Purification and characterization of an enkephalin aminopeptidase from rat brain.
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 mumol 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 molecular weight of 115000 on Sephadex G-200 and a molecular weight of 102000 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 degrees C. Enzyme activity was inhibited by metal chelators, and it was found that 1 mol of Zn2+ was associated with 1 mol of enzyme (102000 Mr). 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.
D000626:Aminopeptidases / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004744:Enkephalin, Methionine; D004745:Enkephalins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature; D015032:Zinc / Q000032:analysis
D004745:Enkephalins; D004744:Enkephalin, Methionine; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme; D015032:Zinc