Uncoupling of Occlusion From ATP Hydrolysis Activity in Sarcoplasmic Reticulum (Ca2++Mg2+)-ATPase

The uncoupling of Ca²⁺ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu³⁺ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Co²⁺ and at 25°C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca²⁺, 3 mM ATP, and at 25°C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu³⁺. Digest past TD2 in the presence of 5 mM Ca²⁺ and 3 mM AMP-PNP. (a non-hydrolyzable ATP analog) at 25°C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca²⁺ transport from ATPase activity is possible by tryptic digestion of the (Ca²⁺ + Mg²⁺)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyte ghosts

Incubation of erythrocyte ghosts with carbonylcyanide m-chlorophenylhydrazone (CCCP) plus Ca²⁺ resulted in inactivation of the Ca²⁺-stimulated ATPase activity. Omission of Ca²⁺ or lowering of the temperature below 25 °C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of β-mercaptoethanol did not influence the Ca²⁺-dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca²⁺-containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(β-aminoethyl acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca²⁺-stimulated, Mg²⁺-ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg²⁺-ATPase slightly. CCCP was much more specific for the Ca²⁺-stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca²⁺ for inactivation was also quite specific.     

Effect of cadmium on ciliary and ATPase activity in the gills of freshwater mussel Anodonta cygnea

1. Cadmium (≤ 50 μM) decreases the heat resistance (39°C) of the activity of frontal cilia in the Anodonta cygnea gills incubated in dechlorinated tap water, while in the presence of added 2 mM Ca²⁺ the minimal acting concentration of cadmium rises up to 100 μM.2. The inhibitory effect of Cd²⁺ (1.5 mM) on the ATPase activity measured in the gill microsomal fraction is temperature dependent and increases as follows: ouabain insensitive Na²⁺- or K⁺-ATPase (no inhibition), Ca²⁺-ATPase (50% inhibition), Mg²⁺-ATPase (100% inhibition).3. Cadmium itself (≤ 50 μM) added to microsomal suspension stimulates the H⁺-sensitive ATP hydrolysis resembling on its pH-dependence the Mg²⁺- but not Ca²⁺-ATPase activity.4. Cd²⁺ can mimic the effect of Mg²⁺ as a cofactor required for activation of the ouabain-insensitive Na⁺- or K⁺-ATPase. Monovalent cations fail to activate the ATPase when Mg²⁺ is substituted by Ca²⁺.5. One of the mechanisms underlying the toxicity of Cd²⁺ to Anodonta gills could be based upon an interaction of Cd²⁺ with Mg²⁺-ATPase followed by suppression of the ciliary activity.     

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.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Affinity chromatography of H+-translocating adenosine triphosphatase isolated by chloroform extraction of Rhodospirillum rubrum chromatophores. Modification of binding affinity by divalent cations and activating anions

1. ATPase isolated from Rhodospirillum rubrum by chloroform extraction and purified by gel filtration or affinity chromatography shows three bands (α, β and γ) upon electrophoresis in sodium dodecyl sulphate.2. Ca²⁺-ATPase activity of the preparation is inhibited by aurovertin and efrapeptin but not by oligomycin. Activity may be inhibited by treatment with 4-chloro-7-nitrobenzofurazan and subsequently restored by dithiothreitol.3. The enzyme fails to reconstitute photophosphorylation in chromatophores depleted of ATPase by sonic irradiation.4. Most of the active protein from the crude chloroform extract binds to an affinity chromatography column bearing an immobilised ADP analogue but not to a column bearing immobilised pyrophosphate.5. In the absence of divalent cations, a component with a very high specific activity for Ca²⁺-ATPase is eluted from the column by 1.6 mM ATP. This protein migrates as a single band on 5% polyacrylamide gel electrophoresis and only possesses three subunits. At 12 mM ATP an inactive protein is eluted which does not run on acid or alkali polyacrylamide gels and shows a complex subunit structure.6. ATPase preparations prepared by acetone extraction or by sonic irradiation of chromatophores may also be purified 10-fold by affinity chromatography.7. The inclusion of 5 mM MgCl₂ or CaCl₂ during affinity chromatography of chloroform ATPase increases the capacity of the column for the enzyme and demands a higher eluting concentration of ATP.8. When the enzyme is more than 90% inhibited by efrapeptin or 4-chloro-7-nitrobenzofurazan, the binding characteristics of the enzyme are not affected.9. 10 mM Na₂SO₃, which greatly stimulates the Ca²⁺- and Mg²⁺-dependent ATPase activity of the enzyme and increases K_i (ADP) for Ca²⁺-ATPase from 50 to 850 μM, prevents binding to the affinity column. Binding may be restored by the addition of divalent cations.10. Na₂SO₃ increases the rate of ATP hydrolysis, ATP-driven H⁺ translocation and ATP-driven transhydrogenase in chromatophores.11. It is proposed that anions such as sulphite convert the chromatophore ATPase into a form which is a more efficient energy transducer.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

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.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

Reconstitution and Characterization of a Calmodulin-Stimulated Ca-Pumping ATPase Purified from Brassica oleracea L

Purification and functional reconstitution of a calmodulin-stimulated Ca²⁺-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca²⁺, together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropyl]-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca²⁺ uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 μmol min⁻¹ (mg protein)⁻¹, and the Ca²⁺/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca²⁺-ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca²⁺) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca²⁺-pumping ATPase in Brassica oleracea inflorescences.     

Stimulatory effect of regucalcin on mitochondrial ATP‐dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca²⁺ signaling, on Ca²⁺‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca²⁺‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent ⁴⁵Ca²⁺ uptake by the mitochondria. Ruthenium red (10⁻⁶ M) or lanthunum chloride (10⁻⁶ M), an inhibitor of mitochondrial Ca²⁺ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca²⁺‐ATPase activity and ⁴⁵Ca²⁺ uptake. The effect of regucalcin (100 nM) in elevating Ca²⁺‐ATPase activity was completely prevented by the presence of digitonin (10⁻²%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca²⁺‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10⁻⁴ M), which could increase Ca²⁺‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca²⁺‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca²⁺‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca²⁺‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc.     

Effects of chromium compounds on plasma membrane Mg2+-ATPase activity of BHK cells.

An ATPase activity stimulated by divalent ions (Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺) has been observed in intact hamster fibroblasts cultured in vitro (BHK line). Such activity has been determined by the incubation (30 min at 37°C) of washed cell suspensions (about 1 mg of proteins) in a medium containing 100 mM NaCl, 20 mM KCl, 15 mM Tris—HCl (pH 7.4), 10 mM NaHCO₃, 5 mM glucose and equimolar concentrations of ATP and divalent cation. Mg²⁺-ATPase activity is insensitive to ouabain and lacks specificity towards nucleoside triphosphate substrates. AMP and ADP are not hydrolyzed under these conditions. Apparent K_m of 0.76 mM and V_max of 1.46 μmol Pi · mg proteins^?1 · h^?1 have been calculated for Mg-ATP complex. This ATPase is an ectoenzyme, therefore its activity could be used as a suitable index of the action of chemicals like chromium compounds known for their cytotoxic effects on membrane functions.Salts of trivalent (CrCl₃) and hexavalent (K₂Cr₂O₇) chromium at concentrations ranging from 1 mM to 5 mM inhibit Mg²⁺-ATPase. The inhibition by K₂Cr₂O₇ is observed after pretreatment of the cells with this compound followed by its absence from the assay medium “per se” for Mg²⁺-ATPase, and it is referred to the alterations of membrane bound enzyme structures by the oxidizing hexavalent chromium. The inhibition by CrCl₃ is mainly evident when this compound is present in the incubation medium, and is referred to the interaction of trivalent chromium with Mg²⁺-ATP as it is partially reversed by increasing Mg²⁺-ATP concentration.     

Biochemical characteristics of the Ca2+ pumping ATPase in the peribacteroid membrane from broad bean root nodules

Ca²⁺-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH 7.0. Translocation of Ca²⁺ through the PBM by the Ca²⁺-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP?>?ITP???GTP???UTP???CTP, the K _m of the enzyme for MgATP being about 100 μM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca²⁺-ATPase for Ca²⁺ of about 0.1 μM. The transport activity of Ca²⁺-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca²⁺-ATPases which are present in other plant membranes.     

The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin,ATP and partial proteolysis

(1) Depending on the assay conditions, the ability of the Ca²⁺-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg²⁺ and K⁺ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca²⁺ so that the activity becomes negligible above 30 μM Ca²⁺. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca²⁺. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca²⁺ are needed and phosphatase activity is maximal in media without Ca²⁺. This is the first report of an activity sustained by the Ca²⁺-ATPase of red cell membranes in the absence of Ca²⁺. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca²⁺. These results strongly suggest that during activation by calmodulin, Ca²⁺ is needed only to form the calmodulin-Ca²⁺ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca²⁺ and the induction of phosphatase activity by ATP + Ca²⁺ suggests that activation of the phosphatase by Ca²⁺ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E₂, the conformer of the Ca²⁺-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca²⁺ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E₁ and E₂ towards E₂. On the other hand, ATP + Ca²⁺ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E₂ predominates over other conformers of the ATPase.     

The effect of calcium ionophore A23187 on the ATP level of human erythrocytes

Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca²⁺ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca²⁺ and decreases as Ca²⁺ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca²⁺ influx into the cell. The biphasic nature of Ca²⁺-stimulated ATP depletion in whole cells reflects the activity of Ca²⁺-ATPase in membrane preparations at varying Ca²⁺ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr.     

A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl₂ (5 mm) and a Ca²⁺ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca²⁺ (K_0.5 ~0.4 μm); but not by similar concentrations of Mn²⁺, Zn²⁺, Co²⁺, or La²⁺. This stimulation was specific for ATP; there was little or no effect of Ca²⁺ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca²⁺ (10 to 100 μm) in the presence or absence of Mg²⁺ stimulated transfer of ³²P from [γ-³²P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K⁺. The specific activity of Ca²⁺-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca²⁺-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.     

Biochemical characterization of the plasma membrane Ca2+ pumping ATPase activity present in the gill cells of Mytilus galloprovincialis LAM

• 1.1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca²⁺-activated ATPase activity is present. The ATPase has high Ca²⁺ affinity (K_ma = 0.3 μM).
• 2.2. The optimum assay conditions to evaluate the enzymatic activity of the Ca²⁺-stimulated ATPase at 19°C are: 120–300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca²⁺ ATPase activity is stimulated by DTT (0.5–1 mM) and it is inhibited by low concentrations of vanadate (10–50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive.
• 3.3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca²⁺ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La³⁺; (c) the mol. wt of Ca²⁺ ATPase is about 140 kDa.
• 4.4. Low Ca²⁺ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca²⁺ uptake by plasma membrane inside-out vesicles.
• 5.5. The results indicate that the Ca²⁺ pump present in the gill plasma membranes could be responsible for Ca²⁺ extrusion and therefore involved in maintaining the cytosolic Ca²⁺ concentration within physiological levels.

     

K+-and Mg2+-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca²⁺ + Mg²⁺-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg²⁺ and EGTA and is stimulated by Ca²⁺. The Mg²⁺-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5mM MgCl₂, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca²⁺ causes no further increase in the rate of hydrolysis and Ca²⁺ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca²⁺ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by P_i unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect P_i inhibition of the Mg²⁺-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a P_i-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and P_i. In this conformation the enzyme is transformed from a Ca²⁺- and Mg²⁺-dependent ATPase into a (K⁺ + Mg²⁺)-ATPase.