Dimeric and high molecular weight forms of the large external transformation-sensitive protein on the surface of chick embryo fibroblasts.

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The subunit structure of the large external transformation-sensitive (LETS) protein of chick embryo fibroblasts has been examined. When cells were labeled by lactoperoxidase-catalyzed iodination and analyzed on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agent, no more than 1% of the 1251labeled LETS protein migrated as monomer (M, = 230,000 to 240,000). About 20 to 23% of LETS migrated as dimer and the remainder failed to enter the 3.5% stacking gels (LETS high molecular weight (HMW) complex). These proportions did not appear to be an artifact resulting from disulfide bond formation induced by the lactoperoxidase labeling technique. Additional experiments rule out a second possible artifact, namely, that LETS HMW complex was actually LETS dimer that was retained at the top of the gel because of trapping: 1) the measured proportion of LETS dimer was unaffected by the concentration of the cell lysate sample; and 2) ‘251-labeled LETS dimer added exogenously to nonlabeled, nonreduced cell lysates migrated almost entirely as dimer with less than 6% trapped at the top of the gel in the presence of cell lysate concentrations routinely employed in our assays. Pulse-labeling experiments demonstrated the conversion of LETS dimer to HMW complex. The kinetics were biphasic, with rapid conversion ( TI,~ = 0.8 to 1.9 h) of most of the LETS dimer followed by slow conversion ( TI,Z = 14.5 h) of the remainder. The proportion of LETS present as dimer predicted from the kinetic parameters agreed well with the experimental value determined by lactoperoxidase labeling. LETS dimer and HMW complex were shed into the medium at similar slow rates, and culture conditions seemed to have little influence on the subunit structure of LETS. The proportions of LETS dimer and HMW complex were the same for culture times between 1 and 3 days and for 2day cultures at final cell densities ranging from 1 to 18 x lo4 cells/cm’.