Proteolytic conversion of [Met]enkephalin-Arg6-Gly7-Leu8 by brain synaptic membranes. Characterization of formed peptides and mechanism of proteolysis.
The degradation of the enkephalin-containing octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu (YGGFMRGL) was systematically investigated by incubating the peptide with synaptic membranes from rat striatum or with purified peptidases. The degradation products were derivatized with 4-dimethylamino-azobenzene-4'-isothiocyanate and then analyzed by high pressure liquid chromatography and by amino-terminal analysis. The incubation of YGGFMRGL with synaptic membranes yielded YGG, YGGF, YGGFM, and MR in a manner that was linear with respect to time. The corresponding carboxyl-terminal fragments FMRGL, MRGL, and RGL could not be detected, which suggests that the degradation of YGGFMRGL by synaptic membranes occurs by carboxypeptidase activity. The incubation of YGGFMRGL with different purified peptidases produced cleavage patterns unique from that seen with synaptic membranes. Enkephalinase recognized only the Gly-Phe bond to produce YGG and FMRGL. Thermolysin recognized the Gly-Phe bond and the Phe-Met bond to yield YGG, YGGF, FMRGL, and MRGL. Angiotensin-converting enzyme (ACE) produced primarily YGGF, MR, and lesser amounts of YGGFMR and YG. The formation of YGG, YGGF, and YGGFM by synaptic membranes could be stimulated 3-fold by the addition of 30 mM NaCl and inhibited by MK-422, an ACE inhibitor, with an IC50 of 3 nM. These data suggest that ACE, a dipeptidyl carboxypeptidase, is the primary enzyme involved in the degradation of YGGFMRGL in brain. ACE apparently works in concert with another carboxypeptidase in brain to yield YGGFM and YGG since the carboxyl-terminal peptides RGL and FMRGL could not be detected.
Norman
J A
JA
Chang
J Y
JY
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000494:pharmacology; D015773:Enalaprilat; D010450:Endopeptidases / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D008297:Male; D015260:Neprilysin; D010446:Peptide Fragments / Q000032:analysis; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology; D013570:Synaptic Membranes / Q000378:metabolism*
D004151:Dipeptides; D010446:Peptide Fragments; D012965:Sodium Chloride; D004744:Enkephalin, Methionine; C033453:enkephalin-Met, Arg(6)-Gly(7)-Leu(8)-; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; D015260:Neprilysin; D015773:Enalaprilat