Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss).
1. Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). 2. The approximate molecular weights of chymotrypsin I and II were 28,200 (+/- 1200) and 28,800 (+/- 900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. 3. The pH optima of the enzymes were centered around nine, when assayed for succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. 4. The amidase activity of both enzymes increased with temperature up to about 55 degrees C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by stronger calcium binding of the former. 5. The trout chymotrypsins were significantly more active than bovine alpha-chymotrypsin when assayed against Suc-AAPF-NA at 25 degrees C and casein at low temperatures (10-20 degrees C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.
Kristjánsson
M M
MM
Marine Biotechnology Center, Technical University of Denmark, Lyngby.
Nielsen
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology*; D008969:Molecular Sequence Data; D012474:Salmon; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013696:Temperature
D002364:Caseins; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D002118:Calcium