We have identified a novel cDNA encoding a protein highly homologous to the mammalian brown fat uncoupling protein (UCP). Unlike the known UCP, which is expressed specifically in brown adipose tissue, the UCP homolog (UCPH) mRNA is expressed in a variety of tissues, with predominant expression in human white adipose tissue and skeletal muscle. In the white adipose tissue of ob/ob and db/db mice, the UCPH transcript is induced approximately fivefold relative to lean littermate controls. Expression of murine UCPH in yeast results in growth inhibition under conditions that require aerobic respiration, but does not affect growth under anaerobic conditions. Furthermore, UCPH expression in yeast causes a decrease in the mitochondrial membrane potential, as judged by staining with the potential-sensitive dye DiOC 6 . These observations suggest that UCPH, like UCP, uncouples oxidative phosphorylation. The possibility that the UCPH protein is an important mediator of human thermogenesis is discussed. Diabetes 46:900-906, 1997

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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.

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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...