Open AccessPurification and properties of aminopeptidase H from chicken skeletal muscle
Author(s) -
RHYU Mee Ra,
NISHIMURA Toshihide,
KATO Yutaka,
OKITANI Akihiro,
KATO Hiromichi
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17158.x
Subject(s) - leupeptin , phenylmethylsulfonyl fluoride , biochemistry , pepstatin , chemistry , aminopeptidase , leucine , sepharose , chromatography , puromycin , enzyme , amino acid , pmsf , protease , protein biosynthesis
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 β‐naphthylamide and α‐ N ‐benzoyl‐DL‐arginine β‐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 β‐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.