Description of a protein kinase derived from insulin-treated 3T3-L1 cells that catalyzes the phosphorylation of ribosomal protein S6 and casein

Particulate preparations from insulin-treated 3T3-L1 cells retain the enhanced ability to incorporate 32P from [gamma-32P]ATP into ribosomal protein S6 (Smith, C. J., Rubin, C. S., and Rosen, O. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2641-2645). A cyclic AMP-independent protein kinase that phosphorylates S6 and casein and that may be involved in the increase in S6 phosphorylation produced by insulin has been isolated based upon the observation that there is 1.5-3.0-fold higher activity in particulate preparations derived from insulin-treated cells than there is in comparable preparations from control cells. The enzyme activity was purified 2071-fold by KCl extraction, phosphocellulose chromatography, and gel filtration. The S6 phosphorylating activity was also characterized by its behavior on casein-Sepharose and DEAE-cellulose chromatography and its sedimentation in glycerol gradients. None of these procedures resolved the S6 and casein kinase activities. Some of the properties of this kinase, including a molecular weight of about 35,000, inhibition by F- or phosphate, chromatography on DEAE-cellulose and phosphocellulose, and insensitivity to inhibition by GTP, are similar to those of a previously described enzyme, casein kinase I (Dahmus, M. E. (1981) J. Biol. Chem. 256, 3319-3325; Hathaway, G. M., and Traugh, J. A. (1979) J. Biol. Chem. 254, 762-768).     

Adenylate kinase of Escherichia coli, a component of the phage T4 dNTP synthetase complex

Adenylate kinase, which catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720-5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phage-coded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.     

Escherichia coli nucleoside diphosphate kinase interactions with T4 phage proteins of deoxyribonucleotide synthesis and possible regulatory functions

In both prokaryotic and eukaryotic organisms, nucleoside diphosphate kinase is a multifunctional protein, with well defined functions in ribo- and deoxyribonucleoside triphosphate biosynthesis and more recently described functions in genetic and metabolic regulation, signal transduction, and DNA repair. This paper concerns two unusual properties of nucleoside diphosphate (NDP) kinase from Escherichia coli: 1) its ability to interact specifically with enzymes encoded by the virulent bacteriophage T4 and 2) its roles in regulating metabolism of the host cell. By means of optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and glutathione S-transferase pull-down assays, we have shown that E. coli NDP kinase interacts directly with T4 thymidylate synthase, aerobic ribonucleotide reductase, dCTPase-dUTPase, gene 32 single-strand DNA-binding protein, and deoxycytidylate hydroxymethylase. The interactions with ribonucleotide reductase and with gp32 are enhanced by nucleoside triphosphates, suggesting that the integrity of the T4 dNTP synthetase complex in vivo is influenced by the composition of the nucleotide pool. The other investigations in this work stem from the unexpected finding that E. coli NDP kinase is dispensable for successful T4 phage infection, and they deal with two observations suggesting that the NDP kinase protein plays a genetic role in regulating metabolism of the host cell: 1) the elevation of CTP synthetase activity in an ndk mutant, in which the structural gene for NDP kinase is disrupted, and 2) the apparent ability of NDP kinase to suppress anaerobic growth in a pyruvate kinase-negative E. coli mutant. Our data indicate that the regulatory roles are metabolic, not genetic, in nature.     

Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes.

The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.     

Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitutions and frameshifts in mismatch-repair-deficient strains

Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools. Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors. We show here that in E. coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system. Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient. A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T --> G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T --> T:A transversions.     

Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus.

The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.     

Competition between nucleoside diphosphates and triphosphates at the catalytic and allosteric sites of phosphorylase kinase

The interactions of nucleotides at the allosteric and catalytic sites of phosphorylase kinase were examined. Binding of nucleoside triphosphates at the nucleoside diphosphate allosteric activation site inhibited enzymatic activity; this was observed with either ATP or GTP. Increasing concentrations of ADP caused a biphasic response: low concentrations activated and higher concentrations inhibited. Inhibition was due to the binding of ADP at the catalytic site, as opposed to an allosteric inhibitory site. GDP activated at low concentrations, but did not inhibit even at relatively high concentrations, and is therefore a specific probe for the allosteric site. Maximal activity of the nonactivated holoenzyme at pH 6.8 is achieved at an optimal ratio of ATP to ADP, such that the inhibitory actions of ATP at the allosteric site and of ADP at the catalytic site are balanced. Various potential molecular mechanisms to explain the allosteric activation by ADP were examined and ruled out, thus strengthening our previous conclusion that the activation is predominantly caused by a conformational transition in the beta subunits directly induced by the binding of ADP (Cheng, A., Fitzgerald, T. J., and Carlson, G. M. (1985) J. Biol. Chem. 260, 2535-2542; Trempe, M. R., and Carlson, G. M. (1987) J. Biol. Chem. 262, 4333-4340; Cheng, A., Fitzgerald, T. J., Bhatnager, D., Roskoski, R., Jr., and Carlson, G. M. (1988) J. Biol. Chem. 263, 5534-5542). The catalytic site exhibited high stereospecificity for inhibition by the Rp and Sp epimers of adenosine 5'-O-(1-thiodiphosphate), with the Rp epimer (Ki = 0.5 microM) being 136-fold more effective than its Sp counterpart. This can readily explain the inability of the Rp epimer to be an effective allosteric activator.     

Nucleoside diphosphate kinase from Escherichia coli.

Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993).     

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.     

ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.

Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.     

Reversible autophosphorylation of a cyclic 3':5'-AMP-dependent protein kinase from bovine cardiac muscle.

Purified cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase of bovine cardiac muscles catalyzes the incorporation of 2 mol of 32P from [gamma-32P]ATP to seryl residues in its cAMP-binding protein. The reaction appears to be catalyzed by the protein kinase itself rather than by a protein kinase kinase and is enhanced by cAMP and by the addition of polyarginine. Phosphorylation of the purified enzyme facilitates its dissociation by cAMP (Erlichman, J., Rosenfeld, R., and Rosen, O.M. (1974) J. Biol. Chem. 249, 5000-5003) but does not affect cAMP binding. At equilibrium, 2 mol of cAMP are bound to both the phospho- and dephospho-enzymes. Phosphorylation of protein kinase is reversible. Upon addition of ADP and Mg2+, phosphate is transferred from the protein to ADP, and ATP is formed. The reverse reaction is optimal at pH 5.5 unlike the forward reaction which has a broad, more alkaline pH activity optimum. It is activated by polyarginine and dependent upon the addition of cAMP to a much greater degree than the forward reaction. The data suggest that the catalytic subunit of protein kinase catalyzes the forward and reverse reactions but do not exclude the possibility that the holoenzyme may also be active. Autophosphorylation by protein kinase and dephosphorylation by phosphrprotein phosphatases of by reverals of the autophosphorylation reaction may regulate the sensitivity of certain protein kinases to activation by cAMP in vivo.     

Creatine phosphate inhibition of heart lactate dehydrogenase and muscle pyruvate kinase is due to a contaminant

Previously reported inhibitions of heart lactate dehydrogenase (Guppy, M., and Hochachka, P.W. (1978) J. Biol. Chem. 253, 8465-8469) and muscle pyruvate kinase (Kemp, R.G. (1973) J. Biol. Chem. 248, 3963-3967) by creatine phosphate are due to oxalate which is a contaminant found in some commercial preparations of creatine phosphate.     

Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA.

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.     

Hormonal regulation of a calcium-activated, phospholipid-dependent protein kinase in bovine adrenal cortex

The calcium-activated, phospholipid-dependent protein kinase (C kinase) and its proteolytic product (M kinase), originally discovered in central nervous tissue (Takai, Y., Kishimoto, A., Inoue, M., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7603-7610) were characterized in bovine adrenal cortex cytosol. An endogenous calcium-dependent protease able to generate M kinase from the isolated C kinase in vitro was also present in adrenocortical extracts. Bovine adrenocortical cells in suspension as well as in primary culture contain the C and the M kinase activities. Treatment of these cells by steroidogenic concentrations (nM to microM) of ACTH resulted in a time and dose-dependent increase of cytosolic C kinase activity, whereas no change in M kinase activity was detected. This apparent activation appears to result mostly from an intracellular shift of the membrane-associated C kinase to a soluble cytosolic form of the enzyme. These observations open the question of the possible implication of the calcium, phospholipid-dependent protein phosphorylation system in hormone-dependent cellular regulatory processes.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle.

The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.     

Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.

Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the 32P label. The 32P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of 32P label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein kinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KCl did not decrease the amount of 32P labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calcium uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings (Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Tada, M., Kirchberger, M.A., Repke, D.I., and Katz, A.M. (1974) J. Biol. Chem. 249, 6174-6180) that cyclic AMP-dependent protein kinase-catalyzed phosphorylation is associated with stimulation of calcium transport in the cardiac sarcoplasmic reticulum, and further indicate that this phosphorylation occurs at a component of low mass (22,000 daltons) of the cardiac sarcoplasmic reticulum which, while separable from the calcium transport ATPase protein (100,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the ability to regulate calcium transport by the cardiac sarcoplasmic reticulum.     

Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli

Acetate kinase from Salmonella typhimurium and Escherichia coli was purified to electrophoretic homogeneity. The amino acid compositions of both proteins were similar, and the apparent molecular weights were the same, about 40,000 for the putative monomers. The native proteins gave higher molecular weights, suggesting that the enzymes may be oligomers, perhaps with two polypeptide subunits. Steady-state kinetic studies were performed with the enzymes isolated from both organisms and the kinetic constants were determined. The Km values were 0.07 and 7 mM for ATP and acetate, respectively. In contrast to earlier studies using less pure preparations, the homogeneous enzymes from both strains were active only with acetate but not with propionate or butyrate. The enzyme activity was cold-labile, and the length of reactivation time in the presence of Mg X ATP and acetate was dependent on protein concentration, suggesting that the monomer may not be catalytically active. The enzyme was phosphorylated with [gamma-32P]ATP and the phosphoprotein was isolated. Phosphoacetate kinase was capable of transferring the phosphate group to either ADP or acetate. The accompanying paper (Fox, D. K., Meadow, N. D., and Roseman, S. (1986) J. Biol. Chem. 261, 13498-13503) shows that the phosphoryl group of phosphoacetate kinase can also be reversibly transferred to Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system.     

Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDP<-->ADP+(d)NTP. This reaction, suggested to occur by the transfer of the gamma-phosphoryl from ATP to the nucleoside diphosphate, is overall similar to that normally carried out by nucleoside diphosphate kinase (Ndk). Accordingly, Adk was proposed to be responsible for residual Ndk-like activity measured in a mutant strain of Escherichia coli, where the ndk gene was disrupted. We present data supporting a mechanism for the synthesis of nucleoside triphosphates by Adk that unlike the previously suggested mechanism mentioned above are in complete agreement with the current knowledge about the Adk enzyme and its various catalytic properties. We propose that nucleoside triphosphate synthesis occurs by beta-phosphoryl transfer from ADP to any bound nucleoside diphosphate. Our results point to the fact that the proposed Ndk-like mechanism of Adk originated from an erroneous interpretation of data, in that contamination of ATP preparations with AMP and ADP was not taken into account. Our results also address the proposed role of Adk in restoring a normal growth rate of mutant strains of E. coli lacking Ndk. These mutant strains apparently, in spite of a mutator phenotype, are able to synthesise nucleoside triphosphates by alternative pathways to maintain the same growth rate as the wildtype.     

The (Na,K)-ATPase of Friend erythroleukemia cells is phosphorylated near the ATP hydrolysis by an endogenous membrane-bound kinase

Friend murine erythroleukemia cells (MEL cells) contain a cAMP-independent protein kinase which phosphorylates the 100,000-Da catalytic subunit of the (Na,K)-ATPase both in living cells and in the purified plasma membrane (Yeh, L.-A., Ling, L., English, L., and Cantley, L. (1983) J. Biol. Chem. 258, 6567-6574). We have taken advantage of the selective phosphorylation of the 100,000-Da subunit in purified plasma membranes and the similarity between the proteolysis patterns of the MEL cell and dog kidney (Na,K)-ATPase to map the site of kinase phosphorylation on the MEL cell enzyme. The chymotryptic and tryptic cleavage sites of the dog kidney (Na,K)-ATPase have previously been located (Castro, J., and Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228). The 100,000-Da catalytic subunits of the dog kidney and MEL cell enzymes were specifically labeled at the active site aspartate residue by incubation with (32P)orthophosphate in the presence of Mg2+ and ouabain. Digestion of these two enzymes with chymotrypsin or trypsin revealed similar active site aspartate containing proteolytic fragments indicating a similar structure for the two enzymes. Chymotryptic digestions of MEL cell (Na,K)-ATPase labeled in vitro with [gamma-32P]ATP localize the region of kinase phosphorylation to within a 35,000-Da peptide derived from the middle of the 100,000-Da subunit. Tryptic digestion of the MEL cell plasma membranes degraded the 100,000-Da subunit to an NH2-terminal 43,000-Da peptide which contained the active site aspartate but which did not contain the kinase-labeled region. These results further locate the region of kinase phosphorylation to the COOH-terminal half of the 35,000-Da chymotryptic peptide. This location places the site of phosphorylation between the active site aspartate residue which accepts the phosphate of ATP during turnover and an ATP-binding site which has previously been located by labeling with fluorescein 5'-isothiocyanate (Carilli, C. T., Farley, R. A., Perlman, D. M., and Cantley, L. C. (1982) J. Biol. Chem. 257, 5601-5606). Phosphorylation of the (Na,K)-ATPase in this region may serve to regulate the activity of this enzyme.