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The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan. 1986

J G Verburg, and M Yoshida, and W S Allison

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.

UI MeSH Term Description Entries
D009327 4-Chloro-7-nitrobenzofurazan A benzofuran derivative used as a protein reagent since the terminal N-NBD-protein conjugate possesses interesting fluorescence and spectral properties. It has also been used as a covalent inhibitor of both beef heart mitochondrial ATPase and bacterial ATPase. Chloronitrobenzoxadiazole,NBD Chloride,7-Chloro-4-nitrobenzofurazan,NBF-Cl,Nitrobenzoxadiazole Chloride,4 Chloro 7 nitrobenzofurazan,7 Chloro 4 nitrobenzofurazan,Chloride, NBD,Chloride, Nitrobenzoxadiazole,NBF Cl
D010069 Oxadiazoles Compounds containing five-membered heteroaromatic rings containing two carbons, two nitrogens, and one oxygen atom which exist in various regioisomeric forms. Oxadiazole
D010084 Oxidation-Reduction A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471). Redox,Oxidation Reduction
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004227 Dithionite Dithionite. The dithionous acid ion and its salts. Hyposulfite,Sodium Dithionite,Dithionite, Sodium
D006180 Proton-Translocating ATPases Multisubunit enzymes that reversibly synthesize ADENOSINE TRIPHOSPHATE. They are coupled to the transport of protons across a membrane. ATP Dependent Proton Translocase,ATPase, F0,ATPase, F1,Adenosinetriphosphatase F1,F(1)F(0)-ATPase,F1 ATPase,H(+)-Transporting ATP Synthase,H(+)-Transporting ATPase,H(+)ATPase Complex,Proton-Translocating ATPase,Proton-Translocating ATPase Complex,Proton-Translocating ATPase Complexes,ATPase, F(1)F(0),ATPase, F0F1,ATPase, H(+),Adenosine Triphosphatase Complex,F(0)F(1)-ATP Synthase,F-0-ATPase,F-1-ATPase,F0F1 ATPase,F1-ATPase,F1F0 ATPase Complex,H(+)-ATPase,H(+)-Transporting ATP Synthase, Acyl-Phosphate-Linked,H+ ATPase,H+ Transporting ATP Synthase,H+-Translocating ATPase,Proton-Translocating ATPase, F0 Sector,Proton-Translocating ATPase, F1 Sector,ATPase Complex, Proton-Translocating,ATPase Complexes, Proton-Translocating,ATPase, H+,ATPase, H+-Translocating,ATPase, Proton-Translocating,Complex, Adenosine Triphosphatase,Complexes, Proton-Translocating ATPase,F 0 ATPase,F 1 ATPase,F0 ATPase,H+ Translocating ATPase,Proton Translocating ATPase,Proton Translocating ATPase Complex,Proton Translocating ATPase Complexes,Proton Translocating ATPase, F0 Sector,Proton Translocating ATPase, F1 Sector,Triphosphatase Complex, Adenosine
D006358 Hot Temperature Presence of warmth or heat or a temperature notably higher than an accustomed norm. Heat,Hot Temperatures,Temperature, Hot,Temperatures, Hot
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001419 Bacteria One of the three domains of life (the others being Eukarya and ARCHAEA), also called Eubacteria. They are unicellular prokaryotic microorganisms which generally possess rigid cell walls, multiply by cell division, and exhibit three principal forms: round or coccal, rodlike or bacillary, and spiral or spirochetal. Bacteria can be classified by their response to OXYGEN: aerobic, anaerobic, or facultatively anaerobic; by the mode by which they obtain their energy: chemotrophy (via chemical reaction) or PHOTOTROPHY (via light reaction); for chemotrophs by their source of chemical energy: CHEMOLITHOTROPHY (from inorganic compounds) or chemoorganotrophy (from organic compounds); and by their source for CARBON; NITROGEN; etc.; HETEROTROPHY (from organic sources) or AUTOTROPHY (from CARBON DIOXIDE). They can also be classified by whether or not they stain (based on the structure of their CELL WALLS) with CRYSTAL VIOLET dye: gram-negative or gram-positive. Eubacteria
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining

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