Nonrandom turnover of actin and tubulin in cultured rabbit cardiac fibroblasts

Abstract

Total protein fractional rates of growth, synthesis, and degradation were assessed in primary cultures of rabbit cardiac fibroblasts. Differences in fractional growth rates were produced by subculturing cells at low density and growing them to confluence. Total protein fractional degradative rates were then derived by subtracting fractional growth rates from measured fractional synthetic rates (obtained in [3H]leucine pulse-labeling experiments). Actin and tubulin degradation were studied in similar rapidly and slowly growing cultures. [35S]methionine pulse-chase experiments, followed by dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, and densitometry were used to determine the amount of labeled actin and tubulin remaining in cultures at various times during the chase (0-96 h). The indirect study showed a substantially lower total protein fractional degradative rate during rapid vs. slow growth (0.04 +/- 0.13 vs. 0.42 +/- 0.01 d-1 at 2 and 15 days after subculture, respectively; P less than 0.01). At both growth rates, the disappearance of labeled actin and tubulin was delayed, suggesting a more complex model for their degradation than random decay. Serum deprivation of slowly growing fibroblasts increased the rate of disappearance of both proteins by eliminating the delay in their breakdown. Thus the suppression of protein degradation during rapid growth appears to result from the presence of relatively greater amounts of “new” actin and tubulin (and possible other long-lived proteins) that are kinetically distinct from the total intracellular pools of these proteins with respect to their susceptibility to proteolysis.