Characterization of the Insulin-regulated Membrane Aminopeptidase in 3T3-L1 Adipocytes (*)
- Stuart A. Ross,
- Hazel M. Scott,
- Nicholas J. Morris,
- Wai-Yee Leung(1),
- Fei Mao(1),
- Gustav E. Lienhard and
- Susanna R. Keller(§)
- From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 and
- Molecular Probes, Inc., Eugene, Oregon 97402
- §To whom correspondence should be addressed. Tel.: 603-650-1619; Fax: 603-650-1128
Abstract
A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant DK 25336 (to G. E. L.) and National Institutes of Health Fellowship Training Grant T32 AR07576 (to S. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- BSA
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bovine serum albumin
- AMC
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7-amino-4-methylcoumarin
- DAPI
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4,6-diamidino-2-phenylindole.
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↵2This value is based upon an estimate of the ng vp165 per mg protein in the low density microsomes from rat adipocytes (3) and the relative intensities of vp165 in known amounts of rat adipocyte low density microsomes and 3T3-L1 adipocyte lysates on an immunoblot.
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- Received October 12, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
