Human monocyte carboxylesterase. Purification and kinetics.

Area of Science:

• Biochemistry
• Enzymology
• Cell Biology

Background:

• Carboxylesterase (CBE) is an enzyme found in human monocytes.
• Understanding CBE's structure and function is crucial for its role in cellular processes.

Purpose of the Study:

• To isolate and purify membrane-localized carboxylesterase (CBE) from human peripheral blood monocytes.
• To characterize the biochemical properties, including molecular weight, quaternary structure, isoenzymes, substrate specificity, and inhibitor interactions of purified human monocyte CBE.

Main Methods:

• Monocyte isolation using density gradient and counterflow centrifugation elutriation.
• Enzyme purification via nonionic detergent extraction, ion exchange, gel filtration, hydroxylapatite chromatography, and HPLC.
• Analysis of enzyme properties using SDS-PAGE, native gel filtration, and spectrophotometric assays.

Main Results:

• Purified human monocyte CBE has a molecular weight of 60,000 Da as a monomer and 200,000 Da as a trimer.
• At least four major isoenzymes were identified with isoelectric points between 7.5 and 7.8.
• The enzyme specifically hydrolyzes short-chain alpha-naphthyl, o-nitrophenyl, and p-nitrophenyl esters, activated by short-chain alcohols, and inhibited by organophosphorus compounds and other agents.

Conclusions:

• The purification and characterization of human monocyte CBE provide a foundation for further studies on its structure and regulatory mechanisms.
• The identified isoenzymes and substrate specificities offer insights into CBE's diverse functions.