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D J Prockop

Publications and source records attributed to D J Prockop.

At least 307 records · Page 17Linked to original sources
PubMed1981

Comparison of the NH2-Terminal sequences of chick Type I preprocollagen chains synthesized in an nRNA-dependent reticulocyte lysate.

RNA was extracted from 17-day-old chick embryo calvaria and translated by an mRNA-dependent reticulocyte lysate. Procedures were developed for purifying intact pro alpha 1(I) and pro alpha 2 translation products from the lysate. These products were identified by comparing tryptic peptide elution patterns of the translation products with those obtained from secreted pro alpha chains. Partial sequence data from the amino terminus of each translation product demonstrated that each chain begins with a sequence that is different from that of the corresponding pro alpha chain, and that the two sequences are not the same. Also, the bacterial collagenase-resistant peptide from the amino terminus of prepro alpha 1(I) had an apparent molecular weight which was 10 000 greater than the peptide from pro alpha 1(I).

Amino Acid Sequence↗
PubMed1981

Prevention of bleomycin-induced pulmonary fibrosis in the hamster by cis-4-hydroxy-l-proline.

We investigated the effect of the proline analogue cis-hydroxyproline on pulmonary fibrosis produced by intratracheal administration of bleomycin in hamsters. Two groups of animals received a single dose of bleomycin; one of these groups was also injected with cis-hydroxyproline twice daily for 30 days. Control animals received intratracheal saline. Lungs were examined on day 30. In the bleomycin group, the collagen content of lungs was 131% of the control value (p less than 0.05). Administration of cis-hydroxyproline decreased collagen accumulation to 114% of the control value (p less than 0.05). Pressure-volume curves were significantly shifted downward and to the right of control in the bleomycin group; this shift was partially prevented by cis-hydroxyproline. Morphometric studies showed that alveolar walls comprised 15% of the lung parenchyma in the control group and 19% in the bleomycin group (p less than 0.05). Administration of cis-hydroxyproline significantly decreased alveolar walls to 16% of the lung parenchyma (p less than 0.05). We conclude that cis-hydroxyproline partially prevents the chemical, structural, and functional changes of bleomycin-induced pulmonary fibrosis in the hamster.

Animals↗
PubMed1980

Evidence for a structural mutation of procollagen type I in a patient with the Ehlers-Danlos syndrome type VII.

We have found that the collagen from a patient with the Ehlers-Danlos syndrome type VII contained a polypeptide chain, pN alpha 2, not present in collagen prepared from normal tissue. Fibroblasts cultured from the patient's skin produced type I procollagen in which the NH2-terminal propeptide of pro alpha 2 was cleaved to about half of normal values by chick procollagen neutral protease which removes the NH2-terminal propeptides from procollagen (N-protease). The NH2-terminal propeptide on the pro alpha 2 chain of the patient's procollagen was also more resistant than procollagen from control fibroblasts to digestion by pepsin or alpha-chymotrypsin. assays for procollagen N-protease indicated that the patient's fibroblsts contained about the same level of enzymic activity as normal fibroblasts. These results suggest that the patient's fibroblasts synthesize both an abnormal pro alpha 2 chain and a normal pro alpha 2 chain. The abnormality probably consists of a structural mutation in or near the site at which procollagen N-protease cleaves the pro alpha 2 chain. The results presented here appear to provide the first example of a mutation in a structural gene for collagen. Since equal amounts of pN alpha 2 and alpha 2 are found in the protein in neutral salt extracts of the patient's tissue, as well as in newly synthesized collagen produced by cultured skin fibroblasts, and since both parents are phenotypically normal and express exclusively normal collagen chains, the patient is likely to be a sporadic heterozygote, arisen by new mutation, with one normal and one abnormal gene coding for pro alpha 2.

Cells, Cultured↗
PubMed1980

Inhibitors of procollagen N-protease. Synthetic peptides with sequences similar to the cleavage site in the pro alpha 1(I) chain.

A series of peptides was synthesized with amino acid sequences identical with the cleavage site at which the procollagen N-protease cleaves the N-terminal propeptide from the pro alpha 1 chain of type I procollagen. Peptides up to 11 residues in length did not serve as substrates for the enzyme, an observation consistent with the demonstration that the N-protease will not cleave denatured procollagen or dissociated pro alpha chains. Several of the peptides, however, served as effective inhibitors of the cleavage of procollagen. Comparison of the inhibitor activities of peptides of varying lengths suggested that the L-phenylalanine found three residues to the left of the cleavage site was important for inhibitor activity. This suggestion was confirmed by synthesis of analogues of inhibitory peptides in which L-phenylalanine was replaced by D-phenylalanine, tyrosine, lysine, aspartic acid, or glycine.

Animals↗
PubMed1980

Thermal stability of type I and type III procollagens from normal human fibroblasts and from a patient with osteogenesis imperfecta.

Type I and type III procollagens were isolated from the medium of human fibroblast cultures in amounts adequate for examination by circular dichroism. Type I procollagen had a spectrum similar to that of type I procollagen and collagen from chicken embryos. The human type III procollagen showed a red shift not seen in type III collagen from calf skin. The midpoint (tm) for the helix-to-coil transition for both human procollagens was 40 degrees C. the same tm values were obtained with type I and type III procollagens synthesized by fibroblasts from a patient with osteogenesis imperfecta. Type I procollagen synthesized by the patient's fibroblasts, however, tended to aggregate more readily than type I procollagen from normal human fibroblasts, apparently because of a structural alteration of the protein.

Cells, Cultured↗
PubMed1980

A defect in the structure of type I procollagen in a patient who had osteogenesis imperfecta: excess mannose in the COOH-terminal propeptide.

Fibroblasts from normal human subjects and from a patient who had osteogenesis imperfecta were incubated with [3H]mannose, and types I and III procollagens were isolated from the culture medium. The type I procollagen from the patient's fibroblasts contained 2-3 time more [3H]mannose than the type I procollagen from the normal fibroblasts. In contrast, there was no difference in the [3H]mannose content of the type III procollagen simultaneously synthesized and secreted by the same cells. Isolation of a collagenase-resistant peptide fragment from the type I procollagen showed that the excess mannose was located in the COOH-terminal propeptide of the protein. Radioimmunoassays of the medium and the cell layer showed that the type I procollagen synthesized by the patient's fibroblasts was secreted into the medium more slowly than the type I procollagen synthesized by normal fibroblasts. These results appear to provide evidence for an alteration in the structure of procollagen in osteogenesis imperfecta.

Cells, Cultured↗
PubMed1980

Folding mechanism of the triple helix in type-III collagen and type-III pN-collagen. Role of disulfide bridges and peptide bond isomerization.

The kinetics of triple-helix formation in type III pN-collagen, type III collagen and a quarter fragment of type III collagen was followed by optical rotation and circular dichroism. Kinetic intermediates were detected by trypsin digestion and polyacrylamide gel electrophoresis. The end products of refolding at 25 degrees C were identical to the native molecules according to their melting profiles, molecular weights and sedimentation behavior. Only at low temperatures (4-15 degrees C) were mismatched structures of lower stability formed. At 25 degrees C helix formation started exclusively at the set of three disulfide bridges which link the three chains at the carboxy-terminal end. The growth of the triple helix proceeds from this single nucleus at a rather uniform rate in a zipper-like fashion. This gives rise to zero-order kinetics over a large fraction of the conversion. Consequently the time of half conversion is proportional to the length of the molecule. From the appearance and disappearance of intermediates the growth of the triple helix could be observed directly. The rate of helix propagation is determined by the rate of cis leads to trans isomerization of peptide bonds. A model mechanism was devloped which quantitatively described the overall kinetics as well as the time course of the intermediates with a single set of parameters: the rate constant of cis leads to trans isomerization k = 0.015 s-1 and an average number of 30 tripeptide units in uninterrupted stretches of residues with all peptide bonds in trans configuration.

Animals↗
PubMed1980

Prevention of collagen deposition following pulmonary oxygen toxicity in the rat by cis-4-hydroxy-L-proline.

Exposure of rats to high oxygen tensions causes increased collagen content of lungs and alveolar enlargement in 3-6 wk. We tested whether cis-hydroxyproline, a proline analogue that inhibits collagen synthesis, could prevent the collagen accumulation and alveolar enlargement. Rats were exposed to hyperoxia for 60 h and then to room air and hyperoxia for alternate 24-h periods for 11.5 d. Treated oxygen-exposed rats received 200 mg/kg cis-hydroxyproline twice daily over the 14-d exposure period. Control rats breathed room air. Examination of lungs on day 14 showed collagen content of oxygen-exposed lungs to be 48% greater than control (P < 0.05). The collagen content of the treated oxygen-exposed lungs was -12% of control (NS). Total lung volume was 16% greater than control in oxygen-exposed rats (P < 0.05) and 8% greater than control in treated oxygen-exposed rats (NS). Morphometric studies showed alveolar size was greater than control in oxygen-exposed rats (188+/-11 [SE] vs. 143+/-6 mumul [P < 0.05]). Oxygen-exposed, treated rats had a mean alveolar volume of 150+/-7 mumul. Lung pressure-volume curves were significantly shifted to the left of control in the oxygen-exposed rats, whereas the curves of the oxygen-exposed, treated group were identical to control. These data suggest that cis-hydroxyproline prevented the accumulation of collagen in the lungs in pulmonary oxygen toxicity. In addition, there was apparent protection from airspace dilatation and decreased lung elasticity, suggesting that alveolar enlargement after oxygen toxicity is linked to the deposition in lung tissue of new connective tissue fibers.

Animals↗
PubMed1980

Demonstration by immunofluorescence that the same cells from chick embryo aortas synthesize elastin and collagen types I and III.

Cells were isolated from the aortas of 17-day old chick embryos and they were stained with fluorescent antibodies specific for Type I collagen, Type I procollagen, Type III collagen, elastin and prolyl hydroxylase. The results indicated that the same cells simultaneously synthesize Type I procollagen, Type III procollagen and elastin. The synthesis of procollagens, and the presence of prolyl hydroxylase, in the same cells which synthesize elastin may well explain why elastin contains hydroxy-proline.

Animals↗
PubMed1979

Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells.

Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes.

2,2'-Dipyridyl↗
PubMed1978

Partial purification and characterization of a neutral protease which cleaves the N-terminal propeptides from procollagen.

A rapid assay procedure was developed for cleavage of the N-terminal propeptides of procollagen. With the assay a neutral procollagen N-protease was purified about 300-fold from chick embryo tendon extract. The enzyme had an apparent molecular weight of 260 000 and a pH optimum of 7.4. Ca2+ was required for enzymic activity but this requirement was partially replaced by Mg2+ or Mn2+. The enzyme was bound to concanavalin A-agarose and therefore was presumably a glycoprotein. The N-propeptides released from type I procollagen were of about 23 000 and 11 000 daltons as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The partially purified enzyme was also found to cleave type II procollagen and the N-propeptide obtained was about 18 000 daltons. Heat denaturation of either type I or type II procollagen decreased the rate at which the proteins were cleaved by the N-protease.

Animals↗