Temperature effects on kinetic properties of plasma membrane ATPase from the yeast Saccharomyces cerevisiae

The reaction of plasma membrane ATPase from yeast with Mg²⁺ and Mg · ATP was studied in a temperature range of 10 – 30°C. The random mechanism of activation by Mg²⁺ and the pseudocompetitive inhibition at higher concentrations was not altered when the temperature was varied, nor were the kinetic constants representing substrate binding. However, at low temperature, the affinity of the enzyme for Mg²⁺ is greatly reduced. The Arrhenius plot of log V vs. 1/T shows straight lines with an inflection point at 24°C, which disappears in the presence of detergent. Calorimetric studies of the plasma membranes show a transition point at the same temperature. From these findings we suppose that Mg²⁺ is bound at a regulatory site of the ATPase, which is influenced by the surrounding phospholipids.     

Altered kinetic properties of liver mitochondrial membrane-bound enzyme activities following paracetamol hepatotoxicity in the rat

The effects of treatment with subtoxic (375 mg/kg) and toxic (750 mg/kg) doses of paracetamol on NADH oxidase, succinoxidase and Mg²⁺-ATPase activities in rat liver submitochondrial particles were examined. In the NADH oxidase system, treatment with subtoxic doses of paracetamol resulted in a 37% increase in activation energy in the high temperature range (E¹) while the phase transition temperature (Tt) for this system decreased by 9‡C. Subtoxic doses caused a 43% decrease in E₁. For the succinoxidase system, Tt decreased by 2.4 to 3.4‡C after paracetamol administration. E₂ increased by 42% only in the subtoxic-treatment group while E₁ remained unaltered in both paracetamol-treated groups. For the Mg²⁺-ATPase system, subtoxic doses of paracetamol treatment did not change the values of E₁ E₂ and Tt whereas toxic dose treatment resulted in a 29% decrease in E₂ with a concomitant increase in Tt by 2.4‡C without any change in the value of E₁ The results thus suggest that treatment with toxic and subtoxic doses of paracetamol results in possible differential alterations in the membrane lipid milieu.     

An oligomycin-resistant Mg2+-dependent ATPase in rabbit bone marrow mitochondria

Summary Rabbit bone marrow mitochondria isolated by differential centrifugation showed typical oxypolarographic tracings with glutamate oxidation with ADP:O ratio of 2.9. Similar results were obtained with liver mitochondria of the same animal. When marrow mitochondria were oxydizing a substrate such as glutamate, added MgCl₂ markedly stimulated state-4 respiration giving a respiratory rate identical to that of state-3. In contrast, no Mg²⁺-stimulation was observed with liver mitochondria. Oligomycin completely blocked the stimulation by Mg²⁺ but further addition of 2,4-dinitrophenol reactivated the oxygen consumption by uncoupling. Further purification of marrow mitochondria by density gradient centrifugation in Percoll provided identical oxypolarographic results. Moreover, when marrow mitochondria were incubated without Mg²⁺, they showed a low ATPase activity that was stimulated by 2,4-dinitrophenol and blocked by oligomycin. The presence of Mg²⁺ in the incubation medium uncovered an additional ATPase activity which was resistant to oligomycin and apparently unaffected by 2,4-dinitrophenol. It is concluded that bone marrow mitochondria possess two types of ATPase activity distinguished on the basis of their reactivity with oligomycin, 2,4-dinitrophenol and Mg²⁺.Abbreviations EDTA ethylenediamine tetraacetate - DNP 2,4-dinitrophenol - BSA bovine serum albumin - BMM bone marrow mitochondria - LM liver mitochondria - Oligo. oligomycin - Anti A antimycin A Howard Hughes Investigator.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg²⁺ and in the presence of Mg²⁺ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl^->HCO ₃ ^- >Br^->malate>acetate>SO ₄ ^2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH ₄ ⁺ Na⁺>K⁺). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na⁺ and K⁺. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in V_max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO₃ but other inhibitors tested (azide, oligomycin, orthovanadate, K₃[Cr(oxalate)₆] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATP_F free ATP - ATP_T total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - K_m apparent Michaelis constant - MgATP complex of Mg²⁺ and ATP - Mg _F ²⁺ free Mg²⁺ - Mg _T ² total Mg²⁺ - MES 2-(N-Morpholino)ethanesulphonic acid - Na₂EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - P_i inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - V_max maximum velocity     

Temperature Response and Effect of Ca2+ and Mg2+ on ATPases from Roots of Oats and Wheat as Influenced by Growth Temperature and Nutritional Status

Activation by Ca²⁺, Mg²⁺, Zn²⁺, or Mn²⁺ of adenosine triphosphatases in a microsomal fraction from wheat roots depends upon the growth temperature when the plants are grown under low salt conditions, but not when the plants get a full-strength culture medium. At low ionic strength, cultivation at 25°C gives only half as high activation as cultivation at 18°C or at high ionic strength at both temperatures. Corresponding data for activation of ATPases from oats also show that low ionic strength during growth gives the highest temperature dependence. Low temperature together with low salt conditions during growth gives the highest ATPase activity after stimulation with divalent cations. High growth temperature and full-strength medium decrease the ATPase activity. Activation energies (Ea) were calculated for the two temperature intervals 35–20°C and 20–5°C. The dominating ATPase stimulation (Ca²⁺ in wheat, Mg²⁺ in oats) is characterized by high specific activity combined with a low Ea value. The differences in ATPase activity between oats and wheat can be correlated with different cultivation requirements known from agriculture.     

Alterations in Ca2+/Mg2+ ATPase activity upon treatment of heart sarcolemma with phospholipases

In order to examine the role of phospholipids in the activation of membrane bound Ca²⁺/Mg²⁺ ATPase, the activities of Ca²⁺ ATPase and Mg²⁺ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg²⁺ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg², ATPase activity by each phospholipase treatment was associated with a decrease in the V_max value without any changes in the K_a value. The depression of Mg²⁺ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg²⁺ ATPase, the sarcolemmal Ca²⁺ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg^- ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg²⁺ ATPase activity to a greater extent in comparison to that for the Ca²⁺ ATPase activity and the phospholipids associated with Mg²⁺ ATPase are predominantly exposed at the outer surface of the membrane.     

Molecular mechanisms of temperature adaptation in fish myofibrillar adenosine triphosphatases

Summary Studies have been carried out on the Mg²⁺ Ca²⁺-myofibrillar ATPase from the muscles of fish adapted to different environmental temperatures. The thermal stability of the ATPase is strongly correlated with mean habitat temperature. Activities of Antarctic fish ATPases are significantly higher at low temperatures than those of temperate and tropical water species. The effects of ionic strength on ATPase activity have also been studied. The Gibbs free energy of activation (G ^#) was found to increase and enzyme activity decrease with increasing ionic strength within the physiological temperature range of each species. Significantly lower values of G ^#, of around 1 Kcal/mole, are obtained for the ATPase of cold-adapted compared to tropical fish. Enthalpic and entropic activation energies were also reduced in the cold adapted ATPases. It is postulated that the reduction of the enthalpic activation term in the cold adapted enzyme confers the advantage of reducing the temperature sensitivity of the rate limiting step thus partly compensating for the low heat content of the cellular environment. Possible molecular mechanisms of temperature compensation in fish myofibrillar ATPase are discussed.     

Role of Mg2+ ions in the subunit structure and membrane binding properties of bacterial energy transducing ATPase.

ATPase extracted from $\text{Streptococcus faecalis}$ membranes was purified by preparative slab gel electrophoresis in the presence of Mg⁺⁺ (plus Mg²⁺ ATPase) and without Mg²⁺ (minus Mg²⁺ ATPase). The subunit composition and membrane binding capacity of both preparations was then examined. The plus Mg²⁺ ATPase had 5 types of subunits (αβγδ?) and reattached normally to depleted membranes. The minus Mg²⁺ ATPase had the αβγ and ? chains, but no δ chain, and failed to reattach to membranes. These data indicate that Mg²⁺ or a similar cationic ligand anchors the δ chain to the core enzyme complex and that the δ chain in turn is needed for membrane attachment. For the plus Mg²⁺ ATPase the data are consistent with the subunit stoichiometry and arrangement, (α₃β₃ γ ?)-Mg²⁺)_n?(δ).     

Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria

A23187, a new antibiotic with ionophore properties, uncoupled oxidative phosphorylation in mitochondria which oxidized either malate plus glutamate or succinate. Ca²⁺, but not Mg²⁺, enhanced the uncoupling effect. Fluorescence of ANS¹ was increased by A23187 suggesting the mitochondrial membranes were de-energized. This de-energization was presumably by activation of the energy-dependent uptake of Ca²⁺. The steady-state measurements of murexide-divalent cation complexes showed that A23187 caused mitochondria to release the accumulated Ca²⁺ to the medium. This reduced the transmembrane Ca²⁺ gradient even though normal active Ca²⁺ uptake could take place. A23187 inhibited activity of ATPase induced by 2,4-dinitrophenol, valinomycin, and Ca²⁺. The addition of Mg²⁺ could prevent this inhibition presumably by maintaining the endogenous Mg²⁺ concentration. The above metabolic events could be explained by the fact that molecules of A23187 function in the mitochondrial inner membrane as mobile carriers for divalent cations.     

K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells

Differential centrifugation of oxyntic cell homogenates yielded microsomal fractions which contained large amounts of mitochondrial membrane. The presence of marker enzymes (succinate dehydrogenase and cytochrome c oxidase) indicated that mitochondrial contamination of crude microsomes ranged from 20 to 60% in different preparations. A discontinuous sucrose density gradient procedure was developed for the routine preparation of purified oxyntic cell microsomes. A K⁺-stimulated, Mg²⁺-requiring ATPase was localized in these purified membranes and coincided with the presence of a K⁺-stimulated p-nitrophenylphosphatase. Na⁺ and ouabain had no effect on the K⁺ stimulation of the microsomal ATPase. The apparent activation constant for K⁺ was approximately 1 mM at pH 7.5, the optimal pH for stimulation.An anion-sensitive ATPase has been widely studied in gastric microsomal preparations. We found that the basal microsomal ATPase (i.e. without K⁺) and the mitochondrial ATPase were inhibited by SCN^? and enhanced by HCO₃^?, however, the K⁺-stimulated component of the microsomal ATPase was virtually unaffected by these anions.     

Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide–adenine dinucleotide by succinate in Escherichia coli

1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD⁺ by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg²⁺, P_i and temperature have been studied.     

Mg2+-induced ADP-dependent inhibition of the ATPase activity of beef heart mitochondrial coupling factor F1.

Incubation of F₁ in the presence of Mg²⁺ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg²⁺ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F₁ treated to remove tightly bound nucleotides was preincubated in the presence of Mg²⁺. Mg²⁺-induced inhibition of ATPase activity of nucleotide-depleted F₁ can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg²⁺-induced inhibition of the ATPase activity of F₁ is due to the formation of an inactive F₁. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, $\text{86}$, 434/ is directed to F₁ component of the complete mitochondrial ATPase system.     

Characteristics of the Mg-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg²⁺-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na₂SO₃-dependent Mg²⁺-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg²⁺-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg²⁺-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg²⁺-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t_½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.     

The sensitivity of ATPases from mouse tissues to DDT at different temperatures.

Oligmoycin-sensitive (O-S) Mg²⁺ ATPase from mouse brain has a higher sensitivity to DDT at a low temperature, 17°C than at 27° or 37°. The I₅₀ value for 17° was 0.24 μM DDT. The DDT sensitivity did not differ significantly at 27° and 37°C. This negative temperature correlation is similar to results in brain and muscle tissues of insects. Oligomycin-insensitive Mg²⁺ ATPase, also was inhibited by DTT more effectively at cooler temperatures. In contrast, O-S Mg²⁺ ATPase from mouse muscle showed no significant sensitivity difference to DDT at the 3 temperatures. Na⁺-K⁺ ATPase, inhibited to a lesser degree by DDT, was inhibited to a much greater extent (61%) at 37° than at 17° (23%). This positive temperature correlation is similar to findings in insect homogenates.     

Tertiary structure and energy coupling in Ca2+-pump system

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca²⁺ Mg²⁺)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H₂O molecules surrounding the Ca²⁺ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca²⁺ - Ca²⁺ intersite distance is estimated to be 8–9 Å and the average distance from the Ca²⁺ sites to CrATP is about 18 Å.Digestion of the (Ca²⁺ + Mg²⁺)-ATPase at the T₂ site (Arg 198) causes uncoupling of Ca²⁺-transport from ATPase activity while calcium occlusion due to E₁-P formation remains unchanged. Further tryptic digestion beyond T₂ and in the presence of ATP diminishes Ca²⁺ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T₂ and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca²⁺ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.     

Cholesterol affects divalent cation-induced fusion and isothermal phase transitions of phospholipid membranes

The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10–40°C. The fusogenic activity of the cations decreases in the sequence Ca²⁺ > Ba²⁺ > Sr²⁺ Mg²⁺ for cholesterol concentrations in the range 20–40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca²⁺ and Ba²⁺ at 25°C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25°C, Mg²⁺ is ineffective in causing fusion at all cholesterol concentrations. However, at 30°C, Mg²⁺-induced fusion is observed with vesicles containing cholesterol. At 40°C, Mg²⁺ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca²⁺, Ba²⁺ and Sr²⁺. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na⁺ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (T_m) in Na⁺ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the T_m upward, with a sequence of effectiveness Ba²⁺ > Sr²⁺ > Mg²⁺. The T_m of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na⁺, Sr²⁺ or Mg²⁺, the addition of Ba²⁺ reveals endotherms with T_m progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the T_m, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg²⁺ concentrations. With Ca²⁺, saturation is not observed for cholesterol-containing vesicles. The highest rate of fusion for both Ca²⁺- and Mg²⁺-induced fusion is observed with vesicles containing 30 mol% cholesterol.     

Effects of Alkalinization and ATPase Inhibition on Stromal Free Mg2+ Concentration in Spinach Chloroplasts

Our earlier studies indicate that stromal alkalinization is essential for light-induced increase in free Mg²⁺ concentration ([Mg²⁺]) in chloroplast. Stromal [Mg²⁺] was increased by dark incubation of chloroplasts in the K⁺-gluconate medium (pH 8.0), or by NH₄Cl. These results indicate that stromal alkalinization can induce an increase in stromal [Mg²⁺] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H⁺ efflux inhibited the alkalinization-induced increase in [Mg²⁺].     

Gastric HCO3-stimulated ATPase: Evidence against its microsomal localization in rat fundus mucosa

Rat gastric mucosa was shown to contain a Mg²⁺-dependent ATPase which is stimulated by HCO₃⁻ at pH 8–9.Triton X-100 solubilizes this HCO₃⁻-stimulated, Mg²⁺-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3).The gastric mucosa was resolved into five subcellular fractions by differential centrifugation. A large granule fraction (Fraction M), 28 000 g · min, was characterized by cytochrome c oxidase (marker enzyme for mitochondria). A microsomal fraction (Fraction P), 2 760 000 g · min, was characterized by 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) (plasma membrane).The Mg²⁺-dependent ATPase was demonstrated to have a bimodal mitochondrial membranous localization: 24% of its activity is associated with cytochrome c oxidase, and 75% with 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) at pH 8.The HCO₃⁻ addition resulted in two opposite effects: (1) a strong stimulation (84%) in Fraction M; (2) a slight inhibition (12%) in Fraction P.Fraction M was subfractionated by equilibration on a sucrose gradient. It gave rise to a homogeneous mitochondrial (d, 1.17–1.21) Mg²⁺-dependent ATPase, closely associated with cytochrome c oxidase. This ATPase is strongly stimulated (×2) by HCO₃⁻. The subfractionation of Fraction P gave rise to two distinct ATPases: (1) the major one is associated with membranous (d, 1.10–1.15) material marked by 5′-nucleotidase and is slightly inhibited by HCO₃⁻; (2) the other is associated with denser (d, 1.17–1.21) material and is stimulated by HCO₃⁻.The bicarbonate-stimulated fraction of the Mg²⁺-dependent ATPase activity found in the gastric microsomal fraction is assumed to arise from mitochondrial cross-contamination. Further support comes from the optimal HCO₃⁻ concentration. In addition, SCN⁻ is shown to specifically inhibit the ATPase of Fraction M.From these results it appears that the implication of HCO₃⁻-stimulated ATPase in the gastric secretion of H⁺ is not as clear as had been suggested. However, in the view of an ATPase-supported model for H⁺ secretion, attention can be directed towards the Mg²⁺-dependent ATPase found to be associated with microsomes.     

A Mg2+-stimulated ATPase in platelet alpha-granule membranes: possible involvement in proton translocation

Isolated porcine platelet α granules display a Mg²⁺-stimulated ATPase activity. The enzyme is membrane bound and several criteria suggest that it is intrinsic to the α granules, rather than arising from contamination with other structures. Characterization of the ATPase revealed an apparent K_m for ATP of 198 μm. Other nucleotides are also hydrolyzed by the enzyme, though at a slower rate. The enzyme has an absolute requirement for divalent cations, and both Mg²⁺ (apparent K_m 0.93 mm) and Ca²⁺ (apparent K_m 0.95 mm) can activate it. Maximal hydrolysis rates are higher with Mg²⁺ than with Ca²⁺. Micromolar Ca²⁺ in the presence of maximally stimulating Mg²⁺ concentrations produces a small additional enhancement of activity. The Mg²⁺ ATPase has a broad activity maximum between pH 6.5 and 8.5, and an activation energy of 11.8 Kcal/mol. Several independent observations suggest that the ATPase could be involved in H⁺ translocation across the granule membrane: (a) the activity is stimulated upon disrupting membrane continuity by either hypotonic lysis or addition of nondenaturing detergents; (b) proton ionophores enhance the activity in intact but not in disrupted α granules; (c) permeating anions stimulate the ATPase more than slowly permeant or impermeant ones; (d) addition of NH₃ (as either NH₄Cl or (NH₄)₂SO₄) activates enzyme activity; (e) silicotungstate and disulfonic stilbene derivatives, which are inhibitors of other H⁺-transporting ATPases, also inhibit the α-granule enzyme. These findings are compared with the reported properties of H⁺ pumps of other storage and secretory organelles.