The identification and characterization of two separate carboxylesterases in guinea-pig serum

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Abstract
The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 .mu.mol of substrate/min per mg of protein, respectively. The esterases were labeled with [3H]DFP and separated electrophoretically on sodium dodecyl sulfate[SDS]/polyacrylamide gels. Two main labeled bands were detected: bands I had MW 80,000 and bound 18-19 pmol of [3H]DFP/mg of protein, and band II had MW 58,000 and bound 7 pmol of [3H]DFP/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labeling of bands I and II. The residual labeling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DFP labeling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DFP-labeled and uninhibited partially purified enzyme preparation revealed that there were at least 2 separate carboxylesterases, which had pI [isoelectric point] 3.9 and pI 6.2, a cholinesterase enzyme (pI 4.3) and an unidentified protein that reacts with [3H]DFP and has a pI 5.0. SDS/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI 3.9 and pI 6.2 corresponded to the 80,000-MW subunit (band I) and 58,000-MW subunit (band II). The cholinesterase enzyme was also composed of 80,000-MW subunis (i.e., the residual labeling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI 5.0 corresponded to the residual labeling in band II (MW 58,000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. The carboxylesterase activity of guinea-pig serum is the result of at least 2 separate and distinct enzymes.