Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.14 (ATP synthase)
 7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gamma-subunit of chloroplast coupling factor 1 contains a disulfide bond which is involved in the redox regulation of the enzyme. In all the sequence plant gamma-subunits this disulfide bond is separated by a five amino acid spacer region. To investigate the regulatory significance of this region genetic transformation experiments were performed with Chlamydomonas reinhardtii. C. reinhardtii strain atpC1 (nitl-305, cw 15, mt-), which does not accumulate the CF1 gamma-subunit polypeptide, was independently transformed with two constructs, each bearing mutations within the disulfide bond spacer region between Cys198 and Cys204 of the gamma-subunit. Successful complementation was confirmed by phenotypic selection, Northern blot analysis, and reverse transcription polymerase chain reaction. Whereas wild-type thylakoid membrane particles catalyze in vitro, PMS-dependent photophosphorylation that is stimulated 2-fold by the addition of DTT, similar particles from each of the mutant strains exhibit rates of ATP synthesis that are independent of DTT. Consistent with these results, wild-type CF1 ATPase activity is stimulated by DTT which is in contrast to the ATPase activities of both the mutant strains which are independent of DTT addition. These results suggest a role of the gamma-subunit disulfide bond spacer region in the redox regulation of chloroplast ATP synthase.
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PMID:A role for the disulfide bond spacer region of the Chlamydomonas reinhardtii coupling factor 1 gamma-subunit in redox regulation of ATP synthase. 878 38

We have isolated the F0F1-ATP synthase complex from oligomycin-sensitive mitochondria of the green alga Chlamydomonas reinhardtii. A pure and active ATP synthase was obtained by means of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and PMSF [corrected]. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase alpha subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58-70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase beta subunit from C. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771-780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15-18 residues longer than in ATP synthase alpha subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.
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PMID:Isolation and characterization of the mitochondrial ATP synthase from Chlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the alpha subunit. 891 27

The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of 50% of the protein spots in the gels, but also resulted in losses of some spots.
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PMID:An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE. 1772 85

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).
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PMID:Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system. 1787 May 88

Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.
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PMID:Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis. 1799 21