Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes.
Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9-22 independent cultures of amniocytes.
Chowdhury
S K
SK
Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 4, Prague, Czech Republic.
Drahota
Z
Z
Floryk
D
D
Calda
P
P
Houstek
J
J
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000653:Amniotic Fluid / Q000166:cytology* / Q000201:enzymology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002950:Citrate (si)-Synthase / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005260:Female; D005470:Fluorometry; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000378:metabolism; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption; D011048:Polarography; D011190:Potassium Cyanide / Q000494:pharmacology; D011247:Pregnancy; D013053:Spectrophotometry; D013384:Succinate Cytochrome c Oxidoreductase / Q000378:metabolism
D003574:Cytochrome c Group; D000244:Adenosine Diphosphate; D013384:Succinate Cytochrome c Oxidoreductase; D009245:NADH Dehydrogenase; D003576:Electron Transport Complex IV; D002950:Citrate (si)-Synthase; D014450:Electron Transport Complex III; D011190:Potassium Cyanide