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F_1-ATPase Purification and structure of an atomic force microscope

Author: ZhuHuaZe
Tutor: SunRunGuang
School: Shaanxi Normal University
Course: Biophysics
Keywords: ATP synthase Chloroplast Mitochondria An atomic force microscope
CLC: TH742
Type: Master's thesis
Year: 2008
Downloads: 54
Quote: 1
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Abstract

ATP synthase (F ₁ F ₀-ATPase) is a key enzyme in energy conversion organisms. It is located in the inner mitochondrial membrane (MF ₁ F ₀-ATPase), the chloroplast thylakoid film (CF ₁ F _{0 < / sub>-ATPase) and the plasma membrane of bacteria (BF ₁ F ₀-ATPase) on. F ₁ F ₀-ATPase not only be able to take advantage of the transmembrane proton motive force synthesis of ATP, can hydrolyze ATP to form the the organisms life activities proton gradient. The various sources F ₁ F ₀-ATPase has a similar structure: that contain transmembrane F ₀ and prominent on the outside of the membrane The F ₁ two parts. Specific subunits F ₀ part of the subunits a, b and c (chloroplasts corresponding to subunit Ⅰ, Ⅱ, Ⅲ and Ⅳ) subunits of the quasi-volume relationship ab ₂ c _9-12 (Ⅰ Ⅱ Ⅲ ₁₄ Ⅳ): F ₁ subunits α, β, γ, δ and ε, the relationship of quasi amount of each subunit α ₃ β _{3 / sub> γδε the addition γεC _9-12 (γε Ⅲ _12-14) subunits synthase \δ (α ₃ β ₃ δ Ⅰ Ⅱ Ⅳ) constitutes a \In the working process of the ATP synthase, F ₀ part play a transshipment proton transmembrane proton gradient potential, due to the need to establish the synthesis of ATP synthase catalytic sites located in the α and β subunits , so F ₁ is the site of the catalytic synthesis of ATP. In this thesis work is mainly mitochondria and chloroplasts F ₁-ATPase were isolated and purified using atomic force microscopy (Atomic Force Microscopy, AFM) 1} structure of the ATP synthase F _{were studied. (1) CF ₁-ATPase separation and purification: separation spinach chloroplast CF ₁-ATPase, first with sodium pyrophosphate solution was eluted from the thylakoid membrane CF ₁, respectively, then its purified cellulose DE-52 column chromatography and ammonium sulfate fractionation and separation; separation of liver mitochondria MF ₁, the first by ultrasonic treatment method makes MF ₁ dissociating from the inner mitochondrial membrane, and then were separation and heat treatment method of their purified by ammonium sulfate fractionation. It was found that the use of these two methods get of a purity better the MF ₁-ATPase; isolated from chloroplasts and mitochondria, respectively (2) gel electrophoresis experiments: the separation of the two F _l-ATPase are respectively made by polyacrylamide gel electrophoresis analysis and gel enzyme activity analysis. It was found that the isolated sample purity, all a major subunit ingredients containing F ₁ part, the gel activity assays showed that separated F ₁ part still has strong ability to hydrolyze ATP. (3) activity assays: temperature, pH, KCl concentration of F ₁-ATPase activity of ATP hydrolysis. Was found that the ice bath treatment causes the enzyme molecules to decompose, so that loss of activity, but the 20% methanol solution, can effectively protect the integrity of the enzyme molecules, presumably with alcohols hydroxy outside in the enzyme to form a hydrophobic area, thereby play a protective role related; two different sources of F ₁-ATPase are stable at pH 7.4 to 8.0 range, but chloroplast origin CF ₁ in pH a value of 8.0 to maintain a higher hydrolytic activity, with mitochondrial sources MF ₁ slightly different, the latter has a relatively high hydrolytic activity at pH 7.4; at high concentrations the presence of KCl, _f is the dissociation into active subunits, its dialysis desalting recombinant found that the enzyme molecules can be restored again to the hydrolysis activity, having activity to obtain subunit unit provides a good method. (4) AFM observation experiment: contact mode atomic force microscopy observations F ₁-ATPase molecules, a high-resolution AFM topography image. Two enzyme molecules were shown in the image obtained spherical structure, particle size analysis of the AFM software measured the enzyme molecule an average diameter of 18nm and can be divided into three categories according to their diameter, speculated which respectively correspond to the ATP synthase three states, namely vacant state, loose-bound and tightly bound. The multiple scans found that the source of the enzyme molecule chloroplast probe be enzyme molecules cause irreversible damage. Showing a comet-like shape of the enzyme molecule. Through this study, not only to explore the set for this laboratory F ₁-ATPase extraction and purification methods, and the AFM is used to the morphology study which of the enzyme molecule for further use of the AFM The structure provides the basis of the physiological state F ₁ F ₀-ATPase molecules.}}

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