Purification and characterization of esterase 6A, a trimeric esterase of the house mouse (Mus musculus).

Abstract

Esterase 6A was isolated from mouse lung and purified 440-fold by ion-exchange chromatography, inverse ammonium sulphate gradient solubilization, gel filtration and isoelectric focusing. The resultant product was apparently homogenous by the criteria of polyacrylamide gel electrophoresis and immunodiffusion, and consisted of the electrophoretic form 6A3. A single species of subunit was present on sodium dodecyl sulphate gel electrophoresis. The molecular weight of the native protein was found to be about 178,000 with a subunit molecular weight of about 60,000. The equivalent weight obtained by active-site titration with diethyl-p-nitrophenyl phosphate was approximately 178,000 g/mol, indicating a functional asymmetry in the trimer. The enzyme was shown to have a high affinity for 4-nitrophenyl hexanoate (Michaelis constant Km = 4.4 mumol/l) with a relatively low catalytic efficiency (catalytic constant kcat = 12 s-1). Esterase 6A was immunologically related to esterase 1 and esterase 9, with which it is genetically closely linked. Further properties of the three esterases were compared.