Mechanism of Action of Pseudomonas syringae Phytotoxin, Syringomycin : Stimulation of Red Beet Plasma Membrane ATPase Activity

Syringomycin, a peptide toxin produced by the phytopathogen Pseudomonas syringae pv syringae preferentially stimulated (2-fold) the vanadate-sensitive ATPase activity associated with the plasma membrane of red beet storage tissue. The toxin had a very slight effect on the tonoplast ATPase and had no detectable effect on the mitochondrial ATPase. Optimal stimulation was achieved with 10 to 50 micrograms of syringomycin per 25 micrograms of membrane protein. Treatment of membranes with 0.1% (weight/volume) deoxycholate eliminated the activation effect, and enzyme solubilized with Zwittergent 3-14 was not affected by syringomycin. ATPase activity was activated to the same extent at KCl concentrations ranging from 0 to 50 millimolar. Valinomycin, nigericin, carbonylcyanide p-trifluoromethoxyphenylhydrazone, and gramicidin did not increase the plasma membrane ATPase activity. However, these ionophores did not hinder the ability of syringomycin to stimulate the activity. We suggest that syringomycin does not increase ATPase activity by altering membrane ion gradients nor directly interacting with the enzyme, but possibly through regulatory effectors or covalent modification of the enzyme.     

Purification and Properties of the Plasma Membrane H-Translocating Adenosine Triphosphatase of Phaseolus mungo L. Roots

The plasma membrane ATPase of mung bean (Phaseolus mungo L.) roots has been solubilized with a two-step procedure using the anionic detergent, deoxycholate (DOC) and the zwitterionic detergent, zwittergent 3-14 as follows: (a) loosely bound membrane proteins are removed by treatment with 0.1% DOC; (b) The ATPase is solubilized with 0.1% zwittergent in the presence of 1% DOC; (c) the solubilized material is further purified by centrifugation through a glycerol gradient (45-70%). Typically, about 10% of the ATPase activity is recovered, and the specific activity increases about 11-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the peak fraction from the glycerol gradient contains three major polypeptides of M_r = 105,000, 67,000, and 57,000 daltons. The properties of the purified ATPase are essentially the same as those of membrane-bound ATPase, with respect to pH optimum, substrate specificity, inhibitor sensitivity, and ion stimulation.     

Phosphorylation of the adenosine triphosphatase in a deoxycholate-treated plasma membrane fraction from corn roots

The ATP phosphohydrolase (ATPase) activity of a corn (Zea mays L., WF9 × Mo17) root plasma membrane fraction was enriched almost 2-fold by selective extraction with 0.1% (w/v) deoxycholate. The detergent treatment solubilized about 30% of the total membrane protein and some ATP hydrolyzing activity that was not K⁺-stimulated, but the major portion of the ATPase activity could be pelleted with membranes. The properties of the ATPase associated with the detergent-extracted plasma membrane fraction were similar to those for the ATPase of the untreated plasma membrane fraction with respect to substrate specificity, pH optimum, kinetics with MgATP, ion stimulation, and inhibitor sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only minor differences in protein composition resulting from the detergent treatment.

The plasma membrane fraction from corn roots contained an endogenous protein kinase activity. This was shown by the time course of phosphate incorporation and by the labeling of a number of protein bands on SDS-polyacrylamide gel electrophoresis. The deoxycholate treatment removed measurable protein kinase activity and allowed the demonstration of a rapidly turning over covalent phosphorylated intermediate associated with the detergent-extracted plasma membrane fraction. The phosphorylated intermediate was present as a 100,000 dalton polypeptide and may represent the catalytic subunit of the plasma membrane K⁺-ATPase.

     

Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction

The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-beta-d-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg(2+), was further stimulated by K(+), was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K(+)-stimulated ATPase activity. Low concentrations of each detergent, including octyl-beta-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity.     

The purification and subunit structure of a membrane-bound ATPase from the Archaebacterium Halobacterium saccharovorum

A membrane-bound ATPase from Halobacterium saccharovorum was solubilized using sodium deoxycholate and Zwittergent 3-10 and purified by hydrophobic and ammonium sulfate-mediated chromatography. The enzyme, which had a molecular mass of 350 kDa, was composed of two major (87 and 60 kDa) and two minor (29 kDa and 20 kDa) subunits. The halobacterial ATPases appear to be unlike any other ATPase described to date.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

Plasma membrane ATPase of sugarbeet

A membrane fraction enriched with magnesium-dependent ATPase activity was isolated from sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI and sucrose density gradient centrifugation. This activity was inhibited by vanadate, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol, but was insensitive to molybdate, azide, oligomycin, ouabain, and nitrate, suggesting enrichment in plasma membrane ATPase. The enzyme was substrate specific for ATP, had a pH optimum of 7.0, but showed little stimulation by 50 mM KCl. The sugarbeet ATPase preparation contained endogenous protein kinase activity which could be reduced by extraction of the membranes with 0.1% (w/v) sodium deoxycholate. Reduction of protein kinase activity allowed the demonstration of a rapidly turning over phosphorylated intermediate on a M_r 105000 polypeptide, most likely representing the catalytic subunit of the ATPase. Phosphorylation was magnesium dependent, sensitive to diethylstilbestrol and vanadate but insensitive to oligomycin and azide. Neither the ATPase activity nor phosphoenzyme level were affected by combinations of sodium and potassium in the assay. These results argue against the presence of a synergistically stimulated NaK-ATPase at the plasma membrane of sugarbeet.     

Purification and characterization of the plasma membrane ATPase of Neurospora crassa

The plasma membrane of Neurospora crassa contains a proton-translocating ATPase, which functions to generate a large membrane potential and thereby to drive a variety of H+-dependent co-transport systems. We have purified this ATPase by a three-step procedure in which 1) loosely bound membrane proteins are removed by treatment with 0.1% deoxycholate; 2) the ATPase is solubilized with 0.6% deoxycholate in the presence of 45% glycerol; and 3) the solubilized enzyme is purified by centrifugation through a glycerol gradient. This procedure typically yields approximately 30% of the starting ATPase activity in a nearly homogeneous enzyme preparation of high specific activity, 61-98 mumol/min/mg of protein. The membrane-bound and purified forms of the ATPase are very similar with respect to kinetic properties (pH optimum, nucleotide and divalent cation specificity, sigmoid dependence upon Mg-ATP concentration) and sensitivity to inhibitors (including N,N'-dicyclohexylcarbodiimide and vanadate). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified ATPase displays a single major polypeptide band of Mr = 104,000, which is essentially identical in its electrophoretic mobility with the large subunit of [Na+, K+]-ATPase of animal cell membranes and [Ca2+]-ATPase of sarcoplasmic reticulum. The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.     

Solubilization and immunochemical identification of mitochondrial glycerol-3-phosphate dehydrogenase

Lysophosphatidylcholine (contrary to Lubrol WX, Triton X-100, digitonine and deoxycholate) solubilizes hamster brown fat mitochondrial glycerol-3-phosphate dehydrogenase without inactivation. Optimal ratio of lysophosphatidylcholine and membrane protein for solubilization of the enzyme was found to be 0.25 mg of lysophosphatidylcholine per mg protein. The activity of solubilized enzyme, however, was not affected by low concentrations of Lubrol WX, Triton X-100, digitonine, Zwittergent TM 314. Deoxycholate exhibited a pronounced inactivating effect. One-dimensional immunoelectrophoresis of the solubilized membrane proteins revealed 10 protein bands, 3-4 of which exhibited the enzyme activity. Two-dimensional immunoelectrophoresis revealed only a single main band of glycerol-3-phosphate dehydrogenase. This technique thus appears to be the best means for the identification of glycerol-3-phosphate dehydrogenase in the mixture of solubilized membrane proteins and for concentration of the enzyme activity in one major precipitating band.     

Mechanisms of detergent effects on membrane-bound (Na+ + K+)-ATPase

Because the nonionic detergent octaethylene glycol dodecyl ether has been used extensively for studies on active solubilized preparations of (Na+ + K+)-ATPase, we tried to see if the detergent alters the properties of the membrane-bound enzyme prior to solubilization. Addition of the detergent, at concentrations below its critical micellar concentration, to reaction mixtures containing the highly purified membrane-bound enzyme reduced the K0.5 of ATP for (Na+ + K+)-dependent ATPase activity without affecting the maximal velocity or abolishing the negative cooperativity of the substrate-velocity curve. Under these conditions, however, the enzyme was not solubilized as evidenced by complete sedimentation of the membrane fragments containing the enzyme upon centrifugation at 100,000 X g for 30 min. Other nonsolubilizing effects of the detergent included an increase in K0.5 of K+, inhibition of Na+-dependent ATPase with no effect on K0.5 of ATP for this activity, and reductions in the spontaneous decomposition rates of the K+-sensitive phosphoenzyme obtained from ATP and the phosphoenzyme obtained from Pi. The nonsolubilizing effects of the detergent on the purified enzyme were obtained with no detectable lag, were readily reversible, and could be distinguished from its vesicle-opening effects on crude membrane preparations. Several other nonionic and ionic detergents had similar effects on the enzyme. The findings indicate (a) detergent binding to hydrophobic sites on extramembranous segments of enzyme subunits; (b) that occupation of these sites mimics the effects of ATP at a low-affinity regulatory site with no effect on high-affinity ATP binding to the catalytic site; and (c) that in studies on detergent-solubilized preparations, it is necessary to distinguish between the effects of solubilization per se and detergent effects at the regulatory site.     

Solubilization of UDPglucose-ceramide glucosyltransferase from the Golgi apparatus

The choice of a suitable detergent for solubilization of UDPglucose-ceramide glucosyltransferase from Golgi membranes has been investigated. Among the various classes of detergent, CHAPS, a zwitterionic detergent, was used as it produced a substantial activation of the enzyme activity. 30% of the enzyme activity and 50% of proteins were solubilized in the first attempts. Further experiments were conducted with addition of a second detergent, Zwittergent 3-14 which increased enzyme recovery to 45%. Lastly, reducing the concentrations of buffer and divalent cations Mn2+, Mg2+ and introducing glycerol (20%, v/v) allowed 80% of proteins to be solubilized together with 68% of the ceramide glucosyltransferase activity.     

Bacterial phytotoxins, syringomycin, syringostatin and syringotoxin, exert their effect on the plasma membrane H+-ATPase partly by a detergent-like action and partly by inhibition of the enzyme

Syringostatin is a newly discovered phytotoxin produced by a phytopathogenic bacterium Pseudomonas syringae pv. syringae lilac isolate. The effects of syringostatin and the similar phytotoxins, syringomycin and syringotoxin, on H⁻-ATPase activity were investigated using cultured mung bean ( Vigna radiata L. cv. Ryokuto) cells or plasma membrane vesicles isolated from mung bean hypocotyls. ³¹P-NMR analysis of cultured cells treated with syringostatin revealed that the cytoplasmic pH was decreased. When plasma membrane was prepared by a two-step method (Dextran gradient followed by a sucrose gradient). syringostatin, syringomycin and syringotoxin inhibited the H⁺-ATPase activity in a dose-dependent manner. In contrast, these toxins stimulated H⁺-ATPase activity when plasma membrane was prepared by a one-step method (sucrose gradient). While these toxins inhibited the H⁺-ATPase activity of inside-out plasma membrane vesicles, the H⁺-ATPase activity of right-side-out vesicles was stimulated. The detergent. Triton X-100, abolished this stimulatory effect of the toxins on the H⁺-ATPase of right-side-out vesicles and of one-step purified plasma membrane. The toxins also inhibited the activity of the plasma membrane H⁺-ATPase solubilized with deoxycholate and Zwittergent 3–14. Taken together, these results indicate that these toxins exert their effects partly by a detergent-like action on the plasma membrane and partly by inhibition of the enzyme.     

Effects of solubilization on the inhibition of the p-type ATPase from maize roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.     

Selective delipidation of the plasma membrane by surfactants : enrichment of sterols and activation of ATPase

The influence of plasma membrane lipid components on the activity of the H⁺-ATPase has been studied by determining the effect of surfactants on membrane lipids and ATPase activity of oat (Avena sativa L.) root plasma membrane vesicles purified by a two-phase partitioning procedure. Triton X-100, at 25 to 1 (weight/weight) Triton to plasma membrane protein, an amount that causes maximal activation of the ATPase in the ATPase assay, extracted 59% of the membrane protein but did not solubilize the bulk of the ATPase. The Triton-insoluble proteins had associated with them, on a micromole per milligram protein basis, only 14% as much phospholipid, but 38% of the glycolipids and sterols, as compared with the native membranes. The Triton insoluble ATPase could still be activated by Triton X-100. When solubilized by lysolecithin, there were still sterols associated with the ATPase fraction. Free sterols were found associated with the ATPase in the same relative proportions, whether treated with surfactants or not. We suggest that surfactants activate the ATPase by altering the hydrophobic environment around the enzyme. We propose that sterols, through their interaction with the ATPase, may be essential for ATPase activity.     

Extraction of 11 beta-hydroxysteroid dehydrogenase from rat liver microsomes by detergents

In these studies our goal was to solubilize the microsomal enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD) as the first step in its purification. Enzyme was extracted from rat liver microsomes with representative detergents (Zwittergents, Tritons, modified sterols). Oxidation-reduction (O-R) ratios of extracts varied with detergent used and ranged from 0.18 (CHAPS) to 3.8 (Zwittergent 3-14) relative to a ratio of 1.7 in intact microsomes. All detergents solubilized 11-HSD using lack of sedimentation during high speed centrifugation as criterion. With Triton DF-18 and Triton X-100, optimum extraction of 11-HSD occurred in the detergent-protein ratio range of 0.1 to 0.2 O-R ratios decreased with increased Triton X-100, but were constant as Triton DF-18 was varied. The pH optimum of enzyme extraction was 9 at a detergent-protein ratio of 0.05 and 7.5-8.0 at a ratio of 0.2. Sodium chloride increased enzyme extraction by detergents; in the absence of detergent, salt extracted protein, but not enzyme. In aqueous solution at 0 degrees C or -15 degrees C, microsomal 11-oxidation activity rose within 24 h, then decreased; reductase activity consistently decreased. Oxidation and reduction activities were inversely related in the microsomal bound enzyme. No relationship between these activities appeared in detergent-solubilized enzymes. Possible mechanisms to account for the unexpected behavior of this enzyme are discussed.     

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.     

Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum.

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37 degrees C, with 50% inhibition occurring at 22 microM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 micrograms/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 microM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.     

Role of lipids in sarcoplasmic reticulum: A higher lipid content is required to sustain phosphoenzyme decomposition than phosphoenzyme formation

Enzyme preparations with variable phospholipid contents were obtained by removing lipids from sarcoplasmic reticulum with deoxycholate. Preparations containing from 90 to 37 phospholipids per enzyme showed normal values of both Ca²⁺-ATPase activity and steady-state phosphoenzyme levels. Fractions containing 37 to 23 phospholipids per enzyme had a reduced ATPase activity but normal phosphoenzyme levels, showing that in this range of lipid content the ATPase reaction is inhibited in a reaction step subsequent to phosphoenzyme formation but prior to phosphoenzyme decomposition. Delipidation below 23 lipids per enzyme caused a marked reduction of the amount of phosphoenzyme formed, so that although both reactions require lipids, fewer lipids are required for phosphoenzyme formation than for decomposition. The effect of lipid removal could be completely reversed by readdition of lipids to fractions containing more than 11 lipids per enzyme. It is proposed that phosphoenzyme formation requires full occupancy of a boundary domain of 23 lipids per enzyme, and that the selective inhibition of phosphoenzyme decomposition at higher lipid contents is caused by a decrease in the rotational mobility of the enzyme.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane.

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).