Identification of serine esterases in tissue homogenates

Serine esterases react with [H-3]diisopropylphosphofluoridate ([H-3]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel el ectrophoresis. To identify an esterase or its catalytic subunit, a potentia l substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP, The nature of the enz yme could be inferred from the character of the substrates that suppress la beling. The validity of this analytical method was tested with two serine p roteases, trypsin and cu-chymotrypsin, and two serine esterases, acetylchol inesterase (AChE) and butyrylcholinesterase (BuChE), and several of their n atural or model substrates or inhibitors. Application of the method to comp lex biological systems was tested with chicken embryo brain microsomes. Try psin labeling with [H-3]DFP was suppressed by alpha-N-benzoyl-L-arginine et hyl ester (BAEE) and poly-L-lysine but not by benzoyl-L-tyrosine ethyl este r (BTEE). [H-3]DFP labeling of chymotrypsin was suppressed by both BALE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [H-3]DFP reacted with brain microsomes t o produce nine distinct radioactive bands, When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of onl y the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 8 5- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity. (C) 1999 Academic Press.