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CPT-11 converting enzyme from rat serum: purification and some properties
Journal of Pharmacobio-Dynamics
|June 1, 1991
Summary
Researchers purified a rat serum enzyme that converts the pro-drug CPT-11 into the anticancer drug SN-38. This carboxylesterase enzyme
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- The pro-drug 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11) is converted to its active anticancer form, 7-ethyl-10-hydroxycamptothecin (SN-38), by serum enzymes.
- Understanding the properties of this activating enzyme is crucial for optimizing CPT-11-based cancer therapies.
Purpose of the Study:
- To purify and characterize the rat serum enzyme responsible for CPT-11 activation.
- To determine the enzyme's kinetic properties and identify its class.
Main Methods:
- Enzyme purification using various chromatography techniques (DEAE-Toyopearl, QAE-Sephadex, Sephadex G-150, Con A-Sepharose, ion-exchange HPLC).
- Characterization of enzyme activity, stability, molecular weight, and isoelectric point.
- Kinetic analysis using CPT-11 and p-nitrophenylacetate (p-NPA) as substrates, and inhibition studies with specific reagents.
Main Results:
- The purified enzyme exhibits optimal activity at pH 7.5 and is stable between pH 4-9.
- Molecular weight was estimated at 60 kDa (gel filtration) and 57 kDa (SDS-PAGE); isoelectric point is 4.6.
- The enzyme is identified as a carboxylesterase (EC 3.1.1.1), with a Km of 0.28 µM for CPT-11, inhibited by DFP and PMSF, and shows a burst phenomenon in hydrolysis kinetics, indicating deacylation as the rate-limiting step.
Conclusions:
- A rat serum carboxylesterase was successfully purified and characterized.
- This enzyme plays a key role in converting CPT-11 to SN-38 and can hydrolyze other SN-38 ester derivatives.
- The findings provide insights into the enzymatic activation of CPT-11 and potential targets for drug metabolism studies.