Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules.
Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.
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D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D001358:Avian Sarcoma Viruses; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000032:analysis; D002472:Cell Transformation, Viral; D002645:Chickens; D007425:Intracellular Membranes / Q000378:metabolism*; D015688:Oncogene Protein pp60(v-src); D014764:Viral Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
D000914:Antibodies, Viral; D014764:Viral Proteins; D015688:Oncogene Protein pp60(v-src)