Intrinsic contributions of polar amino acid residues toward thermal stability of an ABC-ATPase of mesophilic origin

The nucleotide-binding subunit of phosphate-specific transporter (PstB) from mesophilic bacterium, Mycobacterium tuberculosis, is a unique ATP-binding cassette (ABC) ATPase because of its unusual ability to hydrolyze ATP at high temperature. In an attempt to define the basis of thermostability, we took a theoretical approach and compared amino acid composition of this protein to that of other PstBs from available bacterial genomes. Interestingly, based on the content of polar amino acids, this protein clustered with the thermophiles.     

Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells

We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.     

The carboxy-terminal end of the peptide deformylase from Mycobacterium tuberculosis is indispensable for its enzymatic activity

The peptide deformylase in bacteria is involved in removal of N-formyl group from newly synthesized proteins. The gene encoding this enzyme from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The enzyme activity of the recombinant protein (mPDF) was insensitive to modulation by common monovalent/divalent cations. Kinetic analysis, using N-formylmethionine-alanine as the substrate, yielded K(cat)/K(m) of approximately 1220 M(-1)s(-1). Actinonin, a naturally occurring antibiotic, and 1,10-ortho-phenanthroline strongly inhibited the enzyme activity. The mPDF was very stable at 30 degrees C with a half-life of approximately 4h and exhibited resistance to oxidizing agents, like H(2)O(2). Thus, the mPDF achieved distinction in its behavior among any reported iron-containing peptide deformylases. Furthermore, amino acid sequence analysis of mPDF revealed the presence of an unusually long carboxy-terminal end (residues 182-197), which is atypical for any gram-positive bacteria. Our results, through deletion analysis, for the first time established the role of this region in mPDF enzyme activity.     

Coupled kinetics of ATP and peptide hydrolysis by Escherichia coli FtsH protease

FtsH from Escherichia coli is an ATP- and Zn(2+)-dependent integral membrane protease that is involved in the degradation of regulatory proteins such as sigma(32) and uncomplexed subunits of membrane protein complexes such as secY of the protein translocase. We describe a protocol for solubilizing the recombinant enzyme from inclusion bodies and its subsequent refolding and purification to near homogeneity. This is a high-yield protocol and produces in excess of 20 mg of purified FtsH per liter of E. coli culture. We found that refolded FtsH has biochemical properties similar to detergent extracted overexpressed protein described previously. FtsH forms a large complex with an apparent mass of 1200 kDa as determined by gel filtration. Both ATPase and protease activities are coincident with this large complex; smaller forms of FtsH do not exhibit either activity. While FtsH-catalyzed hydrolysis of ATP can occur in the absence of protein substrate (k(c) = 22 min(-1); K(m) = 23 microM), proteolysis shows an absolute dependence on nucleoside-5'-triphosphates, including ATP, CTP, and various analogues. In the presence of 5 mM ATP, FtsH catalyzes the hydrolysis of sigma(32) with the following observed kinetic parameters: k(c) = 0.18 min(-1) and K(m) = 8.5 microM. Significantly, this reaction is processive and generates no intermediate species, but rather, approximately 10 peptide products, all of MW <3 kDa. FtsH protease also efficiently hydrolyzes the peptide Phe-Gly-His-(NO)2Phe-Phe-Ala-Phe-OMe. Hydrolysis occurs exclusively at the (NO)2Phe-Phe bond (k(c) = 2.1 min(-1); K(m) = 12 microM), and like proteolysis, shows an absolute dependence on NTPs. We propose a mechanism for the coupled hydrolytic activities of FtsH toward ATP and peptide substrates that is consistent with a recently proposed structural model for FtsH.     

Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies.

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H(+)-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent Vmax for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent Km for ATP was approximately 50 microM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (all inhibitors of vacuolar-type H(+)-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca2+. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-gamma-32P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H(+)-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H(+)-ATPase.     

Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.     

The Autographa californica nuclear polyhedrosis virus p143 gene encodes a DNA helicase

The P143 protein of Autographa californica nuclear polyhedrosis virus is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143. ATPase activity copurified with P143 protein during purification and also during gel filtration at a high salt concentration. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated fourfold by the addition of single-stranded DNA. The ATPase activity of P143 had a K(m) of 60 microM and a turnover of 4.5 molecules of ATP hydrolyzed/s/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.     

Simultaneous bindings of ATPase inhibitor and 9K protein to F1F0-ATPase in the presence of 15K protein in yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F1-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F1F0-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4 x 10(-5) M. The ATPase inhibitor and 9K protein also bound to F1F0-ATPase in the presence of ATP and Mg2+, and the dissociation constants of their bindings were about 3 X 10(-6) M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F0 part and functions to hold the ATPase inhibitor or 9K protein on the F1 subunit.     

Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA

We find that the rate of dsDNA-dependent ATPase activity is biphasic, with a fast component which represents the unwinding of the dsDNA and a slow component which results from the ssDNA-dependent ATPase activity of recBCD enzyme. Comparison of the ATPase and helicase activities permits evaluation of the efficiency of ATP hydrolysis during unwinding. This efficiency can be calculated from the maximum rates of ATPase and helicase activities and is found to range between 2.0 and 3.0 ATP molecules hydrolyzed per base pair of DNA unwound. The number of ATP molecules hydrolyzed per base pair unwound is not altered by temperature but does increase at low concentrations of DNA and high concentrations of sodium chloride and magnesium acetate. The apparent Km values for the DNA and ATP substrates of recBCD enzyme dsDNA-dependent ATPase activity at 25 degrees C were determined to be 0.13 nM DNA molecules and 85 microM ATP, respectively. The observed kcat value is approximately 45 microM ATP s-1 (microM recBCD enzyme)-1. If this rate is corrected for the measured stoichiometry of recBCD enzyme binding to dsDNA, the kcat for ATPase activity corresponds to an ATP hydrolysis rate of approximately 740 ATP molecules s-1 (functional recBCD complex)-1 at 25 degrees C.     

In situ assembly states of (Na+,K+)-pump ATPase in human erythrocytes. Radiation target size analyses

The in situ assembly state of the (Na+,K+)-pump ATPase of human erythrocytes was studied by applying the classical target theory to radiation inactivation data of the ouabain-sensitive sodium efflux and ATP hydrolysis. Erythrocytes and their extensively washed white ghosts were irradiated at -45 to -50 degrees C with an increasing dose of 1.5-MeV electron beam, and after thawing, the Na+-pump flux and/or enzyme activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose, from which a radiation sensitive mass (target size) was calculated. When intact cells were used, the target sizes for the pump and for the ATPase activities were equal and approximately 620,000 daltons. The target size for the ATPase activity was reduced to approximately 320,000 daltons if the cells were pretreated with digitoxigenin. When ghosts were used, the target size for the ATPase activity was again approximately 320,000 daltons. Our target size measurements together with other information available in literature suggest that (Na+,K+)-pump ATPase may exist in human erythrocytes either as a tetramer of alpha beta or as a dimer of alpha beta in tight association with other protein mass, probably certain glycolytic enzymes, and that this tetrameric or heterocomplex association is dissociable by digitoxigenin treatment or by extensive wash during ghost preparation.     

The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni

Polar membrane in Campylobacter jejuni has been visualized on membrane vesicles. It was composed of doughnut-shaped particles 5-6 nm in diameter, with stalks, arranged in a hexagonal array. This structure was stabilized on the membrane by a high ionic strength buffer in the presence of 2-mercaptoethanol. Histochemical staining indicated localized ATPase activity at the poles of the cells. An ATPase with distinctive properties has been isolated and purified from this organism; it gives a specific activity of approximately 0.3 units/mg of protein. Electron microscopy showed doughnut-shaped particles 5-6 nm in diameter. Nondissociating and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed, respectively, a single band with ATPase activity and a molecular weight of ca. 75,000 Da. The enzyme was cold labile and activity was abolished by trypsin. Dicyclohexylcarbodiimide inhibited the membrane-bound form of the enzyme, but did not inhibit the soluble form. Oligomycin had no inhibitory activity on either form of the enzyme. The enzyme specifically hydrolysed ATP, but other nucleotide substrates were not degraded. The enzyme was activated by Mg2+ and inhibited by Ca2+, whereas other ions had no effect on activity. Antibodies prepared to this enzyme bound to the polar regions of whole cells as shown by protein A - colloidal gold immunoelectron microscopy. The antibodies to this ATPase cross reacted (shown by Western blotting) with four proteins from a whole-cell extract of this organism, two proteins in Aquaspirillum serpens MW5, and three proteins from Escherichia coli K12. They did not cross-react with any proteins from Spirillum volutans, Methanococcus voltae, Vibrio cholerae, or rat liver mitochondria. Antibodies raised against the F1-ATPase of E. coli K12 cross reacted with six proteins in a whole-cell extract of this organism, and one protein species in each of the whole-cell extracts of V. cholera, A. serpens MW5, S. volutans, and rat liver mitochondria. These antibodies did not recognize any whole cell proteins from either C. jejuni or M. voltae. These results along with the ATPase activity localized by histochemical staining suggest that polar membrane is an assembly of ATPase molecules at the poles of the cell and that the ATPase isolated from C. jejuni is serologically and structurally unusual.     

Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: rapid purification with high yield and purity

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116mg of recombinant enzyme was purified to near homogeneity with approximately 80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni-NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His(6)) was approximately 800U/mg of protein. The apparent K(m) value of the active recombinant protein followed Michaelis-Menten kinetics and was 0.27+/-0.03mM. K(i) for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6-9.0 and was stable up to 55 degrees C. rPGI-His(6) exhibited enzyme activity almost equal to that of enzyme without histidine tag.     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Inactivation of the Na,K-ATPase by modification of Lys-501 with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS).

The sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss of ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCl. Only the alpha-subunit was labeled by SITS. The alpha-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the alpha-subunit of Na,K-ATPase.     

Osmoregulation in Bacillus subtilis under potassium limitation: a new inducible K+-stimulated, VO4(3-)-inhibited ATPase

Bacillus subtilis exhibited an inducible K+-transporting ATPase activity with apparent Km and maximum velocity Vmax of 12.9 microM and 25.1 micromol x min(-1) x (g cell protein)(-1), respectively, when cultivated on a synthetic medium containing less than 400 microM K+. Due to this enzyme, the growth rate of the bacterium in synthetic medium was not changed down to 115 microM K+, and the bacterium was able to grow down to 20 microM K+. The limiting K+ concentration was higher in media with osmolarity increased by NaCl or sucrose. The ATPase was inhibited by micromolar concentrations of vanadate (Ki = 1.6 microM). The ATPase activity was not stimulated by any other monovalent cation. The subunit of this ATPase, with an Mr of 52000, covalently bound the gamma phosphate group of ATP. This phosphorylated intermediate was unstable in neutral and basic pH as well as in the presence of potassium and was stable in acid pH. The enzyme did not show immunological cross-reactivity with antibody against Kdp ATPase of Escherichia coli.     

Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.     

Adenosine Triphosphatase Activity at the External Surface of Chicken Brain Synaptosomes

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.     

Nucleotide-induced conformational change in the catalytic subunit of the phosphate-specific transporter from M. tuberculosis: implications for the ATPase structure

The nucleotide binding subunit of the phosphate-specific transporter (PstB) from Mycobacterium tuberculosis is a member of the ABC family of permeases, which provides energy for transport through ATP hydrolysis. We utilized the intrinsic fluorescence of the single tryptophan containing protein to study the structural and conformational changes that occur upon nucleotide binding. ATP binding appeared to lead to a conformation in which the tryptophan residue had a higher degree of solvent exposure and fluorescence quenching. Substantial alteration in the proteolysis profile of PstB owing to nucleotide binding was used to decipher conformational change in the protein. In limited proteolysis experiments, we found that ATP or its nonhydrolyzable analog provided significant protection of the native protein, indicating that the effect of nucleotide on PstB conformation is directly associated with nucleotide binding. Taken together, these results indicate that nucleotide binding to PstB is accompanied by a global conformational change of the protein, which involves the helical domain from Arg137 to Trp150. Results reported here provide evidence that the putative movement of the alpha-helical sub-domain relative to the core sub-domain, until now only inferred from X-ray structures and modeling, is indeed a physiological phenomenon and is nucleotide dependent.     

Properties of the N,N'-dicyclohexylcarbodiimide resistant ATPase of Streptococcus cremoris

1. The specific activity of the membrane-bound ATPase of Streptococcus cremoris HA was 1.30 mumol Pi/mg protein/min. 2. Km for ATP as substrate was 0.8 mM. 3. The pH optimum was 8.0 at +37 degrees C. 4. The ATPase was maximally activated with Mg2+/ATP molar ratio of 1:2. 5. Cations activated the enzyme in order: Mg2+ greater than Co2+ greater than Mn2+ greater than Zn2+ greater than Ca2+ greater than K+ greater than Na+. 6. The enzyme was inhibited by oligomycin (27-77%), sodium azide (13-33%) and ouabain (15-22%). N,N'-dicyclohexylcarbodiimide had no effect on the enzyme activity.     

Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K+ + EDTA or Ca2+ and very little activity in the presence of Mg2+. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K+ + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.