Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis).
An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn(2+). Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K(m) of 32.2 microM and k(cat) of 29.5 s(-1) with Ala-pNA and a K(m) of 11.1 microM and k(cat) of 9.49 s(-1) with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala-Phe-Tyr-Glu.
Umetsu
Hironori
H
Graduate School of Environmental Science, Aomori University, Kobata, Aomori 030-0943, Japan. umetsu@aomori-u.ac.jp
Arai
Mito
M
Ota
Toshinori
T
Kudo
Ryohei
R
Sugiura
Hitomi
H
Ishiyama
Hiroyuki
H
Sasaki
Kazuo
K
D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005753:Gastric Mucosa / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008974:Mollusca / Q000201:enzymology*; D013379:Substrate Specificity
D000626:Aminopeptidases