| Abstrakt: |
Procollagen N-proteinase (EC 3.4.24.14), the enzyme that cleaves the NH2-terminal propeptides from type I procollagen, was purified over 20,000-fold with a yield of 12% from extracts of 17-day-old chick embryo tendons. The procedure involved precipitation with ammonium sulfate, adsorption on concanavalin A-Sepharose, and five additional column chromatographic steps. The purified enzyme was a neutral, Ca2+-dependent proteinase (5–10 mM) that was inhibited by metal chelators. It had a molecular mass of 500 kDa as determined by gel filtration. The enzyme contained unreduced polypeptides of 61, 120, 135, and 161 kDa that were separated by Polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The 135-and 161-kDa polypeptides were catalytically active after elution from the Polyacrylamide gel. Other properties of 500-kDa enzyme are: 1) the Kmfor type I procollagen is 54 nMat pH 7.5 and 35 °C, and the kcatis 350 h-1; 2) the activation energy for reaction with type I procollagen is 7,100 cal mol−1; 3) the isoelectric point is 3.6; and 4) the enzyme specifically cleaves the NH2-terminal propeptides of type I and II procollagen, but not of type III procollagen. |
