Effects of two major activating lesions on the structure and conformation of human ras oncogene products.

ras oncogenes are frequently activated in human tumors by mutations at codon 12 or 61 in their coding sequences. To investigate how these subtle alterations exert such profound effects on the biologic activities of these genes, we studied structural and conformational properties of human ras-oncogene-encoded 21-kDa proteins (p21s). We observed striking differences in the electrophoretic mobilities of the proteins under reducing and nonreducing conditions. These findings imply that intramolecular disulfide bonds affect native p21 conformation. The two activating lesions were shown to induce distinctly different alterations in p21 electrophoretic mobility that were unmasked only under reducing conditions. These results suggest that regions of the molecule containing such alterations are either not exposed or under conformational constraints in the native p21 molecule. We confirmed the opposing effects on protein mobility induced by the two activating lesions by using a recombinant gene containing both lesions. The recombinant gene's high-titer transforming activity further established that the two lesions do not negatively complement one another with respect to transforming-gene function. Our findings of distinct alterations in electrophoretic mobilities of position-12- and position-61-altered p21 molecules should be applicable to the rapid immunologic diagnosis of ras oncogenes in human malignancies.