Kinetic studies on the processing of human beta-lipotropin by bovine pituitary intermediate lobe pro-opiomelanocortin-converting enzyme.
The kinetics of the previously reported paired basic residue-specific pro-opiomelanocortin-converting enzyme from bovine pituitary intermediate lobe secretory vesicles (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) were studied using human 125I-beta-lipotropin as substrate. The enzyme, at a concentration of 20 ng/100 microliters cleaved human beta-lipotropin to yield gamma-lipotropin, a beta-melanotropin linked to beta-endorphin intermediate and beta-endorphin, whereas at an enzyme concentration of 40 ng/100 microliters, the substrate was completely cleaved to yield beta-endorphin and beta-melanotropin. These products were identified by their immunological properties and size on sodium dodecyl sulfate-polyacrylamide gels. The 125I-beta-endorphin product was further shown by high pressure liquid chromatography to contain two forms; the major form co-eluted with 125I-Arg0-beta h-endorphin and the minor form with 125I-beta h-endorphin. No COOH-terminal shortened forms of beta-endorphin were detected. The products formed indicate cleavages at two of the three pairs of basic residues of human beta-lipotropin, at Lys37-Lys38 and Lys57-Arg58, but not at Lys86-Lys87. The cleavage at Lys57-Arg58 occurred primarily in between these basic residues. The Km values for the cleavage of the Lys37-Lys38 and Lys57-Arg58 pairs were 1.9 and 2.5 microM, respectively. The Vmax values for the cleavage of the Lys37-Lys38 and Lys58-Arg58 pairs were 4.8 nmol/micrograms of enzyme/h and 9.1 nmol/micrograms of enzyme/h, respectively.
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010902:Pituitary Gland / Q000201:enzymology*; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D008083:beta-Lipotropin / Q000378:metabolism*
D011480:Protease Inhibitors; D008083:beta-Lipotropin; D010450:Endopeptidases; D043484:Proprotein Convertases