Photoaffinity labelling of mitochondrial NADH: ubiquinone reductase with pethidine analogues.
1. Chemically reactive derivatives of pethidine analogues--novel potent inhibitors of the mitochondrial NADH: ubiquinone reductase (complex I)--were synthesized. 2. Dose-response curves of these components revealed that the photoactivatable aryl azido derivative has retained most of the inhibitory activity displayed by the parent substance. After introduction of a radioactive iodine isotope into the molecule, it was used as a probe for the localization of the inhibitor binding polypeptides within complex I. 3. Photolysis of the radiolabelled derivative bound to isolated complex I both from Neurospora crassa and beef heart resulted in a covalent incorporation of the inhibitor into 6-7 individual subunits of the enzyme. Essentially the same labelling patterns were obtained, when whole mitochondrial membranes were incubated with the reactive derivative. 4. Applying a double isotope labelling technique, the inhibitor-binding polypeptides in N. crassa were identified as mitochondrially synthesized constituents of complex I (ND gene products). In the beef heart enzyme the ND-1 product was detected to be among the polypeptides reacting with the inhibitor. 5. Competition experiments employing either NADH or decylbenzoquinone (DB), together with the pethidine analogue, showed that both enzyme substrates interfere specifically with the inhibitor binding to complex I.
Werner
S
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Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Universität München, Federal Republic of Germany.
D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008614:Meperidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone); D009492:Neurospora crassa; D010777:Photochemistry; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000201:enzymology*; D014451:Ubiquinone / Q000378:metabolism
D000345:Affinity Labels; D046911:Macromolecular Substances; D009243:NAD; D014451:Ubiquinone; D008614:Meperidine; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases