The Structure And Function Of Atp Synthase, Its Activity Changes In Cell Photoautotrophic

F1Fo ATP synthase serves the central catalytic function of ATP synthesis in most organisms. The enzymes can catalyze the synthesis of ATP using the trans-membrane proton motive force. In chloroplast, this enzyme consists of two parts: membrane-embedded CFo and cytoplasmic CF1. CFo conducts protons flux through the thylakoid membrane and provides the binding site for CF1. CF1 contains the nucleotide-binding and catalytic sites. In addition, cultured photoautotrophic cells are a kind of new material to study structure and function of protein complex in thylakoid membrane. Its most important advantage is easy to control the physical and chemical environment and regulate the subsequent growth and differentiation of the tissue. This thesis mainly focuses on the structure and function of ( subunit, induction culture of photoautotrophic callus and the change of photosynthetic characteristics and photophosphorylation activity of callus during changing from heterotrophic into photoautotrophic callus.Effects of site-directed mutation on the functions of chloroplast ATP synthase ( subunitThe Leu at the N-terminal third site (Leu3) of chloroplast ATP synthase ( subunit was replaced with Ile, Phe, Thr, Arg, Glu and Pro by site-directed mutagenesis, forming mutants (L3I, (L3F, (L3T, (L3R, (L3E and (L3P respectively. The inhibitory potency of ( variants on ATP hydrolysis and their abilities to block proton leakage were all lower than that of wild type ( subunit ((WT). The ability of variants (L3I or (L3F to restore the ATP synthesis activity of reconstituted membrane was higher than that of (WT, but the abilities of the other ( variants were still lower than that of (WT. These results indicated that the hydrophobic, nonpolar, neutral characteristic of Leu3 of chloroplast ( subunit was very important for its functions of inhibiting ATP hydrolysis and blocking proton leakage. 2. Effects of truncated mutants of the ( subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability When the ( subunit or the truncated ( proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. But for the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of (-deficient membrane reconstituted with the ( subunit or the truncated ( proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. But for the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ( protein. These results suggested that: (a) the N-terminal domain of the ( subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ( subunit; (b) the C-terminal domain of the ( subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase. 3. Photoautotrophic growth and change of photophosphorylation activity of tobacco callusWe first cultured photoautotrophic tobacco callus by the method of decreasing the sucrose concentration in the culture medium gradually successfully. After the callus was cultured in liquid medium containing 20 g/L sucrose for 4 weeks, its chlorophyll concentration, soluble protein concentration, ATP hydrolysis (ATPase) activity and photophosphorylation activity were all very low. Whe...