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At least 991 records · Page 55Linked to original sources
PubMed1991

The kinetics of the hydroxylation of procollagen by prolyl 4-hydroxylase. Proposal for a processive mechanism of binding of the dimeric hydroxylating enzyme in relation to the high kcat/Km ratio and a conformational requirement for hydroxylation of -X-Pro-Gly- sequences.

Prolyl 4-hydroxylase modifies only approx. 5% of the hydroxylatable prolyl residues in procollagen at a relatively high rate, after which the rate of further hydroxylation rapidly decreases. This suggests that the probability to exist in a defined hydroxylation-committed conformation differs between the numerous -X-Pro-Gly- sequences in the substrate. The enzyme reaction is characterized by the unusually high kcat/Km ratio of 3 x 10(9) M-1 s-1. To explain these kinetic features, an extremely high second-order rate constant for the association of enzyme and the subset of rapidly hydroxylated prolyl residues has to be assumed. A two-step mechanism is proposed in which diffusional constraints on the rate of association of prolyl 4-hydroxylase with hydroxylatable prolyl residues can be overcome. Upon encountering a random coil pro-alpha chain, the dimeric enzyme is first 'aspecifically' bound, followed by rapid transfers between different segments of the flexible peptide substrate via fast transitions between 'aspecific' single and double bound intermediate states. The rate of the second step, the productive (specific) binding of hydroxylation-committed -X-Pro-Gly- sequence to the active site, can be enhanced significantly by such an, in essence, 'one-dimensional' search. This processive mechanisms of binding does not necessarily imply many hydroxylation reactions during one encounter between enzyme and a peptide with several substrate sites as suggested previously in a slightly different model (De Waal, A. and De Jong, L. (1988) Biochemistry 27, 150-155).

Amino Acid Sequence↗
PubMed1997

A recombinant homotrimer of type I procollagen that lacks the central two D-periods. The thermal stability of the triple helix is decreased by 2 to 4 degrees C.

A D-period cassette system was developed that can be used to synthesize a variety of recombinant homotrimers of type I procollagen. A construct lacking the central two D-periods of pro alpha 1(I) chains was assembled and expressed as a recombinant protein in the mammalian cell line. The recombinant protein was purified to homogeneity and the thermal stability of the triple helix assayed by rapid protease digestion. The results indicated that deletion of the central 468 amino acids from the major triple helix lowered the thermal stability of the protein by 2 to 4 degrees C. The results therefore begin to define regions of the molecule that vary in their contributions to helical stability.

Bone Morphogenetic Protein 1↗
PubMed1982

Hydroxylation of prolyl residues in type II procollagen in vitro and in cellulo. Lack of preferential hydroxylation of specific regions of the protein.

[14C]Proline-labeled protocollagen, the unhydroxylated form of procollagen, was isolated from cartilage cells incubated with alpha, alpha'-dipyridyl. For examination of the initial steps in the hydroxylation of the protein, it was incubated in vitro with prolyl hydroxylase so that an average of 1.3-2.7 prolyl residues per chain was hydroxylated. The partially hydroxylated alpha chain were cleaved with cyanogen bromide, and the fragments were separated by polyacrylamide gel electrophoresis or column chromatography. The cyanogen bromide fragments were hydroxylated to the same degree. The results indicated, therefore, that in the initial hydroxylation of alpha chains in vitro, there was no preferential hydroxylation of any specific regions of the protein. In a second series of experiments, cartilage cells were incubated with [14C]proline and alpha, alpha'-dipyridyl so that prolyl hydroxylase in the cells was extensively, but not completely, inhibited. Partially hydroxylated alpha chains were isolated, and cyanogen bromide fragments of the alpha chains from the cells were assayed for hydroxy[14C]proline. The alpha chains contained an average of two residues of hydroxyproline per chain, and the cyanogen bromide fragments were hydroxylated to about the same degree. The results indicated, therefore, that when prolyl hydroxylase activity in cells is low relative to the rate at which pro alpha chains are synthesized, hydroxylation of prolyl residues occurs as it does in vitro, and there is no preferential hydroxylation of a specific region of the protein.

Animals↗
PubMed1981

Effect of prevention of procollagen triple-helix formation on proline 3-hydroxylation in freshly isolated chick-embryo tendon cells.

Inhibition of procollagen triple-helix formation by the addition of cis-hydroxyproline or azetidine-2-carboxylic acid increased the synthesis of 3-hydroxy[14C]proline 1.7-1.8-fold in pulse-chase experiments with freshly isolated chick-embryo tendon cells. The amount of 3-hydroxy[14C]proline, expressed as a percentage of the total 14C radioactivity in hydroxyproline, reached 8.4%. Control experiments indicated that the two analogues had no effect on the prolyl 3-hydroxylase activity of these cells. The data suggest that the time available before triple-helix formation in part regulates the extent of the 3-hydroxylation of proline in the biosynthesis of collagen in intact cells.

Animals↗
PubMed1979

Beta-turns in nascent procollagen are sites of posttranslational enzymatic hydroxylation of proline.

The selective hydroxylation of proline residues in nascent procollagen chains by prolyl hydroxylase (EC 1.14.11.2) can be understood in terms of the conformational feature of the -Pro-Gly-segments in linear peptides and globular proteins. The folded beta-turn conformation in such segments appears to be the conformational requirement for proline hydroxylation. The available data on the hydroxylation of native and synthetic substrates of prolyl hydroxylase are explained on the basis of the extent of beta-turn formation in them. Taken in conjunction with the conformational features of the hydroxyproline residue, our results bring out the conformational reason for the posttranslational proline hydroxylation which, it is proposed, leads to the "straightening" of the beta-turn segments into the linear triple-helical conformation.

Hydroxyproline↗
PubMed1996

Serum amino-terminal propeptide of type III procollagen levels in chronic pancreatitis.

The severity of pancreatic fibrosis, a characteristic feature of patients with chronic pancreatitis (CP), can be assessed only by direct histologic analysis of pancreatic tissue. Since serum levels of the amino-terminal type III procollagen propeptide (PIIIP) can reflect the degree of fibrogenic activity in several diseases associated with fibrosis, the current study was aimed at investigating whether PIIIP are increased in chronic pancreatitis, the relationship between PIIIP and pancreatic fibrogenic activity, and the influence of pancreatectomy, pancreatic exocrine function, and duration of disease on PIIIP levels. Serum PIIIP was measured in 18 patients with CP (15 without liver disease and three with cholestasis) and in 21 healthy controls. The effect of pancreatectomy on PIIIP was evaluated in seven patients, in whom PIIIP was measured immediately before and 2 months after surgery. Prolylhydroxylase (PHase) activity as an index of pancreatic fibrogenesis was evaluated in pancreatic tissue from 11 patients who had undergone subtotal pancreatectomy and from 11 organ donors. The bentiromide (BT)-PABA test as an index of exocrine pancreatic function was measured in all patients. PIIIP was significantly higher in patients who had or had not undergone pancreatectomy (17.3 +/- 4.0 and 25 +/- 11.4 ng/ml, respectively) than in controls (12.3 +/- 3.1 ng/ml) (p < 0.001). PIIIP decreased significantly after pancreatectomy (before, 32.0 +/- 9.3 ng/ml; after, 18.4 +/- 4.8 ng/ml; p = 0.005). PHase was significantly higher in patients (773 +/- 250 cpm/mg protein) than in controls (405 +/- 121 cpm/mg protein) (p < 0.001). PIIIP was correlated with pancreatic PHase (r = 0.7, p = 0.001) but not with BT-PABA or with the duration of the disease. In conclusion, serum PIIIP levels are increased in patients with CP and reflect the severity of pancreatic fibrogenic activity. No relationship between the serum PIIIP levels and the pancreatic exocrine function and duration of disease was found.

Adult↗
PubMed1989

Prolyl hydroxylase and procollagen III peptide levels in the sera from patients with collagen diseases and psoriasis.

Prolyl hydroxylase (PH) and procollagen III peptide (PIIIP) levels in 43 serum samples from various collagen diseases including 19 progressive systemic sclerosis (PSS) cases were simultaneously assayed and compared to those of 22 age-matched psoriasis patients and 20 healthy controls in order to examine whether they can serve as useful parameters of fibrotic changes, especially in the skin. Serum PH levels were determined with enzyme immunoassay and serum PIIIP levels with radioimmunoassay. The average values of PH were 109% (n = 3, (p less than 0.001; generalized morphea, GM), 104% (n = 1; overlap syndrome, OS), 57% (n = 6; mixed connective tissue disease, MCTD), 48% (p less than 0.05; PSS), 33% (n = 5; dermatomyositis, DM), 28% (psoriasis), and 9% (n = 9; systemic lupus erythematosus, SLE), higher than the control levels, while those of PIIIP were 82% (OS), 57% (MCTD), 42% (p less than 0.01; PSS), 11% (GM), and 5% (DM) higher and 22% (psoriasis) and 28% (SLE), lower than the controls. These results indicate that both values were higher in the diseases with skin fibrosis and lower in those without it, suggesting that both assays generally reflect the state of actual progression of fibrosis at the point of determination. These two values had considerable correlation; however, GM, psoriasis and SLE patients had relatively higher values of PH than of PIIIP. This result suggests that the PH assay may also reflect the state of connective tissue activation; therefore the PIIIP assay may be more specific for pathological fibrotic process.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1996

Serum markers for hepatic fibrosis in alcoholic liver disease: which is the best marker, type III procollagen, type IV collagen, laminin, tissue inhibitor of metalloproteinase, or prolyl hydroxylase?

Although various serum markers for the evaluation of hepatic fibrosis have been introduced, it remains unclear which is the best marker to evaluate the hepatic fibrosis observed in alcoholic liver disease (ALD). In this study, we measured serum concentrations of the immunoreactive beta-subunit of prolyl hydroxylase, procollagen type III peptide, the 7S domain (7S-IV) and triple-helix domain (TH-IV) of type IV collagen, laminin, and tissue inhibitor of metalloproteinase (TIMP) in patients with and without ALD (non-ALD), and controls to evaluate the best serum marker reflecting the characteristic histologic features of ALD. After Azan-Mallory and silver-impregnated reticulin staining, histologic specimens were examined; and the degree of hepatic fibrosis was classified as mild, moderate, or severe. Although serum concentrations of all markers, except for TIMP, in patients with each type and stage of liver disease were higher than cut-off values and these concentrations increases with the progression of liver disease, statistical analyses indicate that serum TH-IV concentration is the best marker to distinguish ALD from non-ALD. A good correlation was also found between the hepatic type IV collagen content and serum TH-IV, but not serum 7S-IV concentration. Moreover, after abstinence from alcohol, serum concentrations of TH-IV decreased more quickly than other serum markers. These results clearly suggest that, compared with other markers, serum concentration of TH-IV may more strongly reflect the histologic features of ALD. However, other serum markers, except for TIMP, may be useful in evaluating the degree of hepatic fibrosis.

Alcohol Drinking↗
PubMed1992

[Type III procollagen N-terminal peptide (P-III-P, prolyl hydroxylase (PH), and laminin P1 levels in serum and BALF of radiotherapy patients].

The etiology of pulmonary fibrosis remains unclear, and at present there are no definite biochemical markers of its activity. We measured serum and BALF levels of type III procollagen N-terminal peptide (P-III-P), prolyl hydroxylase (PH), and laminin P1 in patients who had undergone radiotherapy for malignant neoplasms, and investigated their value as biochemical markers in a model of pulmonary fibrosis. The following results were obtained: 1) Patients with abnormal liver function had significantly higher serum P-III-P levels and showed a tendency to have higher serum PH levels. If P-III-P or PH are to be used as markers of pulmonary fibrosis, the effect of liver function must be taken into consideration; however, no significant difference was detected with respect to laminin P1 levels. 2) Serum P-III-P levels were significantly elevated by radiotherapy. 3) Laminin P1 levels rose in a similar manner to P-III-P levels after radiotherapy, but no significant change was detected. 4) In most cases, the levels of all markers in BALF were below the threshold of detection, nevertheless all three markers were elevated in a patient who developed diffuse radiation pneumonitis during radiotherapy. Increases in the lymphocyte count were found in BALF of this patient. 5) BALF hyaluronic acid levels were negative in the 3 cases assayed. 6) A significant correlation between P-III-P and laminin P1 in serum was shown, but no significant correlations could be found between the other combinations of markers in serum. Thus it appears that serum P-III-P and laminin P1 are valid biochemical markers of pulmonary fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchoalveolar Lavage Fluid↗
PubMed1992

A tripeptide deletion in the triple-helical domain of the pro alpha 1(I) chain of type I procollagen in a patient with lethal osteogenesis imperfecta does not alter cleavage of the molecule by N-proteinase.

Dermal fibroblasts from a fetus with perinatal lethal osteogenesis imperfecta synthesized normal and abnormal type I procollagen molecules. The abnormal molecules contained one or two pro alpha 1(I) chains in which glycine, alanine, and hydroxyproline at positions 874, 875, and 876 in the triple-helical region were deleted as the result of a 9-base pair genomic deletion. Molecules that contained abnormal chains were overmodified from the site of the deletion toward the amino-terminal region of the molecule. Secretion of the overmodified molecules was impaired. The thermal stability of molecules containing abnormal chains was lower than that of normally modified molecules. After cleavage of molecules with vertebrate collagenase, the temperature of thermal denaturation of the overmodified A fragments was greater than that of the fragments from the normal molecules. The rates of cleavage of the normal and the abnormal molecules by N-proteinase were indistinguishable. Our findings suggest that the tripeptide deletion introduces a shift in the phase of the chains in the triple helix. This structural change is propagated from the site of the deletion toward the amino terminus of the molecule, but the subsequent alteration in the structure of the N-proteinase cleavage site is not sufficient to cause a decrease in the rate of cleavage by the enzyme.

Adult↗
PubMed1990

[Serum type III procollagen N-terminal peptide in patients with collagen diseases].

The serum concentration of type III procollagen N-terminal peptide (P III P) level is known to reflect the activity of collagen biosynthesis. To analyze the correlation between the disease activity and serum P III P levels in collagen diseases, serum P III P levels in patients with rheumatoid arthritis (RA), progressive systemic sclerosis (PSS), and other collagen diseases were measured. In some patients serum levels of prolyl++-hydroxylase, which is an intra-cellular enzyme of collagen biosynthesis, were measured. Serum P III P levels were elevated in patients with PSS and MCTD/overlap syndrome, suggesting a high rate of collagen biosynthesis by fibroblasts. Patients with RA showed no significant elevation of serum P III P compared with normal control group. But the group of RA patients with elevated ESR and/or serum CRP values showed high levels of serum P III P. The correlation between the disease activity and serum P III P levels was observed in RA patients with positive rheumatoid factor (RF), but not patients without RF. In addition we measured P III P levels in synovial fluid of RA and osteoarthritis patients. The levels were one to three hundred times higher than serum levels, and they showed the positive correlation with serum levels, suggesting that serum P III P might be originated from synovial P III P.

Adult↗
PubMed1986

Serum enzymes of collagen synthesis and type III procollagen aminopropeptide in Nigerian patients with sickle cell disease.

Serum immunoreactive prolyl hydroxylase protein (S-IRPH), galactosylhydroxylysyl glucosyltransferase activity (S-GGT), and the aminoterminal propeptide of type III procollagen (S-Pro(III)-N-P) were measured in 20 patients with sickle cell disease and the values were compared with those in 20 apparently healthy Nigerians. The means for the two enzymes and S-Pro(III)-N-P were significantly elevated in the sickle cell disease patients. Significant correlations were found between the values of the two enzymes and the protein (S-Pro(III)-N-P) within the sickle cell disease patients. The data confirm that collagen formation is found in the bone, liver, or other organs of patients with this disease. The measurement of S-GGT and S-Pro(III)-N-P in prospective studies might be helpful in predicting general and hepatic fibrogenesis in sickle cell disease.

Adolescent↗
PubMed1993

[Expression of type III and IV procollagen, prolyl 4-hydroxylase mRNAs in fibrotic human liver].

In situ localization of type III, IV procollagen and prolyl 4-hydroxylase mRNAs were examined to clarify the collagen production in fibrotic human liver. These mRNAs were localized in hepatocytes and mesenchymal cells in the areas of liver fibrosis. In alcoholic liver disease, the expression was promoted, and positive cells were diffusely observed in nodules. These results suggest that hepatocytes along with mesenchymal cells produce collagens, and correlate with pericellular fibrosis and sinusoidal capillarization in alcoholic liver disease.

Gene Expression↗
PubMed1987

Ehlers-Danlos syndrome. A variant characterized by the deficiency of pro alpha 2 chain of type I procollagen.

The clinical findings and biochemical studies are described for a 30-year-old man with Ehlers-Danlos syndrome. Type analysis of collagen produced by cultured fibroblasts revealed the lack of a detectable pro alpha 2 chain of type I procollagen. The intracellular degradation rate of newly synthesized collagen was higher than that of normal cells, resulting in the reduction of net collagen production. Clinical manifestations characterized by cardiovascular abnormalities due to decreased collagen deposition in the aortic valve and the wall of aorta, hypermobility of the joints, and hyperextensibility of the skin in this patient may be closely related to the observations described above.

Adult↗
PubMed1987

Scleredema and paraproteinemia. Enhanced collagen production and elevated type I procollagen messenger RNA level in fibroblasts grown from cultures from the fibrotic skin of a patient.

An edematous rash developed on the abdominal skin of a 76-year-old woman who had had diabetes mellitus for ten years. Some months later, the affected skin became thickened and indurated. Histopathologic examination revealed marked dermal fibrosis with excessive deposition of collagen. The patient also had IgA (k-type) paraproteinemia. Fibroblast cultures from the affected and unaffected skin were studied for collagen metabolism. Procollagen synthesis was elevated about sixfold on fibroblasts derived from the affected skin. A similar increase was detected in messenger RNA (mRNA) levels using a complementary DNA clone specific for human pro alpha 1(l) collagen mRNA. The elevated mRNA level could be the result of increased transcriptional activity of collagen genes or decreased degradation of collagen mRNAs. Our findings suggest that increased collagen deposition may account for the marked dermal fibrosis that we observed in this patient.

Aged↗
PubMed1992

Procollagen II gene mutation in Stickler syndrome.

Four affected members of a family with Stickler syndrome were found to have a single base-pair deletion resulting in a translational frameshift in exon 40 of the procollagen II (COL2A1) gene on chromosome 12. This mutation was not seen in any of five clinically unaffected family members or in any of 15 unrelated control patients. All affected members had distinctly abnormal vitreous syneresis and all had retinal perivascular pigmentation. Retinal detachments occurred in three of the four affected patients. Three of the four affected patients had peripheral cortical "wedge" cataracts, and the fourth had extensive nuclear sclerosis. Abnormalities of the soft palate were found in all four affected patients. All patients reported severe joint pains, and epiphyseal dysplasia was found radiographically in all patients.

Adolescent↗
PubMed1995

Stickler syndrome. A mutation in the nonhelical 3' end of type II procollagen gene.

BACKGROUND: All of the mutations in the type II procollagen (COL2A1) gene that have been identified in families affected with Stickler syndrome have been located primarily in the triple helical region of the gene. We report what we believe is the first premature stop codon in the globular C-propeptide region encoded by the COL2A1 gene, in a family affected with Stickler syndrome. DESIGN: Genomic DNA from affected and unaffected family members of this three-generation family was amplified using the polymerase chain reaction. The polymerase chain reaction products were directly sequenced for DNA analysis. RESULTS: Direct sequencing showed a single base deletion in exon 50, resulting in a premature stop codon in exon 51 in the globular C-propeptide of COL2A1 gene in all affected members. CONCLUSIONS: These results implicate premature stop codons as a common cause of Stickler syndrome. The location of this premature stop codon in the far end of the nonhelical 3' end of the gene indicates that a truncated C-propeptide of at least 84 amino acid residues is inadequate for the functional gene product.

Adolescent↗
PubMed1987

Growth and phosphorus metabolism in premature infants fed human milk, fortified human milk, or special premature formula. Use of serum procollagen as a marker of growth.

Human milk promotes less than optimal growth and is associated with phosphorus deficiency and decreased bone mineralization in very-low-birth-weight (VLBW) infants. In this study, the effects of feeding premature infants either human milk (HM), fortified human milk (FHM), or special premature formula (Similac Special Care [SSC]) on growth, phosphorus metabolism, and serum type I procollagen (pColl-I-C) were evaluated. Infants fed FHM exhibited a rate of weight gain and an increase in head circumference comparable with infants fed SSC and significantly greater than infants fed HM, despite the fact that both the FHM group and the HM group demonstrated biochemical evidence of phosphorus deficiency. The pColl-I-C concentrations in VLBW infants were tenfold to 20-fold greater than concentrations in normal children older than 2 years of age. The pColl-I-C levels correlated positively with weight gain and were significantly greater in the FHM and SSC groups than in the HM group. By contrast, serum alkaline phosphatase levels did not correlate with weight gain and were significantly lower in the rapidly growing SSC group than in either of the two groups with phosphorus deficiency and presumed poor bone mineralization. We conclude that the serum pColl-I-C concentration is a biochemical marker of growth in VLBW infants and may prove useful as a predictor of growth responses to various nutritional and therapeutic interventions.

Blood Proteins↗