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 glucosyltransferase 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 glycosyltransferase 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 pg/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 x> 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. ~~~ ~ ~~~f37:i.mes and h'ommclrrure. Prolyl 4-hydroxylase or prolylglycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (EC 1.14.1 1.2) has usually been termed prolyl hydroxylase; recently, however, two separate enzymes, prolyl 4-hydroxylase and prolyl 3-hydroxylase have been demonstrated [4]; the present work concerns only the 4-hydroxylase activity, and the term prolyl hqdroxylasc is used exclusively to denote this.The term lysyl hydroxylase is used for peptidyllysine, 2-oxog1utarate:oxygen 5-oxidoreductase (EC 1.14.11.4). The two hydroxylysyl glycosyltransferases. UDPgalactose : 5-hydroxylysine-collagen galactosyl transferase (EC 2.4.1.50) and UDPglucose: 5-hydroxylysine-collagen glucosyltransferase (EC 2.4.1.66) have earlier been known as collagen galactosy...

Catalytic properties of lysyl hydroxylase from cells synthesizing genetically different collagen types

“…On the basis of the above conclusions it seems likely that the differences in the extents of lysyl hydroxylation between the genetically distinct collagen types are due to regulation of the amounts of a single type of the enzyme protein in various cells. This would agree with data indicating that differences in the extent of lysyl hydroxylation between type I, II and IV protocollagens can be explained to a large extent by differences in the amounts of lysyl hydroxylase activity in cells synthesizing exclusively one of these three collagen types (Risteli et al, 1979;Pihlajaniemi et al, 1981), the assays being carried out with type-I protocollagen substrate.…”

“…Lysyl hydroxylase was extracted from the tendons or sterna of 15-day chick embryos by homogenization in a solution containing 0.1 M-glycine, 0.2M-NaCl, 0.1% (w/v) Triton X-100, 0.01% (w/v) soya-bean trypsin inhibitor and 0.02M-Tris/ HCI buffer (pH 7.6 at 4°C) (Risteli et al, 1979) for 0306-3275/82/010215-05$01.50/1 1982 The Biochemical Society 5 x 5 s with an Ultra-Turrax homogenizer. The homogenates were centrifuged at 15 000g for 30min at 4°C and the supernatants used as the 'type I' and 'type II' enzymes. The lysyl hydroxylase referred to as the 'type IV' enzyme was isolated from HT-1080 sarcoma cells grown in 75 cm2 plastic tissue-culture bottles in a total volume of 20ml at 370C in a humidified incubator with an atmosphere of air/CO2 (19:1).…”

Influence of cell density on collagen biosynthesis in fibroblast cultures

“…It is more difficult, however, to control growth in such a way that cell density and extracellular matrix do not differ among the cell strains under investigation. As shown by previous reports, quantitative and qualitative changes in the pattern of collagen synthesis are probably caused by variations in cell density (Green & Goldberg, 1963;Priest & Davies, 1969;Peterkovsky, 1972;Steinberg, 1973Steinberg, , 1978Paz & Gallop, 1975;Kamine & Rubin, 1977;Abe et al, 1979;Kao & Berg, 1979;Risteli et al, 1979;Schneider, 1979;Tolstoshev et al., 1981), which is the basis for cell-cell communication and cell-matrix interaction. Since it is one of our prime interests to study metabolic changes of collagen disorders at the molecular level, we wanted to obtain more information about the cellular properties and biosynthetic capacities of normal human fibroblasts, which are often used as controls.…”

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells

“…The two prolyl hydroxylase activities are not always regulated identically. These activities show similar changes both when cells are grown from the earlyand the late-exponential phase (results not shown; Risteli et al, 1979) and as a function of age in animal tissues (Tryggvason et al, 1979), but they differ in liver tissue after chemically induced liver injury (Risteli et al, 1978b). In the present study the two prolyl hydroxylase activities are also seen to differ not only in sarcoma cells but also in freshly transformed cells.…”