Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Harwood, Richard R.; Grant, Michael E.; Jackson, David S.

doi:10.1042/bj1420641

Cited by 15 publications

(14 citation statements)

References 60 publications

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“…An alternative mechanism for ascorbate action may involve the protein synthesis machinery itself. A shift in the distribution of collagen synthesizing ribosomes toward polysomes has been observed in the presence of ascorbate (38,39). Ultrastructural examination of normal and scorbutic tissues revealed a considerable reduction in rough endoplasmic reticulum in ascorbate deficiency, consistent with this effect of ascorbate (40).…”

Section: Methodssupporting

confidence: 58%

Regulation of collagen synthesis by ascorbic acid.

Murad¹,

Grove²,

Lindberg³

et al. 1981

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

396

221

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After prolonged exposure to ascorbate, collagen synthesis in cultured human skin fibroblasts increased approximately 8-fold with no significant change in synthesis ofnoncollagen protein. This effect of ascorbate appears to be unrelated to its cofactor function in collagen hydroxylation. The collagenous protein secreted in the absence of added ascorbate was normal in hydroxylysine but was mildly deficient in hydroxyproline. In parallel experiments, lysine hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) activity increased 3-fold in response to ascorbate administration whereas proline hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) activity decreased considerably. These results suggest that collagen polypeptide synthesis, posttranslational hydroxylations, and activities of the two bydroxylases are independently regulated by ascorbate.Ascorbic acid is essential for normal collagen formation (1-3) by virtue ofthe fact that it is a required component in the synthesis of hydroxyproline and hydroxylysine in collagen (4). Hydroxyproline serves to stabilize the collagen triple helix (5, 6); its absence results in structurally unstable collagen (7, 8) which is not secreted from cells at a normal rate (9). Hydroxylysine is necessary for formation of the intermolecular crosslinks in collagen (10). In addition, specific carbohydrate residues are linked glycosidically to collagen through hydroxylysine, a process that may be important in the regulation of crosslink formation (11).It is generally believed that ascorbate modulates collagen production through its effect on prolyl hydroxylation (12). There have been indications, however, that ascorbate may have an additional role in collagen biosynthesis (13-16). Notable are the early studies by Jeffrey and Martin (13) who observed a substantial increase in the size of chicken long bones cultured in the presence of ascorbate, concomitant with an increase in the incorporation of proline into peptidyl hydroxyproline.In this study we have examined the long-term effect of ascorbate on collagen production by cultured human skin fibroblasts. The influence ofascorbate on prolyl hydroxylase (prolylglycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) and lysyl hydroxylase (peptidyllysine, 2 oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) levels was also examined simultaneously to understand better the interrelationship of collagen synthesis and posttranslational hydroxylation. The data indicate that ascorbate increases collagen synthesis by acting at a level other than hydroxylation. MATERIALS AND METHODSHuman skin fibroblasts from a normal 3-day-old boy (GM 970) were obtained from the Institute for Medical Research (Camden, NJ) and grown to confluent density in Dulbecco's modified Eagle's medium buffered with 24 mM sodium bicarbonate and 25 mM Hepes and supplemented with 20% fetal calf serum (GIBCO) which had been inactivated for 30 min at 560C. Cultures were growth arrested for 90 hr in ...

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Section: Methodssupporting

confidence: 58%

Regulation of collagen synthesis by ascorbic acid.

Murad¹,

Grove²,

Lindberg³

et al. 1981

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

396

221

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“…The 14C in the fractions was assayed by taking 0.1 ml samples with 0.2ml of 2M-HCI in the solvent system for liquid-scintillation counting as described by Harwood et al (1974a). Hydroxy[14C]proline in the fractions was assayed by the method of Juva & Prockop (1966).…”

Section: Characterization Of [14cjprocollagen Polypeptidesmentioning

confidence: 99%

The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule

Harwood¹,

Merry²,

Woolley³

et al. 1977

Biochemical Journal

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1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.

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“…However, several modifications were necessary to obtain optimal results with the human tissues. Although most of the prolyl hydroxylase activity can be extracted from 12-day-old chick embryos with 0.01 M potassium chloride [6], the use of a higher salt concentration [29] and the addition of Triton X-100 [24,30,31] were found to be necessary for optimal extraction of the enzyme from human foetal tissues. The ammonium sulphate fractionation step [32] was also slightly modified to increase the recovery of enzymic activity.…”

Section: Purification Of Human Prolyl Hydroxylusementioning

confidence: 99%

Human Prolyl Hydroxylase. Purification, Partial Characterization and Preparation of Antiserum to the Enzyme

1975

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Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 pmol to 82.7 pmol hydroxyproline mg protein-' h-' at 37 "C with a saturating concentration of (Pro-Pro-Gly), as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61 000 and 64000 as determined by sodium dodecylsulphate -polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase.Antisera to human and chick prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human and chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments.

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Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Cited by 15 publications

References 60 publications

Regulation of collagen synthesis by ascorbic acid.

Regulation of collagen synthesis by ascorbic acid.

The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule

Human Prolyl Hydroxylase. Purification, Partial Characterization and Preparation of Antiserum to the Enzyme

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