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D J Prockop

Publications and source records attributed to D J Prockop.

At least 325 records · Page 18Linked to original sources
PubMed1977

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

Fibroblasts isolated by enzymic digestion of chick embryo tendons were incubated for several hours in suspension under conditions in which they were in steady state in terms of the synthesis and secretion of procollagen. Under these conditions, the cells synthesized and secreted about 630 microgram of procollagen/10(9) cells/h. The cells were labeled with [14C]proline for 15 to 120 min and then the kinetics of secretion were followed by chasing the label and assaying the 14C-peptides digestible by collagenase in the cells and in the medium. The results demonstrated that secretion of collagenase-digestible peptides did not follow the kinetics of a single first order process but suggested at least two pseudo-first order process with half-times of 14 and 115 min. The [14C]procollagen secreted during 0 to 30 min and 90 to 120 min of chase was the same in terms of the ratio of pro-alpha1 to pro-alpha2 chains, the size of the pro-alpha chains, the extent of interchain disulfide bonding, the extent of prolyl hydroxylation, and the degree of helicity as tested by resistance to pepsin digestion. Addition of ascorbic acid to the incubation medium increased slightly the extent of prolyl hydroxylation but did not alter the kinetics of secretion. The results suggested that the kinetics of secretion are influenced by a two-compartment system in which at least one metabolic pool contributing to the secretory process is present as a "side pocket."

Animals↗
PubMed1977

Hydroxylation of (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 by prolyl hydroxylase. Evidence for an asymmetric active site in the enzyme.

Previous studies with 14C-labeled synthetic peptides demonstrated that prolyl hydroxylase, which synthesizes the hydroxyproline in collagen, preferentially hydroxylates the fourth triplet from the NH-terminal end of the peptide (Pro-Pro-Gly)5. In the experiments reported here, the prolyl hydroxylase reaction was investigated further by preparing chemically modified derivatives of (Pro-Pro-Gly)5 and by synthesizing 14C-labeled preparations of (Pro-Pro-Gly)10. Essentially, the same kcat value was found for the hydroxylation of (Pro-Pro-Gly)5, N-acetyl-(Pro-Pro-Gly)5, (Pro-Pro-Gly)5 methyl ester, (Pro-Pro-Gly)10, and for larger polypeptide substrates of the enzyme. It appeared therefore that preferential hydroxylation of specific triplets in peptides of the structure (Pro-Pro-Gly)n cannot be explained by differences in the kinetic constants for individual triplets. Hydroxylation of 14C-labeled preparations of (Pro-Pro-Gly)10 demonstrated that the ninth triplet was preferentially hydroxylated over any other triplet. The results were best explained by the hypothesis that prolyl hydroxylase has an asymmetric active site in which binding subsites are located adjacent to but not symmetrical with the catalytic subsite.

Amino Acid Sequence↗
PubMed1977

Synthesis of elastin in aortas from chick embryos. Conversion of newly secreted elastin to cross-linked elastin without apparent proteolysis of the molecule.

The biosynthesis of elastin was examined in matrix-free cells isolated by enzymic digestion of aortas from 17-day old chick embryos. After the cells were incubated with [14C]proline and then were rapidly boiled in buffer containing high concentrations of protease inhibitors and sodiumdodecyl sulfate, about one-quarter of the intracellular 14C-labeled protein was recovered as an elastin component with an apparent molecular weight of about 72 000. Examination of the medium from the cell suspension indicated that the largest elastin component secreted by the cells also had an apparent molecular weight of about 72 000. Pulse-chase experiments with intact aortas demonstrated that about two-thirds of the 72 000-dalton component disappeared in 2 h, apparently because it was converted to cross-linked fibers. When cross-linking was inhibited with penicillamine, the 72 000-dalton component persisted in the tissue 5 h. When cross-linking was inhibited with beta-aminopropionitrile, the elastin component of 72 000 daltons persisted for about 2 h, but thereafter it was gradually degraded to small peptides which were recovered in the incubation medium. The results suggest that elastin is secreted by cells in chick aorta as a polypeptide of about 72 000 daltons and that the secreted protein is incorporated into elastin fibers without cleavage to a protein of considerably smaller size.

Animals↗
PubMed1977

Collagen diseases and the biosynthesis of collagen.

Increasing knowledge concerning the molecular structure of collagen and the steps involved in its biosynthesis provides the basis for a new look at the collagen diseases. Some, like the Ehlers-Danlos syndrome, are now known to be the expression of primary molecular defects; others, such as scurvy and scleroderma, appear to be secondary to some process that disrupts normal controls over collagen synthesis or deposition.

Amino Acid Sequence↗
PubMed1976

Effects of the stereo-configuration of the hydroxyl group in 4-hydroxyproline on the triple-helical structures formed by homogenous peptides resembling collagen.

To explore further the recent demonstration that hydroxyproline stabilizes the triple-helical structure of collagen, two peptides containing allohydroxyproline, (aHyp-Pro-Gly)10 and (Pro-aHyp-Gly)10, were synthesized by a modified Merrifield technique which yields products of defined molecular weight. Examination of the peptides by optical rotation and circular dichroism showed that neither of them formed triple-helical structures in aqueous solution. Since the peptides had less tendency than (Pro-Hyp-Gly)10 to become helical, the results demonstrated that the trans-4-hydroxyl group of hydroxyproline makes a specific contribution to stability of the triple helix formed by (Pro-Hyp-Gly)10. Since the peptides also had less tendency than (Pro-Pro-Gly10 to become helical, the results further demonstrated that the cis-4-hydroxyl group on allohydroxyproline decreases the stability of the triple helix. The observations provided direct support for previous data indicating that incorporation of proline analogues such as allohydroxyproline into pro-alpha chains during procollagen biosynthesis prevents the polypeptides from becoming triple helical.

Binding Sites↗
PubMed1976

Segment-long-spacing aggregates and isolation of COOH-terminal peptides from type I procollagen.

Type I procollagen secreted by matrix-free cells from chick embryo tendons was purified by DEAE-cellulose chromatography. Electron microscopy of segment-long-spacing aggregates of the procollagen demonstrated the presence of both NH2-terminal and COOH-terminal extensions not found in collagen. The procollagen was digested with bacterial collagenase and the COOH-terminal fragments were isolated by gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Analysis of tryptic peptides demonstrated that the COOH-terminal extensions on the pro alpha 1 and pro alpha 2 chains had different primary structures.

Amino Acid Sequence↗