Proteins of bacterial membranes. Purification of soluble ATPase from Acholeplasma laidlawii

A purified preparation of ATPase (factor F1) from the Acholeplasma laidlawii was obtained. The purification procedure included extraction of the enzyme complex from the isolated membranes by ultrasonication, chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The specific activity of the ATPase was increased 30-fold as compared to the original activity. The Km value for ATP hydrolysis was 7,4 . 10(-4) M. ADP competitively inhibited the enzyme (Ki = 2,0 . 10(-4) M). Ouabain (2,5 . 10(-4) M) and dicyclohexylcarbodiimide (1,0 . 10(-4) M) did not inhibit the ATPase activity. The enzyme was activated by Mg2+, but was inhibited by a combination of Na+ and K+. The enzyme is cold-labile, but can be stabilized by storage in buffer solutions, containing methanol, glycerol or lecithin.     

Properties of membrane adenosine triphosphatase of the obligately anaerobic bacterium Veillonella alcalescens.

Some properties of membrane ATPase activity in Veillonella alcalescens were examined. Mg2+ is required for the activity of the enzyme, and Ca2+ also activates the enzyme to some degree. Of the nucleotide triphosphates, GTP and ITP were hydrolyzed to a lesser extent than ATP. The apparent Km for ATP hydrolysis was 0.25 to 0.63 mM. ADP inhibited the enzyme and the kinetic data of its inhibition showed that the presence of ADP resulted in positive cooperativity. The enzyme activity was strongly inhibited by DCCD, azide, fusidic acid and the antibody to purified soluble ATPase from the thermophilic bacterium PS3. Oligomycin, dinitrophenol, and ouabain showed no significant effect.     

Energy-transducing membrane-bound coupling factor-ATPase from Mycobacterium phlei. I. Purification, homogeneity, and properties.

The membrane-bound coupling factor from Mycobacterium phlei was solubilized from membrane vesicles by washing with low ionic strength buffer or 0.25 M sucrose. The solubilized enzyme exhibited coupling factor, latent ATPase, and succinate oxidation-stimulating activity. Purification by affinity chromatography using Sepharose coupled to ADP yielded a homogeneous preparation of latent ATPase which was purified about 200-fold with an 84% yield in a single step. Purified latent ATPase exhibited coupling factor activity but no succinate oxidation-stimulating activity. The molecular weight of latent ATPase was determined to be 250,000 +/- 10,000 by Sephadex G-200 chromatography. The ATPase was unmasked by trypsin treatment and activated by Mg2+ ion. However, trypsin treatment inactivated the coupling factor activity in the purified enzyme, indicating that the catalytic sites for ATPase and coupling activity are different. Unlike mitochondrial ATPase, latent ATPase from M. phlei was not cold-labile. Of the nucleoside triphosphates, UTP, ITP, and epsilon-ATP (1-N6-ethenoadenosine triphosphate) were hydrolyzed to a lesser extent compared to ATP. Kinetic data showed that ADP acted as a competitive inhibitor of latent ATPase activity with a Ki of 5 x 10(-3) M. Uncouplers of oxidative phosphorylation and respiratory inhibitors did not affect the latent ATPase activity, while sodium azide (0.1 mM) inhibited the latent ATPase activity.     

Phosphotyrosine phosphatase: a novel phosphatase specific for phosphotyrosine, 2'-AMP and p-nitrophenylphosphate in rat brain

A unique phosphatase that selectively hydrolyzed phosphotyrosine and 2'-AMP at alkaline pH and p-nitrophenylphosphate at neutral pH was isolated from a cytosolic fraction of rat brain. The purified enzyme appeared homogenous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42,000. The molecular weight of the native enzyme was 45,000 as determined by molecular sieve chromatography. These findings indicate that the native enzyme is a monomer protein. At pH 8.6, the enzyme hydrolyzed L-phosphotyrosine, D-phosphotyrosine, 2'-AMP, p-nitrophenylphosphate, 3'-AMP, 2'-GMP, and 3'-GMP; the ratio of its activities with these substrates was 100:96:115:68:39:25:16. Its Km values for L-phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate were 0.8 X 10(-4) M, 1.4 X 10(-4) M, and 1.7 X 10(-4) M, respectively. At pH 7.4, the enzyme hydrolyzed p-nitrophenylphosphate, L-phosphotyrosine, and D-phosphotyrosine; the ratio of its activities with these compounds was 100:17:17, and its Km values for L-phosphotyrosine and p-nitrophenylphosphate were 1.8 X 10(-4) M and 2.0 X 10(-4) M, respectively. The enzyme activity was dependent on Mn2+ or Mg2+, and was strongly inhibited by 5'-nucleotides, pyrophosphate, and Zn2+. The enzyme was not sensitive to inhibitors of some well-characterized phosphatases such as NaF, molybdate, L(+)tartrate, tetramisole, vanadate, and lithium salt. The physiological role of the enzyme is discussed with respect to its activities toward phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate.     

Purification and properties of the ATPase solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

A novel ATPase was solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, with low ionic strength buffer containing EDTA. The enzyme was purified to homogeneity by hydrophobic chromatography and gel filtration. The molecular weight of the purified enzyme was estimated to be 360,000. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate revealed that it consisted of three kinds of subunits, alpha, beta, and gamma, whose molecular weights were approximately 69,000, 54,000, and 28,000, respectively, and the most probable subunit stoichiometry was alpha 3 beta 3 gamma 1. The purified ATPase hydrolyzed ATP, GTP, ITP, and CTP but not UTP, ADP, AMP, or p-nitrophenylphosphate. The enzyme was highly heat stable and showed an optimal temperature of 85 degrees C. It showed an optimal pH of around 5, very little activity at neutral pH, and another small activity peak at pH 8.5. The ATPase activity was significantly stimulated by bisulfite and bicarbonate ions, the optimal pH remaining unchanged. The Lineweaver-Burk plot was linear, and the Km for ATP and the Vmax were estimated to be 1.6 mM and 13 mumol Pi.mg.-1.min-1, respectively, at pH 5.2 at 60 degrees C in the presence of bisulfite. The chemical modification reagent, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, caused inactivation of the ATPase activity although the enzyme was not inhibited by N,N'-dicyclohexylcarbodiimide, N-ethyl-maleimide, azide or vanadate. These results suggest that the ATPase purified from membranes of S. acidocaldarius resembles other archaebacterial ATPases, although a counterpart of the gamma subunit has not been found in the latter. The relationship of the S. acidocaldarius ATPase to other ion-transporting ATPases, such as F0F1 type or E1E2 type ATPases, was discussed.     

Type B nucleoside-diphosphatase of rat brain. Purification and properties of an enzyme with high thiamin pyrophosphatase activity

Type B nucleoside-diphosphatase was purified from membranes of rat brain by solubilization with a non-ionic detergent and successive column chromatographies on DEAE-cellulose DE-52, concanavalin-A-Sepharose, Bio-Gel HT, blue-Sepharose CL-6B, chelating Sepharose 6B, Ultrogel AcA44 and TSK gel G3000 SW. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis and its molecular mass was estimated to be 75 kDa. It hydrolyzed thiamin diphosphate as well as GDP, IDP and UDP. Thiamin diphosphate (TPP) was hydrolyzed twice as efficiently as nucleoside diphosphates in the presence of Mn2+ at pH 7.4. The Km values for TPP, GDP, IDP and UDP were 0.66, 0.40, 0.54 and 1.06 mM respectively. ATP, ADP and pyridoxal 5'-phosphate inhibited thiamin-pyrophosphatase activity competitively and their Ki values were 2.3 mM, 1.0 mM and 0.59 mM respectively. The optimum pH of thiamin-pyrophosphatase activity was 7.4 in the presence of Mn2+ and that of GDP-hydrolytic activity was 6.5 in the presence of Mg2+.     

Characterization of the acetyl-CoA synthetase of Acetobacter aceti.

The acetate activating system of Acetobacter aceti has been studied. The enzyme responsible, acetyl-CoA synthetase, has been purified about 500-fold from crude cell extracts and was approximately 85% pure as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The purified enzyme showed optimal activity at pH 7.6 in both Tris-HCL and potassium phosphate buffers. In its purest form, the enzyme was stable at 4 degrees-C but denatured upon freezing. The Km values for CoA, ATP and acetate were found to be 0.104 mM, 0.36 mM and 0.25 mM respectively; propionate and acrylate were also activated by the enzyme but not butyrate, isobutyrate or valerate. GTP, UTP, CTP and ADP could not replace ATP in the reaction, and cysteine or pantetheine failed to replace CoA. The cationic requirements were studied and of the divalent cations tested, only Mn2+ could significantly replace Mg2+ in the reaction; K+ and NH4+ stimulated enzyme activity but inhibited at high concentrations; Na+ was a poor activator, but did not inhibit at higher concentrations. The effect of a number of glucose and other metabolites on enzyme activity has been tested.     

Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

Isolation and characterization of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum

A new enzyme that hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate has been purified by a factor of 250 from the acellular slime mold Physarum polycephalum. Activity was assayed radioisotopically with [3H]Ap4A. Isolation of the enzyme was facilitated by dye-ligand chromatography. The enzyme symmetrically hydrolyzes Ap4A to ADP and exhibits biphasic kinetics for the substrate with values for the apparent Km of 2.6 micro M and 37 micro M. The two values of Vmax differ by a factor of 10. Mg2+, Ca2+, and other divalent cations inhibit the activity with 40-80% inhibition occurring at 0.5 mM. Mg2+, at 0.5 mM, decreases both values of Vmax by 50%, decreases the low Km value by about 30%, and increases the high Km value by about 100%. (Ethylenedinitrilo)tetraacetic acid (EDTA) and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), at 10 mM, inhibit the activity by 50%. ADP, ATP, Ap4, and Gp4 are equipotent inhibitors with 50% inhibition occurring at 30 micro M. AMP is a relatively weak inhibitor. The molecular weight of the enzyme is 26000 on the basis of elution of activity from a calibrated Sephadex G-75 column.     

Kinetic studies on the membrane-bound and the purified coupling factor-ATPase from Rhodopseudomonas sphaeroides

The activity of membrane-bound and purified ATPase (EC 3.6.1.3) was potentiated by several divalent cations. Highest rates of ATP hydrolysis were obtained when the activity was measured with the (cation-ATP)2- complex. Free ATP and free divalent cations in excess were found to be competitive inhibitors to the complex. The apparent Km (complex) values were lower than the Ki values for free ATP indicating that the (cation-ATP)2- complex is bound more tightly to the enzyme than the free ATP. Based on these results, a binding of the complex to the active site at two points is suggested, namely through the ATP and through the cation. Removal of the coupling factor from the membrane apparently caused conformational changes which resulted in a pronounced alteration of the kinetic parameters of ATPase activity. Whereas highest values in chromatophore-bound ATPase activity were observed in the presence of Mg2+, the purified enzyme became even more active in the presence of Ca2+. The Ki values for free ATP decreased upon solubilization of the enzyme. Free Mg2+ in excess was more inhibitory on the purified ATPase than Ca2+, while free Ca2+ in excess was more inhibitory on the membrane-bound enzyme if compared to Mg2+. Ki values for product inhibition by ADP and Pi were determined. Kinetic analyses of photophosphorylation activity revealed that the (cation-ADP)- complex is the functional substrate. The apparent Km values for the complex and for Pi were estimated. Excess of free cations and ADP inhibited competitively the phosphorylation. Ki(ADP), Ki(Ca2+), and Ki(Mg2+) were calculated by Dixon analyses.     

Sulphur metabolism in Paracoccus denitrificans. Purification, properties and regulation of serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase.

1. Serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase were purified from Paracoccus denitrificans strain 8944. 2. Serin transacetylase was purified 150-fold. The enzyme has a pH optimum between 7.5 and 8.0, is specific for L-serine and is inhibited by sulphydryl-group reagents. The apparent Km values for serine and acetyl-CoA are 4.0 - 10(-4) and 1.0 - 10(-4) M, respectively. Serine transacetylase is strongly inhibited by cysteine. 3. O-Acetylserine sulphydrylase was purified 450-fold. The enzymes has a sharp pH optimum at pH 7.5. In addition to catalysing the synthesis of cysteine, O-acetylserine sulphydrylase catalyses the synthesis of selenocysteine from O-acetylserine and selenide. The Km values for sulphide and O-acetylserine are 2.7 - 10(-3) and 1.25 - 10(-3) M, respectively. The enzyme was stimulated by pyridoxal phosphate and was inhibited by cystathionine, homocysteine and methionine. 4. beta-Cystathionase was purified approx. 50-fold. beta-Cystathionase has a pH optimum between pH 9.0 and 9.5, is sensitive to sulphydryl-group reagents, required pyridoxal phosphate for maximum activity and has an apparent Km for cystathionine of 4.2 - 10 (-3) M. beta-Cystathionase also catalyses the release of keto acid from lanthionine, djenkolic acid and cystine. Cysteine, O-acetylserine, homocysteine and glutathione strongly inhibit beta-cystathionase activity and homocysteine and methionine represses enzyme activity. 5. O-Acetylserine lyase was identified in crude extracts of Paracoccus denitrificans. The enzyme is specific for O-acetyl-L-serine, requires pyridoxal phosphate and is inhibied by KCN and hydroxylamine. The enzyme has a high Km value for O-acetylserine (50--100 mM).     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

Purification and Characterization of β-d-Galactosidase and α-d-Mannosidase from Papaya (Carica papaya) Seeds

β-D-Galactosidase was purified 115-fold from a saline extract of papaya seeds by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel-filtration on Sephadex G-75, G-150, and G-100. The purified β-D-galactosidase (MW, 56,000 daltons) had an isoelectric point (pI) at pH 8.4 and the optimal pH for its activity was 3.5 to 4.5. The enzyme activity was inhibited by Cu²⁺,Ag⁺,Hg²⁺,Pb²⁺,NaAsO₂ and р-chloromercuribenzoate at concentrations of 1x10^-3 M. Among the various mono- and oligosaccharides tested, D-galactose, D-galacturonic acid, D-galactono-γ-lactone and melibiose significantly inhibited the enzyme activities at concentrations of 2xl0^-3 to 1X10^-2M. The purified enzyme hydrolyzed β-nitrophenyl β-D-galactoside (Km = 1.0X10^-3M), methyl β-D-galactoside (Km=1.6x10^-2M), aminoethyl β-D-galactoside (Km =3.3X10^-2M) and lactose (Km = 9.1X10^-2M). β-(l→3)-Linked galactotetraosyl-eryth itol and asialo-glycopeptide isolated from fetuin were also hydrolyzed to the extent of 78 and 75%, 4respectively, on the basis of their galactose contents.

∝-D-Mannosidase from papaya seeds was also purified 130-fold by ammonium sulfate fractionation, DEAE-Sephadex chromatography, gel-filtration on Sephadex G-150 and hydroxylapatite chromatography. The purified enzyme (MW, 156,000 daltons), consisting of two subunits (78,000x2), was inhibited by Hg²⁺,Ag⁺,Cu²⁺, р-chloromercuribenzoate, D-glucose, D-glucosamine and D-mannose at concentrations of lx10^-3 to 1x10^-2M. The ∝-D-mannosidase hydrolyzed р-nitrophenyl ∝-D-mannoside (Km=5.6x10^-3M), methyl ∝-D-mannoside (Km=2.8X10^-2M), ∝-D-mannosyl-D-mannitol (Km=2.2X10^-2M), ∝-(l→2)linked D-mannobiosyl-D-mannitol (Km=6.3x10^-3M) and D-mannotriosyl-D-mannitol (Km=5.3x10^-3 M).     

Adenylate kinase activity in Mycobacterium leprae

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was detected in partially purified preparations of cell-free extracts of Mycobacterium leprae. The apparent Km values of M. leprae adenylate kinase for ADP and Mg2+ were 1 X 10(-4) M, respectively. The enzyme was heat-labile: loss of activity by 80% at 45 degrees C and over 90% at 60 degrees C occurred within 5 min. M. leprae adenylate kinase was distinct from armadillo adenylate kinase in respect of affinity for substrate and heat-sensitivity.     

A potent nucleoside triphosphate hydrolase from the parasitic protozoan Toxoplasma gondii. Purification, some properties, and activation by thiol compounds

A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.     

Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. kinetic properties of the basal and trypsin-stimulated activities

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.     

A membrane-bound ATPase from Halobacterium halobium: purification and characterization

An ATPase was newly identified on the inner face of the plasma membrane of the extremely halophilic archaebacterium Halobacterium halobium. The enzyme was released into an alkaline EDTA solution and purified by several chromatographic steps in the presence of sulfate at 1 M or over. The molecular weight of the native enzyme was around 320,000; it is most likely composed of two pairs (alpha 2 beta 2) of 86,000 (alpha) and 64,000 (beta) subunits. The enzyme hydrolyzed ATP and other nucleoside triphosphates but neither ADP nor AMP. The enzyme required divalent cations, among which Mn2+ was most effective (Mg2+ activated 35% of Mn2+). The ATPase activity was optimum at pH between 5.5 and 6, particularly in a nearly saturated Na2SO4 (or Na2SO3) solution, while it was very low in a chloride salt solution even at 4 M at any pH. The Km value for ATP was 1.4 mM and the K1 value for ADP (competitive to ATP) was 0.08 mM. Neither azide (a specific inhibitor for F0F1-and F1-ATPase) nor vanadate (for E1E2-ATPase) inhibited the enzyme. The ATPase was stable at high concentrations of sulfate. At low concentrations of salts, or at low temperatures even in high NaCl concentrations, the enzyme was inactivated. Although the ATPase isolated here from halobacterial membrane has such unusual characteristics, it is the most probable candidate for the (catalytic part of) halobacterial ATP synthase, which differs from F0F1-ATPase/synthase (Mukohata et al. (1986) J. Biochem. 99, 1-8; Mukohata and Yoshida (1987) J. Biochem. 101, 311-318).     

Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas

An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.     

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%.     

Pyruvate kinase in the bovine adrenal cortex

Pyruvate kinase from bovine adrenal cortex was purified to an electrophoretically homogeneous state. The molecular weight of the native enzyme is about 230 000, that of one subunit is 57 000. The maximal values of the pyruvate kinase initial reaction rate were obtained in 50 mM imidazole-acetate buffer within the pH range of 6.8 to 7.0. The curve of the initial pyruvate kinase reaction rate versus phosphoenolpyruvate (PEP) and ADP concentrations is hyperbolic and obeys the Michaelis-Menten kinetics with Km for PEP and ADP of 0.055 X 10(-3) M and 0.25 X 10(-3) M, respectively. The enzyme is activated by Mn2+ and Co2+ by 43 and 38%, respectively. IDP, GDP, and UDP may be used as analogs of ADP. The enzyme is not activated by fructose-1.6-diphosphate and is inhibited by L-phenylalanine and ATP.