Two trypsin isoforms from the intestine of the grass carp (Ctenopharyngodon idellus).
Two trypsin isoforms (GT-A and GT-B) from the grass carp (Ctenopharyngodon idellus) intestine were isolated and purified. SDS-PAGE electrophoresis showed that GT-A and GT-B had relative molecular masses of 30,740 and 26,400, respectively. Enzyme activity was inhibited by three organic trypsin inhibitors but not by EDTA. They had optimal pH of 8.0 and 8.5, and optimal temperatures of 38.5 and 44.0 degrees C, respectively, when hydrolyzing N-benzoyl-L: -arginine ethyl ester.HCl (BAEE). They lost 95.8 and 93.7% of their activities, respectively, after heating for 20 min at 65 degrees C. Their thermal denaturation temperatures, respectively, were 66.3 and 67.3 degrees C. GT-A has a K(m) value of 21.2 microM and a V(max) of 2.0 x 10(3) min(-1), and GT-B has a K(m) value of 31.7 microM and a V(max) of 3.3 x 10(3) min(-1). Their physiological efficiencies were 94.3 and 105.3 microM(-1) min(-1), respectively. The Arrhenius activation energies of GT-A and GT-B were 4.16 and 4.38 kcal/mol, respectively. The activities of GT-A and GT-B were not activated by Ca(2+), but their thermostability was improved in the presence of Ca(2+). Enzyme activity was reduced in presence of Zn(2+), Cu(2+), Hg(2+) and Al(3+). Thermal stabilities of GT-A and GT-B were intermediate between Arctic and tropical fish species, and consistent with the wide range of water temperatures to which grass carp are exposed in most provinces of China.
Liu
Zhong-Yi
ZY
Food Science and Safety Focal Laboratory of Ministry of Education of China, Food Science Institute of Southern Yangtze University, Wuxi, Jiangsu, China 214036. lzyly@xtu.edu.cn
Wang
Zhang
Z
Xu
Shi-Ying
SY
Xu
Lin-Na
LN
D000535:Aluminum / Q000494:pharmacology; D000818:Animals; D002347:Carps / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology*; D007477:Ions / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011489:Protein Denaturation; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007477:Ions; D007527:Isoenzymes; D014107:Tosyllysine Chloromethyl Ketone; D010664:Phenylmethylsulfonyl Fluoride; D003300:Copper; D014360:Trypsin Inhibitor, Kunitz Soybean; D000535:Aluminum; D014357:Trypsin; D008628:Mercury; D015032:Zinc