Absence Of Other Substrates Research Articles

RADIORESPIROMETRIC STUDIES IN GENUS NEISSERIA

The catabolism of pyruvate and acetate is selected Neisseria species was studied by radiorespirometry. Both substrates were oxidized via the tricarboxylic acid cycle. N. elongata and the "false neisserias" (N. catarrhalis, N. ovis and N. caviae) did oxidize acetate in the absence of other substrates. This can be explained if it is assumed that these species have glyoxylic acid cycle activity. In the "true neisserias" other than N. elongata, acetate was oxidized only in the presence of glutamate, indicating that these species do not possess a glyoxylic acid cycle.

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. d-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only d-glucose and d-mannose were able to stimulate insulin release and cyclic [ 3H]AMP accumulation in the absence of other substrate. d-fructose had a stimulatory effect in the presence of 3.3 mM d-glucose only at a high concentration (38.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. d-Galactose was effective only together with 8.3 mM d-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [ 3H]AMP accumulation, was glucose-mannose-fructose-galactose. l-Glucose and 3- O-methylglucose had no effects at 60 min when incubated together with 8.3 mM d-glucose, whereas at 3 min, 3- O-methylglucose induced a small stimulation of the cyclic [ 3H]AMP response. d-mannoheptulose and d-glucosamine inhibited the insulin and cyclic [ 3H]-AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [ 3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the β-cell.

Effects of ketone bodies on amino acid metabolism in isolated rat diaphragm

1. Diaphragms from 48h-starved rats were incubated in Krebs-Ringer bicarbonate medium at 37degreesC for 30min and then transferred into new medium and incubated for 1, 2 and 3 h. 2. The amount of free amino acids found at the end of each time of incubation was larger than the amount at the beginning of incubation, indicating that in this system proteolysis is prevailing. 3. The diaphragms was releasing mainly alanine and glutamine into the incubation medium. 4. Within the periods of incubation the release and metabolism of free amino acids was proceeding at a constant rate. 5. Addition of sodium DL-3-hydroxybutyrate decreased the tissue content of several amino acids, among which were tyrosine and phenylalanine, suggesting that proteolysis was decreased by ketone bodies. 6. In the presence of glucose (10mM) and branched-chain amino acids (0.5mM), sodium DL-3-hydroxybutyrate at concentrations of 4 or 6 mM resulted in 30% decrease in tissue alanine content and a 20% decline in alanine release. Release of taurine and glutamine was decreased by 19 and 16% respectively with 6 mM-sodium DL-3-hydroxybutyrate. Addition of sodium acetoacetate (1-3mM) also resulted in a 20-35% decrease in tissue content of alanine, glutamine and taurine and in a 15-24% decrease of alanine and glutamine release. Smaller decreases (less than 15%) in the release of glycine, threonine, proline, serine and aspartate were also observed in the presence of sodium DL-3-hydroxybutyrate or sodium acetoacetate. 7. Substitution of pyruvate (1.0mM) for glucose in the presence of acetoacetate restored alanine and glutamine production to control values. In the presence of acetoacetate, pyruvate also increased the tissue content of aspartate by 77% and decreased the tissue content of glutamate by 30%. 8. It is suggested that in diaphragms from starved rats, ketone bodies (a) in the absence of other substrates inhibit protein catabolism and (b) in the presence of glucose and branched-chain amino acids decrease alanine and glutamine production, by inhibiting glycolysis.

Equilibrium and water proton relaxation rate enhancement properties of formyltetrahydrofolate synthetase-manganous ion-substrate complexes.

Binding of Mn(pi)-nucleotide complexes to the enzyme formyltertrahydrofolate synthetase (EC 6.3.4.3) from Clostridium cylindrosporum has been examined in the presence and absence of other substrates by solvent proton relaxation mearurements. MnADP and MnATP form ternary complexes with the enzyme with highly enhanced proton relaxation rates for water. The enhancement parameters, epsilont, for the MnADP and MnATP ternary complexes are 19.8 and 12.5, respectively at 24.3 MHZ and 25 degrees. Titration curves with constant total concentrations of enzyme and Mn(pi) with variable nucleotide concentration are similar to those observed in similar titrations with the endp and MnATP are 175 muM and 64 muM, respectively at 25 degrees. Addition of tetrahydrofolate to solutions of the MnADP OR MnATP ternary complexes lowers the observed relaxation enhancement markedly. An analysis of titration curves with constant total concentrations of enzyme, Mn(pi), and nucleotide with variable tetrahydrofolate concentration gives the dissociation constant for tetrahydrofolate from the respective quaternary complexes. The affinity of the enzyme for tetrahydrofolate is increased 6-fold when MnADP is present at the active site whereas a 3-fold increase is observed with MnATP present. Furthermore, there is a 20-fold increase in the enzyme's affinity for tetrahydrofolate when both MnADP and the third substrate, formate, are present. The observed relaxation rate of water for solutions of the complex, enzyme-MnADP-tetrahydrofolate-formate, is deenhanced with respect to the rate observed for the simple aquo-Mn(pi) solution. Addition of nitrate to solutions of the above complex increases the affinity of the enzyme for tetrahydrofolate and MnADP by an additional factor of 5 and lowers the relaxation rate further to a value which approaches that for solutions of the enzyme and substrates which lack the paramagnetic cation.

Pig liver pyruvate carboxylase. The reaction pathway for the decarboxylation of oxaloacetate.

1. The reaction pathway for the decarboxylation of oxaloacetate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K(+) and acetyl-CoA. 2. Free Mg(2+) binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgADP(-) and P(i) bind randomly, at equilibrium, followed by the binding of oxaloacetate. Pyruvate is released before the ordered steay-state release of HCO(3) (-) and MgATP(2-). 3. These results are entirely consistent with studies on the carboxylation of pyruvate presented in the preceding paper (Warren & Tipton, 1974b) and together they allow a quantitative description of the reaction mechanism of pig liver pyruvate carboxylase. 4. In the absence of other substrates of the back reaction pig liver pyruvate carboxylase will decarboxylate oxaloacetate in a manner that is not inhibited by avidin. 5. Reciprocal plots involving oxaloacetate are non-linear curves, which suggest a negatively co-operative interaction between this substrate and the enzyme.

The Interaction of 8-Anilino-1-naphthalenesulfonate with Creatine Kinase: EVIDENCE FOR COOPERATIVITY OF NUCLEOTIDE BINDING

Abstract Creatine kinase has one tight binding site (Kd = 310 µm) per subunit for 8-anilino-1-naphthalenesulfonate with an enhancement of fluorescence of ≃100 and a blue shift of the emission maximum of 30 nm. Nucleotide substrates non-competitively interact with the dye-binding site causing a 50% decrease in the fluorescence of the bound dye and a concomitant red shift of the emission maximum of 7 nm, while guanidino substrates alone do not affect the fluorescence of the bound dye. The fluorescence of the bound dye is used to determine the dissociation constants of nucleotide substrates from the enzyme in the presence or absence of other substrates or effectors, i.e. divalent metal ion or creatine. In the absence of a small planar anion, the presence of creatine or magnesium (or both) have no significant effect on the Kd of ADP from the enzyme, while in the presence of the nitrate anion creatine induces a drastic tightening and a negative cooperativity in the binding of ADP. With MgADP, the tightening of binding and the cooperativity are further accentuated. Modified inactive forms of the enzyme do not show the tightening nor the cooperativity of ADP or MgADP binding. These results are consistent with the hypothesis that the small planar anion (e.g. nitrate) is acting as an analog of a planar phosphoryl group, thus mimicking the transition state of the enzyme-substrate complex, and further suggest that two of the distinguishing features of this transition-state complex are a substantial interaction between the two protomeric subunits and the formation of a ligand from the protein to the divalent metal ion.

Biosynthesis of dipalmitoyllecithin by the rabbit lung.

The esterification of palmitate by lung homogenates is increased by palmitoyllysolecithin in the presence of CoA and ATP. [32P]lysolecithin can also be converted to [32P]lecithin in the absence of other substrates, suggesting transacylation between two molecules of palmitoyllysolecithin. Some palmitate is esterified in the absence of added lysolecithin, ATP, and CoA. Free palmitate appears to exchange with other esterified fatty acids, especially oleate incorporated into the lecithin of lung tissue slices. The lung contains sufficient 1-palmitoyl-2-oleyllecithin for this exchange to be significant. In homogenates, Ca2+ is required for the exchange and the incorporation of palmitate. The in vivo turnover of the oleate in the lecithin of lung tissues and bronchiolar washings is more rapid than that of the palmitate. This is consistent with a mechanism of dipalmitoyllecithin synthesis involving exchange of esterified oleate for palmitate in lecithin. It is concluded that in addition to de novo synthesis of lecithin in the lung, incorporation of activated palmitate into lysolecithin and exchange of palmitate with oleate esterified in endogenous lecithin also occur. Phospholipase activity is probably involved in the latter two pathways.

Verification of misacylated tRNAphe is apparently carried out only by phenylalanyl-tRNA synthetase.

PREVIOUS results indicate that phenylalanyl-tRNA (Phe-tRNA) synthetase catalyses a reaction, termed verification1, in which tRNAs cognate to the synthetase, but carrying the wrong amino acid, are rapidly deacylated by hydrolysis in the absence of other substrates. Four different enzymes in two species have now been observed to be capable of apparently similar reactions1–3. The process appears to have only limited specificity for the amino acid3,4. Verification is thus a novel and potentially general means of correcting a variety of misacylations before they can reach the ribosome.

Studies on the interaction of tyrosyl-tRNA synthetase from Escherichia coli K12 with tyrosine and with tyrosyl-AMP

Tyrosyl-tRNA synthetase ( l-tyrosine:tRNA ligase (AMP, EC 6.1.1.1) purified from Escherichia coli binds 1 mole of tyrosyl-AMP per mole of enzyme based on quantitative binding of the complex to nitrocellulose filters. The formation of tyrosyl-AMP occurs in the absence of Mg 2+. No binding of either tyrosine or of ATP alone to the enzyme was detected on filters. Tyrosine binding to the enzyme in the absence of other substrates was measured by equilibrium dialysis: one site with an affinity constant of 2.1 · 10 5 l/mole was found. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate demonstrated that tyrosyl-tRNA synthetase is composed of two subunits of mol. wt 48 000. Steady-state kinetics revealed a biphasic curve as a function of tyrosine concentration suggesting the possibility of a second weak binding site for tyrosine.

Inhibition of homogeneous angiotensin-converting enzyme of rabbit lung by synthetic venom peptides of Bothrops jararaca

Seven synthetic peptides, identical to those found in the venom of Bothrops jararaca, inhibited the peptidolytic activity of homogeneous (275-fold purified) angiotensin-converting enzyme from rabbit lung on [Ile 5]-angiotensin I (angiotensin I), and on hippuryl- l-histidyl- l-leucine (Hip-His-Leu), with I 50 values ranging from 0.06 to 76 μM. The nonapeptide, SQ 20 88 i (Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), and decapeptide, SQ 20 858 (Pyr-Asn-Trp-Pro-His-Pro-Gln-Ile-Pro-Pro), were competitive with both substrates (angiotensin I and Hip-His-Leu), but the pentapeptide, SQ 20 475 ∗∗ ∗∗ This pentapeptide was first synthesized by Stewart et al. 8 and named BPP 5a. (Pyr-Lys-Trp-Ala-Pro) was a “mixed” competitive and noncompetitive inhibitor. The K i values (enzyme-inhibitor dissociation constants) calculated for SQ 20 881, SQ 20 858 and SQ 20 475 did not depend significantly on the substrate employed, but were altered by differences in pH. At pH 7.5, with angiotensin I as substrate, the K i values were: SQ 20 475, 0.09 μM; SQ 20 881, 0.8 μM; and SQ 20 858, 4.3 μM. At pH 8.3, with Hip-His-Leu as substrate, the corresponding values were lower: 0.06, 0.10 and 0.74 μM. The most potent of the inhibitors in vitro, SQ 20 475, could be cleaved and inactivated when incubated with homogeneous angiotensin-converting enzyme in the absence of other substrates and of the activator, Cl −; the other six peptides could not serve as substrates for the enzyme. The nonapeptide, SQ 20 881, appears to be the best of the synthetic venom peptides for use in studying the role of the renin (EC 3.4.4.15)-angiotensin system in physiological and pathological processes.

Nicotinate Phosphoribosyltransferase of Yeast: PURIFICATION AND PROPERTIES

Abstract Nicotinate phosphoribosyltransferase from bakers' yeast has been purified about 1,000-fold; the purified material showed only one band on cellulose acetate electrophoresis (pH 5.0 and 8.6). In addition to 5-phosphoribosyl 1-pyrophosphate, adenosine triphosphate (ATP) or an analogous nucleotide is required for enzyme activity and the stoichiometric hydrolysis of ATP to adenosine diphosphate (ADP) and orthophosphate with formation of products, nicotinic acid mononucleotide and pyrophosphate, was verified with this purified preparation. The nucleotide specificity, however, is broader than that of the liver form of this enzyme. The molecular weight of the yeast enzyme was estimated from Sephadex chromatography to be 43,000 (about one-half that of the liver enzyme) whether in the presence or absence of ATP. Phosphate ions greatly stimulate the enzyme activity, giving optimum activity in the range of 10 to 100 mm phosphate in the presence of 5 mm Mg++. At 1 and 5 mm Mg++, optimum enzyme activities are observed with equimolar concentration of ATP. The enzyme catalyzes isotope exchange between ATP and ADP at rates approximately 8 times its molecular activity with all substrates, although no net ATP hydrolysis occurs in the absence of other substrates. ATP, guanosine triphosphate, and the substrates, nicotinic acid and 5-phosphoribosyl 1-pyrophosphate, protect the enzyme from heat inactivation.

Transient-kinetic studies of pig muscle lactate dehydrogenase.

1. The very fast pre-steady-state formation of NADH catalysed by pig M(4) lactate dehydrogenase was equivalent to the enzyme-site concentration at pH values greater than 8.0 and to one-half the site concentration at pH6.8. 2. The rate of dissociation of NADH from the enzyme at pH8.0 (450s(-1)) in the absence of other substrates is faster than the steady-state oxidation of lactate (80s(-1)). The latter process is therefore controlled by a step before NADH dissociation but subsequent to the hydride transfer. 3. The oxidation of enzyme-NADH by excess of pyruvate was studied as a first-order process at pH9.0. There was no effect of NADD on this reaction and it was concluded that the ternary complex undergoes a rate-limiting change before the hydride-transfer step. 4. Some conclusions about the reactions catalysed by the M(4) isoenzyme were drawn from a comparison of these results with those obtained with the H(4) isoenzyme and liver alcohol dehydrogenase.

Role of Pyruvate and S -Adenosylmethionine in Activating the Pyruvate Formate-Lyase of Escherichia coli

The pyruvate formate-lyase activity of extracts of Escherichia coli is stimulated and the dilution effect is abolished by the addition of pyruvate to the extract. The activity can be purified fourfold from pyruvate-supplemented extracts by isoelectric precipitation under anaerobic conditions. The activity of extracts not supplemented with pyruvate has been separated into two fractions by treatment with protamine sulfate-fraction PS, the soluble portion, and fraction N, an extract of the precipitate formed upon the addition of protamine sulfate. After treatment of these fractions with charcoal, pyruvate formate-lyase activity is stimulated by the addition of S-adenosylmethionine. When sodium pyruvate is added to the crude extract before the fractionation, fraction PS has full enzymatic activity and is not stimulated by fraction N or by S-adenosylmethionine. Incubation of the inactive fractions with pyruvate and S-adenosylmethionine in the absence of other substrates similarly results in a highly active preparation, not subject to the "dilution effect" obtained when the fractions are added separately to the assay. These observations suggest that the component in the protamine supernatant fraction is activated by the other fraction and that S-adenosylmethionine and pyruvate are required for the activation reaction. The activating factor present in the protamine precipitate fraction may be further purified by heating for 10 min at 100 C under H(2) atmosphere. The yield of this factor from crude extract is not affected by activation of the pyruvate formate-lyase of the extract, indicating that the factor acts catalytically. The requirement for pyruvate is only partially satisfied by alpha-ketobutyrate and not at all by other alpha-keto acids, acetyl phosphate, or adenosine triphosphate. The rate of activation is maximal at 0.01 m sodium pyruvate and 3 x 10(-4)mS-adenosylmethionine; it is linearly dependent on the amount of activating factor added. The rate of activation is the same when the activation reaction is initiated by addition of any of the four required components, indicating that no slow step of activation can be carried out by any three of the components. A similar pyruvate formate-lyase system was found in extracts of the methionine/B(12) autotroph 113-3, grown with methionine supplement, indicating that vitamin B(12) derivatives do not participate in the system.

Toxic action of cysteine on ascites tumor cells in vitro

Cysteine, especially in the absence of other substrates, has a progressive toxic action on Ehrlich ascites tumor cells. Measurements of cell survival in roller tube suspensions by the dye exclusion method and by rate of substratestimulated respiration indicated permanent cell damage at cysteine concentrations as low as 10 to 20 μg/ml. Substrates such as glutamine, glucose, and bovine albumin when added with the cysteine partially protected the cells against poisoning. Lowered oxygen tension and FeSO 4 at low concentrations had a similar effect. The toxic action of cysteine and some other SH compounds was related to marked or total inhibition of endogenous respiration after an incubation period of one hour, but cysteine-poisoned cells continued to consume O 2 in the presence of glutamine and lactate. Washing of cells after exposure to cysteine reduced their endogenous respiration to a much greater extent than did such treatment of normal cells. The participation of a metal ion in the effect of cysteine is indicated by the protective action of versene (EDTA). Oxidative formation of intracellular disulfide groups and loss of ability to oxidize fats are discussed as possible factors in the inhibitory action of cysteine.

Measurement of oxidative phosphorylation in the cytochrome c oxidase reaction in heart sarcosomes

1. 1. Sarcosomes isolated from cat or rat heart in isotonic sucrose containing ethylenediamine tetraacetate show little phosphorylation associated with the oxidation of the ascorbate in the presence of cytochrome c, in a medium at pH 7.4 (average P:O ratio = 0.12). 2. 2. If the sarcosomes are first treated in a medium of about 0.005 osmolarity, the rate of oxidation and especially the P:O ratios are increased. Most of the preparations gave P:O ratios between 0.3 and 0.4 at pH 7.4. 3. 3. The rate of phosphorylation was constant during an experimental period of 20 min at 25°. 4. 4. The P:O ratio was critically dependent upon the composition of the reaction mixture, in a manner which was rather non-reproducible from preparation to preparation. 5. 5. Oxidative phosphorylation coupled to ascorbate oxidation was not appreciably affected by concentrations of antimycin which maximally inhibited phosphorylation with α-ketoglutarate as substrate. 6. 6. P:O ratios between 0.15 and 0.34 (5 experiments) were obtained with hypotonically pretreated heart sarcosomes oxidizing high concentrations of ferrocytochrome c, in the absence of other substrate. The P:O ratio was not affected by the degree of oxidation of the cytochrome c. 7. 7. A possible explanation of the effect of the hypotonic pretreatment of sarcosomes on the rate of oxidation and reduction of added cytochrome c, and on oxidative phosphorylation, is suggested.