CPT-11 Converting Enzyme from Rat Serum: Purification and Some Properties.

Abstract

A rat serum enzyme that catalyzes the conversion of a pro-drug, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), to an anticancer drug, 7-ethyl-10-hydroxycamptothecin (SN-38), was purified and its properties were characterized. The enzyme was purified by column chromatography on diethylaminoethyl Toyopearl 650M, QAE-Sephadex, Sephadex G-150, Con A-Sepharose and high performance liquid chromatography with an ion-exchanger column. It was most active at pH 7.5 and was stable at pH 4-9 for 1 h at 30 degrees C. The molecular weight was estimated to be 60 and 57 kDa by gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis methods, respectively, and the isoelectric point was 4.6, as determined by isoelectric focusing. The Km value for CPT-11 was 0.28 microM. This enzyme was inhibited by diisopropyl phosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF) but insensitive to eserine, p-chloromercuribenzoate (PCMB) and ethylenediaminetetraacetate (EDTA). The enzyme also hydrolyzed p-nitrophenylacetate (p-NPA), a commonly used substrate for esterases, but was not active toward acetylcholine, suggesting that the enzyme is a carboxylesterase[EC 3.1.1.1]. During the hydrolyses of CPT-11 and p-NPA, an initial burst phenomenon similar to that found in the alpha-chymotrypsin-catalyzed hydrolysis of p-NPA was observed. Kinetic analysis revealed that the deacylation of the enzyme is the rate-limiting step in substrate hydrolysis. This enzyme was found to also split other ester derivatives of SN-38 besides CPT-11.