Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

Body Plasma Membrane Vesicles from Paramecium Contain a Vanadate-Sensitive Ca--ATPase

Paramecia are an excellent model system for studying the mechanisms involved in sensory transductions and intracellular Ca²⁺ regulation. These cells have two functionally distinct plasma membrane domains, body and cilia. The body plasma membrane is responsible for transduction of sensory stimuli into receptor potentials and the ciliary membrane is required for Ca²⁺ action potentials. Although ciliary membrane vesicles (cmv) have been purified and well characterized, body plasma membranes have not. We have generated body plasma membrane vesicles (bmv) by homogenization of deciliated cells and purified them from the microsome fraction by a two-phase aqueous polymer separation. The major criteria for purity of the bmv fraction are: (i) It is enriched 15-fold for a known plasma membrane marker (immobilization antigen) while the marker activities for other membranes were all decreased. The protein banding pattern of bmv is generally similar to cmv on SDS-PAGE. (ii) It contains a vanadate-sensitive Ca²⁺-ATPase activity that has been suggested to be a plasma membrane Ca²⁺ pump. The specific activity of this bmv Ca²⁺-ATPase is increased 4-fold over that of the homogenate. (iii) The phospholipid, fatty acid, and sterol composition of the bmv fraction are indicative of plasma membranes because they are qualitatively similar to cmv. The bmv also contains a membrane-bound NADPH-dependent cytochrome c reductase activity, suggesting that it may play a role in body plasma membrane function. This purified bmv preparation is useful for studying the role of the body plasma membrane in Ca²⁺ regulation, sensory transduction, protein and lipid trafficking, and plasma membrane fusion events.     

Melafen effect on ATP-dependent accumulation of calcium in plasma membrane vesicles from cells of potato tubers

Synthetic growth regulator melafen (10⁻⁵–10⁻¹⁰ M) was tested for aneffect on the Ca²⁺ accumulation in plasma membrane vesicles (PMVs) isolated from potato Solanum tuberosum L. tubers at forced rest and sprouting. Melafen proved to regulate the Ca²⁺ accumulation in PMVs by changing the activity of Ca²⁺, Mg²⁺-ATPase of the plasma membrane, while no effect was observed with respect to Ca²⁺ outflow from vesicles. The melafen effect on Ca²⁺, Mg²⁺-ATPase activity depended on the physiological condition of tubers and the melafen concentration.     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

Activation of calcium-ATPase of liver plasma membrane by dextran sulfates.

Dextran sulfate (DS) with average molecular weight (AMW) of 20,000 and sulfur content of 18%, which has a high lipemia clearing activity, enhanced Ca²⁺ binding to the plasma membrane of rat liver, and the DS itself bound the membrane, whereas there was little binding of DS and Ca²⁺. Various DSs slightly activated Na⁺-K⁺-ATPase, but not Mg²⁺-ATPase activity of the membrane. These results suggest that DSs, especially with high AMW of 20,000, bind the plasma membrane, resulting in enhancements of the Ca²⁺ binding to there and Ca²⁺-ATPase activity.     

Gel electrophoretic and density gradient analysis of the (K + Ca)-ATPase and the (Na + K)-ATPase activities of cardiac membrane vesicles

The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K⁺ + Ca²⁺)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na⁺ + K⁺)-ATPase, an enzymic activity localized to sarcolemma. The (K⁺ + Ca²⁺)-ATPase activity was stimulated 4- to 5-fold by 100 mM K⁺ in the presence of 50 μM Ca²⁺. Phosphorylation of the (K⁺ + Ca²⁺)-ATPase of intact vesicles with [γ-³²P]ATP was Ca²⁺ dependent, and monovalent cations including K⁺ increased the level of [³²P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K⁺ + Ca²⁺)-ATPase was inactivated, as was Ca²⁺-dependent ³²P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na⁺ + K⁺)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na⁺ + K⁺)-ATPase activity of intact membrane vesicles. ³²P incorporation by the (Na⁺ + K⁺)-ATPase of SDS-extracted vesicles was Na⁺ dependent, and Na⁺-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the ³²P-intermediates of the (K⁺ + Ca²⁺)-ATPase and the (Na⁺ + K⁺)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na⁺ + K⁺)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K⁺ + Ca²⁺)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na⁺ + K⁺)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K⁺ + Ca²⁺)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.     

Calcium uptake and Ca2+-ATPase activity in goat spermatozoa membrane vesicles do not require Mg2+

Microsomal membrane vesicles isolated from goat spermatozoa contain Ca²⁺-ATPase, and exhibit Ca²⁺ transport activities that do not require exogenous Mg²⁺ .The enzyme activity is inhibited by calcium-channel inhibitors,e.g. verapamil and diltiazem, like the well known Ca²⁺ , Mg²⁺-ATPase. The uptake of calcium is ATP (energy)-dependent and the accumulated Ca²⁺ can be completely released by the Ca²⁺ ionophore A23187, suggesting that a significant fraction of the vesicles are oriented inside out     

The plasma-membrane H+-ATPase from beet root is inhibited by a calcium-dependent phosphorylation

Several plasma-membrane proteins from beet root (Beta vulgaris L.) have been functionally incorporated into reconstituted proteoliposomes. These showed H⁺-ATPase activity, measured both as ATP hydrolysis and H⁺ transport. The proton-transport specific activity was 10 times higher than in plasma membranes, and was greatly stimulated by potassium and valinomycin. These proteoliposomes also showed calcium-regulated protein kinase activity. This kinase activity is probably due to a calmodulin-like domain protein kinase (CDPK), since two protein bands were recognized by antibodies against soybean and Arabidopsis CDPK. This kinase phosphorylated histone and syntide-2 in a Ca²⁺-dependent manner. Among the plasma-membrane proteins phosphorylated by this kinase, was the H⁺-ATPase. When the H⁺-ATPase was either prephosphorylated or assayed in the presence of Ca²⁺, both the ATP-hydrolysis and the proton-transport activities were slower. This inhibition was reversed by an alkaline-phosphatase treatment. A trypsin treatment (that has been reported to remove the C-terminal autoinhibitory domain from the H⁺-ATPase) also reversed the inhibition caused by phosphorylation. These results indicate that a Ca²⁺-dependent phosphorylation, probably caused by a CDPK, inhibits the H⁺-ATPase activities. The substrate of this regulatory phosphorylation could be the H⁺-ATPase itself, or a different protein influencing the ATPase activities. Received: 1 May 1997 / Accepted: 25 June 1997     

The modulating effects of pH and benzyladenine on the Ca2+– and Mg2+-ATPases in the plasmalemma of wheat root cells

Plant cells frequently and rapidly have to respond to environmental changes for survival. Regulation of transport and other energy-requiring processes in the plasmalemma of root cells is therefore one important aspect of the ecological adaptation of plants. Wheat (Triticum aestivum L. cv. Drabant) was grown hydroponically, with or without 50 nM benzyladenine in the medium, and plasma membranes from root cells of 8-day-old plants were prepared by aqueous polymer two-phase partitioning. The influence of Ca²⁺ and Mg²⁺ on the plasmalemma ATPase activities was investigated. The presence of benzyladenine during growth increased the ATPase activity, that dependent upon Ca²⁺ more than that elicited by Mg²⁺. As a general characteristic, ATP was the preferred substrate, but all nucleotide tri- and diphosphates could be accepted with activities in plasma membranes from control plants of 7-36% (Mg²⁺) and 40-86% (Ca²⁺) and in plasma membranes from benzyladenine-treated plants of 12-47% (Mg²⁺) and 53-102% (Ca²⁺) as compared with activities obtained with ATP. Nucleotidemonophosphates were not hydrolyzed by the preparations. In preparations from benzyladenine-treated plants one peak of Ca²⁺-ATPase at pH 5.2–5.6, with a tail from pH 6 and upwards, and one peak of Mg²⁺-ATPase at pH 6.0–6.5 were observed in the presence of EDTA in the assay media. In preparations from control plants, the addition of EDTA to the assays resulted in a wide optimum between pH 6 and 7 for Mg²⁺-ATPase and low Ca²⁺-ATPase activity with no influence of pH in the range 4.5 to 8. Analysis of the pH dependence in the presence of both Ca²⁺ and Mg²⁺ indicates that the control plants mainly contain Mg²⁺-ATPase corresponding to the proton pump. Preparations from benzyladenine-treated wheat roots show, in addition, activation by Ca²⁺, which, in the slightly alkaline pH range may correspond to a Ca²⁺-extruding (Ca²⁺+ Mg²⁺)-ATPase. In the acidic range, the responses are more complicated: the Mg²⁺-ATPase is inhibited by vanadate, while the Ca²⁺-ATPase is insensitive, and benzyladenine added during growth influences the interaction between Ca²⁺ and Mg²⁺ in a way that parallels the effect of high salt medium.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Interaction of phytohormones and synthetic growth regulator melafen in the control of Ca2+ translocation across the plasma membrane of potato (Solanum tuberosum L.) tuber cells

Interference of phytohormones (jasmonic, gibberellic, and abscisic acids) and synthetic growth regulator melafen on Ca²⁺ translocation across the membrane of plasma membrane vesicles prepared from dormant potato (Solanum tuberosum L.) tubers was studied. The activity of plasma membrane Ca²⁺, Mg²⁺-ATPase was stimulated by melafen and jasmonic and gibberellic acids and suppressed by abscisic acid. These substrances did not change the passive membrane permeability for Ca²⁺. The pattern of the effect of melafen on the activity of Ca²⁺,Mg²⁺-ATPase depended on the presence of phytohormones in incubation medium. When melafen and each phytohormone were simultaneously added to incubation medium, their effects were not additive, which indicates that the effects of the tested compounds on the Ca²⁺ uptake into the plasma membrane vesicles are interdependent. Apparently, the interaction between the phytohormones and plasma membrane components modulates the response to melafen.     

The Ca2+ Pump of the Plasma Membrane of Arabidopsis thaliana: Characteristics and Sensitivity to Fluorescein Derivatives

The transport and hydrolytic activities of the plasma membrane (PM) Ca²⁺ pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two-phase partitioning technique. Ca²⁺ uptake could be energized by ATP and by ITP (at about 70% the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca²⁺-dependent ITPase activity. The PM Ca²⁺ pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half-saturated by 60 μM ITP. It was stimulated by CaM, specially at low, non-saturating Ca²⁺ concentrations. All of these characteristics closely resemble those of the PM Ca²⁺ pump in other plant materials. Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca²⁺ pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM H⁺-ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca²⁺ pump and of the H⁺-ATPase of the PM (eosin Y was about 10000-fold, EB 1000-fold and rose bengal only 50-fold more active on the Ca²⁺ pump than on the H⁺-ATPase); and iv) the effectiveness of EB in inhibiting the Ca²⁺ pump was strongly affected by the protein concentration in the assay medium.     

Evidence that both Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule

• 1.1. Evidence was obtained that activities of both low-affinity Ca²⁺-ATPase and high-affinity (Ca²⁺ + Mg²⁺)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme.
• 2.2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant.
• 3.3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca²⁺-ATPase activity comigrated with that of (Ca²⁺ + Mg²⁺)-ATPase.
• 4.4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated.
• 5.5. These findings suggest that Ca²⁺-ATPase and (Ca²⁺ + Mg²⁺)-ATPase are a single enzyme.

     

Enhancement of plasma membrane (Ca2+-Mg2+)-ATPase activity in regenerating rat liver: Involvement of endogenous activating protein regucalcin

The alteration of (Ca²⁺-Mg²⁺)-ATPase activity in the plasma membranes of regenerating rat liver after a partial hepatectomy was investigated. Liver was surgically removed about two thirds of that of sham-operated rats. The reduced liver weight by partial hepatectomy was restored about 50% at 24 h after the surgery, and it was completely restored at 72 h. Regenerating liver significantly increased calcium content and plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity between 12–48 h after hepatectomy. Those increases were maximum at 24 h after the surgery. The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was completely abolished by the presence of anti-regucalcin IgG (1.0–4.0 g/ml). The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was clearly inhibited by N-ethylmaleimide (2.5 and 5.0 mM) addition into the enzyme reaction mixture. This NEM effect was also seen for the activatory effect with regucalcin (0.25 M) addition on the enzyme activity in the plasma membranes from normal rat liver. The endogenous regucalcin may play a cell physiological role in the activation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase to maintain the intracellular calcium level in regenerating rat liver.     

Streptozotocin-induced diabetes increases (Ca2+-Mg2+)-ATPase activity in hepatic plasma membranes of rats: Involvement of protein kinase C

The alteration in calcium transport in the liver of rats with streptozocin(STZ)-diabetic state was investigated. STZ (6 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 2 weeks later they were sacrificed by bleeding. STZ administration caused a remarkable elevation of serum glucose concentration. Liver calcium content was significantly increased by STZ administration. Hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was markedly elevated by STZ administration. This increase was completely abolished by the presence of staurosporine (10^-7-10^-5 M), an inhibitor of protein kinase C, in the enzyme reaction mixture, suggesting an involvement of protein kinase C signalling. Moreover, the STZ-induced increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was significantly raised by the presence of okadaic acid (10^-5 and 10^-4 M). Meanwhile, the STZ-increased (Ca²⁺-Mg²⁺)-ATPase activity was not appreciably altered by the presence of anti-regucalcin IgG in the reaction mixture, indicating that the activatory protein regucalcin does not participate in the elevation of the enzyme activity. The present study demonstrates that STZ-induced diabetes causes the increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity of rats.     

Reversible changes of extracellular pH during action potential generation in a higher plant Cucurbita pepo

A pH-sensitive electrode was applied to measure activity of H⁺ ions in the medium surrounding excitable cells of pumpkin (Cucurbita pepo L.) seedlings during cooling-induced generation of action potential (AP). Reversible alkalization shifts were found to occur synchronously with AP, which could be due to the influx of H⁺ ions from external medium into excitable cells. Ethacrynic acid (an anion channel blocker) reduced the AP amplitude but had no effect on the transient alkalization of the medium. An inhibitor of plasma membrane H⁺-ATPase, N,N’-dicyclohexylcarbodiimide suppressed both the AP amplitude and the extent of alkalization. In experiments with plasma membrane vesicles, the hydrolytic H⁺-ATPase activity was subjected to inhibition by Ca²⁺ concentrations in the range characteristic of cytosolic changes during AP generation. The addition of a calcium channel blocker verapamil and a chelating agent EGTA to inhibit Ca²⁺ influx from the medium eliminated the AP spike and diminished reversible alkalization of the external solution. An inhibitor of protein kinase, H-7 alleviated the inhibitory effect of Ca²⁺ on hydrolytic H⁺-ATPase activity in plasma membrane vesicles and suppressed the reversible alkalization of the medium during AP generation. The results provide evidence that the depolarization phase of AP is associated not only with activation of chloride channels and Cl^? efflux but also with temporary suppression of plasma membrane H⁺-ATPase manifested as H⁺ influx. The Ca²⁺-induced inhibition of the plasma membrane H⁺-ATPase is supposedly mediated by protein kinases.     

The spinach plasma membrane Ca2+ pump is a 120‐kDa polypeptide regulated by calmodulin‐binding to a terminal region

The spinach (Spinacia oleracea L.) leaf plasma membrane Ca²⁺-ATPase is regulated by calmodulin (3-fold stimulation) and limited proteolysis (trypsin; 4-fold stimulation). The plasma membrane Ca²⁺-ATPase was identified as a 120-kDa polypeptide on western immunoblots using two different antibodies. During trypsin treatment the 120-kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109-kDa band correlated with the increase in enzyme activity following trypsin treatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109-kDa polypeptide represents a Ca²⁺-ATPase lackin a terminal fragment involved in calmodulin regulation. This was confirmed by ¹²⁵I-calmodulin overlay studies where calmodulin labeled the 120-kDa band in the presence of Ca²⁺, while the 109-kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP-dependent accumulation of ⁴⁵Ca²⁺ in isolated inside-out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca²⁺ and ATP. Calmodulin increased the V_max. for Ca²⁺ pumping 3-fold, and reduced K_m for Ca²⁺ from 1.6 to 0.9 µM. The K_m for ATP (11 µM) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca²⁺ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the V_max. for Ca²⁺ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.     

高胆固醇血症病人的红细胞膜ATP酶活性变化

研究表明高胆固醇血症病人的红细胞膜Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性均降低,并且血浆总胆固醇和低密度脂蛋白-胆固醇浓度与这两种酶活性呈高度负相关,而血浆高密度脂蛋白-胆固醇浓度与Na⁺-K⁺-ATP酶活性呈正相关。这些变化的研究,对于进一步探讨动脉粥样硬化的发生机制及其防治可能具有重要意义。     

Synaptosomal non-mitochondrial ATPase activities and drug treatment

Energy-using non-mitochondrial ATPases were assayed in rat cerebral cortex synaptosomes and synaptosomal subfractions, namely synaptosomal plasma membranes and synaptic vesicles. The following enzyme activities were evaluated: Na⁺, K⁺-ATPase; high- and low-affinity Ca²⁺-ATPase; basal Mg²⁺-ATPase; Ca²⁺, Mg²⁺-ATPase. The evaluations were performed after four week-treatment with saline [controls] or -adrenergic agents (-yohimbine, clonidine), energymetabolism interfering compound (theniloxazine), and oxygen-partial pressure increasing agent (almitrine), in order to define the plasticity and the selective changes in individual ATPases. In rat cerebral cortex, the enzyme adaptation to four-week-treatment with -yohimbine or clonidine was characterized by increase in both high- and low-affinity Ca²⁺-ATPase activities. The action involves the enzyme form located in the synaptic plasma membranes. The enzyme adaptation to the subchronic treatments with theniloxazine or almitrine was characterized by increase in Na⁺, K⁺-ATPase or Mg²⁺-ATPase activities, respectively. The action involves the enzymatic forms located in the synaptic plasma membranes. Thus, the pharmacodynamic effects of the agents tested should also be related to the changes induced in the activity of some specific synaptosomal nonmitochondrial ATPases.     

Calix[4]arene C-90 selectively inhibits Ca2+,Mg2+-ATPase of myometrium cell plasma membrane

The supramolecular compound calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenylsulfonylimino)-methylamino-25,26,27,28-tetrapropoxycalix[4]arene) is shown to efficiently inhibit the ATP hydrolase activity of Ca²⁺,Mg²⁺-ATPase in the myometrium cell plasma membrane fraction and also in a preparation of the purified enzyme solubilized from this subcellular fraction. The inhibition coefficient I _0.5 values were 20.2 ± 0.5 and 58.5 ± 6.4 μM for the membrane fraction and the solubilized enzyme, respectively. The inhibitory effect of calix[4]arene C-90 was selective comparatively to other ATPases localized in the plasma membrane: calix[4]arene C-90 did not influence the activities of Na⁺,K⁺-ATPase and “basal” Mg²⁺-ATPase. The inhibitory effect of calix[4]arene C-90 on the Ca²⁺,Mg²⁺-ATPase activity was associated with the cooperative action of four trifluoromethylphenyl sulfonylimine (sulfonylamidine) groups oriented similarly on the upper rim of the calix[4]arene macrocycle (the calix[4]arene “bowl”). The experimental findings seem to be of importance for studies, using calix[4]arene C-90, of membrane mechanisms of regulation of calcium homeostasis in smooth muscle cells and also for investigation of the participation of the plasma membrane Ca²⁺-pump in control of electro- and pharmacomechanical coupling in myocytes.