Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells.

Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with Km and Vmax values similar to those for ATP (0.9-1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gamma S and AMPPNP, and weakly by AMPCPP and ADP-beta S, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-32P]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed to be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.     

Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase

It was demonstrated that the dialdehyde derivative of ATP is a good substrate for Ca-ATPase of heavy meromyosin (Km = (1.2-1.4) X 10(-4) M; V = VATP). At the same time, this compound can induce irreversible inhibition of the enzyme. Since oxo-ATP is rapidly hydrolyzed by myosin to form oxo-ADP, this inhibition is the result of the enzyme interaction with oxo-ADP. It was found that the kinetics of heavy meromyosin inhibition by oxo-ADP are typical of affinity modification; in this case ATP fully protects heavy meromyosin from the activity loss. Similar results on the irreversible inhibition of the ATPase activity under the action of oxo-ADP were obtained in the presence of myosin, heavy meromyosin, subfragment I and natural actomyosin and in the absence of bivalent cations, thus suggesting the modification of the active center of myosin ATPase.     

Reversible inhibition of (Na+, K+) ATPase by Mg2+, adenosine triphosphate, and K+.

Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.     

Calcium release from vesicles of heavy sarcoplasmic reticulum of rabbit skeletal muscles

The release of Ca2+ from vesicles of heavy sarcoplasmic reticulum after its accumulation due to hydrolysis of ATP, GTP, CTP, UTP or ITP has been studied using Antipyrylazo III, a metal-chromic Ca-indicator. All the studied substrates of the Ca-pump provide Ca2+ accumulation inside the heavy sarcoplasmic reticulum vesicles, the spontaneous Ca2+ outflux rate being different for different nucleoside triphosphates. It is only ATP that provides Ca-(caffeine)-induced Ca2+ release, however AMP, ADP, beta, gamma-methylene-ATP induce Ca2+ ejection in the presence of nonadenylic nucleotides. The ruthenium red (10(-7M) inhibits the induced ejection of Ca2+ from vesicles of the heavy sarcoplasmic reticulum, but does not prevent the spontaneous release of Ca2+ in the same concentrations. A conclusion is drawn that besides Ca-channels sensitive to Ca2+ and caffeine in the presence of ATP (or to AMP, ADP, beta, gamma-methylene-ATP in the presence of nonadenylic nucleotides) and possessing high sensitivity to the ruthenium red there is another pathway for Ca2+ in the heavy reticulum membranes along which its spontaneous release occurs after the substrate exhaustion. It is supposed that this release is provided by the presence of the Ca-ATPase protein.     

Characterisation of Ca2+ or Mg(2+)-dependent nucleoside triphosphatase from rat mesenteric small arteries.

When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent Km in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF4-, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N'-dicyclohexylcarbodiimide caused significant (greater than 30%) inhibition. In the presence of micromolecular concentrations of Ca2+ and Mg2+, small (less than 20 mM) concentrations of Na+, K+, Rb+, Cs+ and choline+, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.     

Ca2+- or Mg2+-Dependent Enzymic ATP Hydrolysis Associated with the Microsomal Fraction of Frog Sciatic Nerves

The microsomal fraction of frog sciatic nerves was found to contain Ca2+- or Mg2+-dependent hydrolytic activity toward different nucleoside di- and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p-nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18--24 degrees C in the presence of 1 mM-Ca2+ or 1 mM-Mg2+. Ca2+- and Mg2+-ATP hydrolysis displayed pH maxima around 8.0--8.5 and 7.4--8.0, respectively. Vmax values for Ca2+- and Mg2+-ATP hydrolysis similar: approx. 12 mumol Pi per h per mg protein with a Km value of approx. 0.05 mM. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10(-6)-10(-5) M) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.     

Ectonucleotidase Activities Associated with Cholinergic Synaptosomes Isolated from Torpedo Electric Organ

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.     

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 1. Three-strand exchange reaction.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.     

Characterization of the Ca2+- and Mg2+-Dependent ATPases in Electrophorus Electroplax Microsomes

Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.     

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.     

High affinity, calcium-stimulated adenosine triphosphatase activity in the particulate fraction of rat pancreatic acini

Adenosine triphosphatase activity which is Mg2+-dependent and stimulated by submicromolar concentrations of Ca2+ (as Ca . ATP) was identified in the total particulate fraction of rat pancreatic acini. Half-maximal activity (V0.5) is obtained at 100.1 +/- 6 nM Ca . ATP with a Hill coefficient of 2.2 +/- 0.1 (mean +/- S.E.; n = 4). Maximal activity was 75 +/- 19 pmol of Pi released from ATP minute-1 microgram of membrane protein-1 (mean +/- S.E.; n = 7). High affinity Ca2+-ATPase activity was unaffected by ouabain, Na+, K+, La3+, and added calmodulin. Activity was slightly reduced by ruthenium red (0.1 mM) and by oligomycin (80 micrograms/ml) but was reduced almost 50% by the phenothiazine derivative fluphenazine in a dose-related and Ca2+-dependent manner. Hydrolysis of p-nitrophenyl phosphate was 9% of the rate of ATP hydrolysis and was independent of Ca2+ concentration. However, ADP, GTP, UTP, and ITP were hydrolyzed at 76-93% the rate that ATP was hydrolyzed with V0.5 values and Hill coefficients similar to those of Ca . ATP. We conclude that rat pancreatic acini contain an enzyme for active Ca2+ translocation: ATPase activity that is Mg2+-dependent and stimulated by submicromolar concentrations of Ca . ATP. Substrate hydrolysis appears to involve positive cooperative interactions of multiple ligand-binding sites and may be regulated in part by calmodulin.     

Characterization of Calcium-Activated and Magnesium-Activated ATPases of Brain Nerve Endings

The properties of Ca2+-activated and Mg2+-activated ATPases of nerve endings from mouse brain were investigated. Ca2+ and Mg2+ each can activate ATP hydrolysis in synaptosomes and its subfractions. Both Ca2+-ATPase and Mg2+-ATPase exhibit high and low affinity for their respective cations. At millimolar concentrations of Ca2+ or Mg2+, several nucleoside triphosphates could serve as substrate for the two enzymes and their specific activities were about three to four times higher in synaptic vesicles than in synaptosomal plasma membranes (SPM). Both in SPM and in synaptic vesicles the relative activity in the presence of Ca2+ was in the order of CTP greater than UTP greater than GTP = ATP, but with Mg2+ the activity was higher with ATP than with the other three triphosphates. Mg2+-ATPase was more active than Ca2+-ATPase in SPM, but in synaptic vesicles the two enzymes exhibited similar activity. Kinetic studies revealed that Mg2+-ATPase was inhibited by excess ATP and not by excess Mg2+. The simultaneous presence of Na+ + K+ stimulated Mg2+-ATPase and inhibited Ca2+-ATPase activity in intact synaptosomes and SPM. The stimulation of Mg2+-ATPase by Na+ + K+ was further increased by increasing Mg2+ concentration and was inhibited by Ca2+ and by ouabain. When Ca2+ and Mg2+ are present together in SPM or synaptic vesicles, the total Pi liberated by the two cations may either increase or decrease, depending on their relative concentrations. Kinetic analyses indicate that Ca2+ and Mg2+ bind independently to the enzyme alone or together at different sites. The results suggest that Ca2+-ATPase and Mg2+-ATPase in SPM or synaptic vesicles may be separate and distinct systems.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

Several peculiarities of the purine-base fixation of the substrate in the active sites of myosin Ca2+-ATPase

A comparative study of kinetic parameters (Km and V) of hydrolysis by heavy meromyosin of several synthetic ATP analogs with substituents at positions N(1) and N(C6) of the purine ring was carried out. Analysis of changes in the Km values suggests that the purine base of ATP is fixed in the active center due to the formation of a hydrogen bond between N1 and the proton donor group of the protein as well as between the 6-NH2-amino group of the nucleotide and the proton acceptor group of the protein. It was shown that the rate of catalytic conversion of the substrate is determined by the mode of binding of its purine ring. Depending on the properties of the substituent radical, the latter either prevents the binding by causing little or no increase in the rate of hydrolysis or causes the displacement of the whole substrate molecule in the active center, which leads to the deceleration of hydrolysis.     

Studies of the kinetics of the isolated mitochondrial ATPase using dinitrophenol as a probe

1. Dinitrophenol and maleate anions increase VATP on the 'washed', isolated, mitochondrial ATPase. Hydrolyses of iso-GTP and 2'-deoxy ATP are also stimulated, while hydrolyses of other nucleoside triphosphates (ITP, GTP etc.) are not. 2. Preincubation with ATP, iso-GTP or 2'-deoxy ATP results in a metastable enzyme form with a raised V and a reduced Km. Dinitrophenol stimulates both ATP and ITP hydrolyses by this form. 3. The Arrhenius plot of ATP (but not ITP) hydrolysis by the isolated ATPase shows a break at about 18 degrees C, apparently because the rate limiting step of hydrolysis changes as the temperature rises. 4. Adenylyl beta, gamma-imidodiphosphate (AdoPP[NH]P) inhibits ITP hydrolysis in a pseudofirst order reaction. Its binding is competitive with ITP. If the enzyme is preincubated with ATP, the rate of AdoPP[NH]P binding increases. It is concluded that AdoPP[NH]P inhibits by binding to the hydrolytic site of the enzyme. 5. We conclude that ATP hydrolysis is limited by diphosphate release and ITP hydrolysis by bond splitting. Energy release during ATP hydrolysis is maximal at the ATP binding step, and during ITP hydrolysis at bond splitting.     

Calcium-Activated ATPases in Presynaptic Nerve Endings

We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.     

Deoxythymidine kinase induced in the HELA TK- cells by herpes simplex virus type I and type II. Substrate specificity and kinetic behavior.

Deoxythymidine kinases (EC 2.7.1.--) induced in HeLa TK- cells by Herpes simplex Type I and Type II viruses both had a requirement for divalent cations. The enzymes had the highest activities in the presence of Mg2+, followed by Mn2+, Ca2+, Fe2+, and in that order, whereas they were inactive in the presence of Zn2+ and Cu2+. The amount of Mg2+ required for optimal activity was dependent on the amount of ATP present, so that optimal activities were found when the concentration of Mg2+ was equal to that of ATP; an excess of Mg2+ inhibited the reaction. The activities of various nucleoside triphosphates as phosphate donors for Herpes simplex virus Type I deoxythymidine kinase were in the order: ATP = dATP = ara ATP greater than CTP greater than dCTP greater than UTP greater than dUTP greater than GTP greater than dGTP. Those for Herpes simplex virus Type II deoxythymidine kinase were in the order: CTP greater than dCTP = ara CTP greater than dATP greater than ATP greater than UTP greater than GTP greater than dUTP = dGTP. For both deoxythymidine kinases induced by Herpes simplex virus, the nucleoside triphosphates tested exerted cooperative effects. The Km values of ATP and CTP for the Herpes simplex virus Type I enzyme were 30 and 70 muM respectively; whereas those for the Herpes simplex virus Typr II enzyme were 140 and 450 muM. Studies on binding of various thymidine analogs with free 5'-OH to these deoxythymidine kinases indicated that 5-substituted ethyl-, vinyl-, allyl-, propyl-, iodo- and bromo-dUrd as well as iodo5 dCyd and bromo5 dCyd had good affinity to both enzymes. In contrast, vinyl5 Urd, iodo5 Urd and arabinosylthymidine had good affinity only to the Herpes simplex virus Type I enzyme but not to the Herpes simplex virus Type II deoxythymidine kinase. All of these thymidine analogs were competitive inhibitors, with KI values in the range of 0.25 to 1.5 muM. Herpes simplex virus Type I deoxythymidine kinase was less sensitive to either dTTP or iodo dUTP inhibition than Herpes simplex virus Type II. Both dThd and dCyd could serve as substrates and competed with each other for Herpes simplex viruses Type I and Type II induced kinases, but they differed in their Km values for these enzymes. The Km values of dThd and dCyd were 0.59 muM and 25 muM for Herpes simplex virus Type I deoxythymidine kinase; while they were 0.36 muM and 88 muM respectively for the Herpes simplex virus Type II enzyme.