AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion 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 its association with Na,K-ATPase. The complete cDNA sequence mouse revealed 40% amino acid identity previously cloned beta subunit rat brain Immunoaffinity-purified detergent-purified Na,K-ATPase had identical apparent molecular weights, were...

Abstract 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 identify 1, 2, 3 isozymes, polypeptides made by cell-free translation (Schneider, J.W., Mercer, R.W., Gilmore-Hebert, M., Utset, M.F., Lai, C., Greene, A., Benz, E.J., Jr. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 284-288) analyzed gel electrophoresis...

The Na+,K+-ATPase catalyzes the active transport of ions. It has two necessary subunits, α and β, but in kidney it is also associated with a 7.4-kDa protein, γ subunit. Stable transfection was used to determine effect on Na,K-ATPase properties. When isolated from either or transfected cells, αβγ had lower affinities for both Na+ K+ than αβ. A post-translational modification selectively eliminated affinity, suggesting three configurations (αβ, αβγ, αβγ*) conferring different stable properties...

Two isozymes of the Na,K-ATPase were purified from rat renal medulla and brainstem axolemma, antisera raised in rabbits. When antibody titers measured, two sera showed specificity for either kidney or axolemma Na,K-ATPases had limited cross-reactivity which could be removed by cross-adsorption. In blots polyacrylamide gels, these reacted with only alpha (+) catalytic subunits, while they cross-reacted both types beta subunits. other each recognized (+), indicating that subunit have...

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity the modulation of gradients. Its three isoforms in brain have cell-type development-specific expression patterns. Interestingly, our studies demonstrate late gestation, α2 isoform widely expressed neurons, unlike adult brain, which has been shown to be primarily astrocytes. This unexpected distribution neurons interesting light examination mice lacking fail survive after birth. These...

There are multiple isoforms of the Na,K-ATPase in nervous system, three alpha subunit, and at least two beta subunit. The subunit is catalytic has several roles. It required for enzyme assembly, it been implicated neuron-glia adhesion, experimental exchange modifies kinetics, implying that affects functional properties. Here we describe specificities antibodies against beta1 beta2. These antibodies, along with isoforms, were used to stain sections rat cerebellum cultures cerebellar granule...

The cardiac glycoside sensitivity of the rat heart changes during postnatal maturation and in response to certain pathological conditions. Na,K-ATPase is thought be receptor for glycosides, there are three isozymes its catalytic (alpha) subunit with different affinities: alpha 1 (low affinity) 2 3 (high affinity). We examined developmental expression ventricular membrane preparations by immunoblotting isozyme-specific antibodies. isozyme was present throughout all stages maturation. A switch...

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca2+-ATPase) has recently been determined at 2.6 Å (note 1 = 0.1nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647–655]. Other P-type ATPases are thought to share key features the ATP hydrolysis site a central core transmembrane helices. Outside these most-conserved segments, structural similarities less certain, predicted topology differs between subclasses. In...

There is considerable evidence that protein kinases play a role in regulation of the activity Na,K-ATPase, but characteristics direct kinase phosphorylation Na,K-ATPase subunits are still not well understood. 36 sites could qualify as C motifs rat α1. Here we have used fragmentation with trypsin to localize site α1 subunit within first 32 amino acids N terminus and then sequencing phosphorylated determine which two candidate serine residues was modified. The result at most 25% 32P found on...

An isozyme-specific domain of the catalytic subunit Na,K-ATPase has been identified using a monoclonal antibody, McK1. The antibody's specificity was confirmed by its ability to stain proteolytic fingerprints Na,K-ATPase. antibody recognized alpha I isozyme rat Na,K-ATPase, but not II or III isozymes. It native and sodium dodecyl sulfate-denatured specifically stained basolateral membranes renal tubule. bound with highest affinity, also cross-reacted mouse, monkey, human I. did cross-react...

The seven members of the FXYD protein family associate with Na⁺-K⁺ pump and modulate its activity. We investigated whether conserved cysteines in proteins are susceptible to glutathionylation such reactivity affects function cardiac myocytes <i>Xenopus</i> oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or a GSH antibody. Incubation recombinant resulted competitive displacement native FXYD1. Myocyte oocyte...

There are three isoforms of the catalytic (alpha) subunit Na+,K(+)-ATPase, each derived from a different gene, that differ in their sensitivity to inhibition by cardiac glycosides. Antibodies specific for were used study Na+,K(+)-ATPase isoform expression ventricular myocardium, where an understanding digitalis receptor diversity is most important. In rat heart, there simultaneous two adult ventricle, and immunofluorescence studies demonstrated both expressed uniformly cardiomyocytes....

Phospholemman (FXYD1) is a homolog of the Na,K-ATPase γ subunit (FXYD2), small accessory protein that modulates ATPase activity. Here we show phospholemman highly expressed in selected structures CNS. It most abundant cerebellum, where it was detected molecular layer, Purkinje neurons, and axons traversing granule cell layer. particularly enriched choroid plexus, organ secretes CSF ventricles, colocalized with apical membrane. also enriched, Na,K-ATPase, certain tanycytes or ependymal cells...

Phosphorylation of sodium and potassium ion-activated adenosine triphosphatase (Na,K-ATPase) by protein kinase A (PKA) C (PKC) was investigated in vitro, where substrate conformation, activity, consequent effects on Na,K-ATPase activity could be controlled. With Na, K-ATPase from rat kidney, optimal stoichiometries were close to 1 mol 32P/mol for both kinases. Addition Na+, K+, P(i), or ouabain is known stabilize the different states found affect phosphorylation two kinases a reciprocal way....

The Na,K-ATPase belongs to a family of P-type ion-translocating ATPases sharing homologous catalytic subunits (α) that traverse the membrane several times and contain binding sites for ATP cations. In this family, only Na,K- H,K-ATPases have been shown second subunit, single-span glycoprotein called β. Recently new isoform (β3) has identified in mammals. Here we describe structural features tissue distribution β3 protein, utilizing an antiserum specific its N terminus. was β detected...

The Na,K-ATPase is a dominant factor in retinal energy metabolism, and unique combinations of isoforms its α β subunits are expressed different cell types determine functional properties. We used isoform-specific antibodies fluorescence confocal microscopy to the expression mouse rat retina. In adult retina, α1 was found Müller horizontal cells, α2 some glia, α3 photoreceptors all neurons. β1 largely restricted horizontal, amacrine, ganglion cells; β2 photoreceptors, bipolar glia; β3...

Elevated cAMP in NRK-52E and L6 cells causes a marked reduction the phosphorylation of numerous phosphoproteins, as detected initially with phosphoserine-specific antibodies. Here, we show that elevation NRK by forskolin/3-isobutyl-1-methylxanthine (IBMX) treatment decreased substrates for different protein kinases, pointing to common phosphatase target cAMP-dependent regulation. Forskolin/IBMX completely dephosphorylated selective 2A (PP2A) substrate, elongation factor-2 (EF-2), at its...

Na,K-ATPase activity has been demonstrated to be regulated by a variety of hormones in different tissues. It is known directly phosphorylated on its α-subunit, but the functional effects protein kinases remain controversial. We have developed sensitive, antibody-based assay for detection level phosphorylation α1-isoform rat at serine residue that most readily kinase C (PKC)<i>in vitro</i>, Ser¹⁸. By stimulation endogenous PKC and inhibition phosphatase activity, it was possible...

The gamma-subunit of the Na-K-ATPase is a single-span membrane protein that alters kinetic properties enzyme. It expressed in kidney, but our initial observations indicated it not present all nephron segments (Arystarkhova E, Wetzel RK, Asinovski NK, and Sweadner KJ. J Biol Chem 274: 33183-33185, 1999). Here we used triple-label confocal immunofluorescence microscopy rat kidney with antibodies to alpha1- gamma-subunits segment-specific markers. alpha1-subunit stain was low unambiguous...