CHARACTERIZATION OF A COLLAGENOLYTIC SERINE PROTEINASE FROM THE ATLANTIC COD (GADUS-MORHUA)

A collagenolytic proteinase was purified from the intestines of Atlant ic cod by (NH4)(2)SO4 fractionation, hydrophobic interaction chromatog raphy (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharo se). The proteinase has an estimated molecular weight of 24.1 (+/-0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases, The enzyme cleaves native collagen types I, III , IV and V, and also readily hydrolyzes succinyl-L-Ala-L-Ala-L-Pro-L-P he-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as w ell as succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, a reported ela stase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin, The pH optimum of the e nzyme was between pH 8.0 and 9.5 and it was unstable at pH values belo w 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50 degrees C. Calcium binding stabilized the c od collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30 degrees C .