Human monocyte carboxylesterase. Purification and kinetics.
Human peripheral blood monocytes were isolated by density gradient centrifugation and purified by counterflow centrifugation elutriation. Membrane-localized carboxylesterase (CBE) was extracted with nonionic detergent (Triton X-100) and purified by ion exchange (DEAE-cellulose), gel filtration (Sephacryl S-300), hydroxylapatite column, and high performance liquid chromatography. The purified enzyme migrated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single protein band with a molecular weight of 60,000. Under nondenaturing conditions, monocyte CBE formed a trimer and eluted from a gel filtration column as a protein with an approximate molecular weight of 200,000. Electrophoretic patterns of the enzyme on polyacrylamide gels run a neutral pH did not vary during enzyme purification. At least four major isoenzymes of human monocyte CBE were observed with isoelectric points between 7.5 and 7.8. Pure human monocyte CBE hydrolyzed short chain alpha-naphthyl, o-nitrophenyl, and p-nitrophenyl esters. Amide esters and thioesters were not hydrolyzed by the enzyme. Short chain alcohols activated the enzyme and organophosphorus compounds, diphenyl carbonate, sodium fluoride, and phenylmethylsulfonyl fluoride inhibited the enzyme. EDTA and sulfhydryl reagents had no effect on enzyme activity. The amino acid content of the enzyme was consistent with other CBEs. Inhibitors reacted either with the active or effector site of the enzyme. Purified enzyme now permits the characterization of CBE structure and regulation.
Saboori
A M
AM
School of Hygiene and Public Health, Department of Environmental Health Sciences, Johns Hopkins University, Baltimore, Maryland 21205.
Newcombe
D S
DS
D000438:Alcohols / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes / Q000097:blood*; D007700:Kinetics; D008358:Mannose / Q000032:analysis; D008970:Molecular Weight; D009000:Monocytes / Q000201:enzymology*; D013379:Substrate Specificity
D000438:Alcohols; D000596:Amino Acids; D007527:Isoenzymes; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D008358:Mannose