Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (AppppA) from adenosine 5'-phosphosulfate and adenosine 5'-triphosphate catalyzed by yeast AppppA phosphorylase

A novel way of enzymatic synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), which does not involve aminoacyl-tRNA synthetases, has been discovered. Yeast AppppA alpha, beta-phosphorylase catalyzes irreversible conversion of adenosine 5'-phosphosulfate (APS) and ATP into AppppA according to the equation APS + ATP----AppppA + sulfate. In this reaction, the enzyme exhibits a broad pH optimum (between 6 and 8) and requires Mn2+, Mg2+, or Ca2+ ions for activity, with Mn2+ being twice as effective as Mg2+ or Ca2+ at optimal concentration (0.5 mM). The Km values computed for APS and ATP are 80 microM and 700 microM, respectively. The rate constant for the AppppA synthesis is 3 s-1 (pH 8.0, 30 degrees C, 0.5 mM MgCl2). Some ATP analogues like ppppA, GTP, adenosine 5'-(alpha, beta-methylenetriphosphate), and adenosine 5'-(beta, gamma-methylenetriphosphate), but not dATP, UTP, or CTP, are also substrates for AppppA phosphorylase and accept adenylate from APS with the formation of AppppA, AppppG, Appp(CH2)pA, and App(CH2)ppA, respectively. Functional versatility of yeast AppppA phosphorylase may provide a link between metabolism of AppppA on one hand and metabolism of APS and phosphate on the other and raises the possibility of participation of AppppA in regulation of metabolism of APS and/or inorganic phosphate in yeast.     

Phosphorus-31 nuclear magnetic resonance studies on intact erythrocytes. Determination of intracellular pH and time course changes in phosphorus metabolites

A method to determine the intracellular pH of intact erythrocytes using phosphorus-31 nuclear magnetic resonance spectroscopy is described. Changes in phosphorus metabolites due to the alkalization of intracellular pH were also examined. The normal erythrocytes gave signals of phosphate groups corresponding to 2,3-bisphosphoglycerate, inorganic phosphate, ATP, and NAD. Among them, the separation between alpha and gamma peaks of ATP was shown to be a good indicator of the intracellular pH free from the perturbation caused by hemoglobin. This method enabled us to determine the intracellular pH of the erythrocytes without any pretreatment. The separation between alpha and gamma peaks of ATP was also dependent on the degree of complexation with Mg2+, and was consistent with approximately 80% of total ATP complexing with Mg2+ in the samples investigated here. The pKa value of ATP in the erythrocytes was estimated to be 6.1 at 23 degrees C, which is lower than the value of 6.5 obtained for the Mg2+-free ATP solution. In the alkalized erythrocytes, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were observed in addition to the metabolites found in the normal erythrocytes. Time course changes in these phosphorus metabolites were followed along with the intracellular pH monitored from ATP peaks.     

Binding of guanine nucleotides and Mg2+ to tubulin with a nucleotide-depleted exchangeable site

Binding of GTP and GDP to tubulin in the presence or absence of Mg2+ was measured following depletion of the exchangeable site--(E-site) nucleotide. The E-site nucleotide was displaced with a large molar excess of the nonhydrolyzable GTP analogue, GMPPCP, followed by the removal of the analogue. Using a micropartition assay, the equilibrium constant measured in 0.1 M 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM MgSO4 at 4 degrees C was 9.1 x 10(6) M-1 for GTP and 4.4 x 10(6) M-1 for GDP. Removal of Mg2+ reduced the binding affinity of GTP by 160-fold while the affinity of GDP remained essentially unchanged. Similar values were obtained if 0.1 M Tris, pH 7.0, was used instead of Pipes. Binding of Mg2+ to tubulin containing GTP, GDP, or no nucleotide at the E-site was also examined by the micropartition method. Tubulin-GTP contained one high affinity Mg2+ site (K alpha = 1.2 x 10(6) M-1) in addition to the one occupied by Mg2+ as tubulin is isolated, while only weak Mg2+ binding to tubulin-GDP and to tubulin with a vacant E-site (K alpha = 10(3) M-1) was observed. It is suggested that Mg2+ binds to the beta and gamma phosphates of GTP, and only to the beta phosphate of GDP, as shown for the H. ras p21 protein.     

The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria

The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.     

Human thymidylate kinase. Purification, characterization, and kinetic behavior of the thymidylate kinase derived from chronic myelocytic leukemia.

Thymidylate kinase derived from the blast cells of human chronic myelocytic leukemia was purified 2186-fold to near homogeneity by means of alcohol precipitation, alumina-Cgamma gel fractionation, calcium phosphate gel fraction, ultrafiltration, and affinity column chromatography. The molecular weight was estimated by glycerol gradient centrifugation to be 50,000. This enzyme had an optimal activity at pH 7.1 and required a divalent cation in order to catalyze the reaction. Mg2+ and Mn2+ were found to be the preferential divalent cations. The activation energy was estimated to be 19.1 kcal/mol at pH 7.2. Initial velocity study suggested that the reaction followed a sequential mechanism. Mg2+ ATP had a Km of 0.25 mM and dTMP had a Km of 40 micrometer. The enzyme was unstable even at 4 degrees. In the presence of ATP or dTMP the enzyme maintained its activity. Purine triphosphate nucleosides were found to be better phosphate donors than the pyrimidine triphosphate nucleosides. ATP and dATP had a lower Km and a higher Vmax than GTP and dGTP. dTMP was the only preferred phosphate receptor among all the monophosphate nucleotides tested dTTP and IdUTP competed with both substrates and inhibited the reaction with a Ki of 0.75 mM and 1.1 mM, respectively.     

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.     

Adenosine kinase from human liver

Adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to homogeneity from human liver. The yield was 55% of the initial activity with a final specific activity of 6.3 mumol/min per mg protein. The molecular weight was estimated as about 40 000 by Sephadex G-100 gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 18% of that of adenosine. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad optimum at pH 7.5--8.5. The Km value for adenosine was 0.15 micrometer, and the activity was strongly inhibited at higher concentrations than 0.5 micrometer. ATP, dATP, GTP and dGTP were proved to be effective phosphate donors. Co2+ was more effective than Mg2+, and Ca2+, Mn2+, Fe2+ and Ni2+ showed about 50% of the activity for Mg2+. Some difference in structure between the adenosine kinase from human liver and that from rabbit or rat tissue, was observed by amino acid analysis and peptide mapping analysis.     

Dicyclohexylcarbodiimide-sensitive ATPase in Halobacterium saccharovorum

Membranes from Halobacterium saccharovorum contained a cryptic ATPase which required Mg2+ or Mn2+ and was activated by Triton X-100. The optimal pH for ATP hydrolysis was 9-10. ATP or GTP were hydrolyzed at the same rate while ITP, CTP, and UTP were hydrolyzed at about half that rate. The products of ATP hydrolysis were ADP and phosphate. The ATPase required high concentrations (3.5 M) of NaCl for maximum activity. ADP was a competitive inhibitor of the activity, with an apparent Ki of 50 microM. Dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis. The inhibition was marginal at the optimum pH of the enzyme. When the ATPase was preincubated with DCCD at varying pH values, but assayed at the optimal pH for activity, DCCD inhibition was observed to increase with increasing acidity of the preincubation medium. DCCD inhibition was also dependent on time of preincubation, and protein and DCCD concentrations. When preincubated at pH 6.0 for 4 h at a protein:DCCD ratio of 40 (w/w), ATPase activity was inhibited 90%.     

Identification of a novel casein kinase activity in HeLa cell nuclei

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.     

Properties of H+-translocating adenosine triphosphatase in vacuolar membranes of SAccharomyces cerevisiae

The properties of Mg2+-ATPase in the vacuole of Saccharomyces cerevisiae were studied, using purified intact vacuoles and right-side-out vacuolar membrane vesicles prepared by the method of Y. Ohsumi and Y. Anraku ((1981) J. Biol. Chem. 256, 2079). The enzyme requires Mg2+ ion but not Ca2+ in. Cu2+ and Zn2+ ions inhibit the activity. The optimal pH is at pH 7.0. The enzyme hydrolyzes ATP, GTP, UTP, and CTP in this order and the Km value for ATP was determined as 0.2 mM. It does not hydrolyze ADP, adenosyl-5'-yl imidodiphosphate, or p-nitrophenyl phosphate. ADP does not inhibit hydrolysis of ATP by the enzyme. The activities of intact vacuoles and of vacuolar membrane vesicles were stimulated 3- and 1.5-fold, respectively, by the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile and the K+/H+ antiporter ionophore nigericin. Sodium azide at a concentration exerting an uncoupler effect also stimulated the activity. The activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to sodium vanadate. The ATP-dependent formation of an electrochemical potential difference of protons, measured by the flow-dialysis method, was determined as 180 mV, with contribution of 1.7 pH units, interior acid, and of a membrane potential of 75 mV. It is concluded that the Mg2+-ATPase of vacuoles is a new marker enzyme for these organelles and is a N,N'-dicyclohexylcarbodiimide-sensitive, H+-translocating ATPase whose catalytic site is exposed to the cytoplasm.     

Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins

Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.     

Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima

Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.     

Purification of RNA 3'-terminal phosphate cyclase from HeLa cells. Covalent modification of the enzyme with different nucleotides

RNA 3'-terminal phosphate cyclase has been purified about 6000-fold to near homogeneity from HeLa cells. The purified protein is a single polypeptide with an Mr of 38,000-40,000 and a Stokes radius of 2.66 nm. The cyclase shows a pH optimum of 8.0-9.0. In the presence of Mg2+ and ATP this enzyme catalyzes the conversion of a 3'-phosphate group into the cyclic 2',3'-phosphodiester at the 3' end of RNA, through formation of a covalent cyclase-AMP intermediate. GTP, CTP and UTP (but not dATP or ADP) can also function as cofactors in the cyclization reaction, although less efficiently (apparent Km values for ATP and GTP are 6 microM and 200 microM, respectively). Consistent with this, the enzyme can be covalently labelled with the four [alpha-32P]NTPs.     

The alpha beta-methylene analogues of ADP and ATP act as substrates for creatine kinase. delta G0 for this reaction and for the hydrolysis of the alpha beta-methylene analogue of ATP

The alpha beta-methylene analogues of ATP and ADP, [alpha beta CH2]ATP and [alpha beta CH2]ADP, are substrates for creatine kinase. However, the rate of the phosphoryl transfer reaction catalysed is about 10(-5)-times lower than that with normal ATP. The affinities of the analogues (especially [alpha beta CH2]ADP) for the enzyme are lower than those of the normal substrates. The equilibrium constant at 25 degrees C, measured using 31P NMR, for the reaction Mg[alpha beta CH2]ATP + creatine in equilibrium Mg[alpha beta CH2]ADP + phosphocreatine + H+ is 2.2 X 10(-12) M compared with a value of 2.5 X 10(-10) M for the same reaction with the normal substrates, corresponding to a difference in delta G0 values of 11.7 kJ X mol-1. It follows that delta G0 for the hydrolysis of the terminal phosphate group of Mg[alpha beta CH2]ATP is less favourable by 11.7 kJ X mol-1 than that for MgATP.     

Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH.

The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.     

A phosphorus-magnetic-resonance study of the interaction of Mg2+ with adenyl-5'-yl imidodiphosphate. Binding sites of Mg2+ ion on the phosphate chain.

The interaction of Mg2+ ions with adenyl-5'-yl imidodiphosphate, AMP-P(NH)P, has been studied at basic and acidic pH values by phosphorus magnetic resonance spectroscopy in aqueous solution. The results suggest that Mg2+ binds simultaneously to one (or both) of the two free oxygen atoms of the beta-phosphate moiety and to the nitrogen atom of the phosphate chain (P alpha-O-P beta-N-P gamma). The interaction arises from 1: 1 complexing of Mg2+ to AMP-P(NH)P. The mode of the Mg2+ binding on the phosphate chain remains the same at both basic and acidic pH values. As in the case of ATP and ADP, the association of Mg2+ reduces the pK by about 1.5 units. On the other hand phosphorus titration curves showed that when the phosphate chain does not possess the regular periodicity (O-P alpha-O-P beta-X-P gamma-O,X not equal to O) as in the case of ATP, protonation of the terminal phosphate group may induce a 31P chemical shift variation less important for this group than for the preceding one.     

Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. The aerobic/anaerobic transition: role of arginine phosphate in enzyme control.

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 mumol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 +/- 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6-phosphate, ATP, and Mg2+) at either pH 7.9 OR PH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8, the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate, NAD(P)H,Ca2+, fructose 1,6-bisphosphate, 3-phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K +, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP(0.2- 1.0mM) and to a lesser extent by high concentrations of Pi (20 mM) and adenosine 3' :5'-monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki (ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8, whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values. The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulator of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia, the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.     

Regulation of the microtubule-lysosome interaction: activation by Mg2+ and inhibition by ATP

We developed a sedimentation assay to characterize and quantify the association of purified lysosomes to reconstituted microtubules (Mithieux, G., Audebet, C. and Rousset. B. (1988) Biochim. Biophys. Acta 969, 121-130). In the present work, we have examined the potential regulatory role of ATP and Mg2+ on the microtubule-lysosome interaction. The formation of microtubule-lysosome complexes takes place in the absence of Mg2+, but is activated by the addition of Mg2+; both the rate of the interaction and the amount of complexes formed are increased. The maximal effect is observed between 1.5 and 3.5 mM free Mg2+. Measured at the plateau of the interaction, the proportion of microtubules bound to lysosomes increases as a function of free Mg2+ concentration; at optimal concentration of free Mg2+, 90% of the microtubules present in the incubation mixture are bound to lysosomes. ATP induces a concentration-dependent inhibition of the formation of microtubule-lysosome complexes. The half-maximal effect is obtained at an ATP concentration of 0.83 +/- 0.11 mM (n = 7). The effect of ATP is not related to ATP hydrolysis, since ATP exerts its inhibitory action in the presence of EDTA. The ATP effect is mimicked by GTP, p[NH]ppA and tripolyphosphate, ADP and pyrophosphate, but not by AMP or phosphate. In the presence of 1 mM ATP, a Mg2+ concentration of 3 mM (corresponding to 2 mM free Mg2+) is required to overcome the inhibition caused by ATP; above 3 mM, Mg2+ exerts its activating effect. Since the modulating effects of ATP and Mg2+ are obtained at concentrations closed to those occurring in intact cells, we conclude that the regulation of the microtubule-lysosome interaction reported in this paper could be of physiological significance.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ATP on the Friend Murine leukemia virus-associated endonuclease activity and the endonuclease activity of the avian myeloblastosis virus RNA-directed DNA polymerase

The Mn2+-dependent endonuclease activity associated with the avian myeloblastosis virus RNA-directed DNA polymerase has been shown to be activated by ATP in the presence of Mg2+. In the presence of Mn2+ the endonucleolytic activity was stimulated about 3-fold by the addition of ATP. The earlier identified Mr = 40,000 Friend murine leukemia virus (F-MuLV)-associated endonuclease which functions in the presence of both Mg2+ and Mn2+ has also been shown to be similarly stimulated by ATP. For both endonuclease activities stimulation was only observed at ATP concentrations above 0.5 mM, and it did not increase upon elevating the ATP concentration above 2.5 mM. ADP and dATP also stimulated both activities, although not to the same extent as ATP. GTP had no apparent effect and AMP seemed to inhibit both activities. The effect ATP analogs had on the F-MuLV associated endonuclease activity could suggest that the endonuclease reaction in the presence of ATP might involve the cleavage of beta-gamma phosphate bonds in ATP. Neither adenyl-5'-yl imidodiphosphate nor (beta, gamma-methylene)adenosine 5'-triphosphate stimulated the activity, whereas significant stimulation was observed in the presence of (alpha, beta-methylene)adenosine 5'-triphosphate. Although no ATPase activity could be detected in the purified F-MuLV endonuclease preparation, the data do not exclude the possibility that ATP may be cleaved in amounts which are equivalent to the number of nicks introduced into DNA by the virus-associated endonuclease. In the presence of ATP and Mg2+ the F-MuLV-associated endonuclease nicked both supercoiled and linear DNA duplexes extensively, although the former was nicked more readily than the latter. Single-stranded DNA functioned poorly as a substrate. The nicks introduced by the enzyme contained a 5'-phosphoryl terminus and a 3'-hydroxyl group.