Fumarase assay

Following are a set of assay conditions for marker enzymes of mitochondria (citrate synthetase, malic dehydrogenase, fumarase, and succinate dehydrogenase) and glyoxysomes (citrate synthetase, malate synthetase, and malic dehydrogenase). Some or all of these activities may be assayed across the density gradient. Their quantitative distribution is shown in Table 9-2. 

Fumarase. Fumarase is determined by measuring the conversion to malate to fumarate. This assay was used in the opposite direction by Racker. The mixture contains, in a volume of 1.3 ml, 8.0 x 10 phosphate buffer (adjusted to pH 7.5), 4.0mM dithiothreitol, 8.0mM sodium malate, and 2 to 25 fxg of protein. The reaction is initiated with malate and is followed at 240 nm. The molar extinction coefficient of fumarate is 2.6 X lO cm /mole. 

In addition to assays for the OXPHOS enzymes, assays have been described for many other enzymes involved in the MEGS. For PDHc, various types of diagnostic assays have been described and are widely used (33). Also for the TCA cycle enzymes various assays have been described, although to date, pathogenic defects have only been found in a-ketoglutarate dehydrogenase (38), succinate-CoA ligase (12), and fumarase (39). 

C-IO) Fumarase deficiency. There is a deficit in the transformation of fumarate to malate. The infant has developmental retardation, with abnormal neuromuscular function, lactic acidemia, and fumarate aciduria. The lactic acidosis may result from a backup of Krebs cycle function, all the way to lactate. Lactic acidosis may also be present in rare disorders of cytochrome oxidase activity. Diagnostically, there is a deficit in fumarase activity in assay of liver and skeletal muscle mitochondria. 

Preparation and fractionation of protoplasts were done according to (6). The activities of SHMT, Cytochrome c oxidase and fumarase were assayed according to (6). 

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