title: Prodynorphin processing by rat CNS fractions and purified enzymes: Formation of Dynorphin A 1-8 by sulfhydryl-activated carboxypeptidase and peptidyl dipeptidase.
abstract: The conversion of selected prodynorphin fragments to form the octapeptide Dynorphin A 1-8 was studied in rat brain or spinal cord fractions, and the results compared to the action of purified carboxypeptidases and angiotension converting enzyme. The particulates were shown to convert Dynorphin A or 1-13 to the octapeptide as measured by radioimmunoassay, and by reverse phase high performance liquid chromatography. Detergent extracts of these particulates contained and enzyme converting 1-13 to 1-12 with release of C-terminal lysine, and active over a wide pH range of 4.8-7.6. Purification of these extracts by affinity chromatography (p-amino-benzoyl-arginine-Sepharose-6B) using Bz-Ala-Arg as the substrate led to isolation of a carboxypeptidase converting 1-13 to 1-12 active over the same pH range. Since Dynorphin 1-13 was converted to 1-8 by the consecutive use of purified carboxypeptidase B and angiotensin converting enzyme, the possibility exists that this mechanism might account for some octapeptide production in situ. The properties and substrate specificity of the carboxypeptidase B were compared to a carboxypeptidase A active optimally at pH 5.5 and assayed with Z-Glu-Tyr. The carboxypeptidase B acted only on prodynorphins with C-terminal basic residues as contrasted to a nonspecific action by the carboxypeptidase A. The carboxypeptidase B was characterized by a strong activation by -SH agents and Zn(2+), and thus could be differentiated from other opioid converting enzymes. The enzyme was inhibited by guanidinoethyl succinic acid (GEMSA), and p-chloromercuriphenyl-sulphonic acid (PCMS) but not by benzylsuccinic acid, a potent inhibitor of carboxypeptidase A.
authors.affiliation: The Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York, NY 10035, U.S.A.
authors.forename: N
authors.initials: N
authors.lastname: Marks
authors.forename: M J
authors.initials: MJ
authors.lastname: Berg
authors.forename: M
authors.initials: M
authors.lastname: Benuck
authors.forename: E S
authors.initials: ES
authors.lastname: Lo
authors.forename: H
authors.initials: H
authors.lastname: Novachenko
authors.forename: C A
authors.initials: CA
authors.lastname: Seyfried