Reverse phase chromatography of trypsin digests of a plasma membrane and a cytoplasmic insulin receptor substrate.
The 180,000 molecular weight protein from [32P]phosphorylated wheat germ agglutinin-purified rat liver plasma membranes was digested with trypsin. NIH 3T3 HIR 3.5 cells were [32P]phosphate-labelled in the presence of 10(-7) M insulin, and the 185,000 molecular weight cytoplasmic protein was digested with trypsin. Digests were applied to a C18-mu Bondapak column, eluted with acetonitrile gradients, and radioactivity in the eluate was monitored. The chromatogram for the cytoplasmic protein was similar but not identical to chromatograms of trypsin digests of insulin receptor substrates from other cultured cells. Thirteen and seven phosphopeptides were obtained from the plasma membrane and cytoplasmic substrate, respectively. One phosphopeptide from the two digests eluted at the same acetonitrile concentration; however, dissimilarity in elution profiles and dissimilarity in relative yields of individual phosphopeptides, suggest that the primary structures of tyrosine phosphorylation sites in the two insulin receptor substrates are different.
Goren
H J
HJ
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Boland
D
D
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D014357:Trypsin
D008565:Membrane Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin