Use of a Novel Rapid Preparation of Fat-cell Plasma Membranes Employing Percoll to Investigate the Effects of Insulin and Adrenaline on Membrane Protein Phosphorylation Within Intact Fat-cells

Overview

Journal Biochem J

Specialty Biochemistry

Date 1980 Nov 15

PMID 6263255

Citations 40

Authors

G J Belsham

R M Denton

M J Tanner

Affiliations

Soon will be listed here.

Abstract

1. A rapid method was developed for the preparation of plasma membranes from either isolated rat fat-cells or intact epididymal fat-pads with the use of density-gradient centrifugation in the presence of Percoll. On the basis of 5'-nucleotidase activity, the yield of plasma membranes was about 50% and purification over 10-fold. Activities of marker enzymes indicated that contamination by mitochondria and microsomal fraction was small. 2. Incorporation of 32Pi into proteins associated with plasma membranes within isolated fat-cells was investigated. Four major bands of labelled phosphoproteins were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis; the apparent subunit mol.wts. were 67 000, 61 000, 26 000 and 20 000. None of these phosphoprotein bands corresponded to periodate/Schiff-staining glycoproteins. The extent of phosphorylation of the 61 000 mol.wt phosphoprotein band was increased by about 30 and 60% after exposure of fat-cells for 15 min to insulin or adrenaline respectively.

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