Type I procollagen N-proteinase from chick embryo tendons. Purification of a new 500-kDa form of the enzyme and identification of the catalytically active polypeptides.
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 Km for type I procollagen is 54 nM at pH 7.5 and 35 degrees C, and the kappa cat is 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. A minor form of N-proteinase with a 300-kDa mass was also purified and was found to contain a 90-kDa polypeptide as the major active polypeptide. The enzyme appeared to be a degraded form of the 500-kDa N-proteinase. The properties of the 300-kDa enzyme were similar to those observed for the 500-kDa enzyme.
Hojima
Y
Y
Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
McKenzie
J A
JA
van der Rest
M
M
Prockop
D J
DJ
D000645:Ammonium Sulfate; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D011232:Chemical Precipitation; D002642:Chick Embryo; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D011347:Procollagen / Q000378:metabolism; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013710:Tendons / Q000196:embryology / Q000201:enzymology*; D013816:Thermodynamics
D002614:Chelating Agents; D011347:Procollagen; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010450:Endopeptidases; D011348:Procollagen N-Endopeptidase; D000645:Ammonium Sulfate; D002118:Calcium