Purification and characterization of myofibril-bound serine proteinase from carp Cyprinus carpio ordinary muscle.
1. A novel myofibril-bound serine proteinase (MBP) has been purified from ordinary muscle of the carp Cyprinus carpio. 2. It was solubilized from the myofibril fraction with acid treatment (under the conditions of 0.6 M KCl, pH 4.0), then purified by column chromatographic steps on Ultrogel AcA 54, and Arginine-Sepharose 4B. 3. The purified enzyme revealed a single protein band on SDS-PAGE, and its molecular mass was estimated to be 30 kDa by SDS-PAGE and gel filtration. 4. The optimum pH and temperature of the enzyme were 8.0 and 55 degrees C, respectively, when Boc-Phe-Ser-Arg-MCA and casein were used as substrates. 5. The enzyme hydrolyzed Boc-Gln-Arg-Arg-MCA most rapidly, and also hydrolyzed the substrates for trypsin-type proteinase, but not for chymotrypsin. The enzyme was inhibited by serine proteinase inhibitors such as DFP, STI and leupeptin. These results suggested that the enzyme was a trypsin-type serine proteinase. 6. Boc-Phe-Ser-Arg-MCA hydrolyzing activity of the purified enzyme was reduced by addition of NaCl, but the caseinolytic activity and Boc-Phe-Ser-Arg-MCA hydrolyzing activity of the partially purified enzyme were activated by NaCl.
Osatomi
K
K
Graduate School of Marine Science and Engineering, Nagasaki University, Japan.
Sasai
H
H
Cao
M
M
Hara
K
K
Ishihara
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002347:Carps / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009210:Myofibrils / Q000201:enzymology*; D009218:Myosins / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D012965:Sodium Chloride; D012697:Serine Endopeptidases; D009218:Myosins