Measurement of alpha-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography.
A new method for the determination of the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4 degrees C and for a week when stored at -20 degrees C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added alpha-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 +/- 0.9, 18.1 +/- 2.8, and 2.6 +/- 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.
Shylaja
N
N
Department of Pediatrics, Nagoya University School of Medicine, Japan.
Maehara
M
M
Watanabe
K
K
D000214:Acyl Coenzyme A / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005849:Gerbillinae; D006801:Humans; D007655:Ketoglutarate Dehydrogenase Complex / Q000097:blood / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*
D000214:Acyl Coenzyme A; C012046:succinyl-coenzyme A; D007655:Ketoglutarate Dehydrogenase Complex