Differential expression and enzymatic properties of the Na+,K(+)-ATPase alpha 3 isoenzyme in rat pineal glands.

Abstract

We have used immunoblotting and biochemical techniques to analyze expression of Na',K+-ATPase a and fi subunits in rat pineal glands. Western blot analysis of pineal microsomal membrane fractions with antisera specific for each of the three rat a and two rat I8 subunits revealed similar levels of expression of al and a3 subunits in pineal glands of 5-day-old rats. High levels of a3 and f2 subunits and low levels of al subunits were detected in adult glands. (al, a2, a3) and two ,1 ((31, 132) subunit isoforms have been characterized (3-7).The development of specific molecular probes for each of the subunit components of the Na+,K+-ATPase has permitted a systematic evaluation of Na+,K+-ATPase expression in rat tissues. Substantial differences in the tissue and developmental specificity of expression were found for the genes encoding each of the a and 83 subunits (4,(8)(9)(10). Chromosomal dispersion and tissue-specific expression of the a-and ,3-subunit genes suggest that the enzyme encoded by each gene may have properties selected in response to different physiological demands. However, the functional significance for a-and 1-subunit isoform diversity has not been clearly explained.The existence of multiple a and 13 isoforms has made it inherently difficult to study the enzymatic parameters of individual Na+,K+-ATPase isoenzymes. Two biochemically distinct forms of the catalytic subunit, a (al) and a+ (a2), have been purified from rat kidney and brain axolemma, respectively, and found to differ in their affinities for ouabain and ATP (11). However, it is now known that the a+ isoform derived from axolemma represents a mixture of a2 and a3 subunits (12). To date, purification of Na+,K+-ATPase a2 or a3 isoenzymes has not been achieved. Characterization of the a2 and a3 subunits is further complicated by the fact that these polypeptides exhibit similar mobilities on SDS/PAGE (13).Recent studies have demonstrated that a3 subunits are expressed predominantly in rat brain and copurify with ouabain-inhibitable Na+,K+-ATPase activity (10). Expression of a3 subunits in a membrane fraction enriched for Na+,K+-ATPase activity is consistent with the view that the a3 isoform is a functional component of the brain enzyme. However, the ability to characterize the enzymatic properties of the a3 isozyme is limited because a2 subunits are also expressed at high levels in brain (10). In the course of surveying the tissue distribution of a and ,B subunits, we analyzed the rat pineal gland, a tissue previously shown to express a-and a+-like ATPase activities (25, 26). We identified the pineal gland as a major expression site of the a3 subunit. No a2 subunits were detectable in this tissue. The pineal gland thus provides an opportunity to analyze the enzymatic properties of the a3 isoform in the absence of contaminating a2 isoenzymes.We observed that in the rat there are similar levels of Na+,K+-ATPase al and a3 subunits in 5-day-old pineal glands. Expression of a3 subunits increases relative to the level ofal subunits in a...

Distribution of immunoreactive alpha- and beta-subunit isoforms of Na,K-ATPase in the gerbil inner ear.

“…It is speculated that the a2-subunit in glial cells functions to buffer increased extracellular K+ generated during neuronal activity via regulating its uptake and redistribution into plasma and cerebrospinal fluid. Localization of a3 in pinealocytes and retinal photoreceptors has led to the suggestion that this isoform may be specialized to maintain Na+ and K' gradients during hyperpolarization of the membrane potential (Schneider, 1992;Schneider and Kraig, 1990;Shyjan et al, 1990).…”

“…The physiological significance of the P-subunit remains obscure, but the a-subunit is thought to mediate catalytic activity. Its isoforms have different activities and capacities for movement of Na' and K' (Shyjan et al, 1990;Sweadner, 1985).…”

C-peptide stimulates rat renal tubular Na+, K+-ATPase activity in synergism with neuropeptide Y

“…Na § K+-ATPase activity (pmol of 32p i hydrolysed, mm of tubule -1 -h -1) was then calculated as the difference between the mean value for total ATPase and ouabain-insensitive ATPase activity, and expressed either as absolute values or as per cent above the corresponding control measurement. Kinetic analyses of K0.5(Na + ) were carried out as described by Shyjan et al [18].…”

Two Na,K-ATPase β2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis

“…In humans and rodents, the major expression sites of the ␤2 gene are brain (Shyjan et al, 1990a), pineal gland (Shyjan et al, 1990b), eye (Schneider et al, 1991), and ear (McGuirt and Schulte, 1994;Schulte and Steel, 1994). Together, the expression patterns for atp1b2a and atp1b2b closely parallel that seen for the single mammalian ␤2 gene.…”