The aims of the present study are (i) to purify a mitochondrial glyoxalase II to homogeneity for the first time from any organism and (ii) to compare its kinetic properties with those of the cytoplasmic enzyme. Both the cytoplasmic and the mitochondrial glyoxalases II from Saccharomyces cerevisiae, which are the products of two distinct genes, GLO2 and GLO4, were purified from yeast and in recombinant form from Escherichia coli. To obtain a higher protein yield (compared to wild-type expression) in yeast, the genes were placed under the control of the strong GAL1 promoter on a multicopy plasmid. Amino-terminal sequencing and molecular mass determination by MALDI-TOF mass spectrometry of the mitochondrial Glo4 protein revealed Met-11 of the primary translation product of the gene as the N-terminal amino acid. Judged by enzyme kinetic properties the recombinant and natural proteins were equivalent. The cytoplasmic and the mitochondrial enzyme differed in the pH dependence of the kinetic parameters for the main substrate, S-d-lactoylglutathione. Whereas the cytoplasmic protein showed a pronounced peak of enzyme activity between pH 7-8 and a continuous up to fivefold increase of the K(M) value with increasing pH (from 5. 5-9.0), the mitochondrial protein had a nearly constant K(M) value and an activity maximum over a broad pH range (6.5-9.0). The kinetic parameters (at pH 7.5) of both the cytoplasmic and the mitochondrial enzyme for S-D-lactoylglutathione were of the same order of magnitude as reported recently for the human and Arabidopsis thaliana enzymes which are presumably of cytoplasmic origin. However, both yeast enzymes showed a severalfold lower preference for the more hydrophobic substrate, S-d-mandeloylglutathione.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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