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Subcellular distribution of small GTP‐binding proteins in the intestinal cell line Caco‐2
Author(s) -
STEIN J.,
GERHARD R.,
ZEUZEM S.,
CASPARY W. F.
Publication year - 1995
Publication title -
european journal of clinical investigation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.164
H-Index - 107
eISSN - 1365-2362
pISSN - 0014-2972
DOI - 10.1111/j.1365-2362.1995.tb01959.x
Subject(s) - gtp' , differential centrifugation , brush border , molecular mass , biochemistry , membrane protein , cytoplasm , g protein , chemistry , cell fractionation , rhoa , gtpase , membrane , biology , microbiology and biotechnology , receptor , vesicle , signal transduction , enzyme
. The present study describes the subcellular distribution of low molecular weight GTP‐binding proteins in the human carcinoma cell line Caco‐2. Highly enriched subcellular fractions of basolateral and brush border membranes were prepared by differential density centrifugation and divalent cation precipitation. Small molecular weight GTP‐binding proteins were identified after SDS‐PAGE transfer to nitrocellulose using [α 32 P]‐labelled GTP. Smg‐proteins with molecular masses of 28, 27, 25 and 24 kDa were detectable in all fractions. Homo‐genate and brush border membrane fraction showed specific binding of [α 32 P] GTP to proteins of a molecular mass of 21 kDa, while in the membrane fractions (apical, basolateral) a high enrichment of 24 kDa smg‐proteins was detectable. Western blot analysis identified one of the 21 kDa proteins of the brush border membrane as rhoA. The homogenate of 4, 8, 11 and 14 days old Caco‐2 cells showed different [α 32 P]‐GTP binding to 21, 27 and 28 kDa proteins. In conclusion, this study is the first showing the presence and asymmetrical distribution of smg‐proteins among the various membrane components in the human carcinoma cell line Caco‐2.