ABSTRACT
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.
Subject(s)
Cathepsins/metabolism , Endopeptidases , Osteoclasts/enzymology , Amino Acid Sequence , Animals , Cathepsin K , Cathepsin L , Cathepsins/antagonists & inhibitors , Cysteine Endopeptidases , Escherichia coli/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Protease Inhibitors/pharmacology , Rabbits , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The extent of organic solvent tolerance was variable among strains of Escherichia coli K-12. Genetic analyses of n-hexane-tolerant strains indicated that a number of genes were involved in the solvent-tolerance phenotype. One such gene, designated ostA, was mapped at 1.2 min, close to pdxA. Transduction of ostA from a n-hexane-sensitive strain to a n-hexane-tolerant strain generated n-hexane-sensitive transductants. The sensitive transductant restored n-hexane-tolerance by transduction of ostA from a tolerant strain. Thus, the gene ostA is one of the genes that contributes to deciding the level of organic solvent tolerance in E. coli.