Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.     

(Ca2+ + Mg2+)-ATPase activity in plasma membrane of circulating mononuclear cells. Lack of a direct effect of vitamin D

The in vivo effect of vitamin D on (Ca2+ + Mg2+)-ATPase activity was examined in a plasma membrane fraction of rat circulating mononuclear cells (MPM). Although there was no significant difference in the ATPase activities in red blood cell ghosts, (Ca2+ + Mg2+)-ATPase activity in MPM was significantly higher (p less than 0.05) in long-term vitamin D3-replete rats (100 IU/day for 6 months) than that in vitamin D-deplete rats (for 6 months). In rats maintained on vitamin D-deficient diets for 5-7 weeks, in vivo administration of either vitamin D3, 2,000 IU orally, 5 days prior to killing or 1,25-dihydroxyvitamin D3, 2.4 nmol, intraperitoneally, 24 h prior to killing failed to show any significant effect on (Ca2+ + Mg2+)-ATPase activity in MPM. (Ca2+ + Mg2+)-ATPase activity in MPM from rats maintained on vitamin D-deficient diet with high calcium content (1.8%) was significantly higher (p less than 0.05) than that from rats maintained on vitamin D-deficient diet with low calcium content (0.3%). Moreover, in vitro addition of vitamin D3 metabolites did not show any effect on (Ca2+ + Mg2+)-ATPase activity in MPM. These data suggest that decreased (Ca2+ + Mg2+)-ATPase activity in MPM from long-term vitamin D-deplete rats resulted from an adaptation to low extracellular calcium rather than vitamin D depletion.     

Effect of phospholipase A2 on Ca2+-stimulated adenosine triphosphatase activity in microsomal fraction of rat submandibular gland

The effect of phospholipase A2 on the Ca2+-ATPase (EC 3.6.1.3) activity in the microsomal fraction of rat submandibular gland was kinetically studied in vitro. The Ca2+-ATPase activity was significantly increased by the treatment with phospholipase A2 in the presence of bovine serum albumin as a scavenger for hydrolyzed products. When the microsomal fraction was incubated with phospholipase A2 in the absence of bovine serum albumin, the Ca2+-ATPase activity was not altered. The Vmax and Km values for both ATP and Ca2+ were increased by the phospholipase A2 treatment, respectively. These results indicated that the activation of Ca2+-ATPase by the phospholipase A2 treatment is due to the increase of Vmax.     

Beneficial influence of dietary curcumin, capsaicin and garlic on erythrocyte integrity in high-fat fed rats

In rats rendered hyperlipidemic by maintaining them on a high-fat diet (30%) for 8 weeks, inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet produced significant hypotriglyceridemic effect. Plasma cholesterol remained unaffected in high-fat treatment. Hepatic triglyceride content was significantly higher in high-fat fed rats, and this increase was effectively countered by inclusion of the hypolipidemic spice agents -- curcumin, capsaicin or garlic in the diet. The lipid profile of erythrocyte membranes of hyperlipidemic rats was similar to basal controls. An examination of the osmotic fragility of erythrocytes in various groups indicated that the red blood cells of hyperlipidemic rats display a slight resistance to osmotic lysis. Inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet, which produced the hypotriglyceridemic effect, appeared to beneficially correct this altered osmotic fragility of erythrocytes. Activities of ouabain-sensitive Na(+),K(+)-ATPase as well as acetylcholinesterase of erythrocyte membranes in high-fat fed rats remained unaltered. Activity of Ca(2+),Mg(2+)-ATPase in erythrocyte membrane was significantly decreased in high-fat fed animals, whereas dietary spice principles and garlic countered this reduction in enzyme activity. In the absence of any change in the cholesterol/phospholipid molar ratio in the erythrocyte membrane, a decreased activity of membrane-bound Ca(2+),Mg(2+)-ATPase could have probably contributed to the accumulation of intracellular calcium leading to the diminished deformability of the erythrocytes in high-fat fed rats.     

Influence of certain fish meals on (Na+ + K+)-ATPase and Ca2+-ATPase activity in rat small intestine

The effect of high-protein content fish meal on (Na+ + K+)-ATPase and Ca2+-ATPase activity in rat small intestine was studied. 5 groups of Wistar rats, weighing between 40-60 g, were fed diets with 12% protein content of dry matter for 10 days. The protein source was casein for the control group and fish meal derived from Coryphaenoides rupestris, Chimaera monstruosa and Merluccius merluccius for the test group. The results show a decrease in (Na+ + K+)-ATPase and a rise in Ca2+-ATPase activity in animals fed with fish meal protein compared to those fed on casein. No significant variations were observed between the groups fed on fish meal derived from C. rupestris and Ch. monstruosa. The calcium ion, which is abundant in fish, may be a factor responsible for these variations which produce inhibition of the (Na+ + K+)-ATPase and stimulation of the Ca2+-ATPase.     

Inhibitory effect of sulfhydryl group on Ca(2+)-ATPase activity in the plasma membrane-rich fraction from bovine parotid gland.

1. The present study demonstrated that the Ca(2+)-ATPase activity of the plasma membrane-rich fraction from bovine parotid gland was decreased by the addition of reducing agents. 2. Ca(2+)-ATPase activity staining on SDS-PAGE gels was lost in the presence of 2-mercaptoethanol. 3. Among all the reducing agents tested, GSH was the most effective in inhibiting Ca(2+)-ATPase. 4. The Ca(2+)-ATPase activity decreased by the GSH was restored by the addition of an oxidizing reagent. However, oxidation with an oxidizing reagent subsequent to alkylation of the reduced enzyme with iodoacetamide resulted in no restoration of activity. 5. The decrease of Ca(2+)-ATPase activity by GSH is due to a decrease in the Vmax of the enzyme. 6. These results suggest that the disulfide bond in this enzyme protein is necessary to maintain the activity of this enzyme.     

Effects of vitamin E on microsomal Ca(2+) -ATPase activity and calcium levels in streptozotocin-induced diabetic rat kidney

Vitamin E treatment has been found to be beneficial in preventing or reducing diabetic nephropathy. Increased tissue calcium and abnormal microsomal Ca(2+)-ATPase activity have been suggested as contributing factors in the development of diabetic nephropathy. This study was undertaken to test the hypothesis that vitamin E reduces lipid peroxidation and can prevent the abnormalities in microsomal Ca(2+)-ATPase activity and calcium levels in kidney of streptozotocin (STZ)-induced diabetic rats. Male rats were rendered diabetic by a single STZ injection (55 mg x kg(-1) i.p.). After diabetes was verified, diabetic and age-matched control rats were untreated or treated with vitamin E (400-500 IU kg(-1) x day(-1), orally) for 10 weeks. Ca(2+)-ATPase activity and lipid peroxidation (MDA) were determined spectrophotometrically. Blood glucose levels increased approximately five-fold (> 500 mg x dl(-1)) in untreated-diabetic rats but decreased to 340+/-27 mg x dl(-1) in the vitamin E treated-diabetic group. Kidney MDA levels did not significantly change in the diabetic state. However, vitamin E treatment markedly inhibited MDA levels in both control and diabetic animals. Ca(2+)-ATPase activity was 0.483+/-0.008 U l(-1) in the control group and significantly increased to 0.754+/-0.010 U l(-1) in the STZ-diabetic group (p < 0.001). Vitamin E treatment completely prevented the diabetes-induced increase in Ca(2+)-ATPase activity (0.307+/-0.025 U l(-1), p < 0.001) and also reduced the enzyme activity in normal control rats. STZ-diabetes resulted in approximately two-fold increase in total calcium content of kidney. Vitamin E treatment led to a significant reduction in kidney calcium levels of both control and diabetic animals (p < 0.001). Thus, vitamin E treatment can lower blood glucose and lipid peroxidation, which in turn prevents the abnormalities in kidney calcium metabolism of diabetic rats. This study describes a potential biochemical mechanism by which vitamin E supplementation may delay or inhibit the development of cellular damage and nephropathy in diabetes.     

Separation of active and inactive (nonphosphorylating) Ca(2+)-ATPase in sarcoplasmic reticulum subfractions from low-frequency-stimulated rabbit muscle.

Chronic low-frequency stimulation elicits in rabbit fast-twitch muscle a partial inactivation of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Ca(2+)-uptake activities. Inactive Ca(2+)-ATPase was enriched in a light microsomal fraction by sucrose density gradient centrifugation after calcium oxalate loading in the presence of ATP. This fraction showed a reduced specific activity and phosphoprotein formation of the Ca(2+)-transport ATPase. These results suggest that the inactivation of the Ca(2+)-ATPase as induced by increased contractile activity, is confined to a specific SR vesicle population.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the ?EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F^?, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in ?EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in ?EFA membranes than in +EFA. Total Na⁺, K⁺ (Mg²⁺)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent K_m and V_max. 5′-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Effects of dimethyl sulfoxide and polycationic neomycin on stimulation of purified plasma membrane Ca(2+)-pump by negatively charged phospholipids.

At least two reaction steps are involved in the activation of purified plasma membrane Ca(2+)-transport ATPase by negatively charged phospholipids depending on the type of phospholipids (Lehotsky et al. 1992). The effect of negatively charged phospholipids on Ca(2+)-stimulated ATPase (cycling activity) was compared with that of p-nitrophenylphosphatase (E2-form activity) catalyzed by Ca(2+)-pump. PIP like PS, activated Ca(2+)-ATPase activity by modifying ATP activation curve with increasing Vmax of the high affinity site. Ca(2+)-ATPase activity reconstituted in PC was stimulated by DMSO(10%) by a factor of 1.36. The activity stimulation by DMSO was only weak in PS and activity was inhibited in PIP. Also, phosphatase activity catalyzed by Ca(2+)-pump was strongly stimulated by DMSO and was differentially affected by phospholipid head group. Positively charged neomycin (5 mmol/l) had no effect on Ca(2+)-ATPase activity reactivated in PC or PS, but the stimulatory action of PIP was suppressed. Relative stimulation of phosphatase activity by PS was not influenced. Both hydrolytic activities catalyzed by Ca(2+)-transport ATPase were differentially affected by organic solvents and polycations with respect to the kind of the phospholipid.     

Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities

Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na+ + K+)-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca2+ fluxes via the Na+/Ca2+ exchanger, before and after modification of an imposed Na+ gradient by penetrating or nonpenetrating Na+ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na+ + K+)-ATPase, basal Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca2+ gradient. Thus, the saponin-induced increment in (Ca2+ + Mg2+)-ATPase due only to exposure of occluded sites (as opposed to Ca2+ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na+-channel opening measured indirectly by dissipation of an imposed Na+ gradient utilized to drive a large 45Ca2+ accumulation via the Na+/Ca2+ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na+ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity.     

Regucalcin increases Ca2+-ATPase activity in the mitochondria of brain tissues of normal and transgenic rats

The role of regucalcin, which is a regulatory protein in intracellular signaling, in the regulation of Ca(2+)-ATPase activity in the mitochondria of brain tissues was investigated. The addition of regucalcin (10(-10) to 10(-8) M), which is a physiologic concentration in rat brain tissues, into the enzyme reaction mixture containing 25 microM calcium chloride caused a significant increase in Ca(2+)-ATPase activity, while it did not significantly change in Mg(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca(2+)-ATPase activity was completely inhibited in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), both of which are inhibitors of mitochondrial uniporter activity. Whether the effect of regucalcin is modulated in the presence of calmodulin or dibutyryl cyclic AMP (DcAMP) was examined. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not significantly enhanced in the presence of calmodulin (2.5 microg/ml) which significantly increased the enzyme activity. DcAMP (10(-6) to 10(-4) M) did not have a significant effect on Ca(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not seen in the presence of DcAMP (10(-4) M). Regucalcin levels were significantly increased in the brain tissues or the mitochondria obtained from regucalcin transgenic (RC TG) rats. The mitochondrial Ca(2+)-ATPase activity was significantly increased in RC TG rats as compared with that of wild-type rats. This study demonstrates that regucalcin has a role in the regulation of Ca(2+)-ATPase activity in the brain mitochondria of rats.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Effect of thapsigargin on cardiac muscle cells.

The effect of thapsigargin on the activity of various enzymes involved in the Ca(2+)-homeostasis of cardiac muscle and on the contractile activity of isolated cardiomyocytes was investigated. Thapsigargin was found to be a potent and specific inhibitor of the Ca(2+)-pump of striated muscle SR (IC50 in the low nanomolar range). A strong reduction of the Vmax of the Ca(2+)-pump was observed while the Km (Ca2+) was only slightly affected. Reduction of the Vmax was caused by the inability of the ATPase to form the Ca(2+)-dependent acylphosphate intermediate. Thapsigargin did not change the passive permeability characteristics nor the function of the Ca(2+)-release channels of the cisternal compartments of the SR. In addition, no significant effects of thapsigargin on other ATPases, such as the Ca(2+)-ATPase and the Na+/K(+)-ATPase of the plasma membrane as well as the actomyosin ATPase could be detected. The contractile activity of paced adult rat cardiomyocytes was completely abolished by 300 nM thapsigargin. At lower concentrations the drug prolonged considerably the contraction-relaxation cycle, in particular the relaxation phase. The intracellular Ca(2+)-transients elicited by electrical stimulation (as measured by the changes in Fluo-3 fluorescence) decreased in parallel and the time needed to lower free Ca2+ down to the resting level increased. In conclusion, the results indicate that selective inhibition of the Ca(2+)-pump of the SR by thapsigargin accounts for the functional degeneration of myocytes treated with the drug.     

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.     

Quantification of Ca(2+)-ATPases in porcine duodenum. Effects of 1,25(OH)2D3 deficiency.

Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.     

Calcium transport in basolateral plasma membranes from kidney cortex of Milan hypertensive rats

Ca2+ transport was investigated in basolateral plasma membranes (BLM) isolated from kidney cortex of the Milan strain of genetically hypertensive rats (MHS) and their normotensive controls (MNS) during a pre-hypertensive stage (age 3-4 weeks). It was found that the Vmax of ATP-dependent Ca2+ transport (in the presence of calmodulin) was about 16% lower in MHS than in control rats. In membranes from MNS rats which had been isolated in the presence of EGTA, the ATP-dependent Ca2+ transport showed a hyperbolic Ca2+ concentration dependence, a high Km (Ca2+) and a low Vmax; upon addition of exogenous calmodulin, the kinetics became sigmoidal, the Km (Ca2+) was decreased and the Vmax was increased. In membranes from MHS rats, the Ca2+ concentration dependence of ATP-driven Ca2+ transport was sigmoidal and the Ca2+ affinity was high in the absence of added calmodulin. Addition of exogenous calmodulin to these membranes resulted in an increase in Vmax, but no change in other kinetic parameters. Low-affinity hyperbolic kinetics of Ca2+ transport could only be obtained in MHS rats if the membranes were extracted with hypotonic EDTA and hypertonic KCl. These data suggest that the plasma membrane Ca2+-ATPase, which catalyses the ATP-dependent Ca2+ transport, exists in BLM of pre-hypertensive MHS rats predominantly in an activated, high-affinity form.     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Erythrocyte membrane in rats fed high erucic acid-containing mustard oil: osmotic fragility, lipid composition, and (Na+, K+)- and (Ca2+, Mg2+)-ATPases

Erythrocyte hemolytic properties, cholesterol/phospholipid ratios, fatty acid composition, and activities of the membrane-bound enzymes (Na+, K+)- and (Ca2+, Mg2+)-ATPase were studied in male and female rats fed low erucic acid rapeseed (LEAR) and high erucic acid mustard oils (HEAM) for a period of 16 months. Rats receiving groundnut oil (GNO) served as controls. Erythrocytes from HEAM-receiving male and female rats showed increased resistance to hypotonic hemolysis. In male rats this was associated with an 85% increase (P less than 0.07) in the cholesterol/phospholipid molar ratio. The fatty acid double-bond index showed an increase in male rats receiving HEAM as well as LEAR oils. In the erythrocytes from female rats, the cholesterol/phospholipid molar ratio and double bond index remained unaffected. Specific activity of ouabain-sensitive (Na+, K+)-ATPase showed a small (+20%) but significant (P less than 0.05) increase in male but not female rats of HEAM group. Total (Na+, K+)-ATPase, ouabain-insensitive component, and (Ca2+, Mg2+)-ATPase were not altered in rats receiving LEAR or HEAM.