Purification and some properties of cathepsin H from rabbit skeletal muscle.
Rabbit muscle cathepsin H classified as an aminoendopeptidase was purified and its properties were investigated to clarify its contribution to the proteolysis of postmortem muscle. The purification was performed by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocelluose, DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme gave a single protein band on SDS-PAGE. Its molecular mass was found to be 28 kDa by gel permeation and 30 kDa by SDS-PAGE. The optimum pHs for alpha-N-benzoyl-DL-arginine-beta-naphthylamide (BANA)- and L-leucine-beta-naphthylamide (Leu-NA)-hydrolyzing activities were 6.6 and 7.0, respectively. This enzyme was almost stable in the range of pH 4-5 and up to 50 degrees C at pH 5.0. The Km values of BANA- and Leu-NA-hydrolyzing activities were 0.367 and 0.203 mM, respectively. The enzyme was inhibited by monoiodoacetic acid, antipain, leupeptin, TLCK and TPCK, but was not affected by pepstatin, bestatin, puromycin, PMSF or trypsin inhibitor. This enzyme strongly acted on Arg-, Lys-, Met-, Ala-, Ser- and Leu-NAs, weakly acted on Val- and Glu-NAs, and hardly acted on Pro- and Gly-NAs. The amount of cathepsin H in muscle was estimated to be less than one-fourth of the sum of the amount of aminopeptidases C and H by the Leu-NA-hydrolyzing activity on the chromatography. This enzyme degraded myosin heavy chain, actin, tropomyosin and troponin I clearly at pH 4.0, while it slightly degraded troponin I at pH 5.0-5.6. Therefore, the contribution of cathepsin H to the proteolysis of postmortem muscle is presumed to be relatively small.
Matsuishi
Masanori
M
Department of Food Science and Technology, Nippon Veterinary and Animal Science University, 7-1 Kyonan-cho, 1-Chome, Musashino-shi, 180-8602, Tokyo, Japan. matmas@nvau.ac.jp
Saito
Gun
G
Okitani
Akihiro
A
Kato
Hiromichi
H
D000818:Animals; D056655:Cathepsin H; D002403:Cathepsins / Q000737:chemistry* / Q000302:isolation & purification; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology*; D011817:Rabbits; D013379:Substrate Specificity
D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056655:Cathepsin H