Characterization of type I procollagen N-proteinase from fetal bovine tendon and skin. Purification of the 500-kilodalton form of the enzyme from bovine tendon.
Procollagen N-proteinase (EC 3.4.24.14) is the enzyme that specifically cleaves the NH2-terminal propeptides from type I procollagen. Two forms of N-proteinase with apparent molecular sizes of 300 and 500 kDa were found in partially purified preparations from fetal bovine tendon extracts. The 500-kDa form of enzyme was purified 16,000-fold with a recovery of 8% from the extracts of the tendons by six purification steps. The purified enzyme was a neutral, Ca(2+)-dependent proteinase (5-10 mM) that was inhibited by metal chelators. The 500-kDa enzyme contained unreduced polypeptides of 58, 125, 170, and 190 kDa which were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Electron microscopic study indicated that the enzyme molecules were generally globular and had diameters of 33 +/- 4 nm. Other properties of the 500-kDa enzyme were: 1) the Km for type I procollagen is 35 nM at pH 7.5 and 35 degrees C, and the kcat is 290 h-1; 2) the activation energy for reaction with type I procollagen is 10,050 cal mol-1; 3) the isoelectric point is 3.8; 4) the enzyme cleaves the NH2-terminal propeptides of type II procollagen as well as type I procollagen but not of type III procollagen; and 5) the enzyme specifically cleaves a -Pro-Gln- bond in the pro-alpha 1(I) chain and an -Ala-Gln- bond in the pro-alpha 2(I) chain. The bovine N-proteinase with a mass of 300 kDa was found to be similar to the 500-kDa enzyme and appeared to be a degraded form of the 500-kDa enzyme generated during purification. The N-proteinase from fetal bovine skin extracts also contained 300-kDa and 500-kDa enzyme forms.
Hojima
Y
Y
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Mörgelin
M M
MM
Engel
J
J
Boutillon
M M
MM
van der Rest
M
M
McKenzie
J
J
Chen
G C
GC
Rafi
N
N
Romanic
A M
AM
Prockop
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D005333:Fetus; D005865:Gestational Age; D005973:Glutamine; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011247:Pregnancy; D011347:Procollagen / Q000737:chemistry / Q000378:metabolism; D011348:Procollagen N-Endopeptidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011392:Proline; D011480:Protease Inhibitors / Q000494:pharmacology; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity; D013710:Tendons / Q000201:enzymology*
D011347:Procollagen; D011480:Protease Inhibitors; D005973:Glutamine; D011392:Proline; D011348:Procollagen N-Endopeptidase