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R Blostein

Publications and source records attributed to R Blostein.

At least 37 records · Page 2Linked to original sources
PubMed1997

L antigens of sheep red blood cell membranes and modulation of ion transport.

Sheep are polymorphic with respect to the intracellular Na+ and K+ concentrations of their erythrocytes. Erythrocytes of sheep of the high-K+ (HK) phenotype have high K+ and low Na+ concentrations; erythrocytes from sheep of the allelic low-K+ (LK) phenotype have abnormally low K+ and high Na+ concentrations. The difference is due to differences in rates of cation transport: higher Na+-K+ pump flux in HK cells and higher K+-Cl- cotransport in LK cells. The HK/LK polymorphism is associated with a polymorphism of red blood cell antigens: the L antigen is only on LK cells, and HK cells have only the M antigen. There are two classes of L antigen that assort together: Lp, which is associated with Na+-K+ pumps, and Ll, which is associated with K+-Cl- cotransporters. There are functional consequences of these associations: anti-Lp antibody stimulates the pump and anti-Ll antibody inhibits cotransport. The use of these antibodies has permitted delineation of the roles of the antigens in modulating the function of the transporters. In this review, we summarize the evidence that these antigens are entities distinct from the pump. The Lp antigen reacts reversibly with the Na+-K+ pump; the antigen inhibits the pump, mainly by promoting nonspecific inhibition by intracellular K+. The antigen also modulates pump differentiation in immature cells. In contrast, the Ll antigen stimulates K+-Cl- cotransport. The evidence suggests that the two polymorphisms are controlled by a single genetic locus and that all of the distinct properties of ion transporters in LK cells are attributable to interactions with L antigens.

Animals↗
PubMed1996

Structure/function analysis of the amino-terminal region of the 1 and 2 subunits of Na,K-ATPase.

The alpha2 isoform of the Na,K-ATPase exhibits kinetic behavior distinct from that of the alpha1 isoform. The distinctive behavior is apparent when the reaction is carried out under conditions (micromolar ATP concentration) in which the K+ deocclusion pathway of the reaction cycle is rate-limiting; the alpha1 activity is inhibited by K+, whereas alpha2 is stimulated. When 32 NH2-terminal amino acid residues are removed from alpha1, the kinetic behavior of the mutant enzyme (alpha1M32) is similar to that of alpha2 (Daly, S. E., Lane, L. K., and Blostein, R. (1994) J. Biol. Chem. 269, 23944-23948). In the current study, the region of the alpha1 NH2 terminus involved in modulating this kinetic behavior has been localized to the highly charged sequence comprising residues 24-32. Within this nonapeptide, differences between alpha1 and alpha2 are conservative and are confined to residues 25-27. The behavior of two chimeric enzymes: (i) alpha1 with the first 32 residues identical to the alpha2 sequence, alpha1 (1-32alpha2), and (ii) alpha2 with the first 32 residues identical to the alpha1 sequence, alpha2(1-32alpha1), indicates that the distinctive kinetic behavior of alpha1 and alpha2 is not due to the 24-32 NH2-terminal domain, per se, but rather to its interaction with other, isoform-specific region(s) of the alpha1 protein. We also demonstrate that the distinct K+ activation profiles of either alpha2 or alpha1M32, compared to alpha1 is due to a faster release of K+ from the K+-occluded enzyme, and to a higher affinity for ATP. This was determined in studies using two approaches: (i) kinetic analysis of the reaction modeled according to a branched pathway of K+ deocclusion through low and high affinity ATP pathways and, (ii) measurements of the (rapid) phosphorylation of the enzyme (E1 conformation) by [gamma-32P]ATP following the rate-limiting formation of the K+-free enzyme from the K+-occluded state (E2(K) --> E1 + K+). The observed kinetic differences between alpha2 and alpha1 suggest that these Na,K-ATPase isoforms differ in the steady-state distribution of E1 and E2 conformational states.

Adenosine Triphosphate↗
PubMed1996

Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase.

The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na+ and K+ activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1-transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na+ of alpha1 pumps was axolemma approximately rat alpha1-transfected HeLa > kidney, and for K+, kidney approximately alpha1-transfected HeLa > axolemma. For alpha3, the order of apparent affinities for Na+ was pineal gland approximately axolemma > alpha3*-transfected HeLa, and for K+, alpha3*-transfected HeLa > axolemma approximately pineal gland. In addition, the differences in apparent affinities for Na+ of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K+ concentration was increased. A kinetic analysis of the apparent affinities for Na+ as a function of K+ concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na+ and K+, the latter acting as a competitive inhibitor at cytoplasmic Na+ activation sites. Although the nature of the tissue-specific modulation of K+/Na+ antagonism remains unknown, an analysis of the nature of the beta isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct beta subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances beta1 is the predominant beta isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K+/Na+ antagonism due to distinct betas may apply to alpha3 of axolemma (alpha3beta1) and pineal gland ( alpha3beta2).

Animals↗
PubMed1995

ADP controls the electrogenicity of Na/Na exchange catalyzed by dog kidney Na,K-ATPase proteoliposomes.

Sodium pump mediated Na/Na exchange was studied using Na(+)-loaded proteoliposomes prepared from dog kidney Na,K-ATPase. Measurements of both 22Na+ influx and pump-generated electrical potentials were carried out, the latter using the anionic dye, oxonol VI. In the presence of ATP, the formation of a strophanthidin-sensitive membrane potential confirms that Na/Na exchange associated with ATP hydrolysis can be electrogenic depending on the source of the enzyme. With the addition of varying concentrations of ADP, electrogenic exchange is progressively inhibited and replaced by electroneutral exchange. ADP is equally effective in activating (ATP + ADP)-dependent electroneutral exchange. With sufficient ADP, electrogenic Na/Na exchange is completely replaced by electroneutral exchange.

Adenosine Diphosphate↗
PubMed1994

Functional consequences of amino-terminal diversity of the catalytic subunit of the Na,K-ATPase.

One region of marked sequence diversity among the highly homologous alpha isoforms of the Na,K-ATPase is the lysine-rich NH2 terminus. Expression of a mutant cDNA encoding an alpha 1 protein, minus the 32 NH2-terminal residues, results in a modified enzyme (alpha 1M32), which behaves similarly to alpha 1 in overall Na/K exchange activity (Vmax) and apparent affinities for intracellular Na+ and extracellular K+. However, with membranes isolated from HeLa cells expressing the rat alpha 1M32 mutant, as well as membranes from cells expressing the rat alpha 1 and the ouabain-resistant mutated forms of rat alpha 2 (alpha 2*) and alpha 3 (alpha 3*) developed by Jewell and Lingrel (Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), distinct Na,K-ATPase kinetics are observed. Thus, at 1 microM ATP, the effects of K+ on the Na-ATPase activity of alpha 2* and alpha 1M32 are similar; both are activated, whereas alpha 1 and alpha 3 are inhibited by the addition of K+ at low (0.1 mM) concentration. These effects are attributed to different rates of a step involved in K+ deocclusion (E2(K)<-->E1K<-->E1 + K+) and are consistent with our earlier evidence (Wierzbicki, W., and Blostein, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 70-74) for a role of the NH2 terminus in the K+ deocclusion pathway of the Na,K-ATPase reaction. These differences are not directly related to differences in apparent affinities for ATP, since alpha 3* has alpha 1-like high affinity K+ inhibition but resembles alpha 2* and alpha 1M32 with respect to a lower K'ATP. Na-ATPase activities of alpha 2*, alpha 3*, and alpha 1M32, but not alpha 1, are activated by Li+ but not Rb+, consistent with a relatively faster rate of Li+ deocclusion (Post, R. L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540), as well as higher affinity of alpha 3 for extracellular K+ (Li+) activation of dephosphorylation (E2P + K+<-->E2(K) + Pi). Inhibition of Na-ATPase by higher concentrations (> or = 1 mM) K+ is observed with all isoforms and is attributed to K+ acting at inhibitory cytoplasmic sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1994

Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase.

The objective of this study has been to delineate the side-specific effects of Na+ and K+ on the transport kinetics of tissue-specific Na/K pumps. Two experimental systems have been used. In one, Na/K pumps of exogenous microsomal membrane sources (rat axolemma, kidney) were delivered by membrane fusion into dog erythrocytes, and in the other, the three isoforms of the catalytic subunit of the rat enzyme were individually transfected into HeLa cells as in previous studies (Jewell, E.A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925-16930), with the alpha 2 and alpha 3 isoforms rendered relatively resistant to ouabain by site-directed mutagenesis. Whereas the kidney microsomes comprise the alpha 1 catalytic isoform, the axolemma microsomes were predominantly alpha 3 (approximately 60%) with lesser amounts of alpha 2 (approximately 25%) and alpha 1 (approximately 15%) as measured by the ouabain-sensitive profile of phosphoenzyme as well as by immunoblotting with isoform-specific antibodies using membranes of known specific activity as standards (alpha 1 of kidney, alpha 1 and alpha 2 of muscle). Both systems were analyzed with respect to the effects of varying concentrations of cytoplasmic Na+ and extracellular K+ on pump-mediated 86Rb+(K+) influx. With the individual isoform-transfected HeLa cells and monensin added to vary and control the intracellular Na+ concentration, differences in apparent affinities of the alpha 3 isoform compared with the alpha 1 and alpha 2 isoforms were observed, i.e. a approximately 3-fold higher affinity for extracellular K+ and approximately 4-fold lower affinity for cytoplasmic Na+. Thus, in the presence of 10 mM extracellular Na+, apparent K0.5 values for extracellular K+ activation of K+(Rb+) influxes were 0.22 +/- 0.02 mM for alpha 1, 0.20 +/- 0.02 mM for alpha 2, and 0.09 +/- 0.01 mM for alpha 3. At high intracellular K+ (> or = 100 mM) and saturating extracellular K+ concentrations, apparent K0.5 values for cytoplasmic Na+ activation were 17.6 +/- 1.1 mM for alpha 1, 19.7 +/- 1.0 mM for alpha 2, and 63.5 +/- 9.1 mM for alpha 3. The functional differences observed with the individual isoform-transfected cells were completely consistent with the kinetic differences observed with the axolemma and kidney pumps fused into erythrocytes. Axolemma pumps had a approximately 3-fold lower K0.5 for extracellular K+ and a approximately 2-fold higher K0.5 for cytoplasmic Na+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Decline in number of Na-K pumps on low-K+ sheep reticulocytes during maturation is modulated by Lp antigen.

The number of the Na-K pumps on sheep red blood cells declines markedly during cell maturation. In addition, in red blood cells of the low-K+ (LK) phenotype, there is an increase during maturation in the affinity of the pumps for intracellular K+. This increase does not occur in cells of the high-K+ (HK) phenotype. This HK/LK polymorphism is associated with the M/L blood group antigen system. The Lp antigen, which is on only LK cells, promotes the increase in affinity for K+ [Am. J. Physiol. 265 (Cell Physiol. 34): C99-C105, 1993]. Mature LK cells have fewer pumps than mature HK cells. The present study shows that the Lp antigen also promotes the loss of pumps in LK cells. The evidence was that modification of the Lp antigen of immature LK red blood cells either with anti-Lp antibody or by trypsinization diminished the loss of pumps during culture in vitro (numbers determined from [3H]ouabain binding). Confirmation came from demonstration of the decline during maturation of the amount of the alpha-subunit of the Na-K pump (measured by immunoblotting), which was also retarded by pretreatment with anti-Lp or trypsin. Comparisons of the relative amounts of Lp antigen on immature and mature LK cells showed that there is little decline in number of antigens during maturation. Therefore there is an increase in the antigen-to-pump ratio during maturation even though an association between pumps and antigens is necessary for the loss of pumps.

Animals↗
PubMed1993

Functional properties of an H,K-ATPase/Na,K-ATPase chimera.

Cultured pig kidney epithelial cells were transfected with a chimeric P-type ATPase catalytic subunit composed of the NH2-terminal half of the rat gastric H,K-ATPase and the COOH-terminal half of the rat Na,K-ATPase (alpha 1 isoform). Low concentrations of ouabain (< or = 0.2 mM) were used to inhibit completely the endogenous pig Na,K-ATPase and high concentrations (5 nM) to test the sensitivity of the chimeric rodent pump. In the presence of a low concentration of ouabain, a small but significant inhibition of residual Rb+(K+) influx by 5 mM ouabain was observed in only the transfected cells. Conditions were found in which a similar component of Rb+ influx was inhibited by the gastric H,K-ATPase inhibitor SCH28080, consistent with SCH28080 binding to the extracellular H1-H2 loop of this enzyme. These experiments demonstrate that this chimera behaves as a functional ion pump and indicate that the protein domains involved in cardiac glycoside binding are not confined to the amino-terminal half of the Na,K-ATPase.

Animals↗
PubMed1993

The amino-terminal segment of the catalytic subunit of kidney Na,K-ATPase regulates the potassium deocclusion pathway of the reaction cycle.

Tryptic cleavage of the catalytic subunit of kidney Na,K-ATPase in the E1 conformation effects a change in kinetic behavior apparent at low ATP concentration. Thus, at < or = 10 microM ATP, K+ inhibits Na(+)-dependent ATPase activity of the undigested enzyme but activates activity of the digested enzyme. With time of trypsinolysis, a transient increase followed by a decrease in activity is observed at low [ATP], whereas at high [ATP] (1 mM), activity is progressively reduced. At low [ATP], the trypsin-treated/control activity ratio was > or = 3-fold higher with K+ compared to the ratio observed with the K+ congener Li+. Also, the relative Na/K exchange activity (22Na+ influx into K(+)-loaded inside-out vesicles from erythrocytes) with either 0.01 mM ATP or 1 mM CTP compared to 1 mM ATP was greater for the trypsin-treated than for the control enzyme. The kinetic change is correlated with the initial rapid cleavage of the N-terminal tryptic fragment (< or = 30 residues) from the catalytic subunit. It is concluded that this segment regulates the K+ deocclusion pathway of the reaction; removal of this fragment produces a modified active species having an increased rate of K+ deocclusion.

Animals↗
PubMed1993

Differentiation of Na(+)-K+ pumps of low-K+ sheep red blood cells is promoted by Lp membrane antigens.

Na(+)-K+ pumps of red blood cells from sheep of the low-K+ (LK) phenotype undergo differentiation during circulation, manifested in part by a striking increase in sensitivity to inhibition by intracellular K+ (Ki). Pumps of red blood cells from sheep from the allelic phenotype, high K+ (HK), do not undergo this type of maturation. The hypothesis was tested that the Lp antigen, found on LK but not HK cells, is responsible for the maturation of LK pumps. Lp antigens have been shown to inhibit LK pumps because anti-Lp antibody stimulates the pumps by relieving inhibition by the antigen. Lp antigens were recently shown to be molecular entities separate from Na(+)-K+ pumps [Xu, Z.-C., P. Dunham, J. Munzer, J. Silvius, and R. Blostein. Am. J. Physiol. 263 (Cell Physiol. 32): C1007-C1014, 1992]. The test of the hypothesis was to modify the Lp antigens of immature LK red blood cells with two kinds of treatments, anti-Lp antibody and trypsinization (which cleaves Lp), and to observe the effects of these treatments on maturation of pumps during culture of the cells in vitro. Both of these treatments prevented the maturation of the kinetics of the pumps to the Ki-sensitive pattern, supporting the hypothesis that interaction of the pumps with Lp antigens is responsible for the maturation of the pumps. Strong supportive evidence came from experiments on Na(+)-K+ pumps from rat kidney delivered into immature LK sheep red blood cells by microsome fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle.

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Rat kidney Na-K pumps incorporated into low-K+ sheep red blood cell membranes are stimulated by anti-Lp antibody.

A genetic dimorphism of sheep red blood cells characterized by differences in the intracellular K+ concentration of mature red blood cells (low-K+ or high-K+ cells) reflects differences in their Na-K pumps and is known to be linked to the ML blood group system. We investigated the relationship of Na-K pumps in red blood cells from sheep of the low-K+ phenotype with an antigen, Lp, that is restricted to low-K+ cells. Anti-Lp antibody stimulates the Na-K pumps in these cells presumably by relieving inhibition of the pumps by Lp. The questions addressed were as follows: is Lp a molecular entity distinct from pumps and, if so, can it interact with pumps of exogenous origin? Rat kidney Na-K pumps were incorporated by fusion of microsomes into either low-K+ or high-K+ sheep red blood cells. The activity of the exogenous kidney pumps was distinguished from that of the endogenous red blood cell pumps by the low sensitivity of rodent pumps to ouabain. Anti-Lp stimulated by > 50% rat kidney pumps incorporated into immature low-K+ sheep cells. This indicates that Lp is a distinct molecular entity free to dissociate from endogenous pumps and inhibit exogenous pumps. Anti-Lp did not stimulate kidney pumps incorporated into mature low-K+ cells but did stimulate kidney pumps following in vitro maturation of microsome fused reticulocytes, probably reflecting restriction of lateral movement of pumps and antigens by the cytoskeleton in mature cells.

Animals↗
PubMed1992

Altered stoichiometry of the Na,K-ATPase.

With inside-out membrane vesicles derived from human red cells and incubated at pH > or = 6.6, an altered sodium pump stoichiometry (1Na+:2K+) associated with altered charge transfer is observed when the cytoplasmic Na+ concentration is reduced to very low levels (0.2 mM). With increased proton concentration (approximately pH 6.0), protons can substitute for Na+ or K+ ions such that the Na,K-ATPase can effect either electroneutral Na+/H+ exchange (K+ absent), H+/K+ exchange (Na+ absent), or H(+)-plus-Na+ cotransport in exchange for K+ (low Na+ concentration). Evidence that the stoichiometries of these exchanges are 3Na+/3H+, 2H+/2K+ and 1H(+)-plus-1Na+/2K+, respectively, is presented.

Erythrocytes↗
PubMed1990

Na,K-ATPase isoform expression in sheep red blood cell precursors.

Isoform expression of mammalian red cell Na,K-ATPase was analyzed using messenger RNA isolated from red cell precursor-enriched bone marrow of anemic sheep. Expression of the catalytic alpha subunit was analyzed using rat isoform-specific cDNA probes and expression of the beta 1 subunit, using a sheep beta 1-specific cDNA probe. RNA isolated from sheep kidney and brain were analyzed concurrently. In the red cell, as in the kidney, messenger RNA encoding only one isoform (alpha 1) of the catalytic subunit is detected; neither of the other isoforms (alpha 2 or alpha 3) could be detected. This holds true for bone marrow of sheep of either the low potassium or high potassium phenotype. Relative to the expression of alpha 1, beta subunit-specific message (beta 1) was extremely low in the red cell compared to either kidney (less than 5%) or brain (less than 3%). Using a rat cDNA probe specific for a beta 1-like subunit, beta 2, message was detected in brain but not in either kidney or bone marrow.

Animals↗
PubMed1990

Decrease in Na(+)-K(+)-ATPase associated with maturation of sheep reticulocytes.

Na(+)-K(+)-ATPase of immature and mature sheep red blood cells of both the high-K+ and low-K+ genotype and of immature cells matured in vitro was detected using polyclonal antiserum to purified sheep kidney Na(+)-K(+)-ATPase. This antiserum detects both alpha (alpha 1)- and alpha + (alpha 2 and/or alpha 3)-isoforms of the catalytic subunit as well as the beta-subunit of brain and kidney Na(+)-K(+)-ATPase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, a single major immunologically reactive component corresponding to the alpha-subunit was detected in membranes of immature and mature cells from sheep of both genotypes. Cells separated according to increasing density showed a corresponding decrease in ouabain binding sites on the cells and Na(+)-activated ATP hydrolysis of membranes isolated from the cells. A progressive decrease in immunologically reactive alpha-subunit was also observed. After in vitro culture of reticulocytes, reduction in ouabain binding to the cells was also associated with loss in alpha-subunit. As well, appearance of immunologically reactive alpha-subunit was detected in membranous material shed into the incubation medium, accounting for a fraction (less than or equal to 30%) of the material lost from the cells. Proteolytic sensitivity of the alpha-subunit indicates that, in this material, the cytoplasmic surface of the enzyme is exposed to the medium. The shed material was largely devoid of function as evidenced in little, if any, Na(+)-dependent phosphorylation of Na(+)-K(+)-ATPase. The existence in reticulocytes of an intracellular pool of ouabain binding sites was indicated by the transient appearance on the cell surface of ouabain binding sites after rapid ATP depletion and also after addition of chloroquine to cells during culture. Taken together, these findings indicate that the maturation-associated loss of sodium pump protein involves, at least partly, energy-dependent endocytosis and, presumably, processing whereby inactivation of function occurs as well as release of pump protein into the extracellular milieu.

Anemia↗
PubMed1989

Sodium ions as substitutes for protons in the gastric H,K-ATPase.

In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes.

Adenosine Triphosphatases↗