Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

Ouabain sensitivity of the alpha 3 isozyme of rat Na,K-ATPase

The Na,K-ATPase of rat brainstem axolemma membranes contains two isozymes of its catalytic subunit, alpha 2 and alpha 3. To isolate the alpha 3 isozyme functionally, purified axolemma Na,K-ATPase was treated with trypsin. Immunoblot analysis of trypsin-treated Na,K-ATPase using isozyme-specific antibodies showed that alpha 3 was significantly more resistant to digestion than alpha 2. The trypsin-resistant alpha 3 isozyme fraction, devoid of alpha 2, contained 50-60% of the ATPase activity, and was inhibited by ouabain half-maximally at 0.13 microM. This indicates that the alpha 3 Na,K-ATPase isozyme has a high sensitivity to cardiac glycosides.     

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.     

Structural peculiarities of Na+,K+-ATPase isozymes from the calf brain

Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.     

Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catalytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha-helix of the Na,K-ATPase beta1 subunit to investigate its role in the stabilization of the alpha subunit, the endoplasmic reticulum exit of alpha-beta complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the beta subunits transmembrane domain had no significant effect on the structural maturation of alpha subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane alpha-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha subunit, had no effect on the apparent K(+) affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr(40) and Tyr(44), on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the beta subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the beta subunits transmembrane alpha-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.     

FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.     

Rat brain has the alpha 3 form of the (Na+,K+)ATPase

Multiple forms of the catalytic subunit of the (Na+,K+)ATPase have been identified in rat brain. While two of them (alpha 1 and alpha 2) have been well characterized, the third form (alpha 3) of these catalytic subunits only recently has been described by cDNA cloning; the corresponding polypeptide has not been isolated. In this paper it is shown that rat brain contains the alpha 3 chain. The catalytic subunits of the (Na+, K+)ATPase from rat brain axolemma were purified by SDS-PAGE and subjected to formic acid cleavage. Amino acid sequence analysis of the resulting fragments revealed that axolemma has the alpha 3 form of the catalytic subunit. In addition, alpha 3-specific antiserum was raised in rabbits immunized with a synthetic peptide. Immunoblotting with this antiserum revealed that the alpha 3 form of the (Na+,K+)ATPase is present also in whole brain microsomes. In SDS-PAGE, the mobilities of the three catalytic subunits of brain (Na+, K+)ATPase follow the order alpha 1 greater than alpha 2 greater than alpha 3. Determination of the ouabain-inhibitable ATPase activity indicates that if the alpha 3 form of the (Na+,K+)ATPase is able to hydrolyze ATP, it is present in a form of the enzyme with a high affinity for this cardiac glycoside and is similar to the alpha 2 form in this respect.     

Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart

The present study demonstrates that two forms of the alpha catalytic subunit of the Na,K-ATPase are present in rat heart and originate from cardiomyocytes. They were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and alkylation of the sulfhydryl groups. The two forms were identified on immunoblots using two specific antisera against either the alpha subunit from Bufo marinus kidney and the alpha and beta subunits from lamb kidney. Comparison of the two forms to the alkylated Na,K-ATPase from rat kidney (containing one catalytic subunit) and from rat brain (containing alpha and alpha + subunits) suggested that, in rat cardiac myocytes, the form with a fast migration rate (alpha F) corresponds to the alpha subunit of low ouabain affinity and the one with a slow migration rate (alpha S), to a subunit of high ouabain affinity. Thus, the existence of two isoforms of the catalytic subunit in cardiac myocytes accounts well for the biphasic ouabain inhibition of the Na,K-ATPase activity and for the biphasic inotropic responsiveness to cardiac glycosides of the rat heart.     

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.     

Na+,K(+)-ATPase isozymes in the bovine brain grey matter and brain stem

Active preparations of Na+,K(+)-ATPase containing three types of catalytic isoforms were isolated from the bovine brain to study the structure and function of the sodium pump. Na+,K(+)-ATPase from the brain grey matter was found to have a biphasic kinetics with respect to ouabain inhibition and to consist of a set of isozymes with subunit composition of alpha 1 beta 1, alpha 2 beta m and alpha 3 beta m (where m = 1 and/or 2). The alpha 1 beta 1 form clearly dominated. For the first time, glycosylation of the beta 1-subunit of the alpha 1 beta 1-type isozymes isolated from the kidney and brain was shown to be different. Na+,K(+)-ATPase from the brain stem and axolemma consisted mainly of a mixture of alpha 2 beta 1 and alpha 3 beta 1 isozymes having identical ouabain inhibition constants. In epithelial and arterial smooth muscle cells, where the plasma membrane is divided into functionally and biochemically distinct domains, the polarized distribution of Na+,K(+)-ATPase is maintained through interactions with the membrane cytoskeleton proteins ankyrin and spectrin (Nelson and Hammerton, 1989; Lee et al., 1996). We were the first to show the presence of the cytoskeleton protein tubulin (beta 5-isoform) and glyceraldehyde-3-phosphate dehydrogenase in a high-molecular-weight complex with Na+,K(+)-ATPase in brain stem neuron cells containing alpha 2 beta 1 and alpha 3 beta 1 isozymes. Consequently, the influence of not only subunit composition, but also of glycan and cytoskeleton structures and other plasma membrane-associated proteins on the functional properties of Na+,K(+)-ATPase isozymes is evident.     

Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K-ATPase catalytic subunit

An isozyme-specific domain of the catalytic subunit of the Na,K-ATPase has been identified using a monoclonal antibody, McK1. The antibody's specificity was confirmed by its ability to stain proteolytic fingerprints of the Na,K-ATPase. The antibody recognized the alpha I isozyme of the rat Na,K-ATPase, but not the alpha II or alpha III isozymes. It recognized native and sodium dodecyl sulfate-denatured Na,K-ATPase and specifically stained basolateral membranes of the renal tubule. It bound to rat alpha I with highest affinity, but also cross-reacted with mouse, monkey, and human alpha I. It did not cross-react with sheep, pig, chicken, Torpedo, or dog alpha I. Fine specificity mapping was used to deduce the most likely antibody binding sites, based on comparison of eight amino acid sequences from cDNA clones. Two potential binding sites were found at widely separated locations. Limited tryptic digestion of the native enzyme was then used to demonstrate that the binding site was close to the N-terminal end of the Na,K-ATPase. The binding site is predicted to include the following essential amino acid sequence: Asp-Lys-Lys-Ser-Lys-Lys in rat alpha I or Asp-Lys-Lys-Gly-Lys-Lys in human alpha I. The antibody was found to bind to opened, but not to sealed right-side-out vesicles isolated from the rat renal medulla, demonstrating that the N-terminal end of the Na,K-ATPase is exposed at the interior of the cell.     

Transport and pharmacological properties of nine different human Na, K-ATPase isozymes

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 alpha and beta isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K(+)-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the alpha isoform. On the other hand, variations in external K(+) activation are determined by a cooperative interaction mechanism between alpha and beta isoforms with alpha2-beta2 complexes having the lowest apparent K(+) affinity. alpha Isoforms influence the apparent internal Na(+) affinity in the order alpha1 > alpha2 > alpha3 and the voltage dependence in the order alpha2 > alpha1 > alpha3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, alpha2-beta isozymes exhibit more rapid ouabain association as well as dissociation rate constants than alpha1-beta and alpha3-beta isozymes. Finally, isoform-specific differences exist in the K(+)/ouabain antagonism which may protect alpha1 but not alpha2 or alpha3 from digitalis inhibition at physiological K(+) levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.     

The catalytic subunits of the (Na+,K+)-ATPase alpha and alpha(+) isozymes are the products of different genes

The sequences of the first 14 amino acids of the (Na+,K+)-ATPase catalytic subunits from rat kidney (alpha) and rat brain axolemma (alpha(+)) have been determined. They are: (alpha), NH2-Gly-Arg-Asp-Lys-Tyr-Glu-Pro-Ala-Ala-Val-Ser-Glu-His-Gly; (alpha(+)), NH2-Gly-Arg-Glu-Tyr-Ser-Pro-Ala-Ala-Glu-Val-Ala-Glu-Val-Gly. Although they are highly homologous, it is clear these sequences are also sufficiently different to conclude they are the products of different genes, or at least different exons of the same, differentially spliced, gene. Among mammals, the amino terminal sequence of the kidney alpha chain is essentially invariant. Thus this section of the (Na+,K+)-ATPase molecule is more highly conserved in one tissue between several species than between different tissues in the same species. This may reflect upon the difference in function of the alpha and alpha(+) isozymes of (Na+,K+)-ATPase.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.     

The gamma subunit modulates Na(+) and K(+) affinity of the renal Na,K-ATPase

The Na(+),K(+)-ATPase catalyzes the active transport of ions. It has two necessary subunits, alpha and beta, but in kidney it is also associated with a 7.4-kDa protein, the gamma subunit. Stable transfection was used to determine the effect of gamma on Na, K-ATPase properties. When isolated from either kidney or transfected cells, alphabetagamma had lower affinities for both Na(+) and K(+) than alphabeta. A post-translational modification of gamma selectively eliminated the effect on Na(+) affinity, suggesting three configurations (alphabeta, alphabetagamma, and alphabetagamma*) conferring different stable properties to Na, K-ATPase. In the nephron, segment-specific differences in Na(+) affinity have been reported that cannot be explained by the known alpha and beta subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect gamma in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked gamma, correlating with higher Na(+) affinities in those segments reported in the literature. Selective expression in different segments of the nephron is consistent with a modulatory role for the gamma subunit in renal physiology.     

The sodium pump needs its beta subunit

The sodium pump Na,K-ATPase, located in the plasma membrane of all animal cells, is a member of a family of ion-translocating ATPases that share highly homologous catalytic subunits. In this family, only Na,K-ATPase has been established to be a heterodimer of catalytic (alpha) and glycoprotein (beta) subunits. The beta subunit has not been associated with the pump's transport or enzymatic activity, and its role in Na,K-ATPase function has been, until recently, a puzzle. In this review we describe what is known about the structure of beta and summarize evidence that expression of both alpha and beta subunits is required for Na,K-ATPase activity, that inhibition of glycosylation causes a decrease in accumulation of both alpha and beta subunits, and we provide evidence that pretranslational up-regulation of beta alone can lead to increased abundance of sodium pumps. These findings are all consistent with the hypothesis that the beta subunit regulates, through assembly of alpha beta heterodimers, the number of sodium pumps transported to the plasma membrane.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain

Molecular genetic evidence indicates that there should be three different (Na+ + K+)-stimulated ATPase (Na,K-ATPase) alpha subunit isozymes in the brain where previously only two ("alpha" and "alpha(+)") were resolved as proteins. To detect and identify alpha 1, alpha 2, and alpha 3 isozymes, polypeptides made by cell-free translation (Schneider, J.W., Mercer, R.W., Gilmore-Hebert, M., Utset, M.F., Lai, C., Greene, A., and Benz, E.J., Jr. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 284-288) were analyzed by gel electrophoresis and proteolytic fingerprinting. Synthetic alpha 1 comigrated with tissue alpha 1, while alpha 2 and alpha 3 comigrated with the leading and trailing edges, respectively, of tissue "alpha(+)." Proteolytic fingerprints of newborn rat brain Na,K-ATPase labeled in vivo with L-[35S]methionine indicated the presence of alpha 1 and alpha 3, and a low level of alpha 2. Monoclonal antibodies were characterized by the electrophoretic mobility of their antigens and by their ability to recognize the Na,K-ATPases of kidney, brain, and skeletal muscle. The antibodies were used to assess isozyme expression in the brain. All three isozymes increased in abundance during development from the 18-day fetus to the adult. Small changes were seen in the relative level of expression of alpha 1 and alpha 3 at different developmental ages, while alpha 2 expression increased markedly between the neonate and adult. In adult brain, all three isozymes were found in all brain regions examined. We conclude that all three isozymes are expressed as proteins and that their expression and distribution must be under complex control. No single developmental age or macroscopic brain region provides an exclusive source of any of the isozymes.     

Immunodetection of Na,K-ATPase α3-isoform in renal and nerve tissues

At least three types of mRNA of the catalytic subunit of Na,K-ATPase namely alpha-,alpha+- and alpha 3-isoforms are identified in different tissues. Only two of them alpha and alpha+ have well known structural and catalytic properties. Here we present immunochemical data indicating that the alpha 3 protein really exists in pig and human kidney, and human brain. Crude membrane fractions and purified membrane-bound Na,K-ATPases were immunoblotted with alpha 3-specific antibodies raised against the synthetic peptide corresponding to the unique sequence of this isoform. The mature alpha 3-subunit is shown to include the sequence GDKKDDKSSPK followed by the initiating methionine residue. Nephron collecting tubules are proposed to specifically contain Na,K-ATPase alpha 3-isoform.     

The effect of micromolar Ca2+ on the activities of the different Na+/K(+)-ATPase isozymes in the rat myometrium.

In the present work we show the existence of two Na+/K(+)-ATPase isozymes in rat myometrial microsomes and suggest that they have different Ca2+ sensitivities. The catalytic subunits (alpha 1, alpha 2) of Na+/K(+)-ATPase were labelled by fluorescein-isothiocyanate and separated by SDS gel electrophoresis. The two isozyme Ca2(+)-sensitivities were studied by comparing the kinetics of Ca2+, strophantidin, ouabain and N-ethylmaleimide inhibitions. Our results indicate that the activity of the high ouabain-sensitive part (alpha 2 type) of Na+/K(+)-ATPase enzyme could only be inhibited by micromolar Ca2+. Furthermore, treatment of the microsomal preparation with 1mM N-ethylmaleimide selectively inactivated the high Ca2+ sensitive isoform of myometrial Na+/K(+)-ATPase.