Quantitation of the endopeptidase activity generating gamma-endorphin from beta-endorphin in rat brain synaptic membranes by a radiometric assay.
gamma-Endorphin is a naturally occurring biologically active peptide that is produced by an endopeptidase activity cleaving its precursor beta-endorphin. This enzyme was termed gamma-endorphin generating enzyme (gamma-EGE). In order to quantitate gamma-EGE activity by means of a simple and sensitive assay two synthetic peptides derived from the sequence surrounding the gamma-EGE cleavage site in beta-endorphin were tested as substrates. One of these peptides Ac-Val-Thr-Leu-Phe-Lys-NHCH3 fulfilled all criteria for a suitable gamma-EGE substrate. The peptide was exclusively cleaved at the correct bond for gamma-EGE upon incubation with brain synaptic membranes, and this cleavage was inhibited by the naturally occurring substrate beta-endorphin. The peptide was insensitive to cleavage by exopeptidases and cathepsin D. Addition of a 14C-labeled methyl group at the lysine residue of this peptide by reductive methylation did not alter its properties as a substrate for gamma-EGE activity. The use of the 14C-labeled peptide allowed sensitive quantitation of its radioactive products after simple separation by hydrophobic chromatography on minicolumns containing polystyrene beads. gamma-EGE activity increased linearly with a protein concentration and incubation time. This assay can be used for reliable quantitation of gamma-EGE activity and permits investigations on the regulation of gamma-endorphin production.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D004723:Endorphins / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011874:Radiometry; D051381:Rats; D013379:Substrate Specificity; D013570:Synaptic Membranes / Q000201:enzymology*; D001615:beta-Endorphin / Q000031:analogs & derivatives; D018823:gamma-Endorphin
D000596:Amino Acids; D004723:Endorphins; D010446:Peptide Fragments; C043502:beta-endorphin (13-22), Ac-Glu(13)-Glu(22) methylamide-; C043501:beta-endorphin (15-19), Ac-Val(15)-Lys(19) methylamide-; D001615:beta-Endorphin; D018823:gamma-Endorphin; D010450:Endopeptidases; C043537:gamma-endorphin generating enzyme