Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca(2+)-stimulated ATPase (adenosine triphosphatase), a Mg(2+)-stimulated ATPase, and an Na(+)+K(+)-stimulated Mg(2+)-dependent ouabain-sensitive ATPase. 2. The Ca(2+)-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3-4mm-Ca(2+) when 4mm-ATP was present as a substrate, and was not influenced by Na(+), K(+) or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca(2+)-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg(2+), Ca(2+), Mn(2+), Co(2+), Sr(2+), Ba(2+), Zn(2+), Cu(2+).     

Reactivity of sarcoplasmic reticulum from rabbit cardiac muscle with 1-fluoro- and 1,5-difluoro- 2,4-dinitrobenzene

Sarcoplasmic reticulum preparations from rabbit cardiac and fast skeletal muscle react differentially with low concentrations of 1-fluoro- and 1,5-difluoro-2,4-dinitrobenzene. Dinitrophenylation of cardiac sarcoplasmic reticulum by 1-fluoro-2,4-dinitrobenzene is not affected by Ca2+ and is limited to the lipoprotein-lipid region. This contrasts sharply with the predominant Ca2+-dependent dinitrophenylation of the ATPase protein of rabbit skeletal sarcoplasmic reticulum by this reagent. Formation of non-serial high mol. wt. oligomers by 1,5-difluoro-2,4-dinitrobenzene is significantly greater in cardiac than in skeletal vesicles. Substrate MgATP2- does not protect rabbit cardiac sarcoplasmic reticulum ATPase activity or Ca2+ uptake from dinitrophenylation when monofunctional and bifunctional reagents are used. Chemical differences in the overall structure of the two kinds of membrane preparations can be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the reactivity of these reagents.     

Localization of the membrane-associated thiol oxidase of rat kidney to the basal-lateral plasma membrane.

The localization of the membrane-associated thiol oxidase in rat kidney was investigated. Fractionation of the kidney cortex by differential centrifugation demonstrated that the enzyme is found in the plasma membrane. The crude plasma membrane was fractionated by density-gradient centrifugation on Percoll to obtain purified brush-border and basal-lateral membranes. Gamma-Glutamyltransferase, alkaline phosphatase and aminopeptidase M were assayed as brush-border marker enzymes, and (Na+ + K+)-stimulated ATPase was assayed as a basal-lateral-membrane marker enzyme. Thiol oxidase activity and distribution were determined and compared with those of the marker enzymes. Its specific activity was enriched 18-fold in the basal-lateral membrane fraction relative to its activity in the cortical homogenate, and its distribution paralleled that of (Na+ + K+)-stimulated ATPase. This association indicates that thiol oxidase is localized in the same fraction as (Na+ + K+)-stimulated ATPase, i.e. the basal-lateral region of the plasma membrane of the kidney tubular epithelium.     

Characterization and partial isolation of ouabain-insensitive Na(+) -ATPase in MDCK I cells

We show that MDCK I cells express, besides the classical (Na(+)+K(+))ATPase, a Na(+)-stimulated ATPase activity with the following characteristics: (1) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 23.12+/-1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, respectively). This enzyme forms a Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na(+)+K(+))ATPase with monoclonal anti-alpha(1) antibody reduced its activity in the supernatant by 90%; the Na(+)-ATPase activity was completely maintained. In addition, the formation of the Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na(+)-ATPase and (Na(+)+K(+))ATPase activities are independent, with Na(+)-ATPase belonging to a different enzyme entity.     

Inhibition of Na+,K+-ATPase in Penaeus indicus postlarvae by lead

The plasma membrane/mitochondrial fractions of Penaeus indicus postlarvae contain Mg2+-dependent ATPase, Na+,K+-stimulated ATPase, Na+-stimulated ATPase and K+-stimulated ATPase. The Na+,K+-activated, Mg2+-dependent ATPase was investigated further in relation to different pH and temperature conditions, and at various concentrations of protein, ouabain, ATP and ions in the incubation medium. In vitro and in vivo effects of lead were studied on the enzyme activity. In vitro lead inhibited the enzyme activity in a concentration-dependent manner with an IC50 value of 204.4 microM. In correlation with in vitro studies, in vivo investigations (both concentration and time dependent) of lead also indicated a gradual inhibition in enzyme activity. A maximum decrease of 85.3% was observed at LC50 (7.2 ppm) of lead for concentration-dependent experiments. In time-dependent studies, the decrease was maximal (81.7%) at 30 days of sublethal exposure (1.44 ppm). In addition, the substrate- and ion-dependent kinetics of Na+,K+-ATPase was studied in relation to in vitro exposure of lead; these studies suggest a non-competitive type of inhibition.     

Interaction of a new fluorescent reagent with sulfhydryl groups of the (Na+ + K+)-stimulate ATPase.

The (Na+ + K+)-ATPase enzyme of rat brain microsomes can be reversibly inhibited by a new fluorescent sulfhydryl (SH) probe, dimethylaminoaphthalenecysteine-Hg+ (Dn-cys-Hg+). This reagent is more reactive than N-ethylamaleimide (MalNEt) toward membrane sulfhydryl groups. A procedure using the two SH reagents sequentially seems to permit a more selective labelling of the SH groups involved in the (Na+ + K+)-ATPase than is possible by using MalNEt alone. Brain microsomes treated by this method incorporate the fluorescent label within or near the active site of the enzyme. When the probe was bound a blue shift of its fluorescence emission maximum (from 540 to 495 nm) and a 20-fold increase in relative fluorescence occurred. This indicates that the Dn moiety is within a very non-polar region of the membrane.     

Effects of sulfhydryl compounds on the inhibition of erythrocyte membrane Na+(-)K+ ATPase by ozone

Exposure of human erythrocyte membranes to ozone (5 mumol/10 min) resulted in the inhibition of erythrocyte membrane Na+(-)K+ ATPase (EC.3.6.1.39). It was determined that, the degree of enzyme inhibition in the directly ozone exposed membranes was greater than that of membranes obtained from ozone exposed intact erythrocytes. In the presence of varying concentrations (0-1.0 mM) of dithiotrethiol or mercaptoethanol Na+(-)K+ ATPase activities of both types of ozone exposed membranes were increased almost proportionally with the concentration of dithiotrethiol or mercaptoethanol however, the activities were still lower than the normal Na+(-)K+ ATPase value. The results indicate that, dithiotrethiol or mercaptoethanol prevent the enzyme inhibition by ozone in vitro. This suggests that the membrane thiol groups are primary targets for ozone and thereby preventing the oxidation of essential functional groups of enzyme protein.     

Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups.

1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

Inactivation of membrane-bound (Na+ +K+)-ATPase of Yoshida sarcoma cells and cobra venom cytotoxin complex with the glycolipid components of the enzyme system.

The molecular mechanisms involved in the inactivation of (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells by a cytotoxic protein (P6) from cobra venom have been examined. The overall data obtained using purified (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells suggest that cytotoxin P6 combines with phosphatidyl serine and a glycolipid which are closely associated with (Na+ + K+)-stimulated ATPase which in turn may lead to the inactivation of the enzyme in this cell system.     

Renal brush-border-membrane vesicles prepared from newborn rats by free-flow electrophoresis and their proline uptake.

A method for the isolation of brush-border membranes from newborn-rat kidney, employing centrifugation and free-flow electrophoresis, is described. The composition and purity of the preparation was assessed by determination of enzyme activities specific for various cellular membranes. Free-flow electrophoresis resolves the newborn-rat renal membrane suspension into two populations of alkaline phosphatase-enriched brush-border membranes, designated 'A' and 'B', with the A peak also showing activity of (Na+ + K+)-stimulated ATPase, the basolateral membrane marker enzyme, whereas those of the B peak were enriched 11-fold in alkaline phosphatase and substantially decreased in (Na+ + K+)-stimulated ATPase activity. Membranes in the A peak showed a 7-fold enrichment of alkaline phosphatase, and (Na+ + K+)-stimulated ATPase activity similar to that of the original homogenate. Proline uptake employed to assess osmotic dependency revealed 7% binding of proline to the B vesicles and 31% to the A vesicles. This contrasts with 60% proline binding to vesicles prepared by centrifugation alone. Unlike vesicles from adult animals, proline uptake by B vesicles did not show an Na+-stimulated overshoot, but did exhibit an Na+-gradient enhanced rate of early proline entry. proline entry.     

Effects of N-acetylimidazole on human erythrocyte ATPase activity. Evidence for a tyrosyl residue at the ATP binding site of the (Na+ plus K+)-dependent ATPase.

1. Acetylation of human erythrocytes by N-acetylimidazole alters the structure of stroma prepared from these cells and the degree of alteration appears to be dependent upon the level of the initial treatment. These changes do not occur when stroma are acetylated. 2. Deacetylation by hydroxylamine or mild alkaline treatment causes a complete recovery of the (Na+ plus K+)-dependent and the Ca2+ -stimulated ATPase activities and indicates that the inhibition is due to the acetylation of a tyrosyl residue. There is only partial recovery of the Mg2+ -dependent ATPase after deacetylation. 3. ATP or Mg-ATP completely protect the (Na+ plus K+)-dependent ATPase, but not the Ca2+ -stimulated system. 4. The results indicate that the (Na+ plus K+)-dependent and the Ca2+ -stimulated ATPase activities have separate substrate binding sites and most likely are separate enzyme systems. 5. Acetylation of human erythrocytes has no effect on D-glucose transport.     

A model for the reaction pathways of the K+-dependent phosphatase activity of the (Na+ + K+)-dependent ATPase

(Na+ + K+)-dependent ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+ -dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration: (formula; see text) Differences between the three preparations in K0.5 for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in K0.5 produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+ -dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+ -sites are oriented extracellularly, as are those of the (Na+ + K+)-dependent ATPase reaction. Dimethyl sulfoxide accentuated Na+ -stimulated K+ -dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+ -sites of the Na+ -stimulated pathway.     

Comparison of some minor activities accompanying a preparation of sodium-plus-potassium ion-stimulated adenosine triphosphatase from pig brain

1. An ATPase (adenosine triphosphatase) preparation obtained from pig brain microsomes by treatment with sodium iodide showed four apparently different ouabain-sensitive activities under various conditions. They were (a) ouabain-sensitive Mg(2+)-stimulated ATPase, (b) K(+)-stimulated ATPase, (c) (Na(+),K(+))-stimulated ATPase and (d) Na(+)-stimulated ATPase activities. 2. These activities showed the same substrate specificity, ATP being preferentially hydrolysed and CTP slightly. AMP was not hydrolysed. 3. These activities were inhibited by low concentration of ouabain. The concentration producing 50% inhibition was 0.1mum for ouabain-sensitive Mg(2+)-stimulated ATPase, 0.2mum for K(+)-stimulated ATPase, 0.1mum for (Na(+),K(+))-stimulated ATPase and 0.003mum for Na(+)-stimulated ATPase activity. 4. The ouabain-sensitive ATPase activities were inactivated by N-ethylmaleimide but the insensitive ATPase activity was not. 5. The three ouabain-sensitive ATPase activities were inhibited about 50% by 1mm-Ca(2+), whereas the ouabain-sensitive Mg(2+)-stimulated ATPase activity was activated by the same concentration of Ca(2+). The preparation was treated with ultrasonics at 20kcyc./sec. The 2min. ultrasonic treatment inactivated the ATPase activities by 50%. 7. The temperature coefficient Q(10) was 6.6 for K(+)-stimulated ATPase activity, 3.7 for (Na(+),K(+))-stimulated ATPase and 2.6 for Na(+)-stimulated ATPase. 8. Organic solvents inactivated the ATPase activities, to which treatment the K(+)-stimulated ATPase was the most resistant. 9. The phosphorylation of the enzyme preparation became less dependent on Na(+) with decreasing pH. This Na(+)-independent phosphorylation at low pH was sensitive to K(+) and hydroxylamine as well as the Na(+)-dependent phosphorylation at neutral pH.     

Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.     

Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin. Chemical probing of movement of residues during conformational transitions.

The reaction of myosin with three bifunctional sulfhydryl reagents of differing cross-linking span is reported. In the absence of nucleotide only p-N,N'-phenylenedimaleimide with a cross-linking span of 12-14 A can bridge between the two essential sulfhydryls of myosin. The other two reagents, 2,4-dinitro-1,5-difluorobenzene and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone with cross-linking spans of 3-5 and 7-10 A, respectively, react under identical conditions with the SH1 sulfhydryl but do not bridge to the SH2 group. In the presence of MgADP, both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide. These results are discussed in relation to the conformational change induced in myosin by binding of the nucleotide.     

Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.     

Modification of the conformational equilibria in the sodium and potassium dependent adenosinetriphosphatase with glutaraldehyde

Glutaraldehyde treatment of electroplax membrane preparations of Na,K-ATPase leads to irreversible changes in the enzymic behavior of the protein, which are not due to modification of the active site. When the glutaraldehyde treatment is carried out in a medium containing K+ and without Na+, the "K+-modified enzyme" so produced shows the following changes in enzymic properties: The steady-state phosphorylation by ATP and the rate of ATP-ADP exchange are decreased to approximately 40% of control, while Na,K-ATPase activity decreases to approximately 15% of control. Phosphatase activity is decreased very little, but the potassium activation parameters of the reaction are changed, from K0.5 approximately equal to 5 mM and nH = 1.9 in control to K0.5 approximately equal to 0.5 mM and nH = 1 in K+-modified enzyme. KI(app) for nucleotide inhibition of phosphatase activity is increased significantly. Changes in the cation dependence of the ATPase reaction are also observed. All of these effects can be explained by assuming that the cross-linking of surface groups in protein subunits when they are in conformation E2 shifts the intrinsic conformational equilibrium of the enzyme toward E2. We considered the simplest mathematical model for the coupling between K+ binding and the conformational equilibrium, with equivalent potassium sites that must be simultaneously in the same state. If one assumes that the potassium activation of phosphatase activity in the K+-modified enzyme reflects the affinity for K+ of E2, the behavior of the phosphatase activity in the native enzyme can be fit if there are only two potassium sites, whose affinity is 80-fold higher in E2 than in E1, and the equilibrium constant for E2 in equilibrium E1 is about 250. The same sites can explain the activation of dephosphorylation during ATP hydrolysis. Independent of the model chosen, potassium ions must be required for the catalytic action of form E2 and cannot be merely "allosteric activators". The enzyme modified with glutaraldehyde in a medium containing Na+ also has interesting properties, but their rationalization is less straightforward. The Na,K-ATPase activity is inhibited more than the "partial reactions", as in the K+-modified enzyme. We suggest that this is a generally expected result of modifications of the enzyme.     

An essential arginine residue in the ATP-binding centre of (Na+ + K+)-ATPase.

1. Incubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rabbit kidney outer medulla with butanedione in borate buffer leads to reversible inactivation of the (Na+ + K+)-ATPase activity. 2. The reaction shows second-outer kinetics, suggesting that modification of a single amino acid residue is involved in the inactivation of the enzyme. 3. The pH dependence of the reaction and the effect of borate ions strongly suggest that modification of an arginine residue is involved. 4. Replacement of Na+ by K+ in the butanedione medium decreases inactivation. 5. ATP, ADP and adenylyl imido diphosphate, particularly in the presence of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid to complex Mg2+, protect the enzyme very efficiently against inactivation by butanedione. 6. The (Na+ + Mg2+)-dependent phosphorylation capacity of the enzyme is inhibited in the same degree as the (Na+ + K+)-ATPase activity by butanedione. 7. The K+-stimulated p-nitrophenylphosphatase activity is much less inhibited than the (Na+ + K+)ATPase activity. 8. The ATP stimulation of the K+-stimulated p-nitrophenylphosphatase activity is inhibited by butanedione to the same extent as the (Na+ + K+)-ATPase activity. 9. Modification of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) protects partially against the inactivating effect of butanedione. 10. The results suggest that an arginine residue is present in the nucleotide binding centre of the enzyme.     

Thiol-blocking reagents and phosphate acetyltransferase catalysis, and the assessment of protection by adsorbed molecules (Short Communication)

Phosphate acetyltransferase (EC 2.3.1.8) is shown to be unusually sensitive to thiol-blocking reagents, and one or more thiol groups probably catalyse the surface reaction. A novel method for assessing protection by adsorbed species is described. The adsorbed substrate molecules acetyl-CoA and phosphate are found to provide substantial protection against thiol-blocking reagents.