Hydrolysis of S-aryl-cysteinylglycine conjugates catalyzed by porcine kidney cortex membrane dipeptidase

Abstract

1. Following conjugation with glutathione, xenobiotics are converted into cysteinylglycine conjugates, cysteine conjugates, and finally, mercapturic acids. The structural factors determining the activities of dipeptidases for the metabolism of toxicologically-relevant cysteinylglycine conjugates are not well understood.

2. We purified porcine kidney cortex membrane dipeptidase (MDP) to homogeneity, via phosphatidylinositol-specific phospholipase C-mediated cleavage of the protein’s membrane anchor and cilastatin affinity chromatography. The homodimeric structure of the MDP protein was confirmed by mass spectrometry.

3. The cysteinylglycine conjugates of 1-(chloromethyl)naphthalene, 4-nitrobenzyl chloride, and 1-chloro-2,4-dinitrobenzene were synthesized and HPLC separation methods for their quantitation were developed. MDP catalyzed the hydrolysis of all three conjugates, but the rate of this activity was strongly dependent on the nature of the substituent on the cysteine sulfur atom.

Keywords::

• Cilastatin
• glutathione
• mercapturic acid pathway
• affinity chromatography
• menaphthylglutathione
• dinitrophenylglutathione
• enzyme kinetics

Acknowledgements

We gratefully acknowledge the assistance of Nikole Freeman; Joseph Chu and Dr. Frances J. Sharom (protein purification procedures); and Dr. Dyanne Brewer and Dr. Armen Charchoglyan (mass spectrometry). PDJ wishes to thank Karen Barker, who, as an undergraduate student, asked the questions that first prompted this line of research.

Declaration of interest

Research support was provided by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.