acceptor solvents in the order: CCl4 (0.07), water (0.21), acetone (0.43), and N,N-dimethyl-acetamide (1.35 mM- minus 1 cm- minus 2). Comparison of the integrated absorption coefficients for the alpha-104 cysteine SH (2.43), and the beta-112 SH (0.80), of human HbCO with those of ethanetiol solutions suggested specifically H-bonded structures with peptide carbonyl groups 4 (or 3) residues back in the G helices. This was found to agree with a molecular model of the alpha-chain G helix of horse HbO2 built to coordinates from M. F. Perutz.


Equilibrium and kinetics of the reaction of Aplysia myoglobin with azide. The present paper reports a study on the equilibria and kinetics of the acid-alkaline transition and the azide binding reaction by ferric Aplysia myoglobin. A single completely reversible spectrophotometric titration curve is found over the pH range from similar to 5 to similar to 9, with an apparent pK equals to 7.5 for the acid-alkaline transition. The kinetics of the process, followed by the temperature-jump method, gives, at pH values close to the pK of the transition, one single, well-resolved, relaxation independent of protein concentration and of type of buffer used. The pattern accords to a simple pH dependent reaction, in buffered medium, between the two forms of the protein. The results of the azide binding reaction show that the process conforms to simple equilibrium as expected for a single site protein. The méasured association constant is reported as a function of pH. The kinetics of the reaction of Aplysia metMb with N3- minus shows, on the other hand, a complex behavior. The relaxation pattern is found to strongly depend on pH and ligand concentration in such a way to suggest a linkage between ligand binding and acid-alkaline transition. The system is discussed on the basis of two simplifying conditions, i.e., at low and higher pH with respect to the pK of the acid-alkaline transition. At acid pH the reaction corresponds to a single bimolecular process as expected for a simple binding reaction; at alkaline pH, the dependence of relaxation time on ligand concentration implies the existence of a rate-limiting monomolecular step. On the basis of a reaction scheme implying that binding of the ligand can only occur through the acid (aquomet) form of the protein via the displacement of the water molecule, the experimental data are quantitatively accounted for.


L-phenylalanine ammonia-lyase (maize, potato, and Rhodotorula glutinis). Studies of the prosthetic group with nitromethane. Highly purified enzyme (EC 4.1.3.5) from Rhodotorula glutinis was shown by sodium dodecyl sulfate gel electrophoresis to have subunits which if not identical are closely similar in molecular weight. Like the enzyme from maize and potato [Havir, E. A., and Hanson, K. R. (1973), Biochemistry 12, 1583] it is a tetramer of molecular weight similar to 4 times 83,000. Enzyme from all three sources inactivated and labeled at the active site with 14-CH3NO2 gave on HCl hydrolysis 14-CO2, H-14-CO2H, D- and L-[14-C]aspartic acid, and unidentified radioactive products. In addition, the labeled R. glutinis enzyme gave [1,2-14-C2]glycine. The formation of the first three products is compatible with the hypothesis that the electrophilic prosthetic group of the enzyme contains the dehydroalanine imine system greater than C equals to N minus C-alpha(equals to C-beta-H2)COminus and inactivation involves attack on C-beta. The second-order rate constants for CH3NO2 inactivation varied with pH as a simple titration curve. The pKa values calculated from the curves for the three enzymes differed and were lower than the pKa of CH3NO2 by at least 1 pH unit. Apparently the inactivation process is enzyme catalyzed. Both inactivation and addition of the substrate amino group may occur with attack on C-beta.


Acid-volatile selenium formation catalyzed by glutathione reductase. The production of acid-volatile selenide (apparently H2Se) was catalyzed by glutathione reductase in an anaerobic system containing 20 mM glutathione, 0.05 mM sodium selenite, a TPNH-generating system, and microgram quantities of highly purified yeast glutathione reductase. H2Se production in this system was proportional to glutathione reductase concentration and was maximal at pH 7. Significant nonenzymic H2Se production occurred in the system lacking glutathione reductase and TNPH. A concentration of arsenite (0.1 mM) which does not inhibit glutathione reductase inhibited selenide volatilization, as did bovine serum albumin (1.67 mg/ml). Both appear to inhibit Se volatilization by reacting with the selenide product(s). The selenotrisulfide derivative of glutathione (GSSeSG) was readily converted to H2Se by glutathione reductase and TPNH without the addition of glutathione. These results suggest that GSSeSG formed nonenzymically from glutathione and selenic undergoes stepwise reduction by glutathione reductase (or excess GSH) to GSSeH and finally to H2Se. The same pathway operates when glutathione is used as the reducing agent but to a lesser extent.


Terbium as a fluorescent probe for DNA and chromatin. Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.


Interaction of effecting ligands with lac repressor and repressor-operator complex. The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis. Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers. The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand. By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined. Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration. Anti-inducers increase the affinity of repressor for operator at most a factor of five. Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found. No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.


Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli. Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A  for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.


Lipid biosynthesis in sebaceous glands: regulation of the synthesis of n- and branched fatty acids by malonyl-coenzyme A decarboxylase. Crude cell-free extracts isolated from the uropygial glands of goose catalyzed the carboxylation of propionyl-CoA but not acetyl-CoA. However, a partially purified preparation catalyzed the carboxylation of both substrates and the characteristics of this carboxylase were similar to those reported for chicken liver carboxylase. The Km and Vmax for the carboxylation of either acetyl-CoA or propionyl-CoA were 1.5 times 10- minus-5 M and 0.8 mumol per min per mg, respectively. In the crude extracts an inhibitor of the acetyl-CoA carboxylase activity was detected. The inhibitor was partially purified and identified as a protein that catalyzed the rapid decarboxylation of malonyl-CoA. This enzyme was avidin-insenitive and highly specific for malonyl-CoA with very low rates of decarboxylation for methylmalonyl-CoA and malonic acid. Vmax and Km for malonyl-CoA decarboxylation, at the pH optimum of 9.5, were 12.5 mumol per min per mg and 8 times 10- minus-4 M, respectively. The relative activities of the acetyl-CoA carboxylase and malonyl-CoA decarboxylase were about 4 mumol per min per gland and 70 mumoles per min per gland, respectively. Therefore acetyl-CoA and methylmalonyl-CoA should be the major primer and elongating agent, respectively, present in the gland. The major fatty acid formed from these precursors by the fatty acid synthetase of the gland would be 2,4,6,8-tetramethyl-decanoic acid which is known to be the major fatty acid of the gland (Buckner, J. S. and Kolattukudy, P. E. (1975), Biochemistry, following paper). Therefore it is concluded that the malonyl-CoA decarboxylase controls fatty acid synthesis in this gland.


Lipid biosynthesis in the sebaceous glands: synthesis of multibranched fatty acids from methylmalonyl-coenzyme A in cell-free preparations from the uropygial gland of goose. Cell-free extracts from the uropygial gland of goose catalyzed the incorporation of malonyl-CoA and methylmalonyl-CoA into n- and multi-branched fatty acids, respectively, with NADPH as the preferred reductant. Methylmalonyl-CoA was shown to be incorporated almost exclusively into the acyl portion of wax esters by the cell-free extract while malonyl-CoA was incorporated into polar lipids and both the acyl and alcohol portions of the wax. The optimal pH for the synthesis of both n- and multibranched acids was 6.0. Apparent Km and Vmax for malonyl-CoA were 2 times 10- minus-4 M and 250 nmol per min per mg, respectively, while the Km and Vmax for methylmalonyl-CoA were 7.7 times 10- minus-4 M and 0.8 nmol per min per mg, respectively with 105,000g supernatant; but partial purification resulted in a tenfold decrease in Km values. The partially purified synthetase preparation catalyzed the formation of n-C16 acid (80%) and n-C18 acid (20%) from acetyl-CoA and malonyl-CoA. With the same synthetase preparation and the appropriate primer methylmalonyl-CoA was converted into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid which were identified by radio gas-liquid chromatography and combined gas chromatography-mass spectrometry. Experiments with an equimolecular mixture of acetyl-CoA and propionyl-CoA showed that the synthetase preferred acetyl-CoA as a primer. Since malonyl-CoA is known to be rapidly decarboxylated in the gland, acetyl-CoA and methylmalonyl-CoA are expected to be the major primer and elongating agent, respectively, available in the gland and therefore 2,4,6,8-tetramethyldecanoic acid should be the major product. Combined gas-liquid chromatography and mass spectrometry demonstrated that this acid was in fact the major acid of the gland.


Conformations and interactions of histone H2A (F2A2, ALK). Conformational changes in histone H2A (ALK, F2A2, IIbl) as a function of ionic strength and pH have been followed using high resolution nuclear magnetic resonance (NMR), circular dichroism (CD), and infrared (ir). While change in pH from 3 to 7 (no added salt) causes little structural change, added salt induces the formation of both alpha helix (28 percent maximum) and intermolecular associates in the region of the molecule between 25 and 113. No beta structure was observed at high salt. By the use of different salts it was shown that the structural changes were due largely to nonspecific counterion screening by the added anion. Comparison of observed with simulated NMR spectra has led to the proposal that an ionic strength dependent equilibrium exists between largely unstructured coil molecules and fully structured and aggregated molecules. NMR spectra of H2A obtained in the presence of DNA showed that both the N- and C-terminal regions bind to DNA, i.e., not the portion of the chain that is involved in interhistone interactions.


Conformation and cooperativity in hemoglobin. 19-F and 31-P nuclear magnetic resonance (NMR) spectroscopy have been used to study the ligand binding process in human hemoglobin. 19-F nuclear magnetic resonance studies of hemoglobin specifically trifluoroacetonylated at cysteine-beta93 have permitted observation and characterization of molecular species containing two and three ligands. The behavior of these intermediate species in response to changes in pH and organic phosphate concentration is not completely consistent with any of the current theories of allostery. A model consistent with the 19-F and 31-P NMR data is proposed.


Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization. The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the tryptophan residue in apoazurin has the same asymmetric environment both in the ground and excited states. This finding suggests tight packing of the protein at the tryptophan environment. Native azurin does not decay monoexponentially. Moreover, comparison between the quantum yield calculated from the decay kinetics and the one measured directly shows that the majority of the azurin molecules are not fluorescent. There is thus variability in the structure of azurin molecules with an equilibration time that is longer than the fluorescence lifetime. Different asymmetric environment was found for the tryptophan residue in oxidized and reduced holoprotein and in apoazurin, as studied by the circular polarization of the fluorescence. D(2)O increases the fluorescence lifetime of apoazurin by 6 percent, compared to the lifetime in H(2)O solution; therefore water molecules may have access to the tryptophan residue, though the latter is situated in a hydrophobic environment.


Binding kinetics of mercury(II) TO POLYRIBONUCLEOTIDES. Kinetic studies of the interaction of Hg(II) with polyribonucleotides have been used to investigate structural fluctuations of the bases in nucleic acids. The reaction of Hg(II) with poly(A)-poly(U) occurs in two phases which differ in time scale by a factor of about 100. The slow phase is first order and exhibits cooperativity or autocatalytic kinetics. The rate is found to increase as decreasing chain length of poly(U) is used to make the double helical complex. The reaction appears to initiate at the ends of poly(U) strands and may be associated with a molecular rearrangement which results in strand separation with Hg(II) being linked only to uridine. The fast reaction phase is second order ans shows little cooperative behavior. Protons are released at this stage indicating alteration of the double helix. The measured second-order rate constant is nearly three orders of magnitude smaller than that found for poly(U) alone. This rate difference suggests that the reactive sites are blocked by double helix formation, and become available for reaction with Hg(II) only through a structural fluctuation. The ratio of rate constants for the reaction of Hg(II) with poly(U) and poly(A)-poly(U) was used to place an upper limit on the equilibrium constant for the structural fluctuation of 2 times 10- minus 3 at 15 degrees and 0.5 M NaClO4. The heat of the "breathing" reaction can be estimated to be similar to 9 kcal/mol from comparison of the temperature coefficient of the reaction with poly(U) to that with poly(A)-poly(U).


Endoribonuclease from bovine adrenal cortex cytosol. An endoribonuclease which digests a variety of synthetic homoribopolymers and poly(A)-rich mRNA has been identified and purified greater than 500-fold with respect to specific activity from bovine adrenal cortex cytosol. Enzymatic digestion of synthetic poly(riboadenylic acid) was stimulated by Mn-2+ and Mg-2+ and the enzyme exhibited broad pH and salt optima. Poly(cytidylic acid) and poly(uridylic acid), but not poly(guanylic acid), served as substrates for the enzyme preparation; double-stranded RNA, DNA, and DNA-RNA hybrids were not digested by the enzyme. Digestion generated oligonucleotides with 3-hydroxyl and 5'-monophosphoester termini. On isoelectric focusing, the enzymatic activity banded at pH 8.3 plus or minus 0.2. An initial preferential cleavage of the poly(A) tract of poly(A)-rich RNA is suggested by the rapid appearance of a 4-6S digestion product highly enriched for adenylic acid; however, progressive digestion of the RNA occurs with additional incubation.


Photoexcited bacterial bioluminescence. Identity and properties of the photoexcitable luciferase. Properties of photoexcitable luciferase are compared with those of luciferase, both isolated from the bacterium Beneckea harveyi. The proteins have the same molecular weight, are similarly charged at pH 8, and can be inactivated, with comparable efficiencies, by antibodies against either pure luciferase (a heterodimeric protein) or individual subunits thereof. Compared with luciferase, photoexcitable luciferase has a broader pH range for optimal activity, is more stable under acidic conditions, is less stable under alkaline conditions, and is more resistant at neutral pH to inactivation by heat, urea, and trypsin; A flavine-like chromophore, designated B, can be isolated from photoexcitable luciferase. The binding of B to luciferase restores all the properties characteristic of photoexcitable luciferase. Moreover, photoexcitable luciferases from mutants selected to have heat labile luciferases are also thermally unstable. It is concluded that photoexcitable luciferase actually consists of a luciferase-B complex which is conformationally distinct from luciferase under certain conditions.


Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme. The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.


Purification and properties of a yeast protein kinase. A protein phosphokinase (EC 2.7.1.1.37) was isolated from baker's yeast (Saccharomyces cerevisiae) after a 17,000-fold purification; the purified enzyme is homogeneous according to the criteria of gel electrophoresis and ultracentrifuge analysis. The enzyme has a high isoelectric point of ca. 9 and appears to exist as a monomer with a molecular weight of 42,000 plus or minus 1500. It is neither stimulated by cyclic 3',5'-AMP, -GMP, -CMP or -ump nor inhibited by the regulatory subunit of rabbit muscle protein kinase (Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971), J. Biol. Chem. 246, 1986). In the presence of divalent metal ions, preferably Mg-2+ or Mn-2+, the enzyme readily transfers the terminal phosphate group of ATP to phosvitin, alphaS1B- and beta a-casein and an NH2-terminal tryptic peptide derived from beta a-casein, but not to protamine, lysine, or arginine-rich histones or to yeast enzymes such as phosphorylase, phosphofructokinase, or pyruvate carboxylase; serine and polyserine were also inactive as phosphate acceptors. Km values of 0.17 mM for beta a-casein and 0.2 mMfor ATP were determined at 10 mM Mg-2+. The urified yeast protein kinase also catalyzes the reverse reaction, namely, the transfer of phosphate from fully phosphorylated beta a-casein or its NH2-terminal peptide to ADP resulting in the formation of ATP. AMP, GDP, UDP, and CDP did not serve as phosphate acceptors in this reaction. As observed by Rabinowitz and Lipmann (Rabinowitz, M., and Lipmann, F. (1960), J. Biol. Chem. 235, 1043) both reactions have different pHoptima with values of 7.5 for the forward reaction (phosphorylation of the proteins) and ca 5.2 for the formation of ATP; both are differently affected by salts. Phosphorylation of beta a-casein with [gamma-32-P]ATP followed by digestion of the labeled protein with trypsin indicated that all the radioactivity was exclusively introduced in an NH2-terminal peptide possessing the unique sequence: Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu...(Ribadeau-Dumas, B., Brignon, G., Grosclaude, F., and Mercier, J.-C. (1971), eur J. Biochem. 20, 264). By subjecting beta a-casein and its NH2-terminal peptide to the combined action of almond acid phosphatease and purified yeast protein kinase, it was determined that the phosphorylation and dephosphorylation reactions proceed randomly, i.e., all seryl phosphate residues are equally susceptible and that the rate of phosphorylation decreases drastically as the number of bound phosphate groups in the substrate diminishes.


Stable, nonreducible cross-links of mature collagen. During in vivo maturation, and also during in vitro incubation with physiological buffers, native collagen fibers display a progressive increase in tensile strength and insolubility. Paralleling these physiologically important changes is a progressive loss of the reducible cross-links which initially join the triple-chained subunits of collagen fibers. Although there is evidence suggesting that the reducible cross-links are gradually transformed into more stable, nonreducible cross-links during maturation, the nature of the transformation process and the structure of the stable "mature" cross-links has remained a mystery. In order to test the possibility that cross-link transformation involves addition of a nucleophilic amino acid residue to the reducible cross-links, histidine, arginine, glutamate, aspartate, lysine, and hydroxylysine residues were chemically modified, and the effect of each modification procedure on the in vitro transformation of reducible cross-links was ascertained. The results of these experiments indicated that destruction of histidine, arginine, glutamate, and aspartate residues has no measurable effect on the rate and extent of reducible cross-link transformation in hard tissue collagens. In contrast, modification of lysine and hydrocylysine residues with a wide variety of specific reagents completely blocks the transformation of reducible cross-links. Removal of the reversible blocking groups from lysine and hydroxlylysine residues then allows the transformation to proceed normally. These results indicate that collagen maturation involves nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible cross-links, yielding derivatives which are not only more stable but also capable of cross-linking more collagen molecules than their reducible precursors.


NMR studies on phospholipid bilayers. Some factors affecting lipid distribution. 1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phospholipid mixture contains negative lipids. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4-5 Hz and the individual linewidths are about 4Hz. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phosphatidylinositol and phosphatidic acid. 6. In mixtures of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55-58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. It is concluded that both charge and the packing properties of the head group are major factors in determining the distribution of phospholipids in mixed vesicles.


A thermally induced alteration in lysosome membranes: salt permeability at 0 and 37 degrees C. Preparations of radioactive lysosomes were obtained from mouse kidney after injection of radioactive iodine-labeled bovine ribonuclease. Stability of these lysosomes in various media was estimated from measurements of proteolytic activity towards the ribonuclease, and of ribonuclease retention in particles. The lysosomes were stable at 37 degrees C in isotonic, sucrose-free solutions of KCl, NaCl, and potassium acetate, and in mixtures of these with MgCl2, showing that these salts are relatively impermeant through the lysosomal membranes. The membranes were less permeable to Na+ than to K+. Both KCl and NaCl exerted their optimal protective effects over a broad concentration range above 0.125 M in 0.025 M acetate buffer. Mg2+ enhanced the protective effect of both K4 and Na+; the osmotic effect of 0.075 M NaC1-0.05 M MgCl2 was indistinguishable during the entire course of ribonuclease digestion from that of isotonic sucrose. Osmotic protection by KC1-MgC12 was demonstrated over the H range5.5-7.0. A marked alteration in membrane properties occurs at lower temperatures in 0.11 M KC1-0.01 M MgCl2 such that, at 0 degrees C, K+ permeability is much higher than at 37 degrees C, as shown by a several-fold decrease in stability at the lower temperature.


The effect of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide on calcium binding and associated changes in chloroplast structure and chlorophyll a fluorescence in spinach chloroplasts. 1. Chemical modification of carboxyl groups on the chloroplast membrane with a water-soluble carbodiimide plus a nucleophile caused inhibition of Ca-2plus binding. 2. Both binding sites were affected and showed a decrease in the number of binding sites and an increase in the dissociation constant. 3. Cation-induced changes in chlorophyll a fluorescence and structural changes (deltaA540) were inhibited at the same carbodiimide concentrations as Ca-2plus binding, emphasizing the relationship between these processes. 4. Chloroplasts that were illuminated with high intensity light for short time periods showed a decrease in the carbodiimide-mediated inhibition of Ca-2plus binding.


Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor, CF1. Effects of adenylates on chloroplast delayed light emission, at millisecond dark times, are inverse to the previously characterized effects of adenylates on electron transport rates. Either ADP alone or ATP alone increase intensity of delayed light, while ADP plus Pi decrease it. ADP alone requires the presence of an electron acceptor to have this effect on delayed light, but ATP does not. All three adenylate effects are abolished by uncoupling with gramicidin, by partial removal of photophosphorylation coupling factor (CF1) with EDTA, and by antibody to CF1. Readdition of CF1 re-established the adenylate effects in EDTA-stripped membranes. The three adenylate effects are differentially sensitive to pH, and pH differentially affected their abolition by antibody to CF1. The two adenylate effects shown in the absence of Pi are exhibited at lower adenylate concentrations than the ADP plus Pi effect, and are also less sensitive to phloridzin. These results are discussed in terms of probable adenylate effects on membrane-bound chloroplast coupling factor, CF1. At least two ADP binding sites would differ with respect to adenylate concentration for half maximal binding; pH of optimal binding capacity; phloridzin sensitivity; and functional regulation of electron transport, proton uptake, and energy storage within the membrane as measured by delayed light emission. It remains unclear whether the high affinity ADP binding site is identical to a high affinity ATP binding site on CF1.


The respiratory chain in a ubiquinone-deficient mutant of Saccharomyces cerevisiae. 1. Two allelic mutants of Saccharomyces cerevisiae with a deficiency in the biosynthesis of ubiquinone have been isolated. The properties of one particular mutant strain were investigated. Submitochondrial particles of this strain contain maximally 3% of the amount of ubiquinone in wild-type particles; the amounts of other components of the respiratory chain are essentially normal. 2. The respiratory rates of mutant cells, mitochondria and submitochondrial particles are low with ubiquinone-dependent substrates, but are restored to normal levels by addition of Q-1; the restored respiration is antimycin sensitive. Intact cells and mitochondria show respiratory control both in the absence and presence of Q-1. 3. The NADH:Q-1 oxidoreductase of submitochondrial particles of the mutant followspseudo first-order kinetics in [Q-1]. QH2-1 inhibits competitively with respect to Q-1, the Ki for QH2-1 being equal to the Km for Q-1. 4. Succinate dehydrogenase in both wild-type and mutant submitochondrial particles can be activated by NADH. 5. The turnover number of succinate dehydrogenase in the mutant, measured with phenazine methosulphate as primary electron acceptor, is about one-half that of wild-type particles. The turnover numbers measured with Q-1 as electron acceptor are about the same in the two types of particles. 6. The kinetics of redox changes in cytochrome b, in the presence of antimycin and oxygen, are distinctly different in the mutant and wild-type particles. They indicate that ubiquinone plays an important role in the phenomenon of the increased reducibility of cytochrome b induced by antimycin plus oxygen.


Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles. 1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.


Physicochemical properties of flavodoxin from Desulfovibrio vulgaris. Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.


Chloroplast cytochrome b-6. Molecular composition as a lipoprotein. Disc electrophoretically homogeneous spinach-chloroplast cytochrome b-6 was found to be a lipoprotein whose redox potential was essentially unchanged during isolation. These results further support the hypothesis of Triton X-100/4 M urea, pH 8, as a useful extracting medium for membrane lipoproteins. Cytochrome b-6 was found to have a heme equivalent dry weight of 1 mol of heme per 60000 g. Of this, 20000 g was lipid-extractable. The molecular weight was 60000 with a partial specific volume of 0.84 ml/g. The protein portion of the molecule (40000) consisted of 1 polypeptide chain of 20000 daltons, 1 of 9600 daltons and 2 of 6600 daltons. A simple lipid composition (relative to the original membrane) was found consisting of 7 mol of chlorophyll a and 6 mol of cardiolipin per mol of cytochrome; these two lipids thus account for about 75-80% of the lipid content. An unidentified minor neutral lipid and minor polar lipid were also detected. At pH 7.0 in the presence of 0.5% Triton X-100, E'-o was -0.080 V, and in the absence of Triton X-100, E'-o was -0.120 V. At pH 8 in 0.5% Triton X-100, E'-o was -0.084 V, thus indicating that the redox potential is independent of pH in the region 7-8. The redox reaction proceeded via a one-electron-transfer.


The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of photosystem II. The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of -60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.


Studies on acetylcholinesterase and cholinesterase covalently bound to polymaleinic anhydride. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), respectively, were covalently attached to a cross-linked copolymerisate of maleinic anhydride and butanediol-divinylether. Based on the coupling procedure reported by Brümmer et al. (Brummer, W., Hennrich, N., Klockow, M., Lang, H. and Orth, H.D. (1972) Eur. J. Biochem. 2k, 129--135), a simple method is described which requires only 24 h for completion and provides a sufficient yield. Although a polyanionic carrier was used the Km and k2 values as well as the substrate and pH optima of the bound acetylcholinesterase and bound cholinesterase did not differ considerably from the corresponding values of the free enzymes. Bound acetylcholinesterase and to some extent also bound cholinesterase did not lose any enzymatic activity after storage in saline at 4 degrees C for 140 days.


L-ascorbic acid 2-sulphate. A substrate for mammalian arylsulphatases. Ascorbic acid 2-sulphate has a stability in acid comparable to that of phenyl sulphate and is rather more acid-labile than simple carbohydrate sulphates. At its optimum pH of 4.8 sulphatase A(aryl-sulphate sulphohydrolase EC 3.1.6.1.) hydrolyses ascorbic acid sulphate with a specific activity of 90 mumol/mg per min (150 mumol/mg per min with nitrocatechol sulphate at pH 5.6). At pH 4.8 the kinetics are non-Michaelis. At pH 5.6 Michaelis kinetics are obeyed and Km 12 21 mM ascorbic acid 2-sulphate. K2SO4 is a competitive inhibitor with a Ki of 0.2 and 0.6 mM at pH 4.8 and 5.6, respectively. Sulphatase A is converted into a substrate-modified form during its hydrolysis of ascorbic acid sulphate. Sulphatase B also hydrolyses ascorbic acid 2-sulphate. At pH 4.8 and in the presence of 0.15 M NaCl the specific activity is 0.92 mumol/mg per min (90 mumol/mg per min for nitrocatechol sulphate at pH 5.6). In the absence of NaCl the activity is greatly decreased. Km is 8 mM. K2SO4 is a competitive inhibitor with a Ki of 0.1 mM. Ascorbic acid is not hydrolysed at a detectable rate by the arylsulphatases of the mollusc Dicathais orbita or of Aerobacter aerogenes.?


Ribosomal RNA turnover and the level of ribonuclease activity in the liver of the pregnant rat. Studies were undertaken on the turnover of ribosomal RNA and on ribonuclease activity in the liver of the pregnant rat in an attempt to explain the accumulation of liver RNA which occurs during the latter half of pregnancy. Between the 15th and 20th day of gestation the rate constant of degradation, biological half-life and daily rate of synthesis of ribosomal RNA were calculated to be 0.0887, 7.81 days and 6.21 mg per liver per 100g body weight respectively. Corresponding values in non-pregnant rats were 0.123, 5.68 days and 3.47 mg per liver per 100g body weight. The increase in RNA was therefore associated with an increase in its rate of synthesis and a decrease in its rate of breakdown. From the 14th day of pregnancy there was a decrease in alkaline ribonuclease activity and a marked increase in the level of alkaline ribonuclease inhibitor. The activity of acid ribonuclease was found to increase and that of acid phosphatase to decrease during this period.


A trypsin-resistant heme peptide from cardiac cytochrome c1. A tryptic resistant heme peptide has been prepared and purified from cardiac cytochrome c1. This purified peptide is not further hydrolyzed by reactions of other proteolytic enzymes, such as pronase. The peptide contains 2 residues each of serine, cysteine and valine, and 1 residue each of alanine, methionine, tyrosine, histidine, arginine, proline, glutamic acid (glutamine) and aspartic acid. The intensity of the absorption spectrum of the peptide has been found to be dependent upon, but the positions of the absorption maxima do not vary with, concentration. The heme peptide does not show multiple splitting of absorption peaks at liquid N2 temperatures as does the intact cytochrome C1. However, cyanide rapidly reacts with the peptide and causes significant spectral changes. CD spectra of the peptide exhibit a typical profile of a non-structured heme peptide with positive CD bands in the Soret region and around 250 nm, and a broad negative extreme of 320-360 nm. The similarities and differences between the tryptic resistant heme peptides from cytochromes c1 and c have been compared.


The excretion of hydrogen ion by the isolated amphibian skin: effects of antidiuretic hormone and amiloride. H+ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo, was studied in order to test for the presence of exchange mechanisms of the type Na+/H+ and Cl-/HCO3-, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na+ transport and the pH-stat techinique was utilize to determine the rates of H+ extrusion under different experimental conditions. The conditions were either the withdrawal of the ions intervening the mentioned exchanges (Cl- or Na+), or the addition of drugs with well-known effects on Na+ up-take and transport (antidiuretic hormone and amiloride). In the frog skin, H+ excretion was detected in solutions containing either Cl- or SO4-2-, with identical rates. Again, Na+ substitution by Mg-2+ had no effect on H+ excretion rates, neither did the suppression of Na+ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase. Experiments in toad skin that H+ excretion could not be detected whan Cl- was present in the outer medium, but became apparent if an impermant anion, SO4-2-, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl-/HCO3-. Secondly, in these preparations H+ extrusion increased after stimulation with antidiuretic hormone and  decreased when amiloride was used or when Na+ was substituted by Mg+, suggesting that a least a fraction of the total H+ efflux is linked to Na+ influx. In the isolated frog skin this mechanism does not seem to be operative.


Raman and resonance-Raman scattering by erythrocyte ghosts. 1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.


Gastric acid secretion in the lizard. Ionic requirements and effects of inhibitors. 1. The effects of ion substitution and various inhibitors on the transmucosal potential, short circuit current, mucosal resistance and acid secretion of the lizard gastric mucosa, incubated in an Ussing chamber, have been determined. 2. Ion substitution experiments indicate that the serosal potential step consists of a combined C1- and K+ diffusion potential, and that the mucosal potential step is Na+ dependent and behaves primarily as a Na+ diffusion potential. 3. Experiments with ouabain indicate that the major (Na+, K+)-ATPase activity responsible for maintenance of cation gradients is located on the serosal side of the mucosal cells, and that this pump activity is non-electrogenic. 4. Experiments with amiloride indicate that a passive sodium influx on the mucosal side is essential for the maintenance of the transmucosal potential and short circuit current. 5. Acid secretion requires the presence of sodium and chloride on the serosal side and the maintenance of a high intracellular potassium level through the (Na+, K+)-ATPase system. 6. The effects of acetazolamide and thiocyanate are compatible with an involvement of carbonic anhydrase and anion-dependent ATPase in acid secretion. 7. Upon initiation of acid secretion the serosal membrane permeability for chloride increases and that for potassium decreases.


A poly(U) polymerase in tobacco leaves.

A poly(U) polymerase in tobacco leaves. A poly(U) polymerizing enzyme has been found in healthy and tobacco mosaic virus-infected tobacco leaves and has been partially purified by affinity chromatography on a gel prepared from agarose with chemically coupled RNA. The enzyme is stimulated by Mn-2+ and dependent on a polynucleotide, preferentially poly(A). The synthesis proceeds optimally at pH 7.6 and 25 degrees C. The enzyme is highly specific for UTP and is inhibited by other ribonucleoside triphosphates. The product was partly sensitive to pancreatic ribonuclease. The synthetic reaction is inhibited in the presence of pyrophosphate but insensitive to 10 mM orthophosphate and high levels of cordycepin, rifampicin and actinomycin D. A molecular weight of about 40,000 has been estimated by sucrose gradient analysis and partition cell ultracentrifugation.


Cyclization of three satellite components of calf thymus DNA. Cyclization of denatured and reannealed satellite components of calf thymus DNA was studied by electron microscopy. All three satellite DNA components studied (1.707g/cm-3, 1.714g/cm-3 and 1.721g/cm-3) form circular structures indicating that the sequences of the calf thymus satellite DNAs are arranged in a tandemly repetitious manner. Under appropriate annealing conditions the amount of circular structures is reproducible and practically no aggregates are formed. By comparison of cyclization experiments under defined conditions it is demonstrated that individual satellite components differ in the amount of circular structures formed during reassociation and in the distribution of linear and circular molecules. From the distribution of the contour lengths of circular molecules we conclude that the length of the repetitive sequence decreases with increasing buoyant density of the satellite components. The average lengths of the repetitive sequences calculated from electron microscopy measurements are in good agreement with those from renaturation kinetics.


The conversion of glyceraldehyde-3-phosphate dehydrogenase to an acylphosphatase by trinitroglycerin and inactivation of this activity by azide and ascorbate. Trinitroglycerin oxidizes the essential sulfhydryl group, Cys-149, of pig muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(phosphorylating) EC 1.2.1.12) TO A SLUFENIC ACID, NOT TO A DISULFIDE. This conclusion is based on the observation that the inactivation of the dehydrogenase activity of the enzyme by the organic nitrate induces the acylphosphatase activity which is catalyzed by the sulfenic acid form of the enzyme. Inorganic nitrite is released during this process which is stoichiometric with the degree of inactivation of the dehydrogenase. The acylphosphatase activity induced by trinitroglycerin, unlike the dehydrogenase activity, is sensitive to CN-. Treatment of the enzyme oxidized with trinitroglycerin with 14-CN- leads to the incorporation of protein-bound 14-CN-, which is stoichiometric with the degree of inactivation of the dehydrogenase. Treatment of the sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase at pH 5.3 with a 10-fold molar excess of azide over the concentration of enzyme subunit completely inactivates the acylphosphatase reaction catalyzed by the oxidized enzyme. Concomitantly, the dehydrogenase activity catalyzed by the sulfhydryl form of the enzyme reappears which indicates that excess azide reduces the sulfenic acid which is required for the acylphosphatase. Treatment of the oxidized enzyme with a stoichiometric amount of azide at pH 5.3 stimulates the acylphosphatase activity and does not lead to the reappearance of dehydrogenase activity. When the sulfenic acid form of the enzyme is incubated with 20 mM L-ascorbate at pH 5.3, the acylphosphatase activity is completely inactivated and the dehydrogenase activity catalyzed by the reduced form of the enzyme is recovered. Thus, L-ascorbate also reduces the protein sulfenic acid which is required for the acylphosphatase activity.


Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs. Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).


Udp-galactose: glycoprotein galactosyltransferase activity in a clonal line of rat brain. 1. UDPgalactose:glycoprotein galactosyltransferase (EC 2.4.1.-) activity was demonstrated in homogenates from whole rat brain, isolated neuromal perikarya, enriched glial cell fractions, and cultured rat glial tumor cells (clone C6).  2. Galactosyltransferase activity was enriched 3-9-fold in neuronal perikarya and 1.4--1.8-fold in the glial cell fraction over the activity in whole brains from 19- and 40-day-old rats.  The activity of galactosyltransferase in neuronal perikarya decreased with age.  Extensive contamination of the glial cell fraction with membranous fragments appeared to obscure the precise specific activity of this fraction.  3. The specific activity of the enzyme in glial tumor cells was 4--8-fold higher than in brain tissue when the enzyme was assayed under identical conditions using endogenous and different exogenous acceptors.  4. Galactosyltransferase activities from adult brain and glial tumor cells had similar properties.  They both required Mn-2 plus and Triton, and exhibited pH optima between 5 and 7.  The apparent Km of the enzyme for UDPgalactose was 1.3-10-minus 4 M for brain tissue and 2.2-10-minus 4 M for glial tumor cells.  5. The high galactosyltransferase activity in glial tumor cells and in neuronal perikarya of younger rats is compatible with the possibility of a role of this enzyme in developing brain.


Purification and characterization of a cyclic nucleotide-regulated 5'-nucleotidase from potatoe. A procedure is presented for the rapid purification of a 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from potato tubers, involving ammonium sulphate fractionation and chromatography on phosphocellulose, DEAE-cellulose and Sephadex G-75. Application of this procedure results in a 6000-fold purification of the 5'-nucleotidase and the final preparations are virtually homogeneous, yielding only one protein band on electrophorsis in polyacrylamide gels in non-dissociating or dissociating conditions. The 5'-nucleotidase has a molecular weight of 50 000 from gel filtration experiments. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified 5'-nucleotidase reveals one major band of molecular weight 25 000. The 5'-nucleotidase is competitively inhibited by cyclic nucleotides, having micromolar Ki values for cyclic AMP and cyclic GMP at pH 5.0 and pH 8.0. The enzyme has a pH optimum of 5.0 with 5'-GMP as substrate. While 5'-AMP and 3'-AMP are hydrolyzed at comparable rates at pH 5.0, at pH 8.0 the rate of hydrolysis of 3'-AMP is only 4% of that with 5'-AMP. ADP, ATP and 2'-AMP are very poor substrates for the enzyme. The nucleotidase has micromolar Km values for nucleoside 5'-monophosphates other than 5'-NMP. A wide variety of divalent cations activate the 5'-nucleotidase.


Epidermal nucleases: purification and characterization of ribonuclease from mammalian epidermis. The major ribonuclease of adult guinea pig epidermis has been isolated and purfied over 1000-fold by a combination of ammonium sulfate fractionation, affinity and ion-exchange chromatography, and electrophoresis. The purified enzyme is free from phosphodiesterase and phosphatase activities. The ribonuclease is optimally active near neutrality in phosphate buffer, with a Km of 3mu g/ml toward [14-C]RNA from Erhlich ascites tumor cells. (here are no metal requirements for activity. The enzyme catalyzes the endonucleolytic hydrolysis of high molecular weight yeast RNA and it also hydrolyzes polycytidylic and polyuridylic acids, but not polyadenylic, polyguanylic, and polyinosinic acids. The apparent molecular weight of the active enzyme is 28 500.


Production of 1, 3-beta-glucanase by Bacillus No. 221.  An alkalophilic microorganism. A 1, 3-beta-glucanase of Bacillus No. 221 has been extensively purified by a DEAE-cellulose column followed by a Sephadex G-75 gel filtration, and crystallized in ammonium sulfate solution.  The crystalline enzyme is homogenous on the basis of polyacrylamide gel electrophoresis, sedimentation in ultracentrifuge (3.2 S), Ampholine electrofocusing (pI=4.1) and dodecylsulfate-polyacrylamide gel electrophoresis (Mr=36 000). The enzyme has an optimum pH for enzyme action at 8.5 which is higher than those of other 1, 3-beta-glucanases so far reported.  The enzyme is very thermostable; about 90% of activity remains after being heated at 70 degrees C for 10 min, and no effect of Ca-2's obversed.  The enzyme does not hydrolyse laminaritriose, but hydrolyses laminaritetraose, and yields glucose and laminaritriose.  The enzyme splits laminaran at random and yields glucose, laminaribiose, laminaritriose and higher oligosaccharides.  From these results, this enzyme is a type of endo-1, 3-beta-glucanase.


The regulation of myo-inositol-1-phosphate-synthase activity from Neurospora crassa by pyrophosphate and some cations. The effect of several cations on the inhibition by PPi of the enzyme myo-inositol-1-phosphate synthase (1L-myo-inositol-1-phosphate lyase (isomerizing) EC 5.5.1.4) from Neurospora crassa was studied. The study wastol biosynthesis can occur in the presence of an intracellular PPi concentration which exceeds the Ki for the enzyme by 350-fold. The inhibition of enzyme activity by PPi,at pH 7.7, was reversed, in decreasing order of effectiveness, by Mn-2, Fe-3


Protein kinases in brown adipose tissue of developing rats.  I. GTP as nucleotide substrate for histone phosphorylation. 100 000 times g soluble extracts from interscapular brown adipose tissue catalyzed the transfer of the terminal phosphoryl group from GTP to histone.  Maximal velocity was achieved only with both cyclic AMP and ATP present.  The cyclic AMP dose-response curve was the same as for the ATP-utilizing enzyme, with maximum stimulation at 0.5 muM.  ATP (1--100muM) increased the rate of histone phosphorylation with GTP as the radioactive substrate.  Higher concentrations had a dilution effect similar to that of GTP on the ATP-utilizing enzyme.  Similar effects were observed with ADP and AMP.  The apparent Km values for histone were the same with both GTP and ATP as nucleotide substrates.  The effects of pH, purified beef muscle kinase inhibitor and of NaCl were also the same.  Maximum velocities of histone phosphorylation from ATP and those from GTP were almost the same in brown fat of all age groups testes,  Separated on histone-Sepharose, the GTP-utilizing activity was absolutely dependent on the re-addition of the ATP-utilizing enzyme (a linear relationship with a slope of approx. 0.95).  An extremely active nucleotide phosphotransferase activity was found in the same subcellular fraction.  The rate of equilibration of the gamma-32-P between GTP and ATP could account for all the histone phosphorylation with [gamma-32-P] GTP.  It is concluded that, in spite of the presence of nucleotide phosphotransferase and ATP-protein kinase activities, a direct transfer from GTP to a protein substrate cannot be excluded.  Also, histone may not be the natural protein acceptor for GTP-linked phosphorylation.


Glycolysis in human erythrocytes containing elevated concentrations of 2, 3-P2-glycerate. In studies on the mechanism of the inhibitory effect of 2, 3-diphosphoglycerate on glycolysis in human erythrocytes, the following results were obtained: 1) Glucose consumption and lactate production are reduced by 70 and 40 per cent relative to normal erythrocytes in red blood cells containing five times the normal amount of 2, 3, -P2-glycerate ("high-diphosphoglycerate" cells) at an extracellular pH of 7.4. The marked dependency of glycolysis on the extracellular pH observed in normal erythrocytes is almost completely lost in the "high-diphosphoglycerate" cells.  2) About 50 per cent of the inhibition of glycolysis in "high-diphosphoglycerate" cells can be accounted for by the 2, 3-P2-glycerate-induced decrease of the red-cell pH.  This fall of the red-cell pH which occurs as a conswquence of the Donnan effect of the non-pentrating 2, 3-P2-glycerate anion leads to a reduction of the glycolytic rate due to the properties of the enzyme phosphofructokinase.  3) The remaining part of the inhibitory effect must be attributed to an inhibition by 2, 3-P2-glycerate of glycolytic enzymes.  From measurements of glycolytic rates and of the concentrations of glycolytic intermediates in the absence and presence of methylene blue it is concluded that the hexokinase reaction is inhibited by an elevation of 2, 3-P2-glycerate concentration in "high-diphosphoglycerate" cells suggests that also the enzyme pyruvate kinase is inhibited by 2, 3-P2-glycerate.  4) The dependencies of net-change of 2, 3-P2-glycerate concentration on the red-cell pH are identical in normal and "high-diphosphoglycerate" cells indicating that the balance between formation and decomposition of 2, 3-P2-glycerate is the same in erythrocytes with normal and very high compositions of 2, 3-P2-glycerate.


Endogenous purine metabolism in the conidia of wild type and certain adenine mutants of Neurospora crassa. I. The nature of the reserve pools and pool utilization during adenine starvation. Conidia of four adenine auxotrophs (ad 9, ad 3B, ad 8 and ad 4) of Neurospora crassa differ in their ability to germinate on adenine-deficient medium. A large percentage of the ad 9 and ad 3B mutant conidia germinate while those of ad 8 and ad 4 mutant do not. No correlation was found between the size of the conidial purine reserves and the conidial ability to germinate. In all the strains the major fraction of the conidial purine reserve pools was inosine. The ad 8 and ad 4 mutants are blocked after IMP formation in the adenine biosynthetic pathway and therefore cannot use the stored inosine for germination. Pool-utilization studies indicated that in all strains investigated some of the purine reserves were lost from the conidia during incubation. In the most readily germinating strain, ad 9, only small amounts of the purine pool were lost from the conidia and a large portion of the reserve pool was used for nucleic acid synthesis. The nature of the purine reserves present in the conidia, and the ability of the strains to prevent loss of the stored purines from the conidia appear to be among the factors influencing the conidial germination of the adenine mutants of N. crassa.


The mechanism of liver microsomal lipid peroxidation. In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.


Effect of chorionic gonadotropin, triamcinolone, progesterone and estrogen on enzymes of placenta and liver in rats. The activity of several enzymes of regulatory importance for the pathways of glycolysis, gluconeogenesis and lipogenesis was investigated in the placenta and liver of pregnant rats and in the liver of non-pregnant female rats. The rats received daily hormonal treatments on Days 15 to 17 of pregnancy and enzyme activities were measured on Day 18. Chorionic gonadotropin induced minor changes in enzyme activity, apart from a decrease in the activity of hepatic enzymes of lipogenesis in non-pregnant rats. Triamcinolone induced a marked increase in enzymes of gluconeogenesis and a decrease in the activity of pyruvate kinase in the liver of pregnant and non-pregnant rats; in contrast, inverse changes in activity, these enzymes were observed in the placenta. This response in the placenta was considered to arise not from direct hormone effect, but from the accompanying hyperglycemia and hyperinsulinemia. Triamcinolone also increased the activity of hepatic acetyl-CoA carboxylase in pregnant and non-pregnant rats, whereas it reduced the activity of this enzyme in the placenta. Estrogen produced changes similar to those of triamcinolone in the liver and placenta, except that it depressed the activity of acetyl-CoA carboxylase in both tissues. Progesterone had little effect on placental and hepatic enzymes. In general, the changes induced by these hormones in the placenta affected fewer enzymes than in the liver, were less extensive in magnitude and not necessarily in the same direction as in the liver. This indicates that the regulatory placental enzymes are subject to specific control mechanisms not necessarily influenced by direct hormone action.


Dihydrothiamine-like substances as experimental artefacts. Experimental controls have shown that the dihydrothiamine-like substance previously reported as isolated from animal tissues was an artefact. In addition, no evidence is found for the reduction of thiamine by NADH.


Studies on fungal polysaccharide. XVII. A new glucuronan "protuberic acid" produced by a fungus Kobayashia nipponica. A water-soluble glucuronan "protuberic acid", [alpha] 22-D minus 83.6 degrees, was isolated and purified from Kobayashia Nipponica, and its physicochemical properties were investigated. The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1, 4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [alpha] 22-D minus 44 degrees, is also described.


Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. I. Evidence for superoxide anion involvement in the oxidation of NADPH2. 1. The oxidation of NADPH2 by leucocyte granules, as measured at acid pH in the presence of Mn-2+, was found to be inhibited by superoxide dismutase. 2. Omission of Mn-2+ markedly lowered the oxidase activity at acid pH, which was still inhibited by superoxide dismutase. 3. At alkaline pH the oxidase activity was lower than at acid pH. 4. During oxidation of NADPH2 by leucocyte granules, reduction of cytochrome c occurred which was partially inhibited by superoxide dismutase. 5. It was concluded that NADPH2 oxidation occurs through an enzymatic reaction and a nonenzymatic chain reaction. Superoxide anion (O-minus-2 and NADPH- free radical would be involved in the chain reaction. The differential sensitivity of NADPH2 oxidation to superoxide dismutase in different experimental conditions (see above 1, 2 and 3) was explained on the basis of changes in the properties of the chain reaction.


Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. II. Presence of the NADPH2 oxidizing activity in a myeloperoxidase-deficient subject. 1. The biochemical properties of leucocytes from a myeloperoxidase-deficient subject were compared with those of leucocytes from healthy subjects. 2. Myleoperoxidase-deficient leucocytes responded to phagocytosis of heat-killed bacteria with increased respiration, increased oxidation of glucose through the hexose monophosphate shunt and increased production of H2O2 as normal leucocytes do. 3. The ability of granules isolated from myeloperoxidase-deficient leucocytes to oxidize nicotinamide coenzymes was comparable to that of granules isolated from normal leucocytes. 4. The results argue against the hypothesis that oxidation of NADPH2 in leucocytes is performed by myeloperoxidase.


Metabolism of D-mannosamine in bovine thyroid gland slices. 1. The nature of the acid-soluble mannosamine intermediates was investigated in bovine thyroid gland slices after incubation with a tracer amount of [14C]mannosamine plus 10 mM carrier mannosamine. 2. These radioactive intermediates were separated by ion exchange, columns, characterized by paper chromatography and were identified as N-acetylmannosamine-6-P. The radioactivity associated with these acid-soluble compounds constituted about 17 percent of the added [14C]mannosamine. 3. [14C]Mannosamine was incorporated as sialic acid only into thyroglobulin.


[Purification of human gastric glycoprotein and a study of its carbohydrate components (author's transl)]. Mucus was extracted from human gastric mucosa by homogenization in distilled water. The crude extract was purified from plasma, salivary and tissue contaminants by different steps involving chromatography on Sepharose 6B, Sepharose 2B and immunosorbents. The native glycoprotein so prepared was found to be immunologically pure; it migrated as a single band in acrylamide agarose gel electrophoresis at pH 8.6 and 3.5. The molecule exhibits blood group acitivity, contains 0.71 per cent sialic acid but no sulphate. The carbohydrate moiety contains glucosamine, galactosamine, fucose, galatose and mannose in the molar proportions 3: 1.95: 2.92:4.53:0.05.


Bovine luteinizing hormone. Circular dichroism and thermal difference spectra. The circular dichroism of bovine luteinizing hormone in the far ultraviolet (200-240 nm) and near ultraviolet (240-320 nm) is reported as a function of pH, reduction and denaturation. The significance of side-chain ellipticity in the native and fully randomised hormone is critically examined, using denatured and reduced ribonuclease and insulin as bases for comparison. Disulphide groups make no measurable contribution to the side-chain ellipticity. Judged by the spectra of the subunits both isolated and recombined, those treatments which promote subunit disociation without causing chain unfolding, reversibly decrease the ellipticity in the near ultra-violet with minimal effect in the far ultraviolet. Thermal perturbation difference spectroscopy of bovine luteinizing hormone and its subunits shows that, in common with the ovine and porcine hormones, there are two tyrosine residues located at the interface between the subunits and inaccessible to water. Only two or three of the five water-accessible tyrosine residues are reactive to N-acetylimidazole.


Temperature and pH dependent changes of immunoglobulin G structure. 1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning microcalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 degrees C in the narrow temperature interval (3-4 degrees C). 4. The "acid state" is not accompanied by S-like transition at 25-35 degrees C. The thermal denaturation of the "acid state" occurs at 54 degrees C in the wide temperature interval (8-9 degrees C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 degrees C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.


Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12. Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).


Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing. A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.


Platelet fibrinogen. Identity and initial observations on the mode of its degradation by plasmin. Fibrinogen has been purified from human platelets. Platelet fibrinogen exhibits a characteristic pattern in agar gel immunoelectrophoresis different from that of plasma fibrinogen. Stepwise plasmin degradation has been used in further elucidation of the molecular properties of the platelet protein. Examination of comparative digests by immunologic and gel electrophoretic methods has revealed that (1) the platelet protein is more resistant to plasmin degradation, (2) the plasmin-produced fragments of platelet fibrinogen differ consistently from those of its plasma counterpart, and (3) platelet fibrinogen is different from fragment X of plasma fibrinogen. It is suggested that platelet fibrinogen may contribute to the stability of the thrombus.


On the reaction of papain and succinylpapin with diazo-1-H-tetrazole. 1. The reaction of papain and succinylpapain with diazo-1-H-tetrazole was investigated under different conditions. The extent of modification of the amino acids histidine, tyrosine, tryptophan and lysine was determined spectrophotometrically and/or by amino acid analysis. 2. Only one of the two histidine residues present in the enzyme reacts with diazo-1-H-tetrazole forming a monoazo derivative. The pH dependence of the coupling reaction reveals a normal pK of this reactive histidine. There are several arguments suggesting that this may be histidine 159 near the essential SH-group of papain. 3. All five tryptophan residues of the protein react with the diazonium ion below pH 7 forming a monoazo derivative with an absorption maximum at 370 nm, above pH 7 only four residues couple with diazo-1-H-tetrazole. The reaction of one tryptophan and one histidine are correlated as can be concluded from the pH dependence of the coupling rate of both amino acids and the parallel impairment of the catalytic acitivity. 4. 10-11 tyrosine residues out of 19 react with diazo-1-H-tetrazole to give bisazo compounds. 5 residues involved in hydrogen bridges form monoazo compounds. Only 12 tyrosines can be acylated by acetylimidazole. A relationship between the extent of modification of tyrosine and the activity of the enzyme could not be found.


Purification of a carboxypeptidase B-like enzyme from the starfish Dermasterias imbricata. A carboxypeptidase B-like enzyme which catalyses the hydrolysis of synthetic esters of lysine and arginine has been isolated from the starfish Dermasterias imbricata. This carboxypeptidase B-like enzyme has a molecular weight of approximately 34 000 and shares this and other properties with bovine pancreatic carboxypeptidase B. The existence of zymogen for this activity in the pyloric caeca of the starfish is demonstrated. This zymogen has a molecular weight near 40 000 and appears to be analogous to other monomeric procarboxypeptidases B. The zymogen possesses an intrinsic low-level activity toward synthetic substrates of carboxypeptidase B and is activated by trypsin.


Interaction of amino acids with silver(I) ions. The reactivity of silver(I) ions towards twenty amino acids has been studied in aqueous unbuffered solutions using an ion-selective electrode as a highly sensitive monitor. Contrary to general belief, silver ions are not completely specific for cysteine, but also react with lysine, arginine, methionine and, to a minor extent, cystine. Nevertheless the interaction with all of these species except cysteine is too weak to affect significantly the determination of thiol in proteins by the customary argentometric titration method. The possible origin of errors arising in such titrations is discussed.