J. Biol. Chem., Vol. 263, Issue 14, 6613-6618, May, 1988

A novel dipeptidyl aminopeptidase in rat brain membranes. Its isolation, purification, and characterization

KS Hui
Peptide Research Laboratory, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York 10962.

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.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K.-S. Hui, M. Saito, and M. Hui A Novel Neuron-specific Aminopeptidase in Rat Brain Synaptosomes. ITS IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION J. Biol. Chem., November 20, 1998; 273(47): 31053 - 31060. [Abstract] [Full Text] [PDF]