Pharmacological and biochemical study on the mechanism of enkephalin production in rat dental pulp.
The production of enkephalin (EK) in the rat dental pulp was studied in pharmacological and biochemical aspects of EK-producing enzyme, EK precursor protein and the regulation of EK production. The EK precursor protein was primarily distributed in the microsomal fraction, and a common precursor protein (Mr about 58,000) was partially purified by Sephadex G-100 chromatography. Since the EK-producing enzyme, however, was mainly localized in the lysosomal fraction, and was found to be a cysteine proteinase, the lysosomal cysteine proteinases, cathepsins H, B and L, were separated by CM Sephadex C-50 ion exchange chromatography, and identified in respects to substrate specificity, pH optimum and inhibitor sensitivity. The EK-producing activity of the cathepsin B was demonstrated using the partially purified EK precursor protein from the pulp tissue as a substrate. The cathepsin B was further purified by Sephadex G-75 gel filtration to a 400-fold purity, and SDS polyacrylamide gel electrophoresis of the enzyme showed a distinct homogeneity (Mr about 23,600). The purified enzyme cleaved BAM-12P, a met-EK-containing peptide from bovine adrenal medulla, to met-EK-Arg6, but did not convert met-EK-Arg6 to met-EK, suggesting an endopeptidase activity of the enzyme. On the other hand, a concentration-dependent activation of the enzyme by bradykinin (BK) and des-Arg9-BK was found to be mediated through B1 receptor in intact pulp tissue. It was also demonstrated that intact structure of lysosomes and Ca++ were necessary for the activation of the enzyme by BK.
Wei
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Osaka University Faculty of Dentistry.
Kudo
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Inoki
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D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D003782:Dental Pulp / Q000201:enzymology*; D004745:Enkephalins / Q000096:biosynthesis*; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D004745:Enkephalins; D002401:Cathepsin B; D001920:Bradykinin