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Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts.

ABSTRACT: Hyaluronate synthase activity is localized exclusively in plasma-membrane fractions of cultured human skin fibroblasts. The enzyme activity of plasma membranes prepared from exponential-growth-phase cells was about 6.5 times that of stationary-growth-phase cells. Hyaluronate synthase from exponential-growth-phase cells exhibited lower Km and higher Vmax. values for both UDP-N-acetylglucosamine and UDP-glucuronic acid and higher rate of elongation of hyaluronate chains compared with the enzyme from stationary-growth-phase cells. Hyaluronate synthase exhibited an extremely short half-life, 2.2 h and 3.8 h respectively when cells were treated with cycloheximide and actinomycin D. The cell-growth-phase-dependent variations in hyaluronate synthase activity appear to be due to its high turnover rate as well as due to some post-translational modification of the enzyme protein as cells progress from early exponential to stationary growth phase. The isolated plasma membranes contained a protein (Mr approx. 450,000) that was selectively autophosphorylated from [gamma-32P]ATP in vitro in the presence of hyaluronate precursors in the reaction mixture and that also exhibited some hyaluronate-synthesis-related properties. The 32P-labelled protein isolated from plasma membranes of exponentially growing cells expressed an efficient UDP-[14C]glucuronic acid- and UDP-N-acetyl[3H]glucosamine-binding activity and was able to synthesize oligosaccharides (Mr 5000) of [14C]glucuronic acid and N-acetyl[3H]glucosamine residues. The corresponding protein of stationary-growth-phase cells, which expressed much higher nucleotide-sugar-precursor-binding activity, appeared to have lost its oligosaccharide-synthesizing activity.

SUBMITTER: Mian N

PROVIDER: S-EPMC1146992 | biostudies-other | 1986 Jul

REPOSITORIES: biostudies-other

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Project description:A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.

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Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts.

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