title: 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.
authors.affiliation: School of Pharmaceutical Sciences, Nagasaki University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Tsuji
authors.forename: N
authors.initials: N
authors.lastname: Kaneda
authors.forename: K
authors.initials: K
authors.lastname: Kado
authors.forename: T
authors.initials: T
authors.lastname: Yokokura
authors.forename: T
authors.initials: T
authors.lastname: Yoshimoto
authors.forename: D
authors.initials: D
authors.lastname: Tsuru
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D000077146:Irinotecan; D007526:Isoelectric Point; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011355:Prodrugs / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D011355:Prodrugs; D000077146:Irinotecan; C072888:CPT-11 converting enzyme; D002265:Carboxylic Ester Hydrolases; D002166:Camptothecin