Purification and properties of aminopeptidase H from chicken skeletal muscle.
Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.
Rhyu
M R
MR
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Nishimura
T
T
Kato
Y
Y
Okitani
A
A
Kato
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D004151:Dipeptides; D009842:Oligopeptides; D010450:Endopeptidases; D000626:Aminopeptidases; C030596:hydrolase H