y-Endorphin is a naturally occurring biologically active peptide that is produced by an endopeptidase activity cleaving its precursor flendorphin. This enzyme was termed r-endorphin generating enzyme (r-EGE). In order to quantitate r-EGE activity by means of a simple and sensitive assay two synthetic peptides derived from the sequence surrounding the -r-EGE cleavage site in fl-endorphin were tested as substrates. One of these peptides Ac-Val-Thr-Leu-Phe-Lys-NHCH3 fulfilled all criteria for a suitable r-EGE substrate. The peptide was exclusively cleaved at the correct bond for -y-EGE upon incubation with brain synaptic membranes, and this cleavage was inhibited by the naturally occurring substrate &endorphin. The peptide was insensitive to cleavage by exopeptidases and cathepsin D. Addition of a r4C-labeled methyl group at the lysine residue of this peptide by reductive methylation did not alter its properties as a substrate for -y-EGE activity. The use of the ?I-labeled peptide allowed sensitive quantitation of its radioactive products after simple separation by hydrophobic chromatography on minicolumns containing polystyrene beads. -r-EGE activity increased linearly with a protein concentration and incubation time. This assay can be used for reliable quantitation of r-EGE activity and permits investigations on the regulation of y-endorphin production.
The Leu17-Phe18 bond of beta-endorphin is cleaved by a specific endopeptidase that generates the biologically active peptide gamma-endorphin. gamma-Endorphin-generating endopeptidase (gamma EGE) activity was determined by a radiometric assay, using as substrate a radioactively labeled, N- and C-terminally protected pentapeptide: Ac-Val-Thr-Leu-Lys( [14C]CH3)2-NHCH3, a derivative of beta-endorphin-(15-19). Here we report the tissue distribution of gamma EGE activity and its cellular localization in the testis. gamma EGE activity was present in the cytosolic fraction of most tissues. Highest specific activity occurred in the testis, ovary, and the uterus (10-16 nmol X mg protein-1 X h-1). In testis highest specific gamma EGE activity was found in the tubules (42 nmol X mg protein-1 X h-1) and lowest in Leydig cells (8 nmol X mg protein-1 X h-1). Further fractionation of the tubules showed that the germinal cell fraction had a higher specific activity (24 nmol X mg protein-1 X h-1) than the Sertoli cell fraction (8 nmol X mg protein-1 X h-1). In testis depleted of the germinal cells by prenatal irradiation of the rat or hypophysectomy, specific activity of gamma EGE activity decreased 50-fold and 4-fold, respectively. In testis depleted of Leydig cells by treatment of rats with ethane dimethyl sulfonate, specific gamma EGE activity did not decrease. Adrenalectomy had no effect on the enzyme activity. The results suggest that the germinal cells are sites of processing of beta-endorphin into alpha- and gamma-endorphins. It is concluded that 1) gamma EGE activity is widely distributed in tissues; 2) highest gamma EGE activity is located in reproductive tissues; and 3) in the testis gamma EGE activity is mainly associated in the germinal cells.
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