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The anti-allergic drugs disodium cromoglycate (DSCG) and tranilast are strong inhibitors of hyaluronidase. In the development of new anti-allergic drugs, we studied the inhibitory effects of some natural products on the activation of hyaluronidase. Among the compounds tested, liquiritigenin, isoliquiritigenin and baicalein showed dose-related inhibitory effects. Anti-allergic activities of these compounds were evident from the facts that they inhibited the histamine release from rat peritoneal exudate cells induced by antigen, compound 48/80 and calcium ionophore A-23187, and from their inhibitory effect on Shultz-Dale reaction using sensitized guinea pig ileum.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(Ltrans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10 37 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10 37 M in vitro.z 1999 Federation of European Biochemical Societies.

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The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

We have established a new differential assay method for cathepsin L-type proteinases using specific inhibitors, E-64 for all cysteine proteinases, CA-074 for cathepsin B, and PLCPI for cathepsin L-type proteinases with Z-Phe-Arg-MCA as the substrate. The value of cathepsin B calculated by this method did not coincide with value assayed directly in terms of the hydrolysis of Z-Arg-Arg-MCA, a specific substrate for cathepsin B. The activities of cathepsin L-type proteinases, cathepsins B and J in rat liver and kidney were assayed at the same time using this new assay method as a representative example.

Secretion of procathepsin L into the culture medium from a bone cell mixture was markedly enhanced by addition of parathyroid hormone (PTH), 1 a,25-(OH)2D 3 or tumor necrosis factor a (TNFa). These stimulators of secretion of procathepsin L enhanced bone pit formation, which was inhibited by E-64, but not by CA-074, a specific inhibitor of cathepsin B. Procathepsin L may thus participate in the process of bone collagenolysis during bone resorption. Procathepsin L partially purified from rat long bones under cold conditions was rapidly converted to the mature form under acidic conditions at room temperature. This conversion was inhibited by E-64, suggesting that the procathepsin L secreted into lacunae is catalytically converted to the mature enzyme by cysteine proteinase(s).In the present study, designed to clarify the mechanism and regulation of both the secretion of procathepsin L and its processing in lacunae, we examined the secretion of procathepsin L induced by 10~,25-(OH)2D 3, TNFc~ or by PTH, and also the effects of the cysteine proteinase inhibitors, E-64 and CA-074, on the pit formation induced by these effectors. Furthermore, to clarify the participation of cysteine proteinase(s) in processing from the precursor to the active form, the inhibitory effect of E-64 on the processing of procathepsin L partially purified from rat long bones was also examined. Experimental