Cleavage of type I procollagen by C- and N-proteinases is more rapid if the substrate is aggregated with dextran sulfate or polyethylene glycol.
The enzymes procollagen C- and N-proteinases specifically cleave carboxyl- and amino-terminal propeptides of procollagens. After cleavage of the propeptides, the resulting collagens self-assemble into fibrils. In most previous experiments with the enzymes, the substrate was monomeric type I procollagen. Here we have prepared aggregates of type I procollagen from chick embryo tendons by using 1 to 100 micrograms/ml of 500-kDa dextran sulfate or 3 to 5% (w/v) polyethylene glycol (M(r) 3350). Aggregation of the substrate with dextran sulfate increased its rate of cleavage by purified or crude C-proteinase from chick embryo tendons 10- to 15-fold. Aggregation of the substrate with 25 to 100 microgram/ml of dextran sulfate increased the rate of cleavage by purified N-proteinase about 4-fold. The rate of cleavage by crude N-proteinase was enhanced only about 2-fold, apparently because of partial precipitation of the enzyme by dextran sulfate. Using polyethylene glycol to aggregate the substrate increased the rate of cleavage by procollagen C-proteinases 5- to 20-fold. Aggregation with polyethylene glycol also increased the rate of cleavage by purified procollagen N-proteinases 2- to 5-fold. With crude N-proteinase, the rate of cleavage was increased only 1.5-fold. The results suggest that the rate of cleavage of the substrate by both enzymes is increased by the aggregation of the substrate itself by dextran sulfate or polyethylene glycol. The increased rates of cleavage seen after aggregation of substrate can be used to develop more sensitive assays for the enzymic activities.
Hojima
Y
Y
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Behta
B
B
Romanic
A M
AM
Prockop
D J
DJ
D000818:Animals; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D011232:Chemical Precipitation; D002642:Chick Embryo; D016264:Dextran Sulfate; D010450:Endopeptidases / Q000378:metabolism*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008666:Metalloendopeptidases; D011092:Polyethylene Glycols; D011347:Procollagen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase / Q000378:metabolism*; D013379:Substrate Specificity; D013710:Tendons / Q000378:metabolism
D019485:Bone Morphogenetic Proteins; D007202:Indicators and Reagents; D011347:Procollagen; D011092:Polyethylene Glycols; D016264:Dextran Sulfate; D010450:Endopeptidases; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D055395:Bone Morphogenetic Protein 1