Identification of serine esterases in tissue homogenates.
Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential 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 enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.
Department of Biochemistry, University of Kansas, Lawrence, Kansas, 66049, USA.
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Keshavarz-Shokri
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Suntornwat
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Kitos
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D002091:Butyrylcholinesterase / Q000378:metabolism; D002642:Chick Embryo; D002918:Chymotrypsin / Q000378:metabolism; D004950:Esterases / Q000378:metabolism*; D007531:Isoflurophate / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D007531:Isoflurophate; D004950:Esterases; C022635:serine esterase; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D002918:Chymotrypsin; D014357:Trypsin