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D J Prockop

Publications and source records attributed to D J Prockop.

At least 289 records · Page 16Linked to original sources
PubMed1982

Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases.

A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.

Animals↗
PubMed1982

Isolation and partial characterization of the amino-terminal propeptide of type II procollagen from chick embryos.

The amino-terminal propeptide from type II procollagen was isolated from organ cultures of sternal cartilages from 17-day-old chick embryos. The procedure provided the first isolation of the propeptide in amounts adequate for chemical characterization. The propeptide had an apparent molecular weight of 18000 as estimated by gel electrophoresis in sodium dodecyl sulfate. It contained a collagen-like domain as demonstrated by its amino acid composition, circular dichroism spectrum, and susceptibility to bacterial collagenase. One residue of hydroxylysine was present, the first time this amino acid has been detected in a propeptide. The peptide contained no methionine and only two residues of half-cystine. Antibodies were prepared to the propeptide and were used to establish its identity. The antibodies precipitated type II procollagen but did not precipitate type II procollagen from which the amino and carboxy propeptides were removed with pepsin. Also, they did not precipitate the carboxy propeptide of type II procollagen. The data demonstrated th at the type II amino propeptide was similar to the amino propeptides of type I and type III procollagens in that it contained a collagen-like domain. It differed, however, in that it lacked a globular domain as large as the globular domain of 77-86 residues found at the amino-terminal ends of the pro alpha 1 chains of type I and type III procollagens.

Amino Acids↗
PubMed1982

Partial purification of a procollagen C-proteinase. Inhibition by synthetic peptides and sequential cleavage of type I procollagen.

A procollagen C-proteinase which cleaves the C-propeptides from type I procollagen was purified about 125-fold from membranous bones of chick embryos. As estimated by gel filtration, the enzyme was about 80 000 daltons. When a reaction with modified procollagen was carried out, the enzyme preferentially cleaved the C-propeptides from the pro alpha chains in the order pro alpha 1, pro alpha 1, and then pro alpha 2. The enzyme was inhibited by several metal chelators and high concentrations of dithiothreitol. It was also inhibited by 5% fetal calf serum. A series of inhibitors of serine proteinases and sulfhydryl-containing proteinases were not inhibitory. Four oligopeptides were synthesized with amino acid sequences similar to the amino acid sequences around the sites at which the C-propeptides are cleaved during the conversion of procollagen top collagen in vivo. The peptide Tyr-Tyr-Arg-Ala-Asp-Asp-Ala inhibited the enzyme 35--60% in concentrations of 6--12 mM. Shorter peptides containing the Ala-Asp bond cleaved by the enzyme were less effective. The partially purified enzyme was also found to cleave the C-propeptides from type II and type III procollagens.

Animals↗
PubMed1982

Separation of human pro alpha 1 (I) and pro alpha 2 (I) procollagen chains by reverse phase high performance liquid chromatography.

Human pro alpha 1 (I) and pro alpha 2 (I) procollagen chains have been separated on a large por (33 nm) reverse phase high performance liquid chromatography C18 column using an acetonitrile aqueous gradient containing 9 mM trifluoroacetic acid. The separation of the chains was evaluated by gel electrophoresis. The yield of the chromatography, based on radiolabel recovery, is equal to or better than 80%. Absorbance monitoring at 210 nm permits detection of less than 5 micrograms procollagen.

Chromatography, High Pressure Liquid↗
PubMed1982

Human type I procollagen genes are located on different chromosomes.

A recombinant plasmid containing sequences complementary to human pro-alpha l(I) collagen mRNA was used for the chromosomal assignment of the pro-alpha l(I) collagen gene. Restriction endonuclease analysis of DNA from mouse-human and Chinese hamster-human somatic cell hybrids revealed cosegregation with human chromosome 17. Hybrids containing derivative chromosomes with a t(2;17)(q14;q21) translocation showed cosegregation of the pro-alpha l(I) gene with the segment 17q21 leads to qter. In situ hybridization on human metaphasic chromosomes confirmed this conclusion.

Amino Acid Sequence↗
PubMed1982

How does a skin fibroblast make type I collagen fibers?

We now have an impressive amount of information about several steps in the synthesis of type I collagen fibers by skin fibroblasts: (a) the transcription of the genes for pro alpha 1(I) and pro alpha 2(I) polypeptide chains; (b) the processing of the initial RNA transcripts of the genes to mature mRNAs; (c) translation of the mRNAs to prepro alpha 1(I) and prepro alpha 2(I) chains; (d) processing of the prepro alpha 1(I) and prepro alpha 2(I) chains to procollagen; and (e) the further processing of procollagen in collagen fibers. The speed with which we are currently developing this information about collagen and comparable information about other constituents of skin suggests that skin may well be the first human organ which we fully understand.

Chemical Phenomena↗
PubMed1982

Procollagen N-proteinase. Properties of the enzyme purified from chick embryo tendons.

Procollagen N-proteinase was purified about 3700-fold from chick embryo tendons. Electrophoresis of the protein after iodination and denaturation suggested it was homogeneous. However, the native enzyme could not be examined by gel electrophoresis, and therefore homogeneity of the preparation was not conclusively established. Antibodies to the enzyme completely inhibited activity and gave a single precipitant line by double immuno-diffusion. The Km for a native procollagen substrate was 0.3-0.5 microM. The same protein after denaturation inhibited activity. The enzyme did not cleave type III procollagen from human fibroblasts or a type IV procollagen from a mouse sarcoma. Ca2+ was required for maximal enzymic activity. The data suggested a second metal requirement, but this was not identified. Reducing agents and metal chelators inhibited activity, but there was little if any inhibition from several inhibitors of other neutral metalloproteinases.

Animals↗
PubMed1982

beta-Aminopropionitrile prevents bleomycin-induced pulmonary fibrosis in the hamster.

beta-Aminopropionitrile (beta APN), an agent that prevents collagen accumulation in tissues, was evaluated for its ability to prevent excess collagen formation in bleomycin-induced pulmonary fibrosis in the hamster. Two groups of animals received a single endotracheal dose of bleomycin; one of these was injected with beta APN twice daily for 30 days. A third group received endotracheal saline and a fourth group received saline and beta APN. After 30 days, we measured pressure-volume curves of saline-filled lungs, collagen content, and degree of fibrosis. Endotracheal bleomycin increased collagen content, decreased lung volume, and produced fibrosis and a mortality rate of 51%. The administration of beta APN to bleomycin-treated animals prevented excess collagen accumulation and diminished total protein, reversed volume diminution, produced less fibrosis, and improved the mortality rate to 24%; beta APN alone had no effect on lung mechanics or collagen content. The biochemical, functional, and structural features of bleomycin-induced lung fibrosis are amenable to control with beta APN.

Aminopropionitrile↗
PubMed1982

Kinetics of the incorporation of tropoelastin into elastic fibers in embryonic chick aorta.

Matrix-free cells isolated by enzymic digestion of chick embryo aortas were labeled with [14C]proline for 20 to 60 min and the kinetics of the secretion of tropoelastin were followed by chasing the label and assaying [14C]tropoelastin in the cells and in the medium. The results indicated that secretion of tropoelastin followed the kinetics of a single first order process with a half time of 60 min. In parallel experiments tissue explants of chick embryo aortas were labeled with [14C]proline and the kinetics for the incorporation of tropoelastin into elastic fibers were followed by chasing the label and assaying the soluble [14C]tropoelastin and insoluble [14C]elastin in tissues. It was found that the incorporation of tropoelastin into elastic fibers also followed a single first order process with a half time of 85 min, similar to the secretion of tropoelastin from cells. In further studies, antibodies directed against tropoelastin were utilized to isolate soluble [14C]elastin components in the tissues after 0 to 4 hr chase of the 14C label. The results demonstrated that all soluble elastin components were recovered as monomeric tropoelastin and no soluble oligomeric elastin could be detected. These results are consistent with the proposition that elastic fiber growth occurs by addition of individual tropoelastin molecules to existing fibers and that oligomers of elastin were not intermediates in the process.

Animals↗
PubMed1982

Assignment of the human pro alpha 2(I) collagen structural gene (COLIA2) to chromosome 7 by molecular hybridization.

A cDNA for the pro alpha 2 chain of human type I collagen has been recently cloned and amplified. We have used this specific probe to identify the human chromosome carrying the pro alpha 2(I) collagen gene. The DNA from 17 independent human/hamster and human/mouse somatic cell hybrids was digested by Eco RI and the restriction pattern analyzed in Southern blot experiments, using the 32P-labeled cDNA as a hybridization probe. The gene coding for the pro alpha 2 collagen subunit could be unambiguously assigned to human chromosome 7. All the other chromosomes, including chromosome 17, were excluded.

Animals↗
PubMed1981

Formation of the triple helix of type I procollagen in cellulo. A kinetic model based on cis-trans isomerization of peptide bonds.

The kinetics of the triple helix formation of type I procollagen within chick embryo fibroblasts were measured in a pulse-chase experiment in which the appearance of fully-aligned triple-helical molecules was assayed by proteolytic digestion. Production of triple-helical molecules required 8-9 min after complete synthesis of the pro alpha chains, an observation which was inconsistent with helix formation being a co-translational process. The experimental data were in good agreement with the predictions derived from the following model: triple helix formation is initiated immediately after the completion of the synthesis of the polypeptide chains and after the formation of the interchain disulfide bonds within the C-propeptide; folding of the protein starts from a single nucleus located at one end of the three polypeptide chains, probably the carboxyl terminus, and propagates throughout the chain by a stepwise mechanism limited by the cis-trans isomerization of peptide bonds.

Animals↗
PubMed1981

Cloning a cDNA for the pro-alpha 2 chain of human type I collagen.

Poly(A)-RNA enriched for type I procollagen sequences was isolated from normal human fibroblasts and used as template to synthesize double-stranded cDNA with avian myeloblastosis virus (AMV) reverse transcriptase. After the ends had been blunted with nuclease S1 and dGMP tails had been added with terminal deoxynucleotidyltransferase, the double-stranded cDNA was annealed with pBR322 DNA that had previously been cleaved with EcoRI, blunted with AMV reverse transcriptase, and dCMP-tailed with terminal deoxynucleotidyltransferase. The chimeric molecule was used to transform Escherichia coli strain HB101. Ninety-five recombinant clones were obtained and screened by dot hybridization analysis using 32P-labeled cDNA synthesized from the original poly(A)-RNA collagen-enriched population. Three positive clones were isolated and further characterized by blot hybridization techniques and by EcoRII digestion. One clone with an insert of 2.2 kilobases was shown to contain sequences encoding for the pro-alpha 2 chain of human type I procollagen. DNA sequence analysis of a 172-nucleotide fragment demonstrated that the cloned cDNA extends from amino acid position 450 of the alpha 2 chain to the middle of the COOH-terminal propeptide.

Base Sequence↗