Type I procollagen carboxyl-terminal proteinase from chick embryo tendons. Purification and characterization.
Procollagen carboxyl-terminal proteinase, the enzyme which cleaves the carboxyl-terminal propeptides from type I procollagen, was extensively purified in a yield of 25% from pooled culture media of 17-day-old chick embryo tendons using a procedure which involved chromatography on Green A Dye matrix gel, concanavalin A-Sepharose and heparin-Sepharose, and filtration gels of Sephacryl S-300 and S-200. The purified enzyme is a neutral, Ca2+-dependent proteinase which is inhibited by metal chelators, but not by inhibitors for serine and cysteine proteinases. Calcium in a concentration of 5-10 mM is required for optimal activity. The molecular weight of the enzyme was determined to be 97,000-110,000 by gel filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Other properties of the carboxyl-terminal proteinase are: 1) the Km for the type I procollagen is 96 nM at pH 7.5 and 35 degrees C; 2) the activation energy for the reaction with type I procollagen is 21,000 cal mol-1; 3) amino acid sequencing of the released carboxyl-terminal propeptide indicated the enzyme specifically cleaves an -Ala-Asp- bond in both the pro-alpha 1(I) and pro-alpha 2(I) chains; 4) the enzyme specifically cleaves the carboxyl-terminal propeptides of a homotrimer of pro-alpha 1(I) chains and type II and III procollagens, but it does not cleave type IV procollagen. The results suggest that the enzyme is involved in the processing of type I procollagen in vivo.
D000595:Amino Acid Sequence; D000818:Animals; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D002118:Calcium / Q000378:metabolism; D002642:Chick Embryo; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases; D008970:Molecular Weight; D011347:Procollagen / Q000378:metabolism; D013710:Tendons / Q000196:embryology / Q000201:enzymology*
D019485:Bone Morphogenetic Proteins; D011347:Procollagen; D010450:Endopeptidases; D008666:Metalloendopeptidases; D055395:Bone Morphogenetic Protein 1; D002118:Calcium