The F <sub> 1 </ Sub>-atpase In Isolation, Purification And Structure Of Atomic Force Microscopy Studies

ATP synthase(F₁F₀-ATPase) is the critical enzyme in energy conversion.It is found in the mitochondrial inner membrane(MF₁F₀-ATPase) and in the inner membrane of bacteria (BF₁F₀-ATPase),whereas the homologous CF₁F₀-ATP synthase is localized in the thylakoid membranes of chloroplasts(CF₁F₀-ATPase).The general structural features of the enzyme are highly conserved among different organisms.Functionally and structurally it can be divided into two parts, which can be isolated as separate entities:the membrane intrinsic F₀ and the peripheral F₁.F₀, conducts proton flux through the inner membrane(or thylakoid) and provides an affinity binding site for the F₁ complex,composed ofsubunits of a,b and c(Ⅰ,Ⅱ,ⅢandⅣ) with a stoichiometry of ab₂c_9-12(ⅠⅡⅢ₁₄Ⅳ);F₁,contains nucleotide-binding and catalytic sites,can hydrolyze ATP at high rates after appropriate treatment,and consists of five different subunits having a stoichiometry ofα₃β₃γδε.Otherwise,γεc_9-12(γεⅢ_12-14) and ab₂α₃β₃δ(α₃β₃δⅠⅡⅣ) forming rotor and stator respectively.The area I focused on most is the separation and purification of F₁ headpiece,and obtaining its topography structure under Atomic Force Microscopy.The results are following:(1) Separation and purification of CF₁/MF₁:Firstly,CF₁-ATPase was eluted by NaPi buffer from unstacked thylakoid membranes(spinach chloroplasts).Then,it was isolated and purified by DE-52 chromatography and ammonium sulfate fractionation;For MF₁,it was purified by ammonium sulfate fractionation after ultrasonic treatment.The assay followed indicated that the F₁-ATPase we obtained is neat.(2) PAGE of ATPase:The ATPase obtained was applied to PAGE,and found that all the subunits appeared on the gel,and in-gel ATPase activity staining revealed that the ATPase in vitro was hold its native activity.(3) Activity assay:We found that the activity of ATPase in vitro depends upon temperature,pH value and salt concentration.The ressarch results showed that the enzyme could disassemble,and lose the activity in ice bath.But 20%methanol buffer could protect the integral of enzyme molecular effectively.We also found that the activity of the two different ATPase is steady when the pH is between 7.4 and 8.0.We conjecture that this was because the methanol's hydroxide could form a hydrophobic area around the enzyme to protect it.Besides,we discovered that the two F₁-ATPase could be decomposed as active subunits,which would be reconstituted by dialyzing incubated in 1M KCl at 0℃.(4) AFM image assay:At last the topography of the two F₁-ATPase was obtained by AFM in contact mode.Using AFM Particle Software analysis the enzyme molecules showed that the average diameter is 18nm(CF₁) or 20nm(MF₁),and they could be sorted into 3 groups corresponding to three states(Omit state,Loose state and Tight state) of the enzyme respectively.After scanning the sample several times,we found that the enzyme molecules were destroyed,and were presented comet like structure.Through our study,we not only searched after an optimized way to obtain MF₁-ATPase (CF₁-ATPase),but also applied AFM to research topography structure of this enzyme,and provided a method for farther study F₁F₀-ATPase under physiological condition by AFM.