Further characterization of the acid-soluble phosphoprotein (SDS/PAGE apparent molecular mass of 22 kDa) in rat fat-cells by peptide sequencing and immuno-analysis: effects of insulin and isoprenaline.
1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble protein which migrates as a doublet on SDS/PAGE with an apparent molecular mass of close to 22 kDa; agents such as isoprenaline, which increase cell concentrations of cyclic AMP, also increase phosphorylation, but to a lesser extent [Belsham, Brownsey, Hughes and Denton (1980) Diabetologia 18, 307-312; Diggle and Denton (1992) Biochem. J. 282, 729-736]. 2. The protein has been purified from rat epididymal adipose tissue, and the sequences of six tryptic peptides were determined. All six peptides are present in the deduced sequence of a protein of similar properties, designated PHAS-I by Hu, Pang, Kong, Velleca and Lawrence [(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3730-3734]. Hence the proteins are the same or extremely similar. 3. A rabbit anti-peptide antibody has been raised against one of the peptides (AGGDESQFEMD). The antibody was found to be highly specific for the phosphorylated and non-phosphorylated forms of the acid-soluble 22 kDa protein in Western blots and by immunoprecipitation. Studies with the antibody preparation have shown that both phosphorylated and non-phosphorylated forms of the protein appear to be exclusively located in the cytoplasm, and that exposure of cells to isoprenaline causes increased phosphorylation of the same acid-soluble 22 kDa protein as does insulin treatment. 4. Western blots carried out with the antibody preparation indicate that the protein is also present in other insulin-sensitive tissues, including liver, skeletal muscle, heart and brown adipose tissue. The protein was also detected in lung and spleen, but not brain and kidney. It is concluded that the protein may play an important role in some of the actions of insulin.
Diggle
T A
TA
Department of Biochemistry, University of Bristol Medical School, U.K.
Bloomberg
G B
GB
Denton
R M
RM
D000273:Adipose Tissue / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D017421:Sequence Analysis; D012995:Solubility
D007328:Insulin; D010750:Phosphoproteins; D000242:Cyclic AMP; D007545:Isoproterenol