Action of surfactants on porcine heart malate dehydrogenase isoenzymes and a simple method for the differential assay of these isoenzymes.
The cationic surfactant, cetyl (hexadecyl) trimethylammonium bromide (CTAB), completely inactivates porcine heart cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) at concentrations (of surfactant) which do not affect the activity of the mitochondrial isoenzyme. These concentrations are close to, or higher than, the critical micelle concentration of CTAB. An increase in the ionic strength of the medium significantly retards the CTAB-induced inactivation of the cytoplasmic enzyme. The enzyme is also markedly protected against CTAB inactivation by NADH; L-malate on its own has no effect but a combination of NADH and L-malate affords greater protection than NADH alone. The CTAB inactivation is not reversed by dilution of the surfactant. The highly selective action of CTAB on the two malate dehydrogenases, which correlates well with their electrostatic charges, has been exploited for a simple and reliable differential assay of these isoenzymes. The anionic surfactant, sodium dodecyl sulphate (SDS), at concentrations well below the critical micelle concentration, inactivates both isoenzymes, but the mitochondrial enzyme is significantly more sensitive than its cytoplasmic counterpart. There is thus some correlation, though not as strong as with CTAB, between SDS inactivation and the charges of the two malate dehydrogenases. An increase in ionic strength has opposite effects on the two isoenzymes: the mitochondrial enzyme becomes more resistant and the cytoplasmic enzyme less so. Both isoenzymes are rendered more resistant to SDS by the inclusion of NADH. Inactivation of the enzymes caused by short exposure to SDS is largely reversed by dilution of the detergent, but longer exposure leads to progressive irreversible loss of activity. NADH very effectively protects the isoenzymes against irreversible inactivation. It is likely that a reversible phase of inactivation precedes an irreversible phase and that in the former phase SDS acts competitively with NADH. Both malate dehydrogenases possess considerable resistance to the nonionic detergent, Triton X-100.
D000818:Animals; D002021:Buffers; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors*; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology*; D013552:Swine
D002021:Buffers; D002593:Cetrimonium Compounds; D007527:Isoenzymes; D013501:Surface-Active Agents; D009243:NAD; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; D008291:Malate Dehydrogenase; D000077286:Cetrimonium