Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle.
Two cysteine proteinase inhibitors, CPI-L and CPI-H, were purified from rabbit skeletal muscle by means of successive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. The molecular mass of CPI-L was 13 kDa on gel permeation chromatography and SDS-PAGE under reducing conditions and was 15 kDa on SDS-PAGE under non-reducing conditions. The molecular mass of CPI-H was 23 kDa on gel permeation chromatography and it was converted to 13 kDa by SH-reducing agent. Although CPI-H showed single protein band with 13 kDa on SDS-PAGE under reducing conditions, it showed four protein bands with 21, 20, 15 and 13 kDa on SDS-PAGE under non-reducing conditions. Therefore, CPI-H was suggested to have a complicated subunit structure for which S-S bonds and some non-covalent bonds would be responsible. CPI-L and CPI-H were stable in the range of pH 3.0-9.5 and up to 80 degrees C. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (Ki) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B.
Matsuishi
Masanori
M
Department of Food Science and Technology, Nippon Veterinary and Animal Science University, 7-1 Kyonan-cho, 1-chome, Musashino-shi, Tokyo 180-8602, Japan. matmas@nvau.ac.jp
Okitani
Akihiro
A
D000818:Animals; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002850:Chromatography, Gel; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry*; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits
D015853:Cysteine Proteinase Inhibitors; D002403:Cathepsins; D010206:Papain