Mapping of two loci affecting the synthesis and structure of a periplasmic protein involved in arginine and ornithine transport in Escherichia coli K-12.

The map location of two genes, abpR and abpS, was established. The abpR locus is responsible for the synthesis and the abpS locus is responsible for the structure of the arginine-ornithine-binding protein, a required component of the arginine-ornithine transport system of Escherichia coli. Two loci that result in elevated synthesis of the arginine-ornithine-binding protein and in an altered protein were mapped by bacterial conjugation and transduction studies. The mapping showed that the two genes lie in close proximity near the argA genetic marker in the order, with respect to argA, of argA abpR abpS. The maximal influx of arginine into an abpR mutant, which produces the arginine-ornithine-binding protein in an elevated amount, was substantially higher than the value obtained with an isogenic wild-type strain (apbR+). It also was observed that there was a close similarity between the affinity of the transport system for its substrate and the in vitro affinity of the binding protein for arginine both in the case of the isogenic wild type (abpS+) and a mutant (abpS6) carrying an altered protein. These results were consistent with the concept that the binding protein modulates the affinity of the transport system and suggest that it is the step of substrate recognition by the periplasmic protein which is rate-limiting in the entire process of transport at maximal influx.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Phosphorylation of Cl-, HCO3--stimulated Mg2+-ATPase of plasma membranes of carp (Cyprinus carpio L.) brain sensitive to GABA(A)-ergic ligands

Phosphorylation of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--stimulated Mg2+-ATPase of the plasma membranes from fish brain by [gamma-32P]ATP was investigated in the presence of Mg2+. It was established, that formation of the phosphoprotein at 0-1 degrees C is dependent on time incubation and concentration of Mg2+ in the incubation medium. Hydroxylamine (50 mM) and pH (10) completely inhibited formation of phosphorylated intermediate. Ions of Cl- (10 mM)+HCO3- (2 mM) and also GABA (1-100 microM) dephosphorylated the enzyme. The dephosphorylating effect of GABA on the membrane samples did not appear in the presence of bicuculline. o-Vanadate (10 microM) eliminates the dephosphorylating effect of anions and GABA on the phosphoprotein. It was established by SDS-PAAG electrophoresis and autoradiographia that investigated phosphorylation and GABA(A)-induced dephosphorylation is performed by the protein with molecular weight aproximately 56 kDa. Such molecular weight has a subunit which forms oligomer composition of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--ATPase from fish brain. The obtained data demonstrated that Cl, HCO3- ATPase from fish brain can be directly phosphorylated by [gamma-32P]ATP in the presence of Mg2+ and forms the phosphorylation intermediate.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase

Two phosphoproteins are absorbed to protein A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98K phosphoprotein that contains the steroid binding site, and the other is a 90K non-steroid-binding phosphoprotein that is associated with the molybdate-stabilized receptor [Housley, P. R., Sanchez, E. R., Westphal, H. M., Beato, M., & Pratt, W. B. (1985) J. Biol. Chem. 260, 13810-13817]. In this paper we have incubated L-cell cytosol with rabbit antiserum against the mouse glucocorticoid receptor and show that incubation of protein A-Sepharose-bound immune complexes with [gamma-32P]ATP and Mg2+ results in phosphorylation of the 98K steroid-binding protein but not of the 90K receptor-associated protein. Phosphorylation occurs regardless of whether the receptor is unoccupied or is present as the untransformed or transformed steroid-receptor complex. No phosphorylation occurs in the presence of Ca2+ instead of Mg2+. If protein A-Sepharose-bound immune complexes prepared with a monoclonal antibody against the receptor are incubated with [gamma-32P]ATP and Mg2+, neither protein is phosphorylated. If the protein A-Sepharose pellet is obtained from molybdate-stabilized cytosol that has been incubated both with monoclonal antibody to provide the 98K receptor and its 90K associated protein and with preimmune rabbit serum, which causes the nonspecific adsorption of an L-cell protein kinase, then incubation with [gamma-32P]ATP and Mg2+ causes receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a kinase from Escherichia coli K-12 that phosphorylates two periplasmic transport proteins

The phosphorylation in vivo and in vitro of the arginine-ornithine and the lysine-arginine-ornithine (LAO) periplasmic transport proteins of Escherichia coli K-12 was previously reported (Celis, R. T. F. (1984) Eur. J. Biochem. 145, 403-411). The phosphorylative reaction required ATP (as a direct energy donor), Mg2+, and a kinase that can be released by osmotic shock treatment of the cells. The enzyme was purified to electrophoretic homogeneity. The enzyme exhibited an ATPase activity and a kinase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave an apparent molecular weight of 43,000 for the enzyme. The native protein showed the same molecular weight, suggesting that the protein is a monomer. The protein showed an apparent isoelectric point of 4.8 on isoelectric focusing. The two enzymatic reactions required a divalent cation and the apparent Km value for Mg2+ for the kinase activity was 0.5 mM. Mn2+ and Co2+ served as well as Mg2+, whereas Zn2+ and Ca2+ did not support activity. The ATPase activity of the enzyme yielded an apparent Km value for ATP of 50 microM. A similar value, Km of 100 microM, was calculated for the kinase activity with different concentrations of ATP. The enzyme showed a pH optimum of 7.3.     

Brain protein phosphorylation in vitro: selective substrate action of insulin

Insulin action on [32P]-phosphate incorporation into brain membranes was determined. Hippocampal homogenate tissue was phosphorylated with [32P]-ATP, and insulin was introduced at various times before or after ATP addition. With 50 microM Mg++ in the medium, insulin selectively stimulated the phosphorylation of a 47kD phosphoprotein, Protein F1. This effect required the prior presence of ATP. No effect of insulin on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material, was observed under these conditions. At 1 mM Mg++, insulin selectively decreased the phosphorylation of the alpha-subunit of pyruvate dehydrogenase. Insulin had no effect on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material under these conditions. The present study suggests a role for insulin in the modulation of brain protein phosphorylation. Since Protein F1 is phosphorylated by exogenous C kinase, and is likely the CNS-specific B-50 protein, these data also indicate a brain-specific function for insulin, possibly by action on a Ca++/phospholipid protein kinase.     

ATP-dependent calcium transport in rat parotid microsomes. I. Localization, properties, Ca2+-ATPase activity and phosphoenzyme formation

ATP-dependent Ca2+ transport was studied in rat parotid microsomes; the activity appears to be associated with rough endoplasmic reticulum vesicles, as indicated by marker distribution in subcellular fractions and by electron microscopic observations. Purified rough microsomes exhibit an ATP-dependent Ca2+ accumulation and a Ca2+-dependent ATPase activity; these activities are similarly stimulated by K+ and display an apparent Km for free calcium of 0.6-0.7 microM. A phosphoprotein, with a molecular weight of about 110,000, was detected after short incubation with [gamma 32P] ATP and CaCl2; it is suggested that this compound represents a phosphorylated intermediate form of the Ca2+-ATPase.     

Depolarization-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Characterization of Active Protein Kinases by Phosphopeptide Analysis of Substrates

Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.     

Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli

During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP.     

Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase

Pig brain diacylglycerol kinase did not catalyze autophosphorylation. However, the kinase was phosphorylated on serine, when immunoprecipitated from the partially purified enzyme preparation preincubated with Mg2+ and [gamma-32P]ATP. The action of the endogenous protein kinase phosphorylating diacylglycerol kinase was independent of cyclic nucleotides and Ca2+, and became maximum at pH 5.5. Although the extent of enzyme phosphorylation was limited (maximally about 0.25 mol Pi incorporated per mol kinase), the results show that diacylglycerol kinase can be a phosphoprotein.     

Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.     

Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum.

The present study demonstrated the presence within the myocardium of phosphoprotein phosphatase activity which can account for dephosphorylation of a 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum that has been associated with the stimulatory effects of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase on calcium transport (Tada, M., Kirchberger, M. A., and Katz, A. M. (1975) J. Biol. Chem. 250:2640-2647). Dog cardiac microsomes, consisting mainly of fragmented sarcomplasmic reticulum, were phosphorylated by incubation with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, and subsequently washed with trichloroacetic acid or buffered KCl. Phosphorylated microsomes contained approximately 1 nmole of 32P bound per mg of microsomal protein, 32P labeling occurring almost exclusively at the 22,000 dalton component. Soluble phosphoprotein phosphatases, isolated from the cytosol, catalyzed dephosphorylation of 32P-labeled microsomes. The existence of a phosphoprotein phosphatase that is associated with the microsomes was demonstrated by the ability of the microsomes to dephosphorylate 32P-histone. This membrane-associated phosphatase activity can also account for a rapid decrease in the amount of 32P-labeling of the 22,000 dalton protein. The dephosphorylation of the phosphorylated 22,000 dalton protein by phosphoprotein phosphatase satisfies an important requirement for the phosphorylation of the 22,000 dalton protein to serve a physiological role, namely, its reversibility.     

Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca2+ + Mg2+)-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32P from [gamma-32P]ATP is Ca2+-dependent (approximate Km for free [Ca2+] = 2-3 X 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32Pi incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg2+ and monovalent cations such as K+ or Na+ are necessary. Vanadate inhibits both 32P incorporation and 32P liberation dose dependently (Km = 3 X 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca2+, cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca2+ transport into rough endoplasmic reticulum from pancreatic acinar cells (Bayerd?rffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81, 69-82). The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase.     

Rapid release of 42K or 86Rb from two distinct transport sites on the Na,K-pump in the presence of Pi or vanadate

The rate of 86Rb or 42K release from an occluded form of the phosphorylated Na+ pump has been studied using a rapid filtration apparatus described previously. The rate constant of release is 5-15 s-1, and 42K and 86Rb dissociate at approximately the same rate. Mg2+ is required for deocclusion in the presence of Pi at a site which has the same affinity as the site involved in stabilization of E2(K) with ATP; we propose that Na,K-ATPase has only one site for Mg2+ (apart from Mg2+ complexed with ATP), that the affinity of this site for Mg2+ is increased by Pi binding and decreased by ATP binding, and that Mg2+ is bound and released in the normal transport cycle. In the presence of K+, Cs+, Rb+, or Tl+, the release of two distinct 86Rb ions can be observed, the slow release from one site ("s" site) being blocked by occupancy of the site vacated by the other ("f", fast site). By a sequence of incubations, labeled 86Rb can be placed at either site, and the rate of dissociation monitored individually; in the absence of K+, dissociation from the s site proceeds after a lag in which the f site is vacated. The results are consistent with a "flickering-gate" model of deocclusion to the extracellular pump face, in which the site is exposed to the medium only long enough for a single ion to be released. When deocclusion to the intracellular face is promoted with ATP, ions are released from both sites at the same rate, presumably because the E2----E1 conformational change is rate-limiting. Unlabeled ions co-occluded with 86Rb increase the ATP-stimulated rate of release in the order Rb+ less than Tl+ less than Cs+ less than K+; since the same rank order is observed when dissociation from the s site is monitored in the presence of these ions and MgPi we propose that the latter process proceeds toward the intracellular pump face. 86Rb release from the vanadate-inhibited enzyme has the characteristics of Pi-stimulated release but is approximately 25-fold slower. ATP binds to both the phosphorylated and vanadate-inhibited forms of Na,K-ATPase and increases the rate of deocclusion, apparently to both the intracellular and extracellular faces of the pump.     

Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations

Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.     

Energetics of the calcium-transporting ATPase

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.     

Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.     

Demonstration of two different reactive sulfhydryl groups in the ATP-binding sites of Ca2+-ATPase of sarcoplasmic reticulum by disulfides of thioinosine triphosphates

1. The disulfide of thioinosine triphosphate, (SnoPPP)2, is a substrate of the Ca2+-pump and the Ca2+-ATPase of sarcoplasmic reticulum (Km = 400 microM). 2. Inactivation of Ca2+-ATPase by the beta,gamma-methylene diphosphonate analogue of the disulfide of thioinosine triphosphate, (SnoPP[CH2]P)2, in the presence of (Ca2+ + Mg2+ + K+) is preceeded by a dissociable enzyme inhibitor complex with a dissociation constant of 130 microM for a low-affinity binding site. ATP protected Ca2+-ATPase against the inactivation under these conditions with a dissociation constant of 140 microM. 3. Kinetic analysis of the inactivations of Ca2+-ATPase by (SnoPP[CH2]P)2 in the absence of Ca2+ and Mg2+ but the presence of K+ and EGTA led to the appearance of two nucleotide binding sites with two different inactivation velocities. Inactivation rate constants k2 were found for the rapid inactivating part (k2' = 1.44 X 10(-2) s-1) and the slow inactivating part (k2" = 1.15 X 10(-3) s-1). From the protective effect of ATP under these conditions a high-affinity (Kd = 48.78 microM) and a low-affinity ATP binding site (Kd = 114 microM) were apparent. 4. The affinity of the analogues to the enzyme is decreased in the sequence: (SnoPPP)2 > (SnoPP[NH]P)2 > (SnoPP[CH2]P)2 > (SnoP)2. 5. (SnoPPP)2-inactivated Ca2+-ATPase was reactivated by incubation with dithiothreitol. 6. Inactivation of Ca2+-ATPase by [gamma-32P](SnoPPP)2 in the presence of (Mg2+ + K+ + Ca2+) or (EGTA + K+) was accompanied by the incorporation of hydroxylamine-insensitive radioactivity into the acid-precipitable protein. The enzyme-bound [gamma-32P]SnoPPP was cleaved by dithiothreitol. 7. It is concluded that (SnoPPP)2 and its non-hydrolyzable analogues (SnoPP[NH]P)2 and (SnoPP[CH2]P)2 act as ATP affinity labels and form mixed disulfides with a sulfhydryl group within the active site.     

Mechanism and regulation of phosphate transport in Streptococcus pyogenes.

In contrast to results reported with other bacteria, uptake of 32Pi in Streptococcus pyogenes was found to occur rapidly in starved cultures and to be strongly and immediately inhibited by addition of exogenous glycolytic energy sources (such as glucose) and nonglycolytic sources of ATP (such as arginine). Preincubation of starved cells with NaF, iodoacetate, or arsenate eliminated the inhibiting effect of glucose but not that of arginine. In accordance with the hypothesis that transport was attributable to Pi-Pi exchange, uptake and efflux of 32Pi in the presence of trans unlabeled Pi exhibited similar characteristics and were largely eliminated by reduction of the trans Pi concentration. Neither process was inhibited appreciably by pretreatment of cells with ionophores or metabolic inhibitors, but both processes were abolished by exposure to p-chloromercuribenzoate. Inhibition by both exogenous energy sources resulted in a reduction in the maximal velocity of transport (Vmax). Whereas arginine also caused a shift in the apparent Michaelis-Menten constant (Km) to larger values, glucose did not alter the Km. On the basis of the results reported, we propose that the rate of Pi exchange is determined positively by the intracellular and extracellular concentrations of Pi and negatively by ATP or metabolites thereof. The mechanism of ATP action is unknown but could involve either covalent or noncovalent modification of the carrier protein.