A high affinity Ca2+-dependent ATPase in the surface membrane of the bloodstream stage of Trypanosoma rhodesiense

Addition of Ca2+ (0.01-1 mM) to a standard Trypanosoma rhodesiense Mg2+-ATPase assay failed to elicit any increase in activity. However, in the absence of externally added Mg2+ and using calcium-EGTA or calcium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific Ca2+-ATPase. Cell fractionation studies revealed this ATPase to be predominantly associated with subcellular particles having an equilibrium density of 1.22 g cm-3 and identified as surface membrane. Using a discontinuous sucrose gradient, a surface membrane enriched (SME) fraction, only slightly contaminated with mitochondria as judged by dichlorophenolindophenol-linked alpha-glycerophosphate dehydrogenase activity, was prepared. The SME fraction exhibited Ca2+-ATPase activity, using 200 nM free Ca2+, of 90 and 21 mU mg-1 protein, respectively, using CDTA and EGTA as buffering ligands. This latter result was most unexpected and indicated that the Ca2+-ATPase, in addition to having no Mg2+ requirement, was inhibited by submicromolar levels of Mg2+. The Ca2+-ATPase was found to have a K0.5 = 128 +/- 22 nM free Ca2+, the response to increasing Ca2+ concentration displaying an extremely high degree of co-operativity (Hill number (nH) = 4.9). The enzyme was found to be highly substrate-specific for ATP with K0.5 = 6.2 +/- 0.61 microM ATP. A Hill plot of the reaction velocity as a function of ATP concentration indicated two substrate binding sites (nH = 1.55). A range of potential modulators of ATPase activity were investigated, with only vanadate (V2O3-8) having any effect: 47% inhibition at 5.0 microM. The Ca2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (50 microM), whilst addition of calmodulin failed to produce any stimulation of activity. It is concluded that the kinetic properties of this ATPase are compatible with a potential role in the regulation of intracellular Ca2+ in bloodstream T. rhodesiense.     

Influence of ATP on sarcoplasmic reticulum function of vascular smooth muscle

A vesicular fraction isolated from bovine aorta and enriched in fragmented sarcoplasmic reticulum (FSR) exhibited active calcium transport and ATPase activity. By use of a hypotonic NaHCO3 extraction solution, an active preparation was isolated that retained activity for up to 4 days. A small but significant (P less than 0.05) Ca2+-stimulated, Mg2+-dependent ATPase associated with calcium transport was demonstrated with a specific activity of 0.33 mumol inorganic phosphate (Pi).mg-1.min-1. The basal Mg2+ ATPase demonstrated Michaelis-Menten kinetics [Km(Mg2+-ATP) = 0.44 +/- 0.01 X 10(-3) M; Vmax = 2.22 +/- 0.01 mumolPi.mg-1.min-1]. The Ca2+-stimulated, Mg2+-ATPase demonstrated apparent substrate inhibition (Ks approximately 10 mM) with no evidence for end-product (ADP) or excess added Ca2+ contributing to this inhibition. Oxalate-supported active calcium uptake velocities also exhibited quantitatively similar substrate inhibition. These results suggest that FSR from vascular smooth muscle contains either two enzymes or one enzyme with two isomeric forms, one of which is associated with the calcium uptake activity of this structure and the other of unknown function.     

Age-dependent alterations in synaptic membrane systems for Ca2+ regulation

The effects of aging on two neuronal plasma membrane Ca2+ regulating systems have been examined using synaptic membranes isolated from the brains of adult (5-7-month-old) and aged (23-25-month-old) Fisher 344 rats. The kinetic characteristics of the Na+-dependent Ca2+ transport system were found to be altered in the aged animals. The affinity of the transport carrier for Ca2+ was decreased in membranes from aged animals, with very little change in the maximal transport capacity of the system. The activity of the synaptic membrane Ca2+-activated, Mg2+-dependent ATPase was also altered in membranes from aged animals. In this system, however, the Vmax for Ca2+ activation of the enzymatic activity was lower in the aged animals, while there was no change in the K0.5 for Ca2+ activation. The magnitude of the alterations was small, but the differences were consistent. Even small changes in the effectiveness of the synaptic plasma membrane systems which participate in the maintenance of low intraterminal Ca2+ could progressively affect the integrity of synaptic transmission and lead eventually to neuronal cell death.     

钙对心肌ATP酶的损害及镁的保护作用

本工作在大鼠心肌肌膜观察到,Mg~(2+)为心肌Na~+-K~+-ATP酶激活所不可缺少的因素,当Mg~(2+)浓度在0～6mM时,Na~+-K~+-ATP酶及Mg~(2+)-ATP酶活性与其密切相关;当超过8 mM时,ATP酶总活性不变,但Na~+-K~+-ATP酶/Mg~(2+)-ATP酶之比值增高。高浓度Ca~(2+)对于Na~+-K~+-ATP酶及Mg~(2+)-ATP酶均具有抑制作用,而提高Mg~(2+)浓度可部分地拮抗高Ca~(2+)的损伤作用。     

Hypothalamic digoxin--central role in conscious perception,neuroimmunoendocrine integration and coordination of cellular function--relation to hemispheric dominance

A family with a high prevalence of Parkinson's disease, schizophrenia, neoplasms, syndrome-X, rheumatoid arthritis and epilepsy has been described. The psychological behavioural patterns of the family were as follows--creativity and high IQ, hypersexual behaviour, reduced appetite and eating behaviour, insomnia and reduced sleep patterns, increased tendency for spirituality, increased tendency for addiction, less of bonding and affectionate behaviour and left handedness. Digoxin, an endogenous Na(+)-K(+) ATPase inhibitor secreted by the hypothalamus, was found to be elevated and RBC membrane Na(+)-K(+) ATPase activity was found to be reduced in all the disorders and in the indexed family studied. Hypothalamic digoxin can modulate conscious perception and its dysfunction may lead to schizophrenia. Digoxin can also preferentially upregulate tryptophan transport over tyrosine resulting in increased levels of depolarising tryptophan catabolites - serotonin, quinolinic acid, strychnine and nicotine and decreased levels of hyperpolarising tyrosine catabolites dopamine, noradrenaline and morphine contributing to membrane Na(+)-K(+) ATPase inhibition in all the above disorders and the indexed family. Digoxin induced membrane Na(+)-K(+) ATPase inhibition can result in increased intracellular Ca(2+) and reduced Mg(++) levels leading to glutamate excitotoxicity, oncogene activation and immune activation. Digoxin induced altered Ca(++)/Mg(++) ratios, reduced ubiquinone and increased dolichol can affect glycoconjugate metabolism, membrane formation and structure and mitochondrial function leading to the diverse disorders described above including those in the indexed family. The isoprenoid pathway and neurotransmitter patterns were compared in right-handed/left hemispheric dominant and left-handed/right hemispheric dominant individuals. The biochemical patterns in the indexed family and the diverse disorders studied correlated with those obtained in right hemispheric dominance. The hyperdigoxinemic state indicates right hemispheric dominance. Hypothalamic digoxin can thus function as the master conductor of the neuroimmunoendocrine orchestra and co-ordinate the functions of various cellular organelles.     

Ethacrynic acid inhibitable Ca2+ and Mg2+-activated membrane adenosine triphosphatase in rat mast cells.

A crude plasma membrane fraction from the homogenate of purified rat mast cells demonstrates a high degree of Ca2+-dependent and Mg2+-dependent adenosine triphosphatase (ATPase) activity. The microsomal and mitochondrial fractions show negligible amounts of the Ca2+ and Mg2+-activated ATPases. The broad ATPase inhibitor, ethacrynic acid, effectively blocks the mast cell ATPase activity while ouabain demonstrates little inhibitory effect. Correspondingly, ethacrynic acid inhibits histamine release from antigen-challenged mast cells while ouabain does not. Both ATPase inhibition and histamine release inhibition by ethacrynic acid require the presence of the olefinic bond in the ethacrynic acid molecule.     

Calcium movements across the membrane of human red cells

1. A study has been made of the cellular content and movement of Ca across the membrane of human red blood cells.2. The [Ca] in the cellular contents of fresh red cells is 4.09 x 10(-2) mM. The intracellular concentration of free ionic Ca ([Ca(2+)]) is considered to be less than this value and therefore less than extracellular [Ca(2+)] under normal conditions.3. Observation of unidirectional Ca fluxes with (45)Ca confirms previous reports of low permeability of the red cell membrane for Ca. After nearly 1 week of loading in the cold, intracellular (45)Ca content is 1.8% of extracellular (45)Ca content. Appearance in extracellular fluid of (45)Ca from coldloaded cells can be considered to arise from two compartments. Efflux of (45)Ca from the ;slower compartment' is accelerated by the addition of glucose.4. Starved red cells, incubated at 37 degrees C, after reversible haemolysis for loading with Ca and Mg-ATP, exhibit an outward net transport of Ca against an electrochemical gradient. The transport is associated with the appearance of inorganic phosphate (P(i)). Cells treated similarly, but without ATP show no transport and no appearance of P(i).5. During the initial phase of transport, 1.3 mole P(i) appear per mole Ca transported.6. The transport of Ca from ATP-loaded cells is highly temperature-dependent, with a Q(10) of 3.5.7. Cell membrane adenosine triphosphatase (ATPase) activity of reversibly haemolysed cells is stimulated only by intracellular, and not by extracellular Ca.8. Neither Ca transport in reversibly haemolysed cells, nor the Ca-Mg activated ATPase of isolated cell membranes is sensitive to Na, K, ouabain or oligomycin.9. Mg is not transported under the conditions which reveal Ca transport, but Mg appears to be necessary for Ca transport.10. Sr is transported from reversibly haemolysed Mg-ATP-loaded cells. Sr also can substitute for Ca, but not for Mg, in the activation of membrane ATPase.11. It is concluded that, in addition to a low passive permeability, an active extrusion mechanism for Ca exists in the human red cell membrane. This extrusion mechanism, in addition to a low passive membrane permeability for Ca, may represent the means by which intracellular Ca content is maintained at a low level. It is suggested that the Ca-Mg activated membrane ATPase and the active transport of Ca are two manifestations of the same process.     

A (Ca(2+)-Mg2+)ATPase from Schistosoma mansoni is coupled to an active transport of calcium.

The (Ca(2+)-Mg2+)ATPase activity in microsomes of Schistosoma mansoni is fully inhibited by vanadate (I50 = 2.5 microM). 45Ca2+ is accumulated within microsomal vesicles in an ATP-dependent process that is enhanced 5-fold in the presence of 40 mM phosphate. Accumulated 45Ca2+ is rapidly released by 5 microM of the Ca2+ ionophore A23187 (t1/2 less than or equal to 6 s). (Ca(2+)-Mg2+)ATPase activity and Ca2+ uptake share the same subcellular distribution pattern and similar Ca2+ sensitivities (K0.5 = 0.39 microM and 0.15 microM, respectively). The substrate selectivity is high for both ATPase activity and Ca2+ transport. These results indicate the presence of an active transport of Ca2+ coupled to the (Ca(2+)-Mg2+)ATPase activity previously described in this parasite. A plasma membrane localization and physiological role in calcium homeostasis are suggested.     

脑梗塞患者红细胞膜泵活性与血液流变性的相关性研究

目的 :探讨脑梗塞 (CI)病人红细胞膜Na+ K+ ATPase、Ca2 + Mg2 + ATPase与血液流变学指标的相关性。方法 :对 49例非急性期的脑梗塞患者和 2 9例健康人分别测定Na+ K+ ATPase、Ca2 + Mg2 +ATPase、Ox LDL、LDL C、Cho C以及血液流变学指标。结果 :CI组全血粘度、血浆粘度、还原粘度、HCT、EAI、TK与Na+ K+ ATPase、Ca2 + Mg2 + ATPase活力的变化呈明显负相关 ;与Ox LDL、LDL C、Cho C呈明显正相关。结论 :CI患者红细胞膜泵活性的变化能引起血液粘滞性的改变 ,其作用主要是红细胞膜Na+ K+ 泵、Ca2 + 泵的活性降低 ,导致红细胞变形能力下降。     

Magnesium mediated change in physical state of phospholipid modulates membrane ATPase activity

During reconstitution of pig heart mitochondrial H(+)-ATPase in soybean phospholipid liposomes by the cholate dialysis method, Mg2+ greatly enhanced 32Pi-ATP exchange activity, ATPase activity and the sensitivity to oligomycin or DCCD of the reconstituted enzyme complex. The effect of Mg2+ on the lipid packing or fluidity of the reconstituted proteoliposomes was measured by means of spin labels, fluorescent probes and pyrene excimer formation efficiency. A difference in fluidity seemed to be localized near the polar faces of lipid bilayers of the reconstituted enzyme complex. Fluidity was less in the presence of Mg2(+)-containing and the Mg2(+)-'free' samples. Based on the results obtained a hypothetical scheme was proposed for Mg2(+)-mediated change in the physical state of phospholipid modulates incorporating H(+)-ATPase in liposomes. It postulated that Mg2+ may play a role in altering the lipid fluidity of the bilayers, which would induce a change of conformation of F0 portion (buried in the lipid core) of H(+)-ATPase complex. Such change could be transmitted to the soluble F1 portion, the conformation of which is in turn altered, resulting in higher enzymatic activity. Such an assumption was further supported by the results of a series of biochemical and biophysical experiments. Similar to its effect in the reconstitution of porcine heart mitochondrial H(+)-ATPase in liposomes, Mg2+ may also enhance the enzyme activity of reconstituted cytochrome C oxidase, porcine kidney medulla Na,K-ATPase, Ca-ATPase from rabbit sarcoplasmic reticulum and chloroplast H(+)-ATPase, in liposomes. It may be inferred that the structure and function of many membrane proteins are similarly modulated by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of membrane potential on Na+ -dependent Mg2+ extrusion from rat ventricular myocytes

To study Mg2+ transport across the cell membrane, the cytoplasmic concentration of Mg2+ ([Mg2+](i)) in rat ventricular myocytes was measured with the fluorescent indicator furaptra (mag-fura-2) under Ca2+ -free conditions (0.1 mM EGTA) at 25 degrees C. The fluorescence ratio signal of furaptra was converted to [Mg2+](i) using calibration parameters previously estimated in myocytes (Watanabe and Konishi, Pflügers Arch 442: 35-40, 2001). After [Mg2+](i) was raised by loading the cells with Mg2+ in a solution containing 93 mM Mg(2+), the cells were voltage-clamped at a holding potential of -80 mV using the perforated patch-clamp technique with amphotericin B. At the holding potential of -80 mV, the reduction of extracellular Mg2+ to 1.0 mM caused a rapid decrease in [Mg2+](i) only in the presence of extracellular Na(+). The rate of the net Mg2+ efflux appeared to be dependent on the initial level of [Mg2+](i); the decrease in [Mg2+](i) was significantly faster in the myocytes markedly loaded with Mg2+. The rate of decrease in [Mg2+](i) was influenced little by membrane depolarization from -80 to -40 mV, but the [Mg2+](i) decrease accelerated significantly at 0 mV by, on average, approximately 40%. Hyperpolarization from -80 to -120 mV slightly but significantly slowed the decrease in [Mg2+](i) by approximately 20%. The results clearly demonstrate an extracellular Na(+)- and intracellular Mg2+ -dependent Mg2+ efflux activity, which is consistent with the Na(+)-Mg2+ exchange, in rat ventricular myocytes. We found that the apparent rate of Mg2+ transport depends slightly on the membrane potential: facilitation by depolarization and inhibition by hyperpolarization with no sign of reversal between -120 and 0 mV.     

Calcium ATPase in the sarcoplasmic reticulum of muscle from normal and malignant hyperthermia susceptible pigs.

The density of Mg(2+)-dependent Ca2+ ATPase in the terminal cisternae of pig skeletal muscle fibers was investigated to discover whether a reduction in Ca2+ ATPase content impairs Ca2+ sequestration and contributes to the elevated myoplasmic Ca2+ concentration in malignant hyperthermia. Unexpectedly, immunogold electron microscopy showed an increase in Ca2+ ATPase, while densitometry of SDS-polyacrylamide gels suggested that the Ca2+ ATPase content of terminal cisternae vesicles did not change. The affinity of Ca2+ ATPase in vesicles for our monoclonal antibody was not altered. We suggest that the availability of antigenic sites in malignant hyperthermia increases after processing for electron microscopy, perhaps as a consequence of altered sarcoplasmic reticulum membrane properties.     

Ultrastructural localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) in cerebral endothelium

There is increasing interest in the role of calcium in a variety of biological processes. One of the mechanisms that regulate intracellular calcium concentrations is the calcium-activated adenosine triphosphatases (Ca2+-ATPase). The availability of an histochemical method for ultrastructural localization of Ca2+-ATPase has led to a number of studies attempting to localize this enzyme in a variety of cell types. This ultrastructural study was undertaken to localize Ca2+-ATPase in walls of intracerebral cortical vessels of rats. Both capillary and arteriolar endothelium showed discontinuous deposits of Ca2+-ATPase along the outer plasma membrane including the junctional plasma membranes. Patchy distribution of Ca2+-ATPase was also observed on the outer plasma membranes of smooth muscle and adventitial cells. Focal deposits of reaction product were associated with the actin filaments in endothelium. Invaginating pinocytotic vesicles at the outer plasma membrane of endothelium and smooth muscle cells showed Ca2+-ATPase. Intracytoplasmic vesicles showed the enzyme along the inner plasma membrane. Localization of Ca2+-ATPase on endothelial plasma membranes suggests that Ca2+ may be involved in many endothelial reactions. Further studies are required to determine the role of this enzyme and Ca2+ in endothelial reactions in normal and abnormal states.     

Temperature compensation of sodium transport and ATPase activity in frog skin.

Na+ transport across frog skin, measured as short-circuit current (SCC) shows perfect temperature compensation in frogs acclimated to 6 degrees, 12 degrees, and 23 degrees C as SCC values observed at the acclimation temperatures are equal (about 13 muA/cm2). Reacclimation experiments show that this is not a starvation effect. While very little temperature compensation is seen in the activity of Na+, K+-ATPase in epidermal homogenates from frog skins, the activity of Mg2+-ATPase shows inverse compensation at assay temperatures from 4 degrees to 48 degrees C. This ATPase is apparently activated either by Mg2+ or by Ca2+ and it probably controls the passive permeability of epidermal cells. It is suggested that the inverse temperature compensation in the activity of this enzyme is the main mechanism by which the observed perfect temperature compensation of Na+ transport across frog skin occurs.     

Mechanism of Ca2+ transport by Ca2+-Mg2+-ATPase pump: analysis of major states and pathways

Mechanistic studies of Ca2+ transport by the Ca2+-Mg2+-ATPase of skeletal sarcoplasmic reticulum are reviewed, and a unifying model is proposed. The significant steps in the transport cycle are modeled in terms of occupation and disposition of three binding sites on the enzyme: a) two translocation sites capable of binding to Ca2+ or a charge-stoichiometric amount of alkali cation (M+) or H+, b) an ATP-ADP-binding site, and c) a phosphorylation or phosphate-binding site. The normal transport cycle is characterized as the following sequence of steps: a) binding of two Ca2+ and Mg-ATP to external sites with high affinity and random order, b) enzyme phosphorylation, c) inward translocation of the Ca2+-laden sites, d) Ca2+ release to the sarcoplasmic reticulum lumen and ADP release to the external medium (random order), e) binding of Mg2+ or a charge-stoichiometric amount of K+ plus H+ to the translocators, f) dephosphorylation, g) the return of the K+- and H+-laden translocators to the outside, and h) dissociation of K+ and H+ from the translocator and completion of the cycle with step a. The enzyme is characterized as a Ca2+-K+ plus H+ countertransporter. The K+ plus H+ remove Ca2+ from the inwardly oriented translocator, thereby relieving a product inhibition and increasing the rate of enzyme dephosphorylation.     

Excessive intracellular Ca2+ inhibits glutamate-induced Na(+)-K+ pump activation in rat hippocampal neurons.

1. The effects of increased intracellular Ca2+ concentration ([Ca2+]i) on Na(+)-K+ pump activity in CA1 pyramidal neurons of rat hippocampal slices were investigated. The postglutamate hyperpolarization (PGH), which follows glutamate (GLU)-induced depolarization (GD), was used as an index of Na(+)-K+ pump activity, as was a ratio of PGH area to the preceding GD area (PGH ratio). 2. Perfusion of slices with saline containing Ca2+ ionophore (A23187, 10 microM) inhibited the PGH without producing apparent signs of cell deterioration. A 60-100% (85 +/- 15%, mean +/- SD) reduction in the PGH ratio occurred after 20-50 min of A23187 superfusion in 12 of 18 neurons tested. Complete abolition of the PGH occurred in 8 of these 12 cells exposed to A23187 for 30-120 min. 3. Application of A23187 in Ca(2+)-free/high-Mg2+ solution did not abolish the PGH, although small (less than 50%; 37 +/- 10%) reductions in the PGH ratio were observed after perfusion of 50 min or longer in five neurons tested. 4. Intracellular injection of the Ca2+ chelator bis-(o-amino-phenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, 300-400 mM) blocked inhibition of the PGH by A23187. After 50 min of perfusion with Ca2+ ionophore, no reduction of the PGH ratio was observed in five neurons tested. 5. Rundown of the PGH without apparent change in membrane properties was observed in three neurons that were stable for greater than 2-3 h, allowing repetitive GLU applications. 6. Block of the PGH produced by a Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitor (strophanthidin) prolonged the duration of GDs because of a delay in repolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mg2+- and Ca2+-stimulated ATPase at the outer surface of human peripheral lymphocytes and hematopoietic cell lines. Correlation between enzyme activity and immunoglobulin secretion.

A Mg2+- and Ca2+-dependent ATPase at the outer surface of human peripheral lymphocytes and hematopoietic cell lines has been studied. The enzyme activity varied about 60 fold between the extremes of the cell lines tested. There was no simple relationship between surface-ATPase activity and cell surface area. Nor was the presence of villi or any other morphological characteristic at the cell surface decisive for the degree of surface-ATPase activity. A correlation was, however, found between the ATPase activity and a phenomenon probably involving a contractile process namely immunoglobulin secretion, but it was not possible to establish an interdependence of these cell characteristics.     

Effect of external magnesium on intracellular free sodium: Na+ flux via Na+/Mg2+ antiport is masked by other Na+ transport systems in rat cardiac myocytes.

Mg2+ efflux from heart cells on a Na+/Mg2+ antiport has been postulated, but the Na+ flux component of the antiport has not been demonstrated. The study aimed to establish if the Na+ flux component could be measured by following changes in [Na+]i with SBFI during conditions known to reverse the antiport (5 mmol/L Mg2+(o), Na+(o)- & Ca2+(o)-free): and after minimising the activity of other Na+ transport pathways. Resting [Na+]i was 8 +/- 0.7 mmol/L (mean +/- S.E., n = 39 cells) in normal Tyrode's solution. [Na+]i decreased below the normal level in all cells (a decline of 4-5 mmol/L, n = 21) during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free). Controls using 1 mmol/L Mg2+(o) showed similar declines in [Na+]i, but the fall was greatest when Na+(o) was replaced by K+(o) (decline of 6 mmol/L) rather than the tetramethylammonium ion (TMA+). The rate of decrease in [Na+]i during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free) was slowed by 20 microM ouabain (n = 5) or by elevation of pHo to pH 9 (n = 7) so that [Na+]i remained close to the initial value. The decrease of [Na+]i was not affected by 10 microM imipramine (n = 15). These data suggest that the Na+ efflux component of the Na+/Mg2+ antiport is masked in Na+(o)- and Ca2+(o)-free conditions by other Na+(i) efflux pathways.     

Properties of the HCO 3 − -stimulated Mg2+-ATPase activity in red cell membranes

The HCO-3-stimulated Mg2+ -ATPase activity in red cell ghost fragments was investigated. Increasing the HCO-3 concentration in the incubation medium resulted in increased ATPase activity. NaHCO3 appeared to be more effective than KHCO3 in this regard. The ATPase activities were slightly stimulated by increases in ionic strength and utilized ITP almost as readily as ATP. A Mg/ATP ratio of 1.0 and a pH of 7.6 yielded maximum activity. These properties are of interest since the present enzyme is the only unquestionable instance where a HCO-3 ATPase is located in the surface membrane of a cell.     

Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).