Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level.
A radioisotopic method for the assay of reduced or oxidized pyridine nucleotides, based on the interconversion of 2-[U-14C]ketoglutarate or 2-keto[3,4-3H]glutarate and labelled L-glutamate in the reaction catalyzed by glutamate dehydrogenase, was applied to the measurement of lactate dehydrogenase activity in rat pancreatic islet homogenates. Using the tritiated tracer, the limit of sensitivity of the procedure for NAD(P)H assay was close to 1.0 fmol/sample, and lactate dehydrogenase activity could be measured in as little as 0.0005 islet/sample i.e., at a single cell level. This radioisotopic procedure, which can be used for the assay of various metabolites and enzymic activities, thus provides a tool for investigating the heterogeneity in metabolic behaviour of individual cells.
Laboratory of Experimental Medicine, Brussels Free University, Belgium.
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Zähner
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Sener
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Malaisse
D000818:Animals; D002250:Carbon Radioisotopes; D055598:Chemical Phenomena; D002621:Chemistry; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007515:Islets of Langerhans / Q000201:enzymology*; D007656:Ketoglutaric Acids / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000032:analysis*; D009994:Osmolar Concentration; D011874:Radiometry / Q000379:methods; D051381:Rats; D011919:Rats, Inbred Strains; D012680:Sensitivity and Specificity
D002250:Carbon Radioisotopes; D007656:Ketoglutaric Acids; D009249:NADP; D007770:L-Lactate Dehydrogenase; D005969:Glutamate Dehydrogenase