Purification and characterization of cathepsin D from herring muscle (Clupea harengus).
Cathepsin D was purified and concentrated 469-fold from a homogenate of Clupea harengus muscle. The purified enzyme is a monomer with a molecular weight of 38000-39000. It is inhibited by pepstatin and has optimal activity at pH 2.5 with hemoglobin as the substrate. The isoelectric point is at pH 6.8. Glycosidase treatment and binding to Concanavalin A indicated that the enzyme contains one N-linked carbohydrate moiety of the high-mannose type per molecule. The first 21 amino acid residues of the N-terminal showed high similarity to cathepsin D from antarctic icefish liver (Chionodraco hamatus) and trout ovary (Oncorhynchus mykiss). Digestion of the beta-chain of oxidized insulin resulted in preferential cleavage at Leu(15)-Tyr(16), (47%), Tyr(16)-Leu(17) (34%) and Ala(14)-Leu(15) (18%). Incubation with myofibrils from herring muscle at pH 4.23 showed that the enzyme mainly degraded myosin, actin and tropomyosin.
Nielsen
L B
LB
Department of Molecular Characterization, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. lbn@bioteknologisk.dk
Nielsen
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D002402:Cathepsin D / Q000737:chemistry* / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005399:Fishes; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D010053:Ovary / Q000378:metabolism; D010436:Pepstatins / Q000494:pharmacology; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D006454:Hemoglobins; D010436:Pepstatins; D003208:Concanavalin A; C014195:Streptomyces pepsin inhibitor; D006026:Glycoside Hydrolases; D002402:Cathepsin D; C031375:pepstatin