Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits.
A cDNA clone encoding the rabbit cysteine proteinase cathepsin K, which 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., Sato T., Nemoto K., Kamioka H., Hakeda Y., Kumegawa M., J. Biol. Chem., 269, 1106 (1994)], was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at an alkaline pH. Cathepsin K (37-kDa) was purified by gel filtration and its enzymatic characteristics were determined. The enzymatic activity of cathepsin K was strongly inhibited by cysteine proteinase inhibitors and its optimal pH was pH 5.5. Synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumaryl-amide, which is hydrolyzed by cathepsins L and S, was also cleaved by cathepsin K. On the other hand, benzyloxycarbonyl-Gly-Pro-Arg-7-(4-methyl)coumaryl-amide was the most suitable substrate for cathepsin K, but was hardly hydrolyzed by cathepsin L. The substrate specificity of cathepsin K, as determined using various chemogenic substrates, showed different characteristics from cathepsins L and S.
Aibe
K
K
Drug Serendipity Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Ibaraki, Japan.
Yazawa
H
H
Abe
K
K
Teramura
K
K
Kumegawa
M
M
Kawashima
H
H
Honda
K
K
D000595:Amino Acid Sequence; D000818:Animals; D056657:Cathepsin K; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; D056657:Cathepsin K