The assay of Met-enkephalin aminopeptidase with [125I]Met-enkephalin.
Presented here are procedural modifications which permit the utilization of 125I-labeled Met-enkephalin as substrate in the assay of rat brain enkephalin aminopeptidase. Th hydrolysis of enkephalin is monitored by the release of [125I]tyrosine separated on Porapak Q. The release of tyrosine is proportionate with both increasing time and tissue concentration. The estimated Km is near 10(-4) M and the enzyme activity can be inhibited more than 95% with puromycin. The majority of the enzyme activity remains in the 100,000 x g supernatant following differential centrifugation.
D000626:Aminopeptidases / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004723:Endorphins / Q000378:metabolism*; D004744:Enkephalin, Methionine; D004745:Enkephalins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008722:Methods; D011691:Puromycin / Q000494:pharmacology; D051381:Rats; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D004723:Endorphins; D004745:Enkephalins; D014443:Tyrosine; D011691:Puromycin; D004744:Enkephalin, Methionine; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme