Does calmodulin participate in the regulation of the Ca-pump of erythrocytes in vivo?

Using a highly effective chelator of Ca2+ and 45Ca, the concentration of Cai2+ in human and rat erythrocytes was measured both at normal and accelerated Ca2+ influx into the cells. No effect of the calmodulin-dependent reaction inhibitor R24571 was observed. The Ca-ATPase from saponin-treated erythrocytes was characterized by a high affinity for Ca2+ (K 0.5-0.7 microM). This value is 2-3 times as low as that for Ca2+ concentration causing a 50% increase of the Ca-ATPase activity in erythrocyte ghosts obtained during hypoosmotic hemolysis. The Ca-ATPase activity in saponin-treated erythrocytes did not change either under the effect of calmodulin or by R24571. It was assumed that calmodulin did not participate in the regulation of the Ca2+-pump operation in erythrocytes in vivo.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.     

Comparative characteristics of the properties of erythrocyte Na,K-ATPase in man and the carp Cyprinus carpio

Studies have been made on some of the properties of Na,K-ATPase of a nuclear erythrocytes of man and nuclear erythrocytes of the carp Cyprinus carpio. Human erythrocytes yielded the enzymic activity only after their treatment by a detergent Twin-20; under optimal conditions, it amounted to 2.6 mcmole /ml of erythrocytes per 1 h. In carp erythrocytes, Na,K-ATPase activity could be detected without detergent treatment, being 10-fold higher under optimal conditions than that in human erythrocytes. Repetitive washing of carp erythrocytes from the plasma (for more than 3 times), significantly increased their viscosity and resulted in spontaneous hemolysis. Simultaneously , the activity of Na,K-ATPase increased 2-10 times depending on the composition of incubation media. Under these conditions, the pattern of changes in the enzymatic activity, resulting from shifts in Mg2+ and EDTA concentrations, was altered. The presence of latent Na,K-ATPase activity in the erythrocytes in explained by a low permeability of membranes to ATP and ions. Exogeneous ATP cannot be utilized by the enzyme in the intact human erythrocytes, whereas intact carp erythrocytes exhibit significant permeability to the exogeneous substrate. It is suggested that in vivo this fact may be of physiological importance.     

Vanadate-induced inhibition of renin secretion is unrelated to inhibition Na,K-ATPase activity

There is evidence that three inhibitors of Na,K-ATPase activity--ouabain, K-free extracellular fluid, and vanadate--inhibit renin secretion by increasing Ca2+ concentration in juxtaglomerular cells, but in the case of vanadate, it is uncertain whether the increase in Ca2+ is due to a decrease in Ca2+ efflux (inhibition of Ca-ATPase activity, or inhibition of Na,K-ATPase activity, followed by an increase in intracellular Na+ and a decrease in Na-Ca exchange) or to an increase in Ca2+ influx through potential operated Ca channels (inhibition of electrogenic Na,K transport, followed by membrane depolarization and activation of Ca channels). In the present experiments, the rat renal cortical slice preparation was used to compare and contrast the effects of ouabain, of K-free fluid, and of vanadate on renin secretion, in the absence and presence of methoxyverapamil, a Ca channel blocker. Basal renin secretory rate averaged 7.7 +/- 0.3 GU/g/60 min, and secretory rate was reduced to nearly zero by 1 mM ouabain, by K-free fluid, by 0.5 mM vanadate, and by K-depolarization (increasing extracellular K+ to 60 mM). Although 0.5 microM methoxyverapamil completely blocked the inhibitory effect of K-depolarization, it failed to antagonize the inhibitory effects of ouabain, of K-free fluid, and of vanadate. A concentration of methoxyverapamil two hundred times higher (100 microM) completely blocked the inhibitory effects of vanadate, but still failed to antagonize the effects of ouabain and of K-free fluid. Collectively, these observations demonstrate that vanadate-induced inhibition of renin secretion cannot be attributed entirely to Na,K-ATPase inhibition, since in the presence of methoxyverapamil, the effect of vanadate differed from the effects of either ouabain (a specific Na,K-ATPase inhibitor) or K-free fluid. Moreover, it cannot be attributed entirely to a depolarization-induced influx of Ca2+ through potential-operated Ca channels, since methoxyverapamil antagonized K-depolarization-induced inhibition of renin secretion much more effectively than it antagonized vanadate-induced inhibition.     

Effects of trifluoperazine and mitogenic lectins on calcium ATPase activity and calcium transport by human lymphocyte plasma membrane vesicles

The phenothiazine, trifluoperazine, and the mitogenic lectins, phytohemagglutinin (PHA) and Concanavalin A (Con A), were tested for their effects on human lymphocyte plasma membrane Ca-activated Mg-ATPase and ATP-dependent calcium uptake. Trifluoperazine completely inhibited Ca-uptake when present from the start of the assay at concentrations of 100 microM or more. When added during measurement of calcium uptake, trifluoperazine reduced the rate of vesicular calcium accumulation but was unlike the calcium ionophore, A23187, which caused a rapid release of accumulated calcium from the vesicles. Trifluoperazine also inhibited membrane vesicle Ca-ATPase activity, but this inhibition was non-specific since the Mg-ATPase and Na,K-ATPase activities were inhibited to similar extents at the same concentration of the phenothiazine. In contrast, concentrations of PHA and Con A, which are mitogenic for lymphocytes, did not cause any change in Ca-uptake when added to suspensions of membrane vesicles. Con A had no effect and PHA had a weak inhibitory effect on Ca-ATPase activity.     

Omega-3 fatty acids modulate ATPases involved in duodenal Ca absorption

Dietary supplementation with fish oil that contains omega-3 polyunsaturated fatty acids has been shown to enhance bone density as well as duodenal calcium uptake in rats. The latter process is supported by membrane ATPases. The present in vitro study was undertaken to test the effect of omega-3 fatty acids on ATPase activity in isolated basolateral membranes from rat duodenal enterocytes. Ca-ATPase in calmodulin-stripped membranes was activated in a biphasic manner by docosahexanoic acid (DHA) (10-30 microg/ml) but not by eicosapentanoic acid (EPA). This effect was blocked partially by 0.5 microM calphostin (a protein kinase C blocker). DHA inhibited Na,K-ATPase (-49% of basal activity, [DHA]=30 microg/ml, P <0.01). This effect could be reversed partially by 50 microM genistein, a tyrosine kinase blocker. EPA also inhibited Na,K-ATPase: (-47% of basal activity, [EPA]=30 microg/ml, P <0.01), this effect was partially reversed by 100 microM indomethacin, a cyclo-oxygenase blocker. Omega-3 fatty acids are thus involved in multiple signalling effects that effect ATPases in BLM.     

Ratio of brain synaptosome Na, K-ATPase to dopamine receptors

Activating (0.3-3 microM) or inhibitory (0.03-0.3 mM) effects of dopamine (DA) in the absence of Ca2+, and its inhibitory effect in the presence of Ca2+ on Na,K-ATPase activity of synaptosomes from the caudate nucleus of the rat brain were confirmed. Na,K-ATPase was shown to be inhibited by 6 neuroleptics, with the degree of inhibition stronger in the presence of Ca2+. It was found that: 1) the biphasic or monophasic nature of DA action on Na,K-ATPase activity was preserved in the presence of neuroleptics, 2) DA enhances the inhibitory effects of neuroleptics on the enzyme, 3) the inhibitory effects of DA on Na,K-ATPase are enhanced by Ca2+ ions. The mechanisms of the modifying action of DA on synaptosomal Na,K-ATPase are discussed.     

The endogenous inhibitor of NCX1 does not resemble the properties of digitalis compound

In our previous study, we ware successful in isolation and purification of an endogenous inhibitor of the Na/Ca exchanger (NCX1) from the calf ventricle extracts. The purified factor has characterized to have strong positive inotropic effect on isometric contractions of isolated ventricle strips of guinea pig. A possibility is that besides the NCX1 the endogenous factor may also interact with other ion-transport systems (e.g., Na,K-ATPase) involved in modulation of muscle contractility-relaxation. Therefore, a primary goal of the present study was to detect a possible effect of newly found NCX1 inhibitor on Na,K-ATPase and Ca-ATPase activities. The preparations of isolated sarcolemma vesicles were used for this goal. Although the crude extracts of calf ventricles can inhibit both the Na/Ca exchange and Na,K-ATPase, these two inhibitory activities can be separated on the Sephadex G-10 column, meaning that different molecular entities might be responsible for inhibition of Na/Ca exchange and Na,K-ATPase. Addition of 100 U of purified endogenous factor to the assay medium results in nearly complete inhibition of forward (Na(i)-dependent Ca-uptake) and reverse (Na(o)-dependent Ca-efflux) modes of Na/Ca exchange. On the other hand, no effect was detected on activities of Na,K-ATPase and Ca-ATPase even in the presence of 500 U of purified factor in the assay medium. In light of the present data, it is concluded that the endogenous inhibitor of NCX1 does not resemble the targeting properties of digitalis like compound. Obviously, more systematic studies are required in the future for resolving a possible interaction of the endogenous inhibitor of NCX1 with other ion-transport systems involved in calcium homeostasis and action potential.     

Changes of physiological and biochemical characteristics of rat erythrocytes after blood loss

In experiments of Wistar male rats, changes are studied of erythrocyte hematological, biochemical (activitities of transport ATPases), and rheological properties (capability for aggregation and deformability) 7 days after bloodletting of 12-15 % of the total blood mass. It has been shown that alongside with an elevation of erythrocyte volume and of the number of immature cells - reticulocytes, there was a statistically significant increase of Na,K-ATPase and Ca-ATPase activities in the whole erythrocytes and their membrane preparations - ghosts, the increment of activity in the case of Na,K-ATPase being essentially higher in the whole cells. This indicates the appearance of an enzyme activator inside the erythrocytes. There are also revealed a decrease of firmness of erythrocyte aggregates, a deceleration of spontaneous aggregation, and an increase of index of erythrocyte deformability. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed to optimization of blood circulation in large vessels and capillary network.     

Some properties of transport ATPases in functionally different muscles]

The properties of Ca-transporting system in sarcoplasmic reticulum membranes in fast and slow frog muscles as well as some properties of sarcolemma Na, K-ATPase of the same object were investigated. The rate of Ca2+ uptake, Ca-ATPase activity and Ca/ATP ratio for the reticulum of fast muscle demonstrated higher values than those for the reticulum of slow muscle. The rate of Ca2+ accumulation by the fragments of the rectus reticulum and Ca/ATP ratio were found to decrease under the influence of acetylcholine (0.05-5 mM). The transport system of the sartorius reticulum was found to be less sensitive to acetylcholine. The peak activity of Na, K-ATPase in femoral muscles of the frog occurred at 80 mM NaCl and 60 mM KCl, whereas in the rectus abdominal muscle it equalled 100 mM NaCl and 40 mM KCl. Thus, Na, K-ATPase activity in the slow muscle was predominantly higher than that in the mixed (femoral) muscles. If the sarcolemma preparations of the muscles of both types the inhibitory effect of acetylcholine on Na; K-ATPase was registered. The enzyme of slow muscles exhibited higher sensibility to acetylcholine.     

Effect of dopamine and dopamine mimetics on Na, K-ATPase of corpus striatum synaptosomes

Dopamine (DA) and DA-mimetics (apomorphine, midantan, piribedil) have a dual effect on Na, K-ATPase of the rat brain striate synaptosomes: activating at micromolar concentrations and inhibitory at higher concentrations (less than or equal to 30 microM). In the presence of Ca2+ (1 mM EGTA + 2.5 mM Ca2+) DA activating effect completely disappears and the inhibitory effect becomes even more pronounced. In the presence of cAMP (50 microM) which has no effect of its own on Na, K-ATPase, DA activation maximum is shifted towards lower concentrations, and the inhibitory effect remains unchanged. The above mentioned effects of DA persist in the presence of ouabain (1 mM), i.e. during measuring of Na, K-ATPase activity by an "ouabain" method, with DA activation maximum shifted towards higher concentrations.     

Calcium transport and calcium-ATPase activity in human lymphocyte plasma membrane vesicles

We have studied Ca transport and the Ca-activated Mg-ATPase in plasma membrane vesicles prepared from normal human lymphocytes. Membrane vesicles that were exposed to oxalate as a Ca-trapping agent accumulated Ca in the presence of Mg2+ and ATP. ADP, AMP, GTP, UTP, ITP, TTP, or CTP did not substitute for ATP in energizing uptake. The Vmax for Ca uptake was 2.4 pmol of Ca/micrograms of protein/min, and the Km values for Ca and ATP were 1.0 and 80 microM, respectively. One microM A23187, added initially, completely inhibited net Ca uptake and, if added later, caused the release of Ca accumulated previously. Cyanide, oligomycin, ouabain, or varying Na+ or K+ concentrations had no effect on Ca uptake. A Ca-activated ATPase was present in the same membrane vesicles, which had a Vmax of 25 pmol of Pi/micrograms of protein/min at a free Ca concentration of 4-5 microM. This Ca-ATPase had Km values for Ca and ATP of 0.6 and 90 microM, respectively. These kinetic parameters were similar to those observed for uptake of Ca by the vesicles. The Ca-ATPase activity was insensitive to azide, oligomycin, ouabain, or varying Na+ or K+ concentrations. No Ca-activated hydrolysis of GTP or UTP was observed. Both Ca transport and the Ca-ATPase activity of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-treated lymphocyte plasma membranes were stimulated 2-fold by a cytoplasmic component (calmodulin) that was purified 500-fold from lymphocyte cytoplasm. Thus, human lymphocyte plasma membranes have both a Ca transport activity and a Ca-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivities to calmodulin.     

Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.     

Interplay between sodium and calcium dynamics in granule cell presynaptic terminals.

Fluorescent indicators were used to detect stimulus-evoked changes in presynaptic levels of intracellular sodium (Na(i)) and calcium (Ca(i)) in granule cell parallel fibers in brain slices from rat cerebellum. Ca(i) increased during stimulation, and three exponentials were needed to approximate its return to prestimulus levels. Ca(i) decayed to approximately 10% of peak levels with tau approximately 100 ms, to approximately 1% of peak values with tau approximately 6 s, and then returned to prestimulus levels with tau approximately 1-2 min. After stimulation, Na(i) accumulated in two phases; one rapid, the other continuing for several hundred milliseconds. The return of Na(i) to prestimulus levels was well approximated by a double exponential decay with time constants of 6-17 s and 2-3 min. Manipulations that prevented calcium entry eliminated both the slow component of sodium entry and the rapid component of Na(i) decay. Reductions of extracellular sodium slowed the rapid phase of Ca(i) decay. These Ca(i) and Na(i) transients were well described by a model in which the plasma membrane of presynaptic boutons contained both a sodium/calcium exchanger and a calcium ATPase (Ca-ATPase). According to this model, immediately after stimulation the sodium/calcium exchanger removes calcium from the terminal more rapidly than does the Ca-ATPase. Eventually, the large concomitant sodium influx brings the exchanger into steady-state, leaving only the Ca-ATPase to remove calcium. This perturbs the equilibrium of the sodium/calcium exchanger, which opposes the Ca-ATPase, leading to a slow return of Ca(i) and Na(i) to resting levels.     

The effect of the membrane skeleton of non-nucleated erythrocytes on the properties of transport ATPases]

Removal of spectrin and other proteins of membrane skeleton from rat erythrocyte membranes resulted in a significant loss of Na,K-ATPase and Ca-ATPase activities, and even more of respective phosphatase activities. At the same time the modulating influence of ATP and Ca2+ on the enzymes disappeared. These ATPase activities were reconstituted by addition of concentrated spectrin to spectrin-depleted membranes. The activating influence of Ca2+ on ouabain-resistant and ouabain-sensitive phosphatases in ghosts could be discovered only in the presence of ATP. The highest activities of both the phosphatases were revealed when both ATP (0.5 mM) and Ca2+ (10-30 mM) were present simultaneously in the incubation medium. These data show that the functioning of transport ATPases in non-nuclear erythrocyte membranes is related to the membrane skeleton: regulating influence of intracellular ATP and Ca2+ on enzymes seems to be realized through the proteins of the skeleton.     

Modulation of Na, K-ATPase activity by prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin

Adenylyl cyclase is activated by prostaglandin E and inhibited by mu-opioids. Since cAMP-related events influence the activity of the Na Pump and its biochemical correlate Na,K-ATPase in many systems, we tested the hypothesis that prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid agonist, have opposing actions on Na,K-ATPase activity. Studies were conducted with alamethicin-permeabilized SH-SY5Y human neuroblastoma cells. Prostaglandin E1 (1 microM) transiently inhibited Na,K-ATPase activity for 10-15 min. A direct activator of protein kinase A, 8-Br-cAMP (150 and 500 microM), also inhibited, but more rapidly and for a shorter duration. Both DAMGO (1 microM) and Rp-adenosine 3',5'-cyclic monophosphorothioate (500 microM), a protein kinase A-inhibitor, reversed the inhibitory effect of prostaglandin E1. DAMGO alone (1 microM) stimulated Na,K-ATPase activity up to nearly three-fold control activity. The stimulatory action of DAMGO was blocked by cyclosporine A (2 microM), an inhibitor of calcineurin, and was dependent on Ca2+ entry through nifedipine-sensitive Ca2+ channels. In the presence of 1 mM EGTA, DAMGO inhibited Na,K-ATPase activity. DAMGO-induced inhibition was blocked by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C (1 microM). Na,K-ATPase is poised to modulate neuronal excitability through its roles in maintaining the membrane potential and transmembrane ion gradients. The differential effects of prostaglandin E1 and opioids on Na,K-ATPase activity may be related to their actions in hyperalgesia.     

Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells.

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.     

Changes of physiological and biochemical characteristics of rat erythrocytes after blood loss

In experiments on Wistar male rats, changes are studied of erythrocyte hematological, biochemical (activities of transport ATPases), and rheological properties (capability for aggregation and deformability) 7 days after bloodletting of 12–15% of the total blood mass. It has been shown that alongside with an elevation of erythrocyte volume and of the number of immature cells—reticulocytes, there was a statistically significant increase of Na,K-ATPase and Ca-ATPase activities in the whole erythrocytes and in their membrane preparations—ghosts, the increment of activity in the case of Na,K-ATPase being essentially higher in the whole cells. This indicates the appearance of an enzyme activator inside the erythrocytes. There are also revealed a decrease of firmness of erythrocyte aggregates, a deceleration of spontaneous aggregation, and an increase of index of erythrocyte deformability. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are aimed at optimization of blood circulation in large vessels and capillary network.     

Na K ATPase activity in mammalian erythrocytes

The effects of membranotropic substances--nonionic detergent Tween-20 and EDTA--on the activity and some properties of Na,K-ATPase from mammalian erythrocytes were studied. It was shown that pretreatment of whole erythrocytes with Tween-20 (5 mg/ml) allows a detection of the enzyme activity, which cannot be detected in intact cells. It was also found that erythrocyte ghosts with a high and stable activity of Na,K-ATPase can be obtained by injections of EDTA (1-2 mM) into the hemolysis medium. Although the enzyme activity in whole erythrocytes and their ghosts was detected by the use of various membranotropic agents, the type of the dependence of the Na,K-ATPase activity on MgCl2 and EDTA concentration in the incubation medium was essentially the same for both cell preparations, the optimal concentrations of MgCl2 and EDTA being 3 and 1 mM, respectively. A rise in MgCl2 concentration above 3 mM caused a decrease of the enzymatic activity. Simple techniques have been developed for the detection of the Na,K-ATPase activity in mammalian erythrocytes which allow the determination of a higher enzymatic activity than those described in literature.