Giafi, Chrysanthi Foteini; (1998) Characterisation of human D-glycerate dehydrogenase/glyoxylate reductase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The enzyme D-glycerate dehydrogenase (D-GDH), which is also believed to have gloxylate reductase (GR) activity, plays a role in serine catabolism and glyoxylate metabolism and deficiency of this enzyme is believed to be the cause of primary hyperoxaluria type 2 (PH2). The pH optima and kinetic parameters of D-GDH and GR in human liver have been determined and assays developed for their measurement. A reference range was established for the activity of both enzymes in normal liver. D-GDH activity in kidney, leucocytes and fibroblasts fell within the range of values seen in the liver but GR activity was approximately 30% in the kidney and barely detectable in leucocytes and fibroblasts. Analysis of liver and leucocyte samples from patients with PH2 showed that GR activity was either very low or undetectable, while D-GDH activity was reduced in liver but within the normal range in leucocytes. D-GDH was partially purified from human liver. The purification methods included negative adsorption to DEAE Sephadex pH 7, precipitation with ammonium sulphate, elution from CM Sephadex pH 6 with 150 mM sodium chloride, chromatofocusing using a pH gradient 7.2-5 and fractionation on a 2'5' ADP Sepharose column. D-GDH separated into two peaks on the chromatofocusing column. The first peak, with D-GDH activity only, was not retained by the column, whereas the second peak with D-GDH and GR activity eluted with a pI ~ 6.7. These results suggest that liver contains mere than one protein with D-GDH activity. These observations were reinforced by the finding of only a single, non-retained peak of D-GDH activity when a leucocyte preparation was applied to the chromatofocusing column. Antibodies were raised in guinea pigs against the mixture of six proteins which eluted together from ADP sepharose. In non-denatured samples the antibodies recognised a protein of 71 kDa that was observed only faintly in the liver of a patient with PH2. On denaturation the signal at 71 kDa disappeared and a weaker signal at 37 kDa appeared. Peroxisomes and mitochondria of HepG2 cells, rat hepatocytes and human leucocytes were isolated. In all cases over 80% of D-GDH and GR activity was found in the cytosol.

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