[Elimination effect of ethyl alcohol on the DCCD-induced inhibition of hydrolytic activity of H+-ATPase complex].
12.5% ethyl alcohol was added into the reaction system containing mitochondrial H(+)-ATPase complex of pig heart, which was preincubated with 0.5 microgram/ml DCCD dicyclohexylcarbodiimide) at 30 degrees C. Or the DCCD and ethyl alcohol were simultaneously incubated with H(+)-ATPase at 30 degrees C. In either case, the inhibition of the hydrolytic activity of H(+)-ATPase caused by DCCD could be completely eliminated in the presence of ethyl alcohol. If methyl alcohol was instead of ethyl alcohol, the DCCD inhibition could only be partly eliminated. In the replacement of ethyl alcohol by dimethyl sulfoxide, no elimination could be observed. After preincubation of 2 micrograms/ml oligomycin with H(+)-ATPase complex instead of DCCD, the same concentration of ethyl alcohol could not caused elimination effect, which indicates no un-coupling effect happened by ethyl alcohol. The kinetic experimental result showed that ethyl alcohol exhibits non-competitive inhibition to the hydrolytic activity of H(+)-ATPase complex. It was deduced that ethyl alcohol could result in conformational change of F1 of the complex, such as to affect the activity of the enzyme. The measurement of DPH (diphenylhexatriene) fluorescence polarization, the fluorescence labelled with N-(1-pyrenyl) maleimide and intrinsic fluorescence of H(+)-ATPase complex compared with control show that the three cases, i.e. only treated with DCCD, only treated with ethyl alcohol or treated with DCCD and ethyl alcohol, appear different conformations of H(+)-ATPase complex. But the conformation caused by DCCD and ethyl alcohol was more like that by ethyl alcohol. This is consistent with results obtained from activity of DCCD plus ethyl alcohol and only ethyl alcohol. These results mentioned above indicate that the mechanism of ethyl alcohol eliminating the DCCD-induced inhibition of H(+)-ATPase is a conformational interaction caused by DCCD and ethyl alcohol.
Li
S G
SG
Institute of Biophysics, Academia Sinica, Beijing, China.
Lin
Z H
ZH
Feng
Z Y
ZY
Deng
J P
JP
D000818:Animals; D004024:Dicyclohexylcarbodiimide / Q000037:antagonists & inhibitors*; D000431:Ethanol / Q000494:pharmacology*; D008929:Mitochondria, Heart / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013552:Swine
D000431:Ethanol; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases