Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic-stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase, with an estimated IC-50 of 2.5 X 10(-8) M. The present studies were initiated to evaluate the mechanism of inhibition of Ca2+-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca2+-ATPase indicated alteration of Vmax and Km by Plictran (1 and 5 X 10(-8) M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased Vmax of both ATP- and Ca2+-dependent enzyme activities. However, the Km of enzyme was decreased only for Ca2+. Plictran inhibited isoproterenol-stimulated Ca2+-ATPase activity by altering both Vmax and Km of ATP as well as Ca2+-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca2+. Preincubation of enzyme with 15 microM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 X 10(-7) M propranolol and 5 X 10(-8) M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 X 10(-5) M coenzyme A in combination with 5 X 10(-8) M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca2+-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.     

Inhibition of spider mite ATPases by plictran and three organochlorine acaricides

The ATPase enzyme system from two-spotted spider mites, $\text{Tetranychus}$$\text{urticae}$ (Koch) was sensitive, in vitro, to four acaricides. Tricyclohexylhydroxytin (Plictran^R) was an outstanding inhibitor of oligomycin-sensitive (mitochondrial) Mg²⁺ATPase from fish brain and spider mite homogenates. The I₅₀ values were 6.6×10^?11M and 6.2×10^?10M, respectively. Less effective were chlorbenside, chlorfenethol and ovotran. Plictran at a higher concentration (2×10^?7M) was also more effective on Na⁺-K⁺ATPase both in mites and fish brain homogenates as compared to chlorfenethol, chlorbenside and ovotran. Plictran inhibited oligomycin-insensitive Mg²⁺ATPase at concentrations of 10^?8M but stimulated at high concentrations (5×10^?6M and higher).     

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.     

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266?×?10³ units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25?kDa. They showed temperature optima at 50°C and pH optima at 5.0.?km for SPases was reported to be 1.05?mM and 0.186?mM for guaiacol and H₂O₂ respectively. The V_max/K_m value for o-dianisidine was 449 while for H₂O₂ it was 5?×?10⁵. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5???M) but ferulic acid inhibits its activity at a much lower concentration (0.02???M). Enzymes were stimulated by metal cations like Cu²⁺, Ca²⁺ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn²⁺ and Mg²⁺. Alanine stimulated the enzyme activity (up to 33%; at 0?C100???M) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13?C57% at 0?C100???M).     

R 24 571: A potent inhibitor of calmodulin-activated enzymes

R 24 571, a derivative of the antimycotic miconazole, is a very potent inhibitor of several Ca²⁺-calmodulin-dependent enzymes. Depending on the concentration of calmodulin, the I₅₀-values range from 0.5 ? 1.0 × 10^?8 M for brain phosphodiesterase to 1.5 ? 3.5 × 10^?7 M for erythrocyte Ca²⁺-ATPase and 5.0 × 10^?6 M for phosphorylase b kinase. Being much more potent, and devoid of affinity towards several receptors, R 24 571 is proposed as a more specific and useful tool for studying the involvement of calmodulin in biological processes than the currently used compounds.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+/K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM–500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na⁺/K⁺-ATPase activity was studied. The response of enzymatic activity in the presence of various K⁺ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na⁺/K⁺-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K⁺ ions below 20 mM in the medium assay. The IC₅₀ values for high/low isoforms 2.77 × 10^{? 6} M / 8.56 × 10^{? 5} M and 7.06 × 10^{? 7} M /1.87 × 10^{? 5} M were obtained in the presence of optimal (20 mM) and 2 mM K⁺, respectively. However, preincubation in the presence of elevated K⁺ concentration (50 – 500 mM) in the medium assay prior to Na⁺/K⁺-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC₅₀ values for both isoforms was the same as in the presence of the optimal K⁺ concentration. On the contrary, addition of 200 mM K⁺ into the medium assay after 10 minutes exposure of Na⁺/K⁺-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na⁺/K⁺-ATPase by reducing maximum enzymatic velocity (V_max) and K_m, implying an uncompetitive mode of interaction.     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.     

Unique Ca2+-activated ATPase in the nervous ganglia of Phyllocaulis soleiformis (Mollusca)

Nucleotide-metabolizing enzymes play important roles in the regulation of intracellular and extracellular nucleotide levels. We studied ATPase activity in the nervous ganglia of Phyllocaulis soleiformis, a terrestrial slug. The ATPase was divalent cation-dependent, with a maximal rate for ATP hydrolysis at pH 6.0 and 7.2 in the presence of Ca²⁺ (5 mM). Mg²⁺-ATPase activity was only 26% of the activity observed in the presence of Ca²⁺ (5 mM). ZnCl₂ (10 mM) produced a significant inhibition of 70%. Ca²⁺-ATPase activity was insensitive to the classical ATPase inhibitors ouabain, N-ethylmaleimide, orthovanadate and sodium azide. Levamisole, an inhibitor of alkaline phosphatase, was ineffective. Among nucleotides, ATP was the best substrate. The apparent K_{m (ATP)} for Ca²⁺-ATPase was 348±84 μM ATP and the V_max was 829±114 nmol Pi min⁻¹ mg⁻¹ protein. The P. soleiformis ganglial ATPase does not appear to fit clearly into any of the previously described types of Ca²⁺-ATPases.     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca²⁺ or Mg²⁺ and was not affected by F-actin and ouabain. V_max was 2.8 and 10.0 μmol P_i/mg protein per h for Ca²⁺- and Mg²⁺-dependent activity, respectively, and the corresponding K_m was 4.8·10^?4 M and 3.2·10^?4 M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

Effects of some steroid hormones on head-to-head association in bovine spermatozoa

In the presence of 2 × 10^?6 M Ca²⁺ in Tris-buffered medium 0.5 × 10^?6 M, oestradiol-17β or corticosterone significantly increased the head-to-head association of washed bull spermatozoa; in the same concentration, testosterone and 5α-dihydrotestosterone had no significant effects, whereas progesterone significantly dissociated the associated spermatozoa. At 8 × 10^?6 M Ca²⁺ in the same medium, all five hormones increased the association to about the same level. In Tyrode solution with a Ca²⁺ concentration of 1.4 × 10^?3 M, oestradiol-17β and corticosterone acted as above, whereas progesterone and the two testosterones effected dissociation. In Tyrode solution each of the dissociating hormones was combined with oestradiol-17β. In each case a sum of the effects of the two hormones was obtained without any stimulation or inhibition. All five hormones still produced significant effects at 5 × 10^?7 M in Tyrode solution. A corresponding value for ATP was found at 1 × 10^?5 M.     

Molecular Characterization of the Glycated Plasma Membrane Calcium Pump

We have previously demonstrated (Diabetes 39:707–711, 1990) that in vitro glycation of the red cell Ca²⁺ pump diminishes the Ca²⁺-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca²⁺ pump (Biochem. J. 293:369–375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca²⁺ pump, or to a partial inactivation of all the molecules. Glycation decreased the V _max for the ATPase activity leaving unaffected the apparent affinities for Ca²⁺, calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V _max for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca²⁺-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca²⁺-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated. Received: 9 March 1999/Revised: 11 May 1999     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Effects of potassium on vanadate inhibition of sarcoplasmic reticulum Ca2+-ATPase from dog cardiac and rabbit skeletal muscle.

The concentration of vanadate for half maximal inhibition of dog cardiac and rabbit skeletal SR Ca²⁺-ATPase was approximately 5 μM. Preincubation of the enzyme with vanadate resulted in greater inhibition. Effects of potassium on the inhibition were studied under various conditions.     

Influence of Ca++ and Mg++ on the vanadate inhibition of the Ca++- ATPase from pig heart sarcoplasmic reticulum

Vanadate inhibits the Ca⁺⁺-ATPase of sarcoplasmic reticulum from pig heart half maximally at about 10^?5 M. Mg⁺⁺ promotes this inhibition by vanadate whereas increasing Ca⁺⁺-concentrations protect the enzyme against vanadate inhibition. Keeping the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 × 10^?3 M ATP. Whenever the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ was higher than 1:1 the inhibitory effect of vanadate on the Ca⁺⁺-ATPase was increased.     

Cationic dependency of high affinity prostaglandin F2α receptors in bovine corpus luteum cell membranes

The specific binding of [³H] Prostaglandin (PG) F₂α to bovine corpus luteum cell membranes prepared in homogenizing buffer containing either 1 mM EDTA (H-EDTA) or 1 mM Ca²⁺ (HCa²⁺) was examined. The membranes prepared in H-EDTA buffer bound less [³H] PGF₂α and had a single class of PGF₂α receptors with an apparent dissociation constant (Kd) of 2.7 × 10^?8M. The addition of Ca²⁺ to these membranes resulted in increased binding with the appearance of new PGF₂α receptors of Kd = 4.3 × 10^?9M. The membranes prepared in HCa²⁺ buffer contained two classes of receptors with Kds = 2.9 × 10^?9M and 2.9 × 10^?8M. The removal of Ca²⁺ from these membranes resulted in lower binding as well as a complete disappearance of receptors of Kd = 2.9 × 10^?9M. These results suggest the dependency of high affinity PGF₂α receptors, in bovine corpus luteum cell membranes, on cations.     

LHRH-receptor-regulated Ca2+-ATPase activity in murine pituitary gland

Addition of luteinizing hormone releasing hormone (LHRH) in vitro (10^–5–5×10^–9 M) to murine pituitary membranes resulted in a dose-related decrease in Ca²⁺-ATPase activity within 15 min. Inhibitory effects of LHRH (10^–7 M) occurred after 90 sec, and appeared maximal by 120 sec. Eadie-Hofstee analysis at 10^–7 M LHRH, at varying [Ca²⁺]_free, resulted in aK _m=0.89±0.06 M and aV _max=18.8±0.71 nmol/mg per 2 min, compared to aK _m=0.69±0.06 M and aV _max=32.8±1.21 nmol/mg per 2 min for controls. Pre-incubation for 5 min with LHRH antagonist (10^–8 M) significantly attenuated (50%) the inhibitory effects of 10^–7 M LHRH on pituitary Ca²⁺ ATPase activity with aK _m=0.97±0.24 M and aV _max=28.1±2.8 nmol/mg per 2 min. The addition of LHRH (10^–7 M) to pituitary homogenates significantly increased luteinizing hormone (LH) release already at 10 and up to 40 sec compared to basal LH release. Systemic administration of 50 ng LHRH (i.p.), significantly (P<0.05) reduced pituitary Ca²⁺-ATPase after 30, 60 and 90 min, with a return to control levels by 120 min. Pituitary LH content was reduced slightly at 15 min, but was increased significantly at 90 and 120 min post-treatment. Plasma LH levels were elevated by 5 min, reached a peak by 15 min and returned to control within 60 min. The present findings indicate that LHRH receptor activation may influence cytosolic Ca²⁺ transport through effects on membrane Ca²⁺-ATPase activity. These actions may regulate LHRH-induced synthesis, storage and release of LH from pituitary gonadotropes.     

Role of foliar application of 24-epibrassinolide in response of peanut seedlings to iron deficiency

Limited information is available on the role of brassinosteroids (BRs) in response of plants to nutrient deficiency. To understand the functions of BRs in response to iron deficiency, we investigated the effect of 24-epibrassinolide (EBR) on activities of ferric-chelate reductase (FCR), H⁺-ATPase, Ca²⁺-ATPase, nitrate reductase (NR), antioxidant enzymes, Fe and other minerals content and distribution, chlorophylls, soluble protein, free proline, reactive oxygen species, and malondialdehyde in peanut (Arachis hypogea L.) plants subjected to Fe deficiency (10^?5 M Fe(III)-EDTA) with foliar application of EBR (0, 10^?8, 5.0×10^?8, 10^?7, 5.0×10^?7, and10^?6 M). Results show that EBR increased Fe translocation from roots to shoots and increased Fe content in cell organelles. Activities of antioxidant enzymes increased and so the ability of resistance to oxidative stress was enhanced. As result of enhancement of H⁺-ATPase and Ca²⁺-ATPase activities, the inhibition of Fe, Ca, Mg, and Zn uptake and distribution was ameliorated. Chlorophyll, soluble protein, and free proline content also increased and consequently, chlorosis induced by Fe deficiency was alleviated. The results demonstrate that EBR had a positive role in regulating peanut growth and development under Fe deficiency and an optimal concentration appeared to be 10^?7 M.     

Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate

Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca^2 +-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca²⁺-ATPase, with the following IC₅₀ values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb₁₀), is the strongest P-type enzyme inhibitor, after decavanadate (V₁₀). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca^2 +-ATPase stoichiometry. Furthermore, V₁₀ binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H₂VO₄^2 −; V₁₎ to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca^2 +-ATPase, with a dissociation constant, k_d of 1 μM^− 1. The interaction of decavanadate V₁₀O₂₈^6 − (V₁₀) with Ca^2 +-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb₁₀O₂₈^6 − (Nb₁₀), whereas no significant effects were detected with ATP or with heparin, a known competitive ATP binding molecule, suggesting that V₁₀ binds non-competitively, with respect to ATP, to the protein. Finally, it was shown that decaniobate inhibits SR Ca^2 +-ATPase activity in a non competitive type of inhibition, with respect to ATP. Taken together, these data demonstrate that decameric niobate and vanadate species are stronger inhibitors of the SR calcium ATPase than simple monomeric vanadate, tungstate and molybdate oxometalates, thus affecting calcium homeostasis, cell signalling and cell bioenergetics, as well many other cellular processes. The ability of these oxometalates to act either as phosphate analogues, as a transition-state analogue in enzyme-catalysed phosphoryl group transfer processes and as potentially nucleotide-dependent enzymes modulators or inhibitors, suggests that different oxometalates may reveal different mechanistic preferences in these classes of enzymes.     

The Ca2+-pump of sickle cell plasma membranes. Purification and reconstitution of the ATPase enzyme

The sickle cell (Hb SS) membrane-bound Ca²⁺-ATPase was found to have a V_max in the range of 30–100% of the V_max of the normal enzyme. In all sickle cell preparations, the Ca²⁺-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4–26 fold) in the different preparations. The affinity of the ghost sickle cell Ca²⁺-ATPase for Ca²⁺, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca²⁺-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca²⁺-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adynyah, J.T. Penniston and E. Carafoli, 1981, J.Biol.Chem.256,. 395 – 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that of the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca²⁺, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca²⁺ with normal efficiency (about 1 $\text{Ca}{}^{\mathrm{2+}}\text{ATP}$ hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.