Purification and characterization of a collagenase from the mackerel, Scomber japonicus.
Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.
Park
Pyo-Jam
PJ
Department of Chemistry, Pukyong National University, Busan 608-737, Korea.
Lee
Sang-Hoon
SH
Byun
Hee-Guk
HG
Kim
Soo-Hyun
SH
Kim
Se-Kwon
SK
D000096:Acetone / Q000494:pharmacology; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017364:Collagenases / Q000737:chemistry* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D010473:Perciformes; D011480:Protease Inhibitors / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014105:Tosyl Compounds / Q000494:pharmacology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D007477:Ions; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D014105:Tosyl Compounds; D000096:Acetone; D014107:Tosyllysine Chloromethyl Ketone; C003614:4-toluenesulfonyl fluoride; D014360:Trypsin Inhibitor, Kunitz Soybean; D017364:Collagenases; D008628:Mercury; D015032:Zinc