A SOLUBLE METALLOENDOPEPTIDASE FROM RAT BRAIN: POSSIBLE FUNCTION IN NEUROPEPTIDE PROCESSING AND DEGRADATION.
Item
-
Title
-
A SOLUBLE METALLOENDOPEPTIDASE FROM RAT BRAIN: POSSIBLE FUNCTION IN NEUROPEPTIDE PROCESSING AND DEGRADATION.
-
Identifier
-
AAI8501122
-
identifier
-
8501122
-
Creator
-
CHU, THOMAS GERALD.
-
Contributor
-
M. Orlowski
-
Date
-
1984
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biology, General
-
Abstract
-
A metalloendopeptidase, optimally active at a neutral pH, was purified from the soluble fraction of rat brain homogenates. The enzyme (M(,r) about 67,000) is strongly inhibited by metal chelators such as EDTA and o-phenanthroline. An EDTA-treated enzyme can be reactivated by several divalent metal ions. The enzyme preferentially cleaves peptides having hydrophobic amino acid residues in the P(,3)' position and also in the P(,1) and P(,2) position. Substitution of a D-amino acid residue in either position P(,1) or P(,2)' renders the substrate resistant to hydrolysis. Specificity studies suggest the presence of an extended active site, binding a minimum of five amino acid residues. Bioactive peptides are hydrolyzed by the enzyme at sites consistent with the specificity deduced from the studies with synthetic substrates. Dynorphin(1-8), (alpha)-neo-endorphin, (beta)-neo-endorphin and BAM-12P are hydrolyzed to form enkephalins. Kinetic studies indicate a high affinity of the enzyme towards dynorphin(1-8), (beta)-neo-endorphin, bradykinin and neurotensin. The enzyme exhibits high activity in brain, testes, anterior pituitary and posterior pituitary. Other tissues show activity 10 to 28% of that in the brain.;A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site zinc atom were synthesized. One of these, N-{lcub}1(R,S)-carboxy-2-phenylethyl{rcub}-Ala-Ala-Phe-p-aminobenzoate, was found to be a potent competitive inhibitor of the enzyme with a K(,i) of 1.94 uM. Of the two disastereomers, the more potent diastereomer had a K(,i) of 0.81 uM. Effective inhibition requires the presence in the inhibitor of a group binding to the substrate recognition site of the enzyme and a group capable of coordinating with the active site metal atom. A hydrophobic residue in the inhibitor binding to the S(,1) subsite of the enzyme greatly increases inhibitory potency. The high activity of this enzyme in brain and pituitary, its preference for oligopeptides as substrates, its ability to generate enkephalins from several opioid peptides as well as its ability to degrade several neuropeptides, and its absence of activity towards large proteins and peptides suggests that the enzyme functions in the metabolism of neuropeptides.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
-
Program
-
Biomedical Sciences
