Abstract
Pro-opiomelanocortin (adrenocorticotropin/endorphin prohormone) is processed to yield active hormones by cleavages at paired basic amino acid residues. In this study, an enzyme that specifically cleaves at the paired basic residues of this prohormone has been purified from bovine pituitary intermediate lobe secretory vesicles, the intracellular processing site of proopiomelanocortin. This enzyme, named pro-opiomelanocortin converting enzyme, has been characterized as a glycoprotein of Mr approximately 70,000. It has an apparent isoelectric point between 3.5 and 4.0. The pH optimum of the pro-opiomelanocortin converting enzyme is between 4 and 5, but the enzyme is highly active at the intravesicular pH of 5.1-5.6. The enzyme specifically cleaved the Lys-Arg pairs of pro-opiomelanocortin to yield Mr = to 21,000-23,000 ACTH, beta-lipotropin, Mr 13,000 and 4,500 ACTH, beta-endorphin, and a Mr = 16,000 NH2-terminal glycopeptide, the products synthesized by the pituitary intermediate lobe in situ. NH2- and COOH-terminal analysis of the products indicated that the pro-opiomelanocortin converting enzyme cleaves the peptide bond either between the Lys and Arg or on the carboxyl side of the Arg at Lys-Arg pairs of pro-opiomelanocortin. The intracellular localization, pH optimum, and cleavage specificity of the enzyme suggest that it may function as a pro-opiomelanocortin processing enzyme in the pituitary intermediate lobe in vivo.
Highlights
Phin prohormonei)sprocessedto yieldactivehormones In the last 5 years, several groups have reported the presby cleavages at paired basic amino acid residues
The pH optimum enzyme activity that i s specific for the paired basic residues of the pro-opiomelanocortin converting enzymeis be- of pro-opiomelanocortin (POMC’) in rat pituitary secretory tween 4 and 5, but the enzyme is highly active at the vesicles
NH2- and COOH-terminalanalysis of the productsindicated that the pro-opiomelanocortin convertingenthese lobes, POMC is processed at Lys-Arg pairs (2, 6) to yield a M, = 16,000 glycopeptide, adrenocorticotropin (ACTH), @-lipotropin(@-LPH), and @-endorphin(2, 19)
Summary
Nocortin Converting Enzyme-Bovine '261-pro-insulin(-10,000 cpm) (a gift from Dr Bruce Frank, Lilly Research Laboratories) was incubated with purified pro-opiomelanocortin converting enzyme for 5 hat 37 "C in 0.1 M sodium citrate buffer, pH 4.0 (total volume, 100 pl), with and without dithiothreitol(5mM). The sample wasboiled for 2 min, andthe proteins were separated by SDS-gel electrophoresis on a 15%gel using the Laemmli buffer system (25) in 0.5 X 10-cm tube gels. Incubation of Synthetic Peptides with Purified Pro-opiomelanocortin ConvertingEnzyme-Synthetic peptides ACTH1-39 (0.5 mM) and Z-Val-Lys-Lys-Arg-4-MNA (0.5 mM)were incubated individually with purified pro-opiomelanocortin converting enzyme ina total volume of 100 p1 of pH 4.0 or 5.5 buffer for 5 h at 37'C. Were pooled, dialyzed,lyophilized,and subjected to gel electrophoresis using a Tris/glycine buffer system without SDS. Aliquots from fractions pooled from three or six consecutive gel slices were assayed for protein and pro-opiomelanocortin converting enzyme activity. A , protein concentration ineach pooled fraction. The pooled fractions analyzed were from gel slices 1-6, 7-12,13-15,16-18,19-21,22-24,25-27,28-30,31-36,37-42,43-48, 49-51, and 52-60. Lys-Lys-Arg-4-MNAwas assayed by measuring the formation of the fluorescent leaving group using an Aminco SPF-125 spectrofluorometer with an emission wavelength of455 nm and an excitation wavelength of 383 nm
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