An acidic protease from the grass carp intestine (Ctenopharyngodon idellus).
The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.
Liu
Zhong-yi
ZY
Department of Food and Biological Technology, Xiangtan University, Xiangtan, Hunan, 411105, China. lzyly@xtu.edu.cn
Wang
Zhang
Z
Zhang
Jian
J
D000818:Animals; D002347:Carps / Q000378:metabolism*; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000737:chemistry
D029941:Fish Proteins; D011480:Protease Inhibitors; D010447:Peptide Hydrolases