Detection and analysis of neutral endopeptidase from tissues with substrate gel electrophoresis.
Neutral endopeptidase from human or bovine tissues retains enzymatic activity following electrophoresis and immobilization in polyacrylamide gels. Infiltration of the gel with a fluorogenic substrate permits identification of the active enzyme by fluorescence associated with a distinct protein band. This technique both separates and identifies the enzymatically active species from a crude cell membrane fraction or from partially purified extracts that contain contaminating proteins. Enzymatic activity is quantitated by photographing the fluorescent bands and scanning the negatives with a laser densitometer. Because as little as 25 ng of enzyme can be detected by this method, it could be used where the amount of material is limited.
Sullivan
J
J
Department of Biochemistry, University of Texas Health Center, Tyler 75710.
Johnson
A R
AR
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D005333:Fetus; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008022:Ligaments / Q000201:enzymology*; D008970:Molecular Weight; D015260:Neprilysin / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D015260:Neprilysin