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Abstract
Subcellular fractions of high purity (including plasma membrane, endoplasmic reticulum, mitochondria, nuclei, and cytoplasm) were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis. Each fraction except the endoplasmic reticulum exhibited a unique and reproducible complement of major peptides. Although the endoplasmic reticulum was distinctive in its enzymic markers, its peptide components showed striking homologies with certain species in the plasma membrane and cytoplasm. The two major adipocyte glycopeptides appear to be contained in the plasma membrane, inasmuch as they followed the distribution of 5'-nucleotidase. Incubation of adipocytes with extracellular 32Pi led to a uniform rate of incorporation of 32P into cellular peptides, with steady-state incorporation reached by 2 hours. Plasma membrane, mitochondria, nuclei, and cytoplasm all contained a distinctive complement of from two to five major phosphopeptides of different molecular weights. The majority of endoplasmic reticulum phosphopeptides exhibited molecular weights closely similar to those of certain species in the plasma membrane and cytoplasm. The phosphopeptides of the plasma membrane exhibited the highest absolute 32P incorporation of all phosphopeptides, next was the single major mitochondiral phosphopeptide. All fractions except the mitochondria contained, in addition to the few major phosphopeptides, numerous minor 32P-labeled phosphopeptides.
Highlights
Subcellular fractions of high purity were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis
Dependent protein kinase in 1968 [4], it became clear that the regulation of certain of these phospho-dephospho conversions was the site of hormonal control of glycogen metabolism, as well as of adipose tissue lipolysis
Fractions-The method described by McKee1 and Jarett [7] was designed primarily to obtain a purified plasma membrane fraction
Summary
Gofmoor Farms and used within 2 weeks. Isolated fat cells were prepared according to Rcdbell [6]. The washed fat cells, suspended in 10 to 20 ml of iced Medium I, were homogenized 10 strokes at 1,800 rpm (Thomas 3431-E25). Replicate gels were sliced longitudinally, dried as previously described (ll), subjected to radioautography, followed by densitometry. The area subtended by different bands within the same gel should most accurately approximate the counting data; if the same band was to be compared on replicate gels, the peak height served as well. The latter allowed more accurate estimations forphosphopeptides of similar mobility that were incompletely resolved by electrophoresis (Fig. 3).
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