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REMOVAL Of THE EPSILON-SUBUNIT fROM ESCHERICHIA-COLI f1-ATPASE USING MONOCLONAL ANTI-EPSILON ANTIBODY-AffINITY CHROMATOGRAPHY

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ANALYTICAL BIOCHEMISTRY 1986年第1期159卷 35-42页

作者： DUNN, SD UNIV WESTERN ONTARIO DEPT BIOCHEM LONDON N6A 5C1 ONTARIO CANADA

The usefulness of two monoclonal antibodies .epsilon.-1 and .epsilon.-4, which recognize the .epsilon. subunit of Escherichia coli f1-ATPase, for removing that subunit from ATPase was assessed. The .epsilon. subunit is a tightly bound, but dissociable, inhibitor of the ATPase. .epsilon.-1 binds .epsilon. with 10-fold higher affinity than .epsilon.-4. .epsilon.-1 recognizes a site on .epsilon. which is hidden by the quaternary structure of ATPase, while .epsilon.-4 can recognize .epsilon. when it is part of ATPase. Each antibody was purified and coupled to Sepharose to generate affinity columns. Solutions of ATPase in a buffer which was designed to reduce the affinity of .epsilon. for the enzyme were pumped through the columns and the degree of .epsilon. depletion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blotting. Neither column retained ATPase significantly. At low ATPase concentrations and low flow rates, the .epsilon.-1 column was more efficient than the .epsilon.-4 column, removing in excess of 95% of the .epsilon. in a single passage compared with 93% removal by the .epsilon.-4 column. At higher protein concentrations or flow rates, however, the performance of the .epsilon.-1 column was substantially poorer, while that of the .epsilon.-4 column was much less affected. Very little .epsilon. emerged from the .epsilon.-4 column before most of the measured .epsilon.-binding capacity was filled. A second passage through the .epsilon.-4 column reduced residual .epsilon. to less than 2% of that which was originally present. Pure, active .epsilon. was eluted from either column by 1 M NH4OH, pH 11. The relatively poor performance of .epsilon.-1 is discussed in terms of the low availability of the epitope and the tendency of the .epsilon.-depleted complex to compete with .epsilon.-1 for residual .epsilon. subunit. from consideration of these factors it appears likely that antibodies which recognize exposed epitopes will ge

关键词： f 1 -ATPase affinity chromatography monoclonal antibodies protein/enzyme purification subunit removal

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EffECT Of THE WEAKLY ACIDIC UNCOUPLER 2,4-DINITROPHENOL AND DIMETHYL-SULfOXIDE ON THE COORDINATION Of MG-2+ WITH ATP - POSSIBLE MECHANISM Of ACTIVATION Of THE ISOLATED f1-ATPASE BY 2,4-DINITROPHENOL

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BIOPHYSICAL CHEMISTRY 1990年第3期36卷 201-208页

作者： SHINOHARA, Y YOSHIKAWA, K TERADA, H UNIV TOKUSHIMA FAC PHARMACEUT SCI SHOMACHI 1 TOKUSHIMA 770 JAPAN NAGOYA UNIV COLL GEN EDUC NAGOYA 46401 JAPAN

The exchange rate constants between Mg2+-free and Mg2+-bound ATP were determined under various conditions by line shape analysis of the 31P-NMR spectrum based on the exchange reaction, and the thermodynamic parameters of this exchange reaction were determined from the temperature dependence of its rate constants. Analysis of the activation enthalpy change .DELTA.H* showed that Mg2+ is coordinated with the .beta.- and .gamma.-phosphoryl groups of ATP asymmetrically, being in closer proximity to the .beta.-phosphoryl group. The weakly acidic uncoupler 2,4-dinitrophenol increased this asymmetric coordination of Mg2+, and this effect was enhanced by the further addition of dimethyl sulfoxide. The hydrolysis of ATP in aqueous solution correlated well with the degree of asymmetry of Mg2+ coordination. Thus, this asymmetric coordination specifically weakens the ***. bound at which specific cleavage of ATP catalyzed by most ATPases takes place in the presence of Mg2+. In this paper, the mechanism of activation of isolated ATPase (f1-ATPase) by 2,4-dinitrophenol, and that of ATP synthesis by isolated f1-ATPase in the presence of dimethyl sulfoxide are considered on the basis of these results. The essential role of the OH group of Ser-174 of the .beta.-subunit of f1-ATPase in ATP hydrolysis is also discussed.

关键词： ATP hydrolysis dimethyl sulfoxide f 1 -ATPase NMR, 31 P- uncoupler

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INACTIVATION Of BEEf-HEART MITOCHONDRIAL f1-ATPASE BY THE 2',3'-DIALDEHYDE DERIVATIVES Of ADENINE-NUCLEOTIDES

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fEBS LETTERS 1984年第1期169卷 101-106页

作者： DEMELO, Df SATRE, M VIGNAIS, PV CEN DEPT RECH FONDAMENTALEBIOCHIM LABCNRSERA 903 INSERMU191 F-38041 GRENOBLE FRANCE

Beef heart mitochondrial f1-ATPase was inactivated by the 2',3'-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of f1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol f1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2-3 mol per mol f1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the alpha and beta subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.

关键词： f 1 -ATPase Dialnucleotide Affinity labeling Enzyme inactivation f 1 , beef heart soluble mitochondrial ATPase Tra, triethanolamine oAdN, 2′,3′-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP)

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PRECOCENE-INDUCED STERILITY IN f1-GENERATION Of GLOSSINA-MORSITANS-MORSITANS

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JOURNAL Of INSECT PHYSIOLOGY 1982年第6期28卷 559-+页

作者： SAMARANAYAKARAMASAMY, M CHAUDHURY, MfB INT CTR INSECT PHYSIOL & ECOL POB 30772 NAIROBI KENYA

Precocene treatment does not disrupt the events of reproduction in G. m. morsitans or induce any apparent changes in treated tsetse. Some females of the f1 generation are either sterile or show retardations in follicle development. Sterility is not reversed spontaneously or with juvenile hormone analogs. The critical period for precocene action is related to each ovulation. The corpora allata of precocene-treated tsetse are normal, but those of f1 sterile females are degenerate. The occurrence of retardation has enabled the characterization of stages in follicle development in G. m. morsitans.

关键词： Precocene Glossina morsitans morsitans germarium follicle development oöcyte f 1 generation sterile females corpus allatum

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ACTIVATION Of f1-ATPASE ISOLATED fROM POTATO-TUBER MITOCHONDRIA

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fEBS LETTERS 1987年第2期223卷 304-308页

作者： GLASER, E HAMASUR, B NORLING, B ANDERSSON, B Department of Biochemistry Arrhenius Laboratory University of Stockholm S-106 91 Stockholm Sweden

The ATP-hydrolyzing activity of f 1 -ATPase purified from potato tubers mitochondria was stimulated 2- and 3.5-fold by anions, chloride and bicarbonate, respectively, and 5.5- and 6.5-fold by detergents, octyl glucoside and lauryl dimethylamine oxide (LDAO), respectively. The maximal specific activity of the activated f 1 , 129 μmol/min per mg protein is the highest activity of plant mitochondrial f 1 hitherto reported and exceeds several-fold values reported earlier. In the absence of activators f 1 -catalyzed ATP hydrolysis exhibits non-linear double-reciprocal plots of [ATP] −1 vs v −1 indicative of negative cooperativity, while in the presence of activators, linear plots are observed. It is suggested that the activators reduce the cooperativity originating from the interaction between different subunits of the enzyme.

关键词： f 1 -ATPase Anion activation Detergent activation Cooperativity (Plant mitochondrion) f 1 , f 0 , catalytic respectively H + -translocating coupling factor of H + -ATPase Mops, 3-( N -morpholine)propanesulfonic acid

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INVOLVEMENT Of PLASTOQUINONE AND LIPIDS IN ELECTRON-TRANSPORT REACTIONS MEDIATED BY THE CYTOCHROME B6-f COMPLEX ISOLATED fROM SPINACH

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fEBS LETTERS 1985年第2期180卷 321-325页

作者： CHAIN, RK Division of Molecular Plant Biology University of California Berkeley CA 94720 USA

The isolation of a cytochrome b6-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.

关键词： Cytochrome b 6 -f complex Plastoquinone Lipid Reconstitution PC, phosphatidylcholine PG, phosphatidylglycerol MGDG monogalactosyldiacylglycerol DGDG, digalactosyldiacylglycerol SQDG, sulfoquinovosyldiacylglycerol TLC, thin-layer chromatography DNP-INT, 2,4-dinitrophenyl ether of iodonitrothymol, PQ, plastoquinone b 6 , cytochrome b 6 f , cytochrome f

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KINETIC AND SPECTRAL RESOLUTION Of CYTOCHROME-C-553 AND CYTOCHROME-f IN THE PHOTOSYNTHETIC ELECTRON-TRANSfER CHAIN Of HETEROCYSTS

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fEBS LETTERS 1983年第1-2期159卷 267-270页

作者： HOUCHINS, JP HAWKESfORD, MJ HIND, G Biology Department Brookhaven National Laboratory Upton NY 11973 USA

Oxidation of cytochrome c -553 and cytochrome f following single-turnover flashes was measured in isolated heterocysts. Half-times of oxidation for these two cytochromes were determined by measuring the rate of bleaching at 554 nm in heterocysts possessing cytochrome c -553, or in heterocysts in which cytochrome c -553 was replaced by plastocyanin. flash-induced difference spectra demonstrate that a fast bleaching ( t 0.5 = 20–35 μs) corresponds to the oxidation of cytochrome c -553, and a slower bleaching ( t 0.5 = 87 μs) corresponds to the oxidation of cytochrome f .

关键词： Heterocyst Cytochrome f Cytochrome c -553 Plastocyanin Anabaena Photosynthetic electron transfer

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THE INHIBITOR PEPTIDE Of THE MITOCHONDRIAL f1.f0-ATPASE INTERACTS WITH CALMODULIN AND STIMULATES THE CALMODULIN-DEPENDENT CA2+-ATPASE Of ERYTHROCYTES

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BIOCHIMICA ET BIOPHYSICA ACTA 1985年第1期816卷 63-67页

作者： SCHWERZMANN, K MULLER, M CARAfOLI, E UNIV BERN INST MED CHEM CH-3000 BERN 9 SWITZERLAND SWISS FED INST TECHNOL BIOCHEM LAB CH-8092 ZURICH SWITZERLAND

The binding of calmodulin to the [rat] mitochondrial f1 .cntdot. f0-ATPase was studied. [125I]Iodoazidocalmodulin binds to the .epsilon.-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes was also analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the 2 mechanisms of activation are different. These in vitro effects of the 2 regulatory proteins may reflect a common origin of the 2 ATPases considered and/or of the regulatory proteins.

关键词： ATPase inhibitor f 1 · f 0 -ATPase Calmodulin Ca 2+ -ATPase Erythrocyte

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RESONANCE RAMAN-SPECTROSCOPY INDICATES A LYSINE AS THE 6TH IRON LIGAND IN CYTOCHROME-f

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第1期936卷 61-66页

作者： DAVIS, DJ fRAME, MK JOHNSON, DA University of Arkansas Fayetteville AR U.S.A.

The resonance Raman spectrum of turnip cytochrome f is similar to that of other c-type cytochromes with the exception of a single band at 1532 cm-1 which is shifted to lower frequency relative to its position (1542-1545 cm-1) in other c-type cytochromes. Comparison of the frequency of this band with that in alkylated cytochrome c at high pH suggests that the sixth heme iron ligand in cytochrome f is a deprotonated lysine amino group rather than a methionine sulfur. Comparison of the amino-acid sequences of cytochromes f and c1 suggests lysine-145 as a likely candidate for the sixth heme iron ligand in cytochrome f.

关键词： Cytochrome f Raman spectroscopy Heme iron ligand

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INACTIVATION AND THE SITE Of LABELING Of f1-ATPASE fROM MYCOBACTERIUM-PHLEI BY DICYCLOHEXYLCARBODIIMIDE, 7-CHLORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE AND QUINACRINE MUSTARD

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BIOCHIMICA ET BIOPHYSICA ACTA 1984年第1期764卷 105-113页

作者： AGARWAL, N KALRA, VK UNIV SO CALIF SCH MED DEPT BIOCHEM LOS ANGELES CA 90033 USA

The f1-ATPase from M. phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is time- and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time (t1/2) for DCCD, NBD-Cl and quinacrine mustard was 14, 6 and 7 min, respectively. Inactivation of f1-ATPase resulted in the incorporation of [14C]DCCD and [14C]NBD-Cl into .alpha. and .gamma. subunits. The incorporation of label into .alpha. and .gamma. subunits, utilizing [14C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [14C]DCCD and 4 mol [14C]NBD-Cl bind/mol f1-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in f1-ATPase from M. phlei.

关键词： Dicyclohexylcarbodiimide Quinacrine mustard Enzyme inactivation f 1 -ATPase (M. phlei) DCCD dicyclohexylcarbodiimide EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EEDQ N -(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline Mes 4-morpholineethanesulphonic acid NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole TEMED N , N , N ′, N ′-tetramethylethylenediamine

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