Independent ATPase Research Articles

Hexameric structure of the ATPase motor subunit of magnesium chelatase in chlorophyll biosynthesis.

Magnesium chelatase (MgCh) is a heterotrimeric enzyme complex, composed of two AAA+ family subunits that can assembly into a double ring structure and a large catalytic subunit. The small AAA+ subunit has ATPase activity and can self-oligomerize into a ring structure, while the other AAA+ subunit lacks independent ATPase activity. Previous structural studies of the ATPase motor subunit of MgCh from a bacteriochlorophyll-synthesizing bacterium have identified a unique ATPase clade, but the model of oligomeric assembly is unclear. Here we present the hexameric structure of the MgCh ATPase motor subunit from the chlorophyll-synthesizing cyanobacterium Synechocystis sp. PCC 6803. This structure reveals details of how the hexameric ring is assembled, and thus provides a basis for further studying the heterotrimeric complex.

Substrate independent ATPase activity may complicate high throughput screening

Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening.

The plasma membrane Ca 2+ pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca 2+

The plasma membrane Ca 2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca 2+ at a rate near 1.5% of that attained at saturating concentrations of Ca 2+. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca 2+-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca 2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca 2+ and is catalyzed by E 2-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.

Asn792 Participates in the Hydrogen Bond Network Around the K+-binding Pocket of Gastric H,K-ATPase

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.

Impaired Ca2+ Store Functions in Skeletal and Cardiac Muscle Cells from Sarcalumenin-deficient Mice

Sarcalumenin (SAR), specifically expressed in striated muscle cells, is a Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) of the intracellular Ca2+ store. By generating SAR-deficient mice, we herein examined its physiological role. The mutant mice were apparently normal in growth, health, and reproduction, indicating that SAR is not essential for fundamental muscle functions. SAR-deficient skeletal muscle carrying irregular SR ultrastructures retained normal force generation but showed slow relaxation phases after contractions. A weakened Ca2+ uptake activity was detected in the SR prepared from mutant muscle, indicating that SAR contributes to Ca2+ buffering in the SR lumen and also to the maintenance of Ca2+ pump proteins. Cardiac myocytes from SAR-deficient mice showed slow contraction and relaxation accompanied by impaired Ca2+ transients, and the mutant mice exhibited a number of impairments in cardiac performance as determined in electrocardiography, ventricular catheterization, and echocardiography. The results obtained demonstrate that SAR plays important roles in improving the Ca2+ handling functions of the SR in striated muscle.

Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity.

Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.

A Conformation-specific Interhelical Salt Bridge in the K+ Binding Site of Gastric H,K-ATPase

Homology modeling of gastric H,K-ATPase based on the E2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E2 form-specific salt bridge between Glu820 (M6) and Lys791 (M5). In the E820Q mutant this salt bridge is no longer possible, and the head group of Lys791, together with a water molecule, fills the position of the K+ ion and apparently mimics the K+-filled cation binding pocket. This gives an explanation for the K+-independent ATPase activity and dephosphorylation step of the E820Q mutant (Swarts, H. G. P., Hermsen, H. P. H., Koenderink, J. B., Schuurmans Stekhoven, F. M. A. H., and De Pont, J. J. H. H. M. (1998) EMBO J. 17, 3029-3035) and, indirectly, for its E1 preference. The model is strongly supported by a series of reported mutagenesis studies on charged and polar amino acid residues in the membrane domain. To further test this model, Lys791 was mutated alone and in combination with other crucial residues. In the K791A mutant, the K+ affinity was markedly reduced without altering the E2 preference of the enzyme. The K791A mutation prevented, in contrast to the K791R mutation, the spontaneous dephosphorylation of the E820Q mutant as well as its conformational equilibrium change toward E1. This indicates that the salt bridge is essential for high-affinity K+ binding and the E2 preference of H,K-ATPase. Moreover, its breakage (E820Q) can generate a K+-insensitive activity and an E1 preference. In addition, the study gives a molecular explanation for the electroneutrality of H,K-ATPases.

Influence of Glycerol on Nitrogenase Reactions

The influence of glycerol on the ATPase reaction of nitrogenase and reduction of the substrate (acetylene) is studied. Glycerol inhibits the ATPase nitrogenase reaction dependent on an electron donor. The reaction rate is halved at a glycerol concentration of 11% in the medium when the solution viscosity increases only 1.31 times. The electron donor–independent (decoupled) ATPase reaction of nitrogenase is inhibited to a lesser extent. The activation energies (Ea) of reactions studied in the presence of glycerol are determined. Despite the inhibition effect, glycerol in a concentration of 7.5% does not affect the Ea of acetylene reduction. The introduction of glycerol significantly decreases the Ea of the electron donor-dependent ATPase reaction. In the absence of glycerol, this reaction limits the nitrogenase reaction: Ea = 14 ± 1.4 kcal/mol at temperatures higher than 21°C and Ea = 50 ± 10 kcal/mol at temperatures below 21°C, which are close to the Ea of acetylene reduction. In the presence of 7.5% glycerol, the Ea = 0.7 ± 0.6 kcal/mol at temperatures above 21°C and the Ea = 2.4 ± 0.6 kcal/mol at temperatures below 21°C. This indicates that the reactions of substrate-binding and ATPase sites are decoupled in the presence of glycerol, and the step of substrate reduction becomes the limiting step of the nitrogenase reaction. Glycerol also has a noticeable effect on the Ea of the electron donor-independent ATPase reaction and the shape of the plot of logw vs. 1/T for this reaction. The data obtained indicate the specific interaction of glycerol with nitrogenase in the region of the ATPase site perhaps due to the distortion of the structure of hydrogen bonds, and this interaction changes the limiting step of the nitrogenase reaction.

The E1/E2-preference of gastric H,K-ATPase mutants.

Gastric H,K-ATPase has, in the absence of ATP and added ions, a preference for the E(2) conformation. Mutations in the cation-binding pocket often result in a preference for the E(1)-conformation. This can be paralleled by the occurrence of K(+)-independent ATPase activity. These two phenomena could be separated by combined mutagenesis of several residues in and around the cation-binding pocket. Models of the three-dimensional structure of H,K-ATPase visualize the relationship between the E(1)/E(2) preference and the structure.

Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+-ATPase activity.

The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+,K+-ATPase assay (Beltowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+-stimulated and Na+-independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain-sensitive H+,K+-ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H+,K+-ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+,K+-ATPase was stimulated by K+ with Km of 0.26 +/- 0.04 mM and 0.69 +/- 0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 +/- 0.3 microM in the renal cortex and 1.9 +/- 0.4 microM in the renal medulla) and by Sch 28080 (Ki of 1.8 +/- 0.5 microM and 2.5 +/- 0.9 microM in cortex and medulla, respectively). We found that ouabain-sensitive H+,K+-ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+,K+-ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+,K+-ATPase activity by 32.1% at 1 h after injection but had no effect on H+,K+-ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.

K+-independent Gastric H+,K+-ATPase Activity: DISSOCIATION OF K+-INDEPENDENT DEPHOSPHORYLATION AND PREFERENCE FOR THE E1 CONFORMATION BY COMBINED MUTAGENESIS OF TRANSMEMBRANE GLUTAMATE RESIDUES

Several mutations of residues Glu(795) and Glu(820) present in M5 and M6 of the catalytic subunit of gastric H(+),K(+)-ATPase have resulted in a K(+)-independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for the E(1) conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu(343), present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K(+)-activated ATPase activity and an E(2) preference and a Gln giving K(+)-independent ATPase activity and an E(1) preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of the E(2) <--> E(1) equilibrium was still dominated by the 820 residue: a Glu giving E(2) and a Gln giving E(1). This indicates that the preference for the E(1) conformation of the E820Q mutation is independent of an active dephosphorylation process.

Ectonucleotide Diphosphohydrolase Activities in Hemocytes of Larval Manduca sexta

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 ± 0.94 nmol of Pi/h × 106 cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 ± 1.74 nmol of Pi/h × 106 cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP−4 and Mg-ATP2− were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.

Interaction of the Bacteriophage T4 Gene 59 Helicase Loading Protein and Gene 41 Helicase with Each Other and with Fork, Flap, and Cruciform DNA

Interaction of the Bacteriophage T4 Gene 59 Helicase Loading Protein and Gene 41 Helicase with Each Other and with Fork, Flap, and Cruciform DNA

Ectonucleotide Diphosphohydrolase Activities in Entamoeba histolytica

In this work, we describe the ability of living cells of Entamoeba histolytica to hydrolyze extracellular ATP. In these intact parasites, whose viability was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol Pi/h/105 cells). Interestingly, in the presence of 5 mM MgCl2 an ecto-ATPase activity of 300 nmol Pi/h/105 cells was observed. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.23 mM MgCl2. Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl2 and CaCl2 but not by SrCl2, ZnCl2, or FeCl3. In fact, FeCl3 inhibited both Mg2+-dependent and Mg2+-independent ecto-ATPase activities. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.4 and 8.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced concomitantly with the increase in pH. In order to discard the possibility that the ATP hydrolysis observed was due to phosphatase or 5′-nucleotidase activities, several inhibitors for these enzymes were tested. Sodium orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg2+ (basal activity), the apparent Km for ATP4− was 0.053 ± 0.008 mM, whereas at saturating MgCl2 concentrations, the corresponding apparent Km for Mg-ATP2− for Mg2+-dependent ecto-ATPase activity (difference between total and basal ecto-ATPase activity) was 0.503 mM ± 0.062. Both ecto-ATPase activities were highly specific for ATP and were also able to hydrolyze ADP less efficiently. To identify the observed hydrolytic activities as those of an ecto-ATPase, we used suramin, a competitive antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, as well as the impermeant agent 4′-4′-diisothiocyanostylbenzene-2′-2′-disulfonic acid. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents, and the inhibition by both agents was prevented by ATP. A comparison among the ecto-ATPase activities of three amoeba species showed that the noninvasive E. histolytica and the free-living E. moshkovskii were less efficient than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytica is known to have a galactose-specific lectin on its surface, which is related to the pathogenesis of amebiasis, galactose was tested for an effect on ecto-ATPase activities. It stimulated the Mg2+-dependent ecto-ATPase but not the Mg2+-independent ATPase activity.

Measurement of Sarcoplasmic Reticulum Ca2+-ATPase Activity and E-Type Mg2+-ATPase Activity in Rat Heart Homogenates

The presence of a high and nonlinear Ca2+-independent (or basal) ATPase activity in rat heart preparations makes difficult the reliable measurement of sarcoplasmic reticulum (SR) Ca2+-ATPase activity by usual methods. A spectrophotometric assay for the accurate determination of SR Ca2+-ATPase activity in unfractionated homogenates from rat heart is described. The procedure is based on that reported by Simonides and van Hardeveld (1990,Anal. Biochem.191, 321–331) for skeletal muscle homogenates. To avoid overestimation of the Ca2+-ATPase activity of cardiac homogenates that occurs when sequential measurements of total and basal ATPase activities are performed, two parallel and independent assays are required: one with low (micromolar) and other high (millimolar) calcium concentration. Addition of thapsigargin (0.2 μM) blocked totally the activity considered as Ca2+-ATPase activity. Using this method, the rat heart homogenate Ca2+-ATPase activity was 10.5 ± 2.0 μmol · min−1· g−1tissue wet weight (n= 8). Likewise, a spectrophotometric assay for measuring E-type Mg2+-ATPase activity in cardiac total homogenates has been developed, comparing the following characteristics of the enzymatic activity in homogenate and a membrane-enriched fraction: first-order rate constant for ATP-dependent inactivation,Kmfor ATP, and effects of concanavalin A, Triton X-100, and specific inhibitors.

Origin of concurrent ATPase activities in skinned cardiac trabeculae from rat.

1. To determine the rate of ATP turnover by the sarcoplasmic reticulum (SR) Ca2+ pump in cardiac muscle, and to assess the contributions of other ATPase activities to the overall ATP turnover rate, ATPase activity and isometric force production were studied in saponin-skinned trabeculae from rat. ATP hydrolysis was enzymatically coupled to the oxidation of NADH; the concentration of NADH was monitored photometrically. All measurements were performed at 20 +/- 1 degrees C and pH 7.0. Resting sarcomere length was adjusted to 2.1 microns. All solutions contained 5 mM caffeine to ensure continuous release of Ca2+ from the SR. 2. The Ca(2+)-independent ATPase activity, determined in relaxing solution (pCa 9), amounted to 130 +/- 13 microM s-1 (mean +/- S.E.M., n = 7) at the beginning of an experiment. During subsequent measurements in relaxing solution, a decrease in ATPase activity was observed, indicative of loss of membrane-bound ATPase activity. The steady-state Ca(2+)-independent (basal) ATPase activity was 83 +/- 5 microM s-1 (n = 66). 3. Treatment of saponin-skinned preparations with Triton X-100 abolished 50 microM s-1 (60%) of the basal ATPase activity. Addition of ouabain (1 mM) suppressed 14 +/- 5% of the basal activity, whereas 8 +/- 3% was suppressed by 20 microM cyclopiazonic acid (CPA). It is argued that 31 microM s-1 of the basal ATPase activity may be associated with MgATPase from the transverse tubular system. 4. The maximal Ca(2+)-activated ATPase activity, i.e. the total ATPase activity (determined in activating solution, pCa 4.3) corrected for basal ATPase activity, was found to be 409 +/- 15 microM s-1 (n = 66). Experiments with CPA indicated that at least 9 +/- 6% of the maximal Ca(2+)-activated ATPase activity originates from the sarcoplasmic Ca2+ pump. These experiments indicate that the rate of ATP consumption by the SR Ca2+ transporting ATPase amounts to at least 37 microM s-1. 5. Treatment of preparations with Triton X-100 abolished 15 +/- 3% of the maximal Ca(2+)-activated ATPase activity, indicating that 15 +/- 3% of the maximal Ca(2+)-activated ATPase activity is membrane bound. 6. Variation of free [Ca2+] indicated that apart from the actomyosin ATPase activity a second Ca(2+)-dependent ATPase activity contributed to the overall ATP turnover rate. This activity was half-maximal at pCa 6.21, and probably reflects the SR Ca2+ transporting ATPase. It constituted 18 +/- 3% of the Ca(2+)-dependent ATPase activity, yielding an upper limit for the SR Ca2+ transporting ATPase activity of 74 microM s-1.

The effect of lipid peroxidation on the activity of various membrane-bound ATPases in rat kidney

Peroxidation of membrane phospholipids is accompanied by alteration of the structural and functional characteristics of membranes. Lipid peroxidation changes the activities of various enzymes. The present study evaluates the effect of lipid peroxidation on the activity of various ATPases localized on kidney membranes. Our experiments were performed on crude preparation of rat kidney membranes which were exposed to Fe 2+•ADP/NADPH-induced lipid peroxidation. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases were determined and divided according to their ouabain sensitivity and Mg 2+ dependency. We found that 10 min incubation of isolated rat kidney membranes at 37°C with inductors of lipid peroxidation increased the production of thiobarbituric acid-reactive substances from 1.10 ± 0.26 to 7.72 ± 2.55 nmol malondialdehyde/mg prot. (±SD, n = 4). Under these conditions total ATPase activity was decreased from 681 ± 77 to 507 ± 82, ouabain-sensitive Mg 2+-dependent ATPase (Na +,K +-ATPase) activity from 249 ± 54 to 81 ± 21 and ouabain-insensitive Mg 2+-dependent activity from 287 ± 48 to 173 ± 58 whereas apparently Mg 2+-independent ATPase activity was increased from 145± 37 to 253 ± 42 nmoles P/min/mg prot. (±SD, n = 4). The study indicates different mechanisms by which lipoperoxides affect the function of membrane-bound ATPases activities. It is concluded that the ATPases activities are changed during lipid peroxide formation.

Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown.

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (Pi) from the breakdown of extracellular ATP. Enzyme activity involved Ca2+/Mg(2+)-dependent and Ca2+/Mg(2+)-independent dephosphorylation of ATP. Ca2+/Mg(2+)-dependent ecto-ATPase was active over a limited range of 0.01-1.0 mM ATP, while Ca2+/Mg(2+)-independent ATPase activity was active over a range of 0.1-30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0-9.0), but increased in a relatively linear manner with: (1) time of reaction (0-90 min), (2) number of cells (1-20 oocytes), and (3) temperature (10-37 degrees C). Ecto-ATPase activity was unaffected by ouabain (100 microM), sodium azide (100 microM), and oligomycin (5 micrograms/ml) (as inhibitors of endo-ATPases) and beta-glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP>>alpha, beta-methylene ATP, beta, gamma-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P2-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido)napthalene-1,3,5-trisul phonic acid), 100 microM] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca2+/Mg(2+)-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonate-dependent ATP cleavage catalysed by pyruvate carboxylase in the absence of pyruvate.

Preparations of pyruvate carboxylase catalyse the cleavage of MgATP in the absence of pyruvate and acetyl-CoA. The rate of this cleavage is higher in the presence of HCO3- than in its absence. Incubation of the enzyme preparations with an excess of the pyruvate carboxylase inhibitor, avidin, completely abolishes the pyruvate carboxylating activity of the enzyme preparations but only abolishes the HCO3(-)-dependent MgATP cleaving activity, with no effect on the HCO3(-)-independent ATPase activity. The HCO3(-)-dependent MgATP cleavage is also sensitive to inhibition by a pyruvate carboxylase inhibitor, oxamate, and the dependence of the reaction on the free Mg2+ concentration is similar to that of the pyruvate-carboxylation reaction, whereas the HCO3(-)-independent MgATP cleavage is not dependent on the concentration of free Mg2+ in the range tested. This indicates that MgATP cleavage by pyruvate carboxylase is entirely dependent on the presence of HCO3- and that there may be a low level of ATPase contamination in the enzyme preparations. In addition, inhibition of the HCO3(-)-dependent MgATP cleavage by both avidin and oxamate indicate that although biotin does not directly participate in the reaction, its presence is required in that part of the active site of the enzyme. The rate of HCO3(-)-dependent MgATP cleavage is about 0.07% of that of the full pyruvate carboxylation reaction under similar conditions with saturating substrates. The reaction mechanism is sequential with respect to MgATP and HCO3- addition and Mg2+ adds at equilibrium before MgATP. Acetyl-CoA stimulates the HCO3(-)-dependent MgATP cleavage at low MgATP concentrations, with the stimulation being greater at low Mg2+ concentrations. At high levels of MgATP in the presence of acetyl-CoA, substrate inhibition is evident and is more pronounced at increasing concentrations of Mg2+. This inhibition appears to be, at least in part, caused by inhibition of decarboxylation of the enzyme-carboxybiotin complex by the binding to this complex of Mg2+ and MgATP, which probably act to reduce the rate of movement of carboxybiotin from the site of the MgATP cleavage reaction to that of the pyruvate carboxylation reaction where it is unstable and decarboxylates.

Demonstration of the simultaneous activation of Ca 2+-independent and Ca 2+-dependent ATPases from rat skeletal muscle microsomes

The activation of the Ca 2+-indenpendent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca 2+ to provide the simultaneous activation of the Ca 2+ + Mg 2+-ATPase . It was achieved taking advantage of the delayed inorganic phosphate (P i) release due to the formation of a phosphoenzyme complex during the Ca 2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2°C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The P i effluent profile coincides with the pattern of the pulse when it activates only the Ca 2+-independent ATPase, it appears delayed when the pulse activates only extra P i production by the (Ca 2+ + Mg 2+)-ATPase , and it includes a rapid and a delayed component when both Ca 2+-independent and Ca 2+-dependent ATPases are activated simultaneously by the pulse.