Abstract
A new type of dipeptidyl aminopeptidase, which releases basic aminoacyl dipeptides from the NH2-terminal end of oligopeptides, was purified about 2100-fold with 6.8% recovery from rat brain membranes by column chromatography on Cellex D, Arg-Tyr-AH-Sepharose 4B, hydroxylapatite, and Sephadex G-75, after the membranes were solubilized with Nonidet P-40. Activity was assayed by high performance liquid chromatography (HPLC) using Arg0-Met5-enkephalin (Arg0-enk)* as substrate in the presence of bestatin, thiorphan, and captopril. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme is apparently homogeneous with a mass of 64,000 daltons. This thiol enzyme is optimally active at pH 7 and is selectively activated by Mn(II). It loses 94% of its activity after EDTA treatment and can be reactivated by Mn(II), Co(II), and Zn(II). It splits Arg0-enk into equimolar amounts of Arg-Tyr and Gly-Gly-Phe-Met with a Km of 100 microM, and Vmax of 3.8 mumol/mg of protein/min. Dipeptidyl aminopeptidase does not hydrolyze model substrates for dipeptidyl aminopeptidases I, II, III, and IV, aminoacyl beta-naphthylamides, actin, desmin, tubulin, glial fibrillary acidic protein, and cytoskeletal neurofilament proteins. The enzyme is insensitive to puromycin, but is inhibited by several neuropeptides. Angiotensin III is the most potent with a Ki of 0.3 microM. Substrate specificity, pH optimum, molecular weight, activators, and catalytic site demonstrate that this enzyme is distinct from dipeptidyl aminopeptidases previously described.
Highlights
From the PeptideResearch Laboratory, Neurochemistry Division, NathanS
A new type of dipeptidyl aminopeptidase, which re- Based on substrate specificity with unique naphthylamide leases basic aminoacyl dipeptides from the NHder- substrates andspecific pH optima, four distinct enzymes with minal end of oligopeptides, was purified about 2100- DAP activity have been purified from liver, brain, and pituifold with 6.8%recovery from rat brain membranes by column chromatography on Cellex D, Arg-Tyr-AHSepharose 4B, hydroxylapatite, and Sephadex 6-75, after the membranes were solubilized with Nonidet P40
While numerous peptides in the brain may function as neurotransmitters, neurohormones, or hormones (l),the physiological role of particular peptidases in regulating neuropeptide formation and disposition is unclear [2]
Summary
From the PeptideResearch Laboratory, Neurochemistry Division, NathanS. Kline Institutefor Psychiatric Research, Orangeburg,New York 10962 and the Department of Psychiatry, New York University Medical Center, New York,New York 10016. Insodium dodecyl sulfate- the presence of carboxypeptidase B [9].DAP-I11most prefers polyacrylamide gel electrophoresis, the purified enzyme is apparently homogeneouswith a massof 64,000 daltons. This thiol enzyme is optimally active at pH 7 and is selectively activated by Mn(I1). It loses 94%of its activity after EDTA treatment and can be reactivated by Mn(II), Co(II), and Zn(I1) It splits Argo-enk into equimolar amounts of Arg-Tyr and Gly-Gly-PheMet with a K,,, of 100 p ~ an, d V,,, of 3.8 pmol/mg of protein/min. UsingArgoenk as the substratien the presence of the peptidase inhibitors bestatin, captopril, and thiorphan, we have purified to near homogeneity aneutral dipeptidyl aminopeptidase from rat brain membranes which degrades several neuropeptides with a basic amino acid at theNH, terminus.
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