Interaction of tubulin with rat liver mitochondria.
Tubulin purified from rat brain was labeled by conjugation with N-succinimidyl 3-(4-hydroxy[5-125I]iodophenyl)propionate. Mitochondrial fraction prepared by centrifugation on sucrose density gradient was enriched about 4-fold in cytochrome c oxidase as compared to total liver homogenate. Contamination by plasma membranes was estimated to be about 5%. Radioiodinated pure tubulin bound to purified rat liver mitochondria; binding was time- and temperature-dependent: maximum binding was obtained after 45 min of incubation at 37 degrees C. Under conditions of binding, mitochondria retained their normal characteristics for phosphate accumulation. That binding actually occurs on mitochondria was demonstrated by the co-sedimentation of the tubulin binding and cytochrome c oxidase activities on sucrose gradient. Radioiodinated tubulin binding to mitochondria was specific and saturable. Saturation of binding was obtained using tubulin concentration ranging from 0.02 to 200 micrograms/ml. Hill plot and double reciprocal plot of binding data yielded values of 6 X 10(-8) M for an apparent KD and a maximal binding capacity of 1.4 nmol of tubulin/mg of mitochondrial protein. The Hill coefficient was 0.98 indicating that radioiodinated tubulin bound to a single class of noninteracting sites. The interaction between tubulin and mitochondria was reversible. Dissociation of the complex was obtained by dilution and by lowering the temperature. The dissociation of tubulin-mitochondria complexes was insensitive to ionic strength (0.1 to  M NaCl). Mild treatment of mitochondria by trypsin (5 min at 37 degrees C) decreased of tubulin binding, suggesting that protein component(s) of membranes are involved in the interaction of tubulin with mitochondria.
D000818:Animals; D001921:Brain; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D014404:Tubulin