乙醇能消除二环己基碳二亚胺对H^+-ATPase复合体的抑制效应

ELIMINATION EFFECT OF ETHYL ALCOHOL ON THE DCCD-INDUCED INHIBITION OF HYDROLYTIC ACTIVITY OF H^+-ATPase COMPLEX

本文发现线粒体H^+-ATPase复合体先用0.5μg/ml的DCCD(二环己基碳二亚胺预保温处理,再经12.5%(V/V)乙醇进一步保温处理,则乙醇可完全消除DCCD引起的H^+-ATPase的抑制效应。若H^+-ATPase用DCCD和乙醇同时预保温处理,则DCCD同样消失其抑制作用。用相同浓度的甲醇代替乙醇,则仅可部分的消除DCCD的抑制作用。用相同浓度的DMSO(二甲基亚砜)代替乙醇,则不能消除DCCD的抑制作用。同样浓度的乙醇保温处理经预先用2μg/ml的寡霉素(Oligomycin)处理的H^+-ATPase,并不对寡霉素的抑制作用发生任何影响。表明此浓度的乙醇并未对H^+-ATPase产生解偶联效应。动力学研究表明乙醇对H^+-ATPase水解活力呈现非竞争抑制行为,推测乙醇可能导致H^+-ATPase中的F_1构象的改变,因而影响酶的活性。DPH标记的荧光偏振度,N-[1-P]M标记的荧光强度和内源荧光强度的测定结果显示,乙醇消除DCCD抑制作用的机理,是由于乙醇和DCCD所引起的构象间的相互作用的结果。

12.5% ethyl alcohol was added into the reaction system containing mitochon-drial H+-ATPase complex of pig heart, which was preincubated with 0.5μg/ml DCCD (dicyclohexylcarbodiimide) at 30℃ . Or the DCCD and ethyl alcohol were simultaneously incubated with H+-ATPase at 30℃. 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 alco-hlo, the DCCD inhibition could only be partly eliminated. In the replacement of ethyl alcohol by dimethyl sulfoxide, no elimination could be observed. After pre-incubation of 2 μg/ml oligomycin with H+-ATPase complex instead of DCCD, the same concentration of ethly alcohol could not caused elimination effect, which indicates no un-couplig effect happened by ethyl alcohol. The kinetic experimental result showed that ethyl alcohol exhibits non-competitive inhibition to the hydro- lytic activity of H+-ATPase complex. It was deduced that ethly alcohol could result in conformational change of F1 of the complex, such as to affact the activity of the enzyme. The measurment of DPH (di-phenylhexatriene) fluorescence polarization, the fluorescence labelled with N-(l-pyrenly) maleimide and intrinsic fluorescence of H+ -ATPase complex compared with control show that the threee cases, i. e. only treated with DCCD, only trealed with ethyl alcohol or treated with DCCD and ehtyl alcohol, apear differert 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 obained 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 interection caused by DCCD and ethyl alcohol.