Substrate specificity of carp Cyprinus carpio cathepsin H with methylcoumarylamide substrates

被引:6
作者
Aranishi, F
Hara, K
Osatomi, K
Ishihara, T
机构
[1] NAGASAKI UNIV, FAC FISHERIES, BUNKYO KU, NAGASAKI 852, JAPAN
[2] NAGASAKI UNIV, GRAD SCH MARINE SCI & ENGN, BUNKYO KU, NAGASAKI 852, JAPAN
来源
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY | 1997年 / 116卷 / 02期
关键词
carp; cathepsin H; Cyprinus carpio; fluorescent assay; hepatopancreas; kinetic constants; methylcoumarylamide; substrate specificity;
D O I
10.1016/S0305-0491(96)00249-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cathepsin H was purified from the crude extract of carp (Cyprinus carpio) hepatopancreas by a reformed method involving six stages, and the specific activity increased about 11,500-fold with a 23% recovery. Of varying fluorescent synthetic substrates tested, carp cathepsin H possessed an ability to hydrolyze four N-terminal unblocked substrates those are composed of a single amino acid bound to 4-methylfoumaryl-7-amide (MCA), namely Leu-MCA, Arg-MCA, Lys-MCA and Ala-MCA. In contrast, the enzyme was only marginally able to hydrolyze an unblocked substrate such as Pro-Phe-Arg-MCA and totally unable to degrade all blocked derivatives employed. From the kinetic constants with four unblocked substrates, carp cathepsin H had the highest affinity toward Leu-MCA with a K-m value of 35.4 mu M. Besides, both the hydrolytic rates and molecular activities of the enzyme decreased from Lys MCA > Arg-MCA > Ala-MCA > Leu-MCA as judged by their V-max and K-cat values, respectively. Otherwise, optimal pHs for hydrolysis of cathepsin H were different for four substrates. The enzyme exhibited maximum level of the activity at pH 6.5 for Arg-MCA and Lys-MCA and at pH 7.0 for Leu-MCA and Ala-MCA. Copyright (C) 1997 Elsevier Science Inc.
引用
收藏
页码:203 / 208
页数:6
相关论文
共 20 条
[1]   ISOLATION AND ACTIVATION OF CATHEPSIN L-INHIBITOR COMPLEX FROM PACIFIC WHITING (MERLUCCIUS-PRODUCTUS) [J].
AN, HJ ;
PETERS, MY ;
SEYMOUR, TA ;
MORRISSEY, MT .
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 1995, 43 (02) :327-330
[2]   Purification and immunological properties of cathepsin B in carp Cyprinus carpio [J].
Aranishi, F ;
Hara, K ;
Osatomi, K ;
Ishihara, T .
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 1996, 114 (04) :371-376
[3]   PURIFICATION AND CHARACTERIZATION OF CATHEPSIN-H FROM HEPATOPANCREAS OF CARP CYPRINUS-CARPIO [J].
ARANISHI, F ;
HARA, K ;
ISHIHARA, T .
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 1992, 102 (03) :499-505
[4]   NEW ASSAY FOR CATHEPSIN B1 AND OTHER THIOL PROTEINASES [J].
BARRETT, AJ .
ANALYTICAL BIOCHEMISTRY, 1972, 47 (01) :280-&
[5]   IMPROVED COLOR REAGENT FOR USE IN BARRETTS ASSAY OF CATHEPSIN-B [J].
BARRETT, AJ .
ANALYTICAL BIOCHEMISTRY, 1976, 76 (01) :374-376
[7]  
BARRETT AJ, 1981, METHOD ENZYMOL, V80, P535
[9]  
KIRSCHKE H, 1977, ACTA BIOL MED GER, V36, P185
[10]  
LOWRY OH, 1951, J BIOL CHEM, V193, P265