Cofactor and substrate requirements of collagen proline hydroxylase

Hutton, John J.; Tappel, Al L.; Udenfriend, Sidney

doi:10.1016/0003-9861(67)90302-5

Cited by 301 publications

(94 citation statements)

References 27 publications

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“…With purified enzyme the requirement of a reducing agent for the hydroxylation of peptidyl proline is best fulfilled by ascorbate (15,16). The obvious assumption is that ascorbate in vivo serves as a cofactor in a manner analogous to its in vitro function.…”

Section: Resultsmentioning

confidence: 99%

“…Stimulation of prolyl hydroxylation by ascorbate in vivo has heretofore been attributed solely to its functioning as an electron donor in the hydroxylation process itself, since the in vitro requirement for a reducing agent is best fulfilled by ascorbate (15,16). However, it appears that ascorbate plays another role in collagen biosynthesis by stimulating an activation system resulting in the conversion of an inactive precursor of prolyl hydroxylase to active enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…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 unaffected.Stimulation of prolyl hydroxylation by ascorbate in vivo has heretofore been attributed solely to its functioning as an electron donor in the hydroxylation process itself, since the in vitro requirement for a reducing agent is best fulfilled by ascorbate (15,16). However, it appears that ascorbate plays another role in collagen biosynthesis by stimulating an activation system resulting in the conversion of an inactive precursor of prolyl hydroxylase to active enzyme.…”

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

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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“…Ascorbic acid is an important tion can occur, as it has been shown that LO acts on collagen after it assembles into fibrils (41). Because of component of our culture medium that stimulates collagen production (5,16,20). Although copper can reduce this, we elected to grow vessels in 0.2 µg Cu 2+ /ml medium for 4 weeks to allow assembly of collagen molethe half-life of ascorbic acid (40), engineered vessel collagen contents remained consistent with copper ion concules into fibrils, followed by 0.4 µg Cu 2+ /ml medium for the remaining 3 weeks of culture to increase LO accentrations of 0.2-0.6 µg/ml.…”

Section: Discussionmentioning

confidence: 99%

Effects of Copper and Cross-Linking on the Extracellular Matrix of Tissue-Engineered Arteries

2005

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In many cases, the mechanical strengths of tissue-engineered arteries do not match the mechanical strengths of native arteries. Ultimate arterial strength is primarily dictated by collagen in the extracellular matrix, but collagen in engineered arteries is not as dense, as organized, or as mature as collagen in native arteries. One step in the maturation process of collagen is the formation of hydroxylysyl pyridinoline (HP) cross-links between and within collagen molecules. HP cross-link formation, which is triggered by the copper-activated enzyme lysyl oxidase, greatly increases collagen fibril stability and enhances tissue strength. Increased crosslink formation, in addition to increased collagen production, may yield a stronger engineered tissue. In this article, the effect of increasing culture medium copper ion concentration on engineered arterial tissue composition and mechanics was investigated. Engineered vessels grown in low copper ion concentrations for the first 4 weeks of culture, followed by higher copper ion concentrations for the last 3 weeks of culture, had significantly elevated levels of cross-link formation compared to those grown in low copper ion concentrations. In contrast, vessels grown in high copper ion concentrations throughout culture failed to develop higher collagen cross-link densities than those grown in low copper ion concentrations. Although the additional cross-linking of collagen in engineered vessels may provide collagen fibril stability and resistance to proteolysis, it failed to enhance global tissue strength.

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“…The prolyl and lysyl hydroxylases that catalyze this reaction depend on the substrate oxygen [49]. The Km for O 2 of prolyl hydroxylase has been variously estimated at 20, 25, and 100 mmHg [50][51][52]. Even using the most conservative estimate, proline hydroxylation of collagen will be PO 2 -dependent through the range of 0 to at least 200 mmHg, with 90% of the effect occurring by 90 mmHg.…”

Section: Supplemental Oxygen Tissue Oxygenationmentioning

confidence: 99%

Non-pharmacologic Prevention of Surgical Wound Infection

Sessler

2006

Anesthesiology Clinics of North America

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Wound infections are serious and relatively common postoperative complications. They are generally detected five to nine days after surgery and are usually attributed, even by surgeons, to poor surgical technique or failure to maintain sterility. However, it has been known for decades that all wounds become contaminated, often by bacteria from the skin or within the patient, and that it is host defense mechanisms that prevent most contamination from developing into clinical infections. Host defense is especially important during the initial hours following contamination, i.e., the immediate perioperative period.As might thus be expected, factors that improve host defense reduce infection risk. Many of these are under the direct control of anesthesiologists and are at least as important as appropriate use of prophylactic antibiotics, which halve infection risk [1]. This article will review nonpharmacologic methods of reducing infection risk, with special emphasis on methods available to anesthesiologists.

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Cofactor and substrate requirements of collagen proline hydroxylase

Cited by 301 publications

References 27 publications

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

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

Effects of Copper and Cross-Linking on the Extracellular Matrix of Tissue-Engineered Arteries

Non-pharmacologic Prevention of Surgical Wound Infection

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