| Abstract: | Intact rat fat cells exposed to 12.5 microM gamma-32P]ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which by passes the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an Mr of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to tryptic digestion, whereas the unphosphorylated protein is unaffected by digestion with trypsin (15 min), inhibition of phosphorylation of this protein after brief exposure to melittin, and the consistent observation that more label is associated with the 42,000 Mr band in homogenates and permeabilized cells than in comparable numbers of intact cells exposed to the same amount of label. A 42,000 Mr phosphoprotein is also present in mitochondria which is most likely the alpha subunit of pyruvate dehydrogenase. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, 32Pi-labeled and gamma-32P]ATP-labeled cells were solubilized with Triton and chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the 32Pi-labeled sample, and not in the gamma-32P]ATP-labeled sample. Subcellular fractionation studies of intact cells labeled with gamma-32P]ATP showed differences in the recovery of phosphoproteins of 42,000 Mr depending on whether a continuous sucrose gradient (27.6-54.1%, g/ml) or a discontinuous sucrose gradient (16, 35 and 48%, g/ml) was used. Phosphoproteins of 42,000 Mr were located in the mitochondrial and membrane fractions collected by discontinuous sucrose gradient separation, whereas a phosphoprotein of 42,000 Mr was found primarily in the mitochondrial fraction after continuous sucrose gradient separation. By 5'-nucleotidase activity measurements, the latter approach appears to result in the isolation of a heavy fragment of the plasma membrane with the mitochondrial light fraction which is 42,000 in Mr and labeled. Finally, comparison of the autoradiographs of two-dimensional (2D) gels (isoelectric focusing followed by 10% SDS-PAGE) show different isoelectric points for 42,000 Mr components in gamma-32P]ATP- and 32Pi-labeled cells.(ABSTRACT TRUNCATED AT 400 WORDS) |
