Park7 (DJ-1), a Parkinson-associated glyoxalase/deglycase: characterization of enzymatic activity in biological...

Glycation is an inevitable nonenzymatic covalent reaction between nucleophilic groups in proteins and nucleotides, and endogenous reducing sugars or dicarbonyls (e.g., methylglyoxal) leading to protein and/or nucleotide covalent modifications. This process initiates a series of dehydrations, oxidations and rearrangements that lead to a myriad of products called advanced glycation end products (AGEs). Previously, we showed that Park7 (or DJ-1) is a protein deglycase that repairs methylglyoxal-glycated biomolecules by acting on early glycation intermediates and releasing the repaired biomolecule and lactate. Moreover, the PARK7 gene is associated with recessive and sporadic forms of Parkinson's disease (PD). Thus, over the last few years, the function of Park7 has become an important research topic for understanding the etiology of PD. In this context, the development of sensitive enzymatic assays is crucial to quantify the activity of Park7 and develop specific modulators. Here, we describe a new method for measuring Park7 activity based on the separation and quantification of glycated and unglycated fluorescent nucleotides by Liquid Chromatography (LC). Kinetic and mechanistic analyses using human recombinant Park7 confirmed the reliability of this approach. In addition, this assay was further validated using cellular lysates. Our results indicate that this novel Park7 assay is simple, sensitive, and specific.