SUBSTRATE-SPECIFICITY OF RECOMBINANT OSTEOCLAST-SPECIFIC CATHEPSIN-K FROM RABBITS

A cDNA clone encoding the rabbit cysteine proteinase cathepsin K, whic h is predominantly expressed in osteoclasts and is closely related to cathepsins L (EC 3.4.22.15) and S (EC 3.4.22.27) [Tezuka K,, Tezuka Y. , Maejima A., Sate T., Nemoto K., Kamioka R., Hakeda Y., Kumegawa M., J, Biol. Chem., 269, 1106 (1994)], was expressed at high levels in Esc herichia coli in a T7 expression system, The insoluble recombinant enz yme was solubilized in urea and refolded at an alkaline pH. Cathepsin K (37-kDa) mas purified by gel filtration and its enzymatic characteri stics were determined, The enzymatic activity of cathepsin K was stron gly inhibited by cysteine proteinase inhibitors and its optimal pH was pH 5.5, Synthetic substrate zyloxycarbonyl-Phe-Arg-7-(4-methyl)coumar yl-amide, which is hydrolyzed by cathepsins L and S, was also cleaved by cathepsin K, On the other band, oxycarbonyl-Gly-Pro-Arg-7-(4-methyl )coumaryl-amide was the most suitable substrate for cathepsin K, but w as hardly hydrolyzed by cathepsin L. The substrate specificity of cath epsin K, as determined using various chemogenic substrates, showed dif ferent characteristics from cathepsins L and S.