Detection of low molecular mass GTP‐binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells

FEBS Letters

Doucet, Jean-Pierre¹  Trifaró, José-María¹  Fournier, Susan¹  Parulekar, Manohar¹ 

[1] Secretory Process Research Program, Department of Pharmacology, Faculty of Health Sciences, University of Ottawa, Ottawa, Ontario, Canada

关键词: GTP-binding protein;    Chromaffin granule;    Secretory process;    Gene product;    ras;    SDS-PAGE;    SDS-polyacrylamide gel electrophoresis;    GTP;    guanosine triphosphate;    GTP-γ-S;    guanosine 5′-(3-O-thio)-triphosphate;    Gpp(NH)p;    guanosine 5′-(β;    γ-imido)-triphosphate;    DTT;    dithiothreitol;    CDTA;    trans-1;    2-diaminocyclohexane-N;    N;    N′N′-tetraacetic acid;   

DOI  :  10.1016/0014-5793(89)81254-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [α-³²P]GTP in the presence of Mg²⁺. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [α-³²P]GTP was specific and dependent on Mg²⁺. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.

【 授权许可】

Unknown