An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates.
A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.
Luo
Cheng
C
Institute for Nutritional Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, PR China.
Wang
Xuemin
X
Long
Jiangang
J
Liu
Jiankang
J
D000818:Animals; D001681:Biological Assay / Q000379:methods; D001921:Brain / Q000201:enzymology; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000201:enzymology; D006801:Humans; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000032:analysis*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D009243:NAD / Q000737:chemistry*; D009447:Neuroblastoma / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D013778:Tetrazolium Salts / Q000378:metabolism*
D013778:Tetrazolium Salts; D009243:NAD; C013574:iodonitrotetrazolium; D008291:Malate Dehydrogenase