Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus).
Aminopeptidases play important roles in turnover of proteins, metabolism of hormones and neurotransmission, cell maturation and immunological regulations. In the present study, an aminopeptidase was purified to homogeneity from the skeletal muscle of grass carp by ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite and Phenyl-Sepharose. The purified enzyme revealed a molecular mass of approximately 105 kDa both on SDS-PAGE and on gel filtration of Superdex 200. The enzymatic activity toward synthetic substrates was optimal at 40°C and pH 7.0-7.5. Metal-chelating agents such as EDTA and EGTA effectively inhibited the enzyme activity while inhibitors to serine, asparatic and cysteine proteinases did not show much effect, suggesting its belonging to metalloproteinase family. A specific aminopeptidase inhibitor bestatin was most effective in suppressing the enzymatic activity and performed in a competitive fashion. The enzymatic activity was slightly enhanced by metal ions of Mg2+ and Mn2+ while inhibited to different extents by Co2+, Cu2+, Zn2+ and Ca2+. Sulfhydryl reagent was necessary to maintain its activity. Purified enzyme demonstrated amidolytic activity most effectively against synthetic aminopeptidase substrate Leu-methylcoumarylamide (MCA) while N-terminal-blocked substrates and myofibrillar proteins were not hydrolyzed. The enzyme purified in the present study was quite possibly a leucine aminopeptidase (LAP) and functions during muscular protein metabolism.
Zhou
Li-Gen
LG
Institute of Food Processing, Zhejiang Academy of Agriculture Sciences, Hangzhou, 310021, China.
Liu
Bing-Xin
BX
Sun
Le-Chang
LC
Hara
Kenji
K
Su
Wen-Jin
WJ
Cao
Min-Jie
MJ
D000645:Ammonium Sulfate; D000818:Animals; D002347:Carps / Q000378:metabolism*; D005591:Chemical Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000302:isolation & purification*; D018482:Muscle, Skeletal / Q000201:enzymology*; D013696:Temperature
D007931:Leucyl Aminopeptidase; D000645:Ammonium Sulfate