Intracellular Enzymes of Collagen Biosynthesis in 3T6 Fibroblasts and Chick‐Embryo Tendon and Cartilage Cells

Abstract

An increase of about 4–9‐fold was found in prolyl and lysyl hydroxylase activities per cell when 3T6 fibroblasts were grown from the early‐log to the stationary phase, whereas essentially no changes were found in hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyl‐transferase activities. A control experiment using a different assay procedure indicated that the increase in prolyl hydroxylase activity was not due to any methodological artefact. The results demonstrate for the first time that these four intracellular enzymes of collagen biosynthesis are not regulated in an identical manner within one cell type.Freshly isolated chick embryo cartilage cells, which synthesize type I1 collagen, had higher lysyl hydroxylase and hydroxylysyl galactosyltransferase activities than freshly isolated chick embryo tendon cells, whereas no difference was found between them in prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase activities. The greater extent of the hydroxylation of lysyl residues and galactosylation of hydroxylysyl residues in type I1 than in type I collagen is thus probably due in part to a higher activity of the corresponding enzymes in the cartilage cells, but the greater extent of the glucosylation of galactosylhydroxylysyl residues cannot be explained by any higher activity of the glucosyltransferase.Prolyl and lysyl hydroxylase activities were markedly lower in the cultured 3T6 cells than in freshly isolated chick embryo tendon cells, particularly when expressed per unit solubilized cell protein. These data agree with the lower rate of collagen production by the 3T6 cells. Unexpectedly, the two hydroxylysyl glycosyltaransferase activities were somewhat higher in the 3T6 cells than in the tendon cells when expressed per cell, a finding which may explain the pronounced glycosylation of the collagen synthesized by the 3T6 cells.On incubation of the 3T6 cells with 5 μg/ml puromycin for 10 h the intracellular enzyme activities of collagen biosynthesis decreased by about 15–30% in the early‐log phase and by about 10–20% in the late‐log phase. The results suggest that the half‐lives of all four enzyme activities are at least about 24 h in the early‐log phase and possibly even longer in the late‐log phase.