Proctolin degradation by membrane peptidases from nervous tissues of the desert locust (Schistocerca gregaria).
The hydrolysis of the insect neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) by enzyme preparations from the nervous tissue of the desert locust (Schistocerca gregaria) was investigated. Neural homogenate degraded proctolin (100 microM) at neutral pH by cleavage of the Arg-Tyr and Tyr-Leu bonds to yield Tyr-Leu-Pro-Thr, Arg-Tyr and free tyrosine. Arg-Tyr was detected as a major metabolite when the aminopeptidase inhibitors amastatin and bestatin were present to prevent Arg-Tyr breakdown. Around 50% of the proctolin-degrading activity was isolated in a 30,000 g membrane fraction and was shown to be almost entirely due to aminopeptidase activity. The aminopeptidase had an apparent Km of 23 microM, a pH optimum of 7.0 and was inhibited by 1 mM-EDTA and amastatin [IC50 = 0.3 microM], but was relatively insensitive to bestatin, actinonin and puromycin. Phenylmethanesulphonyl fluoride (1 mM) and p-chloromercuriphenylsulphonic acid (1 mM) had no effect on this enzyme activity. Although the bulk of the Tyr-Leu hydrolytic activity was located in the 30,000 g supernatant, some weak activity was detected in a washed membrane preparation. This peptidase displayed a high affinity for proctolin (Km = 0.35 microM) and optimal activity at around pH 7.0. Synaptosome- and mitochondria-rich fractions were prepared from crude neural membranes. The aminopeptidase activity was concentrated in the synaptic-membrane preparation, whereas activity giving rise to Arg-Tyr was predominantly localized in the mitochondrial fraction. The subcellular localization of the membrane aminopeptidase is consistent with a possible physiological role for this enzyme in the inactivation of synaptically released proctolin.
D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006110:Grasshoppers / Q000378:metabolism*; D006868:Hydrolysis; D008297:Male; D009417:Nerve Tissue / Q000378:metabolism*; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D009479:Neuropeptides; D018377:Neurotransmitter Agents; D009842:Oligopeptides; C011536:proctolin; D010447:Peptide Hydrolases; D000626:Aminopeptidases