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Evidence of heterogeneity of protein-turnover states in cultured cells.

ABSTRACT: Previous studies on L-cell cultures [Amenta & Sargus (1979) Biochem. J. 182, 847--859] have suggested: (a) that degradation of slow-turnover proteins occurs in a distinct cell state (D-state); (b) that cells randomly enter the D-state with a first-order transition constant, rapidly degrade cell protein, and return to a quiescent G0-state. In the present study we have tested the hypothesis that the putative D-state exists as a substate within A-state (non-replicating) fibroblasts. Rat-embryo fibroblasts were prelabelled with [14C]leucine and [3H]thymidine, 'chased' for 24 h, and then placed in fresh growth medium containing either vinblastine (10 microM) or colchicine (25 microM) for three successive 24 h periods. Cells trapped in mitosis were separated from the residual non-replicating cells and rates of protein synthesis, degradation and net accumulation were measured in both populations. We observed that significant protein degradation occurred only in the non-replicating population, although both populations showed equally high rates of protein synthesis induced by fresh growth medium. These data support the hypothesis that degradation of slow-turnover protein is heterogeneous, occurring only in A-state cells. A model that proposes a separate D-state within G0-phase successfully accounts for these observations and previous reports on this cell line [Amenta, Sargus & Baccino (1978) J. Cell. Physiol. 97, 267--283] showing no differences in degradation of the slow-turnover protein pool in growth-stimulated and stationary-phase fibroblast cultures.

SUBMITTER: Amenta JS

PROVIDER: S-EPMC1162146 | biostudies-other | 1980 Sep

REPOSITORIES: biostudies-other

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Project description:The chain composition and relative abundance of type VI collagen synthesized by cells cultured from foetal bovine nuchal ligament and skin were compared with those of the type VI collagen present in these foetal tissues. Immunoprecipitation of intact collagen VI from medium and cell layers of nuchal ligament fibroblasts and skin fibroblasts at confluence revealed collagen type VI molecules with a chain composition consistent with an [alpha 1(VI)alpha 2(VI)alpha 3(VI)] monomeric assembly. Maintenance of cells in a post-confluent quiescent state promoted a marked phenotypic change in these ratios, with increased concentrations of assemblies composed of equimolar ratios of alpha 1(VI) and alpha 2(VI) chains detected in the medium of these cultures. Analysis of steady-state concentrations of mRNA for alpha 1(VI) and alpha 2(VI) chains revealed these species to be present in increased abundance at post-confluence in all the cultures, but no corresponding increase was observed in the alpha 3(VI) mRNA. In order to assess the physiological significance of these observations, the chain composition of the collagen VI content of the corresponding foetal tissues was assessed by Western blotting after extraction in guanidinium isothiocyanate under reducing conditions. Extracts of nuchal ligament revealed a collagen VI chain composition consistent with a heterotrimeric chain assembly. In contrast, the skin extracts revealed an abundance of alpha 1(VI) and alpha 2(VI) chains with only traces of the alpha 3(VI) chain detected. Increased equimolar concentrations of the alpha 1(VI)-chain and alpha 2(VI)-chain mRNAs in skin again reflected the increased concentrations of these polypeptide chains. Type VI collagen was present in greater abundance both in the nuchal ligament and in the corresponding nuchal-ligament fibroblast cultures. The results indicate that the chain composition of type VI collagen is subject to modulation at the level of transcription as a result of variations in the proliferative state of the cells, and demonstrate that different isoforms of collagen VI occur in foetal development.

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Evidence of heterogeneity of protein-turnover states in cultured cells.

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