Immunological Evidence for an Inactive Precursor of Collagen Proline Hydroxylase in Cultured Fibroblasts

McGee, J O; Langness, U; Udenfriend, Sidney

doi:10.1073/pnas.68.7.1585

Cited by 53 publications

(16 citation statements)

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“…More direct evidence for this hypothesis was provided by the observation that during lactate treatment or cell crowding the amount of protein that crossreacts with a monospecific antibody against the enzyme (crossreacting protein), remained virtually constant, while the enzyme activity increased several-fold (5). Subsequently, McGee and Udenfriend (6) isolated an enzymatically inactive protein from early log-phase, L929 fibroblasts that crossreacts with antibody to the enzyme and obtained evidence that it may be an enzymatically inactive precursor of prolyl hydroxylase.…”

mentioning

confidence: 94%

“…Enzymatically inactive, crossreacting protein was determined by the enzyme-immunoassay of McGee and Udenfriend (18) with an antiserum to rat-skin prolyl hydroxylase, prepared as described (5). In order to measure the protein in the presence of high enzyme activity, we modified the enzyme-immunoassay by using as a standard, heat-inactivated mouseskin prolyl hydroxylase, separated from crossreacting protein by purification through the DEAE-Sephadex step (19).…”

Section: Methodsmentioning

confidence: 99%

“…Prolyl hydroxylase activity was measured by the tritium release assay of Hutton et al (17), as described earlier (5).…”

Section: Methodsmentioning

confidence: 99%

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Activation of Prolyl Hydroxylase in L-929 Fibroblasts by Ascorbic Acid

Stassen

Cardinale

Udenfriend

1973

Proc. Natl. Acad. Sci. U.S.A.

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The addition of ascorbate to the culture medium of early log-phase mouse fibroblasts (L-929 cells) resulted in a 5-fold increase of prolyl hydroxylase activity. Maximal activity was reached within 2 hr after addition of 5 jM ascorbate. The total amount of enzyme-related antigen did not change on treatment with ascorbate and the activation was shown to be independent of RNA and protein synthesis. The increase in activity caused by ascorbate is therefore due to the activation of a preformed precursor. Enzyme (molecular weight 260,000-300,000) and putative precursor (molecular weight 85,000-105,000) were separated by chromatography on DEAE-Sephadex. Treatment of intact cells with dithiothreitol resulted in an almost quantitative conversion of the enzyme to the smaller inactive protein. When these cells were treated with ascorbate or incubated overnight in fresh medium the enzyme reappeared and precursor concentrations decreased proportionately. Ascorbate may act by bringing about aggregation of enzymatically inactive subunits.Green and Goldberg (1) observed that significant hydroxylation of proline did not occur in fibroblast cultures until late log-phase was reached. Gribble et al. (2) then showed that the appearance of hydroxyproline at the end of the logarithmic growth of L-929 mouse fibroblasts occurred along with a sharp increase in prolyl hydroxylase* activity. In early logphase cells a similar increase of enzyme activity could be induced by concentration of the cells to a high density (3), or by incubation of the cells at 370 in the presence of lactate (4).The fact that this activation is independent of RNA and protein synthesis suggested the presence of an inactive precursor of the enzyme. More direct evidence for this hypothesis was provided by the observation that during lactate treatment or cell crowding the amount of protein that crossreacts with a monospecific antibody against the enzyme (crossreacting protein), remained virtually constant, while the enzyme activity increased several-fold (5). Subsequently, McGee and Udenfriend (6) isolated an enzymatically inactive protein from early log-phase, L929 fibroblasts that crossreacts with antibody to the enzyme and obtained evidence that it may be an enzymatically inactive precursor of prolyl hydroxylase.There have been many reports that the amount of hydroxyproline formed by cultured fibroblasts and osteoblasts is increased by exposure to ascorbate (7-14). Peck et al. (8) reported that hydroxylation of radioactive proline, which had previously been incorporated into the protein of cultured bone cells, was stimulated by ascorbate even in the presence of puromycin or cycloheximide. These findings indicate that ascorbate stimulates the hydroxylation of a prolinerich peptide. Recently, Peterkofsky (14) reported that ascorbate deteriorates rapidly in tissue culture medium and that by maintaining adequate concentrations of ascorbate, hydroxyproline formation was markedly increased in early log-phase cells, while collagen polypeptide formation was unaff...

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confidence: 94%

Section: Methodsmentioning

confidence: 99%

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Activation of Prolyl Hydroxylase in L-929 Fibroblasts by Ascorbic Acid

Stassen

Cardinale

Udenfriend

1973

Proc. Natl. Acad. Sci. U.S.A.

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“…and the cells were separated, the remaining 2 ml being incubated for an additional 3 h. The cell pellets were extracted as described in Methods, the tetramers and the monomer-size protein were separated by gel filtration and immunoprecipitated as described in Methods. The relative amounts of tetramers and the monomer-size protein are given as pg per 10 Table 3. Re-appearance of' pre-luhrlled prolyl hydroxyluse protein in tetrumer ,form gfier reversal of'the dissociation w'itli dithiothreitol About 1.2 x 10' isolated chick-embryo tendon cells were incubated with 60 pCi of universally I4C-labelled protein hydrolysate in a final volume o f 8 ml for 60 min.…”

Section: Labelling Of Prolyl Hydroxylase Tetramers and The Monomer-simentioning

confidence: 99%

Tetramers and Monomers of Prolyl Hydroxylase in Isolated Chick-Embryo Tendon Cells. The Association of Inactive Monomers to Active Tetramers and a Preliminary Characterization of the Intracellular Monomer-Size Protein

1977

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In freshly isolated chick-embryo tendon cells about 65 % of the prolyl hydroxylase protein is present in the form of active enzyme tetramers, and about 35 % in a form corresponding in molecular weight to the enzyme monomers when studied by gel filtration. After incubation of these cells with 0.45 mM dithiothreitol for 30 min, the enzyme activity, the enzyme tetramers as a percentage of total enzyme protein, and the formation of hydroxyl ['4C]proline in the cells after a pulse with[14C]proline, all decreased to about 0-5 % of their original values. When the cells were incubated for an additional 5 h without dithiothreitol, all these parameters again increased to about 50 -60 % of their original values, the increases in enzyme activity and percentage of enzyme tetramers being similar irrespective of whether cycloheximide was present during the reversal period. When the cells were incubated for 1 h with a mixture of radioactively-labelled amino acids, the specific activity of the enzyme tetramers was about one fifth of that of the monomer-size protein. During a chase period of up to 6 h, a small increase occurred in the specific activity of the tetramers and a decrease in that of the monomer-size protein, while the specific activity of the total enzyme protein remained essentially unchanged. When the enzyme protein was first labelled for 1 h and then dissociated with dithiothreitol, re-formation of radioactively-labelled tetramers took place, and the specific activity of these tetramers was markedly higher than that of the tetramers originally present. Preliminary characterization of the radioactively-labelled intracellular monomer-size enzyme protein by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and 2-mercaptoethanol indicated that it consisted of two main polypeptide chains, the molecular weights of which were about the same as those of the enzyme monomers. The data suggest that the monomer-size protein in the cell represents, at least in part, precursors of the enzyme tetramers, and that it can be associated to active tetramers after its ribosomal biosynthesis. It is also suggested that the half-life of prolyl hydroxylase protein in these cells is relatively long, being probably more than one day.Prolyl hydroxylase catalyzes the synthesis of 4-hydroxyproline in collagen by the hydroxylation of prolyl residues in peptide linkages (for recent reviews see [I -31). The enzyme is a tetramer with a molecular weight of about 240000 [4-81 and consisting of two different types of monomers with molecular weights of 60000 and 64000 [5-91.Studies on prolyl hydroxylase protein in cultured cells [lo-131 and in animal [14,15] and human [I61 tissues have demonstrated that the enzyme is present partly in the form of active enzyme tetramers and partly in an inactive form, the molecular weight of

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“…In vitro assays of prolyl and lysyl hydroxylase activity in crude lysates of normal and mutant cells require the presence of oxygen, ca-ketoglutarate, ascorbic acid, and ferrous ion for the hydroxylation reactions to occur at optimal rates. Thus the enzymes in human skin fibroblasts have characteristics in common with prolyl hydroxylases from human and rat liver (4,5), newborn rat skin (6), chick embryos (7), and mouse L-929 fibroblasts (8,9), as well as lysyl hydroxylases from chick embryo (10,11) and fetal rat skin (12). [10][11][12][13][14][15][16][17][18][19][20].…”

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confidence: 99%