title: Pharmacological and biochemical study on the mechanism of enkephalin production in rat dental pulp.
abstract: 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.
authors: Osaka University Faculty of Dentistry.
authors: E Q
authors: EQ
authors: Wei
authors: T
authors: T
authors: Kudo
authors: R
authors: R
authors: Inoki
mesh_terms: 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
chemical_list: D004745:Enkephalins; D002401:Cathepsin B; D001920:Bradykinin