Three deoxyribonucleic acid-dependent adenosine triphosphatases from Bacillus subtilis.

We have isolated from Bacillus subtilis three deoxyribonucleic acid (DNA)-dependent adenosine triphosphatases (ATPases) (gamma-phosphohydrolases). The enzymes were extensively purified, and their physicochemical and functional properties were determined. The three enzymes (ATPases I, II, and III) were shown to be different by several criteria. ATPases II and III showed an absolute requirement for single-stranded DNA as a cofactor, whereas ATPase I had some residual activity also with double-stranded DNA. They required Mg2+ and had a pH optimum of 6.5 to 7. Only adenosine 5'-triphosphate and deoxyadenosine 5'-triphosphate were hydrolyzed. The molecular weights of ATPases I, II, and III were 108,000, 115,000, and 148,000, respectively. Km values for adenosine 5'-triphosphate and DNA were also evaluated and shown to be different for each enzyme. All three enzymes formed physical complexes with single-stranded DNA. We present evidence that ATPases I and II might migrate along DNA during adenosine 5'-triphosphate hydrolysis. On the other hand, this effect was not observed with ATPase III, which exhibited the highest affinity for single-stranded DNA.     

Diglyceride kinase in human platelets

Human platelets contain diglyceride kinase, an enzyme that catalyzes the phosphorylation of diacylglycerol by adenosine 5'-triphosphate to yield phosphatidic acid. The majority of the platelet enzyme is particulate-bound, and membrane fractions of platelet homogenates have a higher specific activity than granule fractions. Both deoxycholate and magnesium are necessary for optimal enzyme activity. The K(m) of the enzyme for adenosine 5'-triphosphate is 1.3 mm, and the apparent K(m) for diacylglycerol is 0.4 mm. The pH optimum is 6.6-6.8 in imidazole-HCl or maleate-NaOH buffer. The enzyme activity of platelets from normal subjects was similar to the activity from patients with renal and hepatic failure.     

Purification, characterization, and regulation of a nicotinamide adenine dinucleotide-dependent lactate dehydrogenase from Actinomyces viscosus.

A nicotinamide adenine dinucleotide-specific L-(+)-lactate dehydrogenase (LDH) (EC 1.11.27) from Actinomyces viscosus T-6-1600 was purified approximately 110-fold by a combination of diethylaminoethyl-cellulose and 0.5 M Agarose A column chromatography. The ldh was stable at 26 C, but was quite labile at temperatures below 5 C. The enzyme had a molecular weight of 100,000 +/- 10,000 as determined by 0.5 M Agarose molecular exclusion chromatography and showed optimum activity between pH 5.5 and 6.2. The A. viscosus LDH exhibited homotropic interactions with its substrate, pyruvate, and its coenzyme, reduced nicotinamide adenine dinucleotide, indicating multiple binding sites on the enzyme for these ligands with some degree of cooperative interaction between them. The enzyme was under negative control by adenosine 5'-triphosphate, and its kinetic response to the negative effector was sigmoidal in nature. Inorganic phosphate reversed the inhibition exerted on the A. viscosus LDH by adenosine. The 5'-triphosphate thermal stability at 65 C of the LDH from A. viscosus was increased in the presence of its negative effector, adenosine 5'-triphosphate, but was markedly decreased in the presence of its coenzyme, reduced nicotinamide adenine dinucleotide. The glycolytic intermediate, fructose-1,6-diphosphate, had no effect on the catalytic activity of the A. viscosus LDH at saturating pyruvate concentrations. However, fructose-1,6-diphosphate was a potent positive effector at low substrate concentrations. Thus the A. viscosus LDH is under positive control by fructose-1,6-diphosphate and inorganic phosphate, but under negative control by adenosine 5'-triphosphate.     

Studies on alkaline phosphatase: Inhibition by phosphate derivatives and the substrate specificity

1. The kinetics of inhibition of calf-intestinal alkaline phosphatase by inorganic phosphate, fluorophosphate, inorganic pyrophosphate, beta-glycerophosphate and adenosine 5'-triphosphate in the range pH8-10 were investigated. The reference substrate was 4-methylumbelliferyl phosphate. 2. The inhibitions were ;mixed' in that both K(m) and V were affected, but the competitive element was by far the stronger. 3. In each case the pH profile for the competitive K(i) was similar to the pH profile for K(m). Since the K(m) and K(i) values both change 100-fold over the pH range 8-10, it is concluded that the inhibitors compete with the substrate for the same active site. 4. It was also found that the enzyme preparation hydrolysed fluorophosphate, pyrophosphate and adenosine 5'-triphosphate as readily as it hydrolysed 4-methylumbelliferyl phosphate and beta-glycerophosphate. Each pH-activity curve, however, had a different shape, but with the exception of pyrophosphate the activity approached the same maximum value at high pH. 5. Attempts to separate the phosphomonoesterase and pyrophosphatase activities by column chromatography were not successful, and the results of other experiments listed suggest that the two activities are a property of the same enzyme. 6. The effect of Mg(2+) ions is briefly mentioned: the phosphomonoesterase activity is enhanced whereas the pyrophosphatase and adenosine triphosphatase activities are strongly inhibited in the presence of excess of Mg(2+) ions.     

Obligatory coupling between proton entry and the synthesis of adenosine 5''-triphosphate in Streptococcus lactis.

Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate.     

Coupling of Energy to Folate Transport in Lactobacillus casei

Lactobacillus casei cells can accumulate folate to an intracellular concentration in excess of 500 muM and to concentration gradients (relative to the extracellular compartment) of several thousand-fold. Maximum rates of folate transport are achieved rapidly (t(1/2) < 1 min) after the addition of glucose to energy-depleted cells and occur at intracellular adenosine 5'-triphosphate concentrations above 625 muM. The rate of folate transport and the adenosine 5'-triphosphate content of cells are both extremely sensitive to arsenate and decrease in parallel with increasing concentrations of the inhibitor, indicating a requirement for phosphate-bond energy in the transport process. The energy source is not a membrane potential or a pH gradient generated via the membrane-bound adenosine triphosphatase, since dicyclohexylcarbodiimide (an adenosine triphosphatase inhibitor) and carbonyl cyanide m-chlorophenylhydrazone (a proton conductor) have little effect on the uptake process. The K(+)-ionophore, valinomycin, is an inhibitor of folate transport, but does not act via a mechanism involving dissipation of the membrane potential. This can be deduced from the facts that the inhibition by valinomycin is relatively insensitive to pH, is considerably greater in Na(+)- than in K(+)-containing buffers, and is not enhanced by the addition of proton conductors. Folate efflux is not affected by valinomycin, glucose, or various metabolic inhibitors, although a rapid release of the accumulated vitamin can be achieved by the addition of unlabeled folate together with an energy source (glucose). These results suggest that the active transport of folate into L. casei is energized by adenosine 5'-triphosphate or an equivalent energy-rich compound, and that coupling occurs not via the membrane-bound adenosine triphosphatase but by direct interaction of the energy source with a component of the transport system.     

Standard thermodynamic formation properties for the adenosine 5'-triphosphate series.

The criterion for chemical equilibrium at specified temperature, pressure, pH, concentration of free magnesium ion, and ionic strength is the transformed Gibbs energy, which can be calculated from the Gibbs energy. The apparent equilibrium constant (written in terms of the total concentrations of reactants like adenosine 5'-triphosphate, rather than in terms of species) yields the standard transformed Gibbs energy of reaction, and the effect of temperature on the apparent equilibrium constant at specified pressure, pH, concentration of free magnesium ion, and ionic strength yields the standard transformed enthalpy of reaction. From the apparent equilibrium constants and standard transformed enthalpies of reaction that have been measured in the adenosine 5'-triphosphate series and the dissociation constants of the weak acids and magnesium complexes involved, it is possible to calculate standard Gibbs energies of formation and standard enthalpies of formation of the species involved at zero ionic strength. This requires the convention that the standard Gibbs energy of formation and standard enthalpy of formation for adenosine in dilute aqueous solutions be set equal to zero. On the basis of this convention, standard transformed Gibbs energies of formation and standard transformed enthalpies of formation of adenosine 5'-trisphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, and adenosine at 298.15 K, 1 bar, pH = 7, a concentration of free magnesium ions of 10(-3) M, and an ionic strength of 0.25 M have been calculated.     

Purification and characterization of cytidine 5''-triphosphate:cytidine 5''-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase

Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5. Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M. respectively, at pH 9.5.     

Is ATP a substrate for 15-lipoxygenase?

Lipoxygenases catalyze peroxidation of polyunsaturated fatty acids containing the 1-cis, 4-cis pentadiene structure. Linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids are the predominant substrates for this class of enzymes. Effects of 15-lipoxygenase on the hydrolysis of adenosine 5'-triphosphate were investigated in vitro using soybean lipoxygenase and adenosine 5'-[gamma-32P]triphosphate. The amount of inorganic phosphate released from adenosine 5'-triphosphate was dependent upon enzyme as well as substrate concentrations, pH, and the duration of incubation. The ATPase activity with a Vmax value of 3.3 mumol.mg protein-1.h-1 and a Km value of 5.9 mM was noted in the presence of different concentrations of ATP at pH = 7.4. Phenidone, a lipoxygenase inhibitor, had no effect on this reaction. These findings suggest that soybean lipoxygenase catalyzes the release of inorganic phosphate from ATP primarily via hydrolysis.     

Accumulation of arsenate, phosphate, and aspartate by Sreptococcus faecalis.

Uptake of arsenate and phosphate by Streptococcus faecalis 9790 is strictly dependent on concurrent energy metabolism and essentially unidirectional. targinine supports uptake only in presence of glycerol or related substances; glycerol is not directly involved in transport but depletes the cellular orthophosphate pool and thus relieves feedback inhibition of transport. Uptake of phosphate and arsenate is stimulated by K+ and by other permeant cations. The results suggest that electroneutrality is preserved by compensatory movement of either H+ or OH minus. Ionophores and N,N'-dicyclohexylcarbodiimide, which prevent establishment of a proton motive force, block the accumulation of thiomethylgalactoside and of threonine but not that of arsenate or phosphate. We conclude that arsenate accumulation requires adenosine 5'-triphosphate but is not driven by the proton-motive force. However, conditions and reagents that lower the cytoplasmic pH do inhibit accumulation of arsenate and phosphate, suggesting that uptake depends on the capacity of the cells to maintain a neutral or alkaline cytoplasm. We therefore propose that phosphate accumulation is an electroneutral exchange for OH driven by adenosine 5'-triphosphate or by a metabolite thereof. Accumulation of aspartate and glutamate also requires adenosine 5'-triphosphate but not the proton-motive force and may involve a similar mechanism.     

Chemiosmotic coupling in Methanobacterium thermoautotrophicum: hydrogen-dependent adenosine 5''-triphosphate synthesis by subcellular particles.

Hydrogenase and the adenosine 5'-triphosphate (ATP) synthetase complex, two enzymes essential in ATP generation in Methanobacterium thermoautotrophicum, were localized in internal membrane systems as shown by cytochemical techniques. Membrane vesicles from this organism possessed hydrogenase and adenosine triphosphatase (ATPase) activity and synthesized ATP driven by hydrogen oxidation or a potassium gradient. ATP synthesis depended on anaerobic conditions and could be inhibited in membrane vesicles by uncouplers, nigericin, or the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The presence of an adenosine 5'-diphosphate-ATP translocase was postulated. With fluorescent dyes, a membrane potential and pH gradient were demonstrated.     

Kinetic studies of recA protein binding to a fluorescent single-stranded polynucleotide

Fluorescence spectroscopy was used to investigate the binding of Escherichia coli recA protein to a single-stranded polynucleotide. Poly(deoxy-1,N6-ethenoadenylic acid) was prepared by reaction of chloroacetaldehyde with poly(deoxyadenylic acid). The fluorescence of poly(deoxy-1,N6-ethenoadenylic acid) was enhanced upon recA protein binding. The kinetics of the binding process were studied as a function of several parameters: ionic concentration (KCl and MgCl2), pH, nature of the nucleoside triphosphate [adenosine 5'-triphosphate or adenosine 5'-O-(gamma-thiotriphosphate)], protein and polynucleotide concentrations, polynucleotide chain length, and order of sequential additions. The observed kinetic curves exhibited a lag phase followed by a slow binding process characteristic of a nucleation-elongation mechanism with an additional slow step governing the rate of the association process. The lag phase reflecting the nucleation step was not observed when the protein was first bound to the polynucleotide before addition of adenosine 5'-triphosphate. Adenosine 5'-triphosphate induced a dissociation of the recA protein, which was immediately followed by binding of the recA-adenosine 5'-triphosphate-Mg2+ ternary complex. The origin of this "mnemonic effect" and of the different kinetic steps is discussed with respect to protein conformational changes and aggregation phenomena.     

Substrate specificity and kinetic properties of a soluble nucleoside triphosphatase from bovine kidneys

Soluble nucleoside triphosphatase differing in its properties from all known proteins with NTPase activity was partially purified from bovine kidneys. The enzyme has pH optimum of 7.5, molecular mass of 60 kDa, as estimated by gel chromatography, and shows an absolute dependence on divalent metal ions. NTPase obeyed Michaelis-Menten kinetics in the range of substrate concentration tested from 45 to 440 microM; the apparent Km for inosine-5'-triphosphate was calculated to be 23.3 microM. The enzyme was found to possess a broad substrate specificity, being capable of hydrolyzing various nucleoside-5'-tri- as well as diphosphates.     

Purification and Properties of Acetate Kinase from Acholeplasma laidlawii

Acetate kinase (EC 2.7.2.1) was purified from Acholeplasma laidlawii cytoplasm by a combination of ammonium sulfate fractionation, gel filtration, diethylaminoethyl-cellulose chromatography, and affinity chromatography on 8-(6-aminohexylamino)-adenosine 5'-triphosphate conjugated to Sepharose 4B. The enzyme was composed of polypeptide chains of about 50,000 molecular weight as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturating conditions, apparent molecular weights between 64,000 and 130,000 were obtained, depending upon mainly the ionic strength of the test solution. The enzyme had a narrow specificity for phosphate acceptor acids, whereas both purine and pyrimidine nucleoside triphosphates were suitable phosphate donors. Na(+) and K(+) inhibited both acetyl phosphate and adenosine 5'-triphosphate synthesis, and the latter was also inhibited by high concentrations of adenosine 5'-diphosphate and acetyl phosphate. This substrate inhibition was partially abolished by 0.5 M NaCl. The enzyme catalyzed the independent adenosine 5'-diphosphate<-->adenosine 5'-triphosphate and acetate<-->acetyl phosphate exchanges. The rate of the latter was enhanced by the addition of cosubstrate Mg(2+)-adenosine 5'-triphosphate. The high affinity for substrates, except for acetate, indicated that under physiological conditions the direction of the enzymic reaction favors adenosine 5'-triphosphate synthesis. Thus, a mechanism for adenosine 5'-triphosphate generation in mycoplasmas is suggested.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization.

Culture medium of exponentially growing Bordetella pertussis (strain 114) contains significant quantities of soluble (100,000 X g for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl-cellulose and Sephadex G-200. The purest material yielded a single band on sodium dodecyl sulfate-disc gel electrophoresis. It is heat labile, has a temperature optimum of 30 degrees C, a pH optimum of pH 7 to 8, and a Km for adenosine 5'-triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate-disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by alpha-keto acids of nonsubstrate nucleoside triphosphates. Thus, but its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to alpha-keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.     

Histidine starvation and adenosine 5'-triphosphate depletion in chemotaxis of Salmonella typhimurium.

Starvation for histidine prevented tumbling in Salmonella typhimurium hisF auxotrophs, including constantly tumbling strains with an additional mutation in cheB or cheZ. However, histidine-starved cheZs hisF strains were not defective in flagellar function or the tumbling mechanism since freshly starved auxotrophs tumbled in response to a variety of repellents. Tumbling in histidine-starved S. typhimurium could be restored in 13 s by addition of adenine or in 4 min by addition of histidine. Chloramphenicol did not prevent restoration of tumbling by these substances. Assays of adenosine 5'-triphosphate were performed based upon previous demonstration of adenine depletion in hisF auxotrophs starved for histidine. The adenosine 5'-triphosphate concentration dropped rapidly during the course of starvation, falling to less than 5% of the initial level as the cells ceased tumbling entirely. The change to smooth motility was prevented by 2-thiazolealanine, which inhibits phosphoribosyltransferase, thereby preventing adenine depletion during histidine starvation. These results suggest that an adenosine 5'-triphosphate deficiency was responsible for the change in tumbling frequency.     

A laser Raman spectroscopic study of the interaction of the methylmercury cation with AMP, ADP and ATP

The interaction of the CH3Hg+ cation with adenosine 5'-monophosphate, adenosine 5'-diphosphate, and adenosine 5'-triphosphate has been studied in aqueous solution at neutral pH by laser Raman spectroscopy. Metal binding is shown to occur preferentially at the N-1 ring position of adenine, with some indication of coordination to the N-7 site and substitution of a proton on the exocyclic NH2 group of the nucleic base. Binding of the cation to phosphate groups also occurs extensively, with both the -PO2-3 and -PO-2 groups. The equilibrium constants for the binding to the phosphate groups and for N-1 coordination are approx. 70 and 600 M-1, respectively.     

Some effects of uncouplers and inhibitors on growth and electron transport in rumen bacteria.

Uncouplers and inhibitors of electron transport affected growth and electron transport of rumen bacteria in various ways. Selenomonas ruminantium was not affected by inhibitor and uncoupler concentrations which affected growth and electron transport of Bacteroides ruminicola, B. succinogenes, and Butyrivibrio fibrisolvens. Inhibitors, when active, led to accumulation of reduced electron carriers before the site of action, but differences were found among organisms in the site of action of these inhibitors. Uncouplers reduced the glucose molar growth yields (Ygluc) of B. ruminicola, B. succinogenes, and B. fibrisolvens compared with those obtained without uncouplers. The extent of Ygluc reduction accompanying inhibitor exposure reflected electron transport chain structure. S. ruminantium appeared to obtain its adenosine 5'-triphosphate from substrate-level processes only. The other organisms studied appeared to obtain adenosine 5'-triphosphate both from substrate-level processes and from electron transport but differed in the amount of adenosine 5'-triphosphate obtained from glucose catabolism and in the proportions of adenosine 5'-triphosphate obtained from substrate-level reactions and electron transport.