Purification and characterization of an enkephalin aminopeptidase from rat brain

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Abstract
Rat brain enkephalin aminopeptidase was purified to apparent electrophoretic homogeneity. Enzyme activity was monitored during the purification by using ([3,5-3H2]Tyr) Met-enkephalin and Tyr-.beta.-naphthylamide as substrates. It was shown that the enzyme activities resulting in hydrolysis of the tyrosine residue of ([3,5-3H2]Tyr)Met-enkephalin and formation of .beta.-naphthylamine from Tyr-.beta.-naphthylamide copurified. The homogeneous enzyme had a specific activity of 10.5 .mu.mol of .beta.-naphthylamide hydrolyzed min-1 mg-1. Hydrolysis of Met-enkephalin yielded the products L-tyrosine and the tetrapeptide Gly-Gly-Phe-Met. Subsequent removal of glycine from Gly-Gly-Phe-Met was not observed with the purified enzyme. The homogeneous aminopeptidase has an apparent MW of 115,000 on Sephadex G-200 and a MW of 102,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enkephalin-degrading enzyme had a pH optimum of 6.5-7.0 and exhibited maximal activity at 40.degree. C. Enzyme activity was inhibited by metal chelators, and it was found that 1 mol of Zn2+ was associated with 1 mol of enzyme (102,000 MW). The enzyme hydrolyzes various neutral and basic amino acid .beta.-naphthylamides but will not utilize acidic, D-amino acid, or N-terminal-blocked amino acid .beta.-naphthylamides as substrates.