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.
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.
D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004837:Epinephrine / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008722:Methods; D010766:Phosphorylation; D011205:Povidone; D051381:Rats; D012822:Silicon Dioxide
D007328:Insulin; D008565:Membrane Proteins; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D004837:Epinephrine