Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases.
A lysed preparation of isolated insulin secretory granules efficiently cleaved murine proopiomelanocortin (mPOMC) at physiologically important Lys-Arg processing sites. This processing was mostly attributed to an activity that co-eluted with the proinsulin processing type-II endopeptidase from anion exchange chromatography (Lys-Arg-directed; Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96). The principal peptide hormone products generated by the insulin secretory granule lysate were identified by specific radioimmunoassay and NH2-terminal microsequencing analysis of high performance liquid chromatography-separated products as alpha-melanocyte-stimulating hormone, corticotropin-like intermediate, gamma-lipotropin, beta-endorphin-(1-31), 18-kDa NH2-terminal fragment and, to a lesser extent, adrenocorticotrophin and beta-lipotropin. This processing had an acidic pH optimum (pH 5-5.5) and was Ca(2+)-dependent (K0.5 activation = 5-80 microM). With increasing Ca2+ concentrations there was an increase in the extent to which mPOMC was processed. The in vitro processing of mPOMC by the insulin secretory granule endopeptidase activity reported here is in excellent agreement with the in vivo processing of this prohormone by a combination of PC2 and PC3, candidates of prohormone endpeptidase, in gene transfer studies with cells that express the regulated secretory pathway (Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. S., Steiner, D. F., and Thomas, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5297-5301).
E. P. Joslin Research Laboratory, Joslin Diabetes Center, Brigham and Women's Hospital, Boston, Massachusetts 02215.
C J
CJ
Rhodes
B A
BA
Thorne
B
B
Lincoln
E
E
Nielsen
J C
JC
Hutton
G
G
Thomas
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007340:Insulinoma / Q000378:metabolism; D008969:Molecular Sequence Data; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D014162:Transfection
D007328:Insulin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D010450:Endopeptidases; C054196:proinsulin endopeptidase I; C061496:proinsulin endopeptidase II