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ISOLATION Of THE PROTON-TRANSLOCATING f0f1-ATPASE fROM RHODOSPIRILLUM-RUBRUM CHROMATOPHORES, AND ITS fUNCTIONAL RECONSTITUTION INTO PROTEOLIPOSOMES

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BIOCHIMICA ET BIOPHYSICA ACTA 1989年第1期975卷 148-157页

作者： SLOOTEN, L VANDENBRANDEN, S VrijeUniversiteit Brussel Faculty of Sciences Laboratory of Biophysics Brussels (Belgium)

The proton-translocating f0f1-ATPase was isolated from Rhodospirillum rubrum chromatophores by extraction with octyglucoside and deoxycholate, and further purified by sucrose gradient centrifugation. The enzyme was reconstituted into sonicated phospholipid vesicles by incubation with cholate, followed by centrifugation through a Sephadex column. ATP-hydrolysis catalyzed by f0f1-proteoliposomes is accelerated approx. 5-10 fold and approx. 15-20 fold by protonophorous uncouplers during assays with or without sulfite, respectively. Maximal turnover numbers are approx. 320 and 60 mol/s during hydrolysis with or without sulfite. The reconstituted enzyme is in, or close to, the native state with respect to the kinetics and regulation of ATP-hydrolysis. It generates a proton gradient (.***) during ATPase activity, and the proteoliposomes are capable of .***-driven ATP-synthesis. Gradient centrifugation of the reconstituted vesicles results in separation of lipid without f0f1 from proteoliposomes. In the purified proteoliposomes the minimum phospholipid/protein weight ratio is around 3. The distribution profiles of ATPase activity and .***-driven ATP synthesis after gradient centrifugation do not completely overlap. It is inferred that the size of the proteoliposomes decreases slightly with decreasing lipid/protein weight ratios. ATPase-induced 9-aminoacridine fluorescence changes, indicative of the generation of .***, were negative or positive, depending on the absence or presence of fluorescence quenchers in the external solution. The fluorescence changes became more positive when the probe concentration was lowered. The reasons for this are discussed.

关键词： ATP hydrolysis ATP synthesis Coupling factor Proteoliposome Membrane reconstitution ATPase, f 0 f 1 - (R. rubrum) CCCP carbonylcyanide m -chlorophenylhydrazone Hepes 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid PMSf phenylmethylsulfonylfluoride Mops 4-morpholinepropanesulfonic acid

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IDENTIfICATION Of A MUTATION IN ESCHERICHIA-COLI f1-ATPASE BETA-SUBUNIT CONfERRING RESISTANCE TO AUROVERTIN

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fEBS LETTERS 1989年第1-2期253卷 269-272页

作者： LEE, RSf PAGAN, J SATRE, M VIGNAIS, PV SENIOR, AE Department of Biochemistry Box 607 University of Rochester Medical Center Rochester NY 14642 USA

A mutation conferring aurovertin resistance on Escherichia coli f 1 -ATPase was identified as R398 → in the f 1 β-subunit. β-subunit from the mutant doe not bind aurovertin; therefore our results suggest the region of sequence around residue β-398 is involved in aurovertin binding. Since nucleotide and aurovertin binding to isolated β-subunit are not mutually exclusive, the data further suggest that the β-subunit catalytic nucleotide-binding domain does not include residue 398. The mutation prevented aurovertin inhibition of ATPase at pH 6 and 8.5, implying charge on the arginine side-chain is not a major determinant of aurovertin binding or that the p K of R398 is shifted due to a peculiar environment. The equivalent residue is usually arginine in f 1 β-subunits of different species; notably in the aurovertin-insensitive thermophilic bacterium PS3 f 1 -ATPase, this residue is phenylalanine.

关键词： Aurovertin resistance f 1 -ATPase β-subunit Mutation

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TEMPERATURE-INDUCED STATES Of ISOLATED f1-ATPASE AffECT CATALYSIS, ENZYME CONfORMATION AND HIGH-AffINITY NUCLEOTIDE BINDING-SITES

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BIOCHIMICA ET BIOPHYSICA ACTA 1989年第1期976卷 77-84页

作者： BARACCA, A AMLER, E SOLAINI, G CASTELLI, GP LENAZ, G HOUSTEK, J UNIV BOLOGNA DEPT BIOCHEM I-40126 BOLOGNA ITALY CZECHOSLOVAK ACAD SCI INST PHYSIOL CS-11142 PRAGUE 1 CZECHOSLOVAKIA SCUOLA SUPER STUDI UNIV & PERFEZIONAMENTO PISA ITALY

Isolated, nucleotide-depleted bovine-heart f1-ATPase exhibits a break in Arrhenius plot with a 2.7-fold increase in activation energy of ATP hydrolysis below ***.C. Analysis of intrinsic tyrosine fluorescence and of the circular dichroism of f1-ATPase showed an abrupt and reversible conformational change occurring at the break temperature, characteristic of a structural tightening at low temperature. Analysis of catalytic nucleotide binding sites using fluorescent ADP analog, 3''-O-(1-naphthoyl)adenosine diphosphate did not show any significant change in affinity of nucleotide binding around the transition temperature but the bound fluorophore exerted a more restricted motion and slower rotation at temperature below the break, indicating a change in the mobility of groups in the close neighbourhood. It is concluded that, as a result of temperature, two kinetically distinct states of f1-ATPase are induced, due to a change in enzyme conformation, which influences directly the properties of catalytic nucleotide binding sites.

关键词： ATPase, f,- Temperature dependence Enzyme conformation Nucleotide binding site fluorescence Enzyme kinetics f 1 f 1 -ATPase (catalytic part of H + -translocating ATP synthase of f 0 f 1 ,-type) Nd-f 1 nucleotide-depleted f 1 -ATPase f 0 membrane sector part of H + -translocating ATP synthase of f 0 f 1 -type N-ADP 3- O -(1-naphthoyl)adenosine diphosphate TNP-ADP 2′,3′- O -(2,4,6-trinitrophenyl)adenosine diphosphate DMAN-ADP 3′- O -1-(5-dimethylamino) naphthoyladenosine diphosphate CD circular dichroism

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EXPLORATION Of SIGMA-SUBUNIT INTERACTIONS IN BEEf-HEART MITOCHONDRIAL f1-ATPASE BY MONOCLONAL-ANTIBODIES

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fEBS LETTERS 1989年第1-2期245卷 223-228页

作者： LUNARDI, J DUPUIS, A fROBERT, Y GRASSI, J VIGNAIS, PV CENS DEPT BIOLPHARMACOL & IMMUNOL SECTF-91191 GIF SUR YVETTEFRANCE

Three monoclonal antibodies (mAbs) recognizing distinct epitopes on the δ-subunit of beef heart mitochondrial f 1 -ATPase were studied for their reactivity towards the δ-subunit both in isolated f 1 and in the f 0 -f 1 complex of submitochondrial particles. Two of the antibodies termed mAb δ 195 and mAb δ239 had free access to δ in f 1 and the f 0 -f 1 complex. Partial hindrance was observed for the third antibody mAb δ22. By a double antibinding assay, it was found that the binding sites for mAb δ195 and mAb δ239 were close to each other and possibly overlapping. Mapping studies conducted with the isolated δ-subunit showed that mAb δ195 and mAb δ239 interacted with the N-terminal portion of δ extending from Ala-1 to Met-16, whereas mAb δ22 interacted with the fragment spanning Ser-17-Glu-68. It was concluded that the Ala-1-Met-16 segment of the δ-subunit in f 1 and the f 0 -f 1 complex is freely accessible from the outside, whereas the Ser-17-Glu-68 segment of δ is partially hidden, possibly as a result of interactions with other subunits.

关键词： Monoclonal antibody ATPase, f 1 − mAb, monoclonal antibody OSCP, oligomycin sensitivity conferring protein f 1 , catalytic sector of the ATPase complex AChE, acetylcholinesterase

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DIETARY CIS-fATTY ACIDS THAT INCREASE PROTEIN f1 PHOSPHORYLATION ENHANCE SPATIAL MEMORY

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BRAIN RESEARCH 1989年第2期505卷 302-305页

作者： WONG, KL MURAKAMI, K ROUTTENBERG, A NORTHWESTERN UNIV CRESAP NEUROSCI LAB 2021 SHERIDAN RD ROOM 311 EVANSTON IL 60201 USA

Activation of protein kinase C (PKC) facilitates long-term potentiation (LTP), a model of memory, and increases its substrate protein f1 (aka GAP43) phosphorylation in direct relation to synaptic enhancement. Unsaturated fatty acids (c-fAs) which activate purified PKC, when injected into hippocampus, enhance LTP. To determine if dietary c-fAs could alter memory itself as well as brain PKC substrate (f1) metabolism, rats were maintained for 10 weeks on fatty diets enriched in mono-unsaturated oleic acid (OA: 20% olive oil, w/w), or a mono- and di-unsaturated mixture of oleate/linoleate (O/L: 20% corn oil), or a saturated fatty acid diet of laurate/myristate (L/M;20% hydrogenated coconut oil). The O/L diet group was superior to the OA and L/M groups in spatial memory performance after the first two weeks of acquisition and in later achievement of criterion performance. The O/L diet had a significantly higher hippocampal protein f1 in vitro phosphorylation than in both the OA and L/M in trained and non-trained animals. Significantly, animals that made made fewer errors showed higher f1 phosphorylation (r = 0.70). Diet both increases brain PKC substrate phosphorylation and enhances maze learning, confirming the feasibility of enhancing learning and memory by dietary regimens derived from basic neurochemical studies of synaptic plasticity.

关键词： cis -fatty acid f 1 -phosphorylation Spatial memory Protein kinase C Long-term potentiation GAP 43

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EffECT Of REDOX POTENTIAL ON RAT-LIVER f1-ATPASE

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fEBS LETTERS 1989年第2期242卷 293-296页

作者： LOPEZZABALZA, MJ fLORES, f SANTIAGO, E LOPEZMORATALLA, N UNIV NAVARRA DEPT BIOQUIMAPARTADO 273E-31080 PAMPLONASPAIN

Redox titration of f1-ATPase from rat liver mitochondria referred to the modification of the hydrolytic activity on Mg-ATP has resulted in a three-step pattern, with three distinct jumps of activity separated by clear plateaus. The measured potentials ranged from -400 mV to +400 mV and were obtained by the addition of dithionite and ferricyanide. Electron exchange was facilitated with a mixture of different redox mediators. At pH 7.4 the midpoint potentials were +210 mV, +40 mV and -230 mV. These three midpoint potentials were displaced towards more negative values by 2,4-dinitrophenol or by an increase of the pH of the medium. The titration curves were described by n = 2 Nernst equations.

关键词： Mitochondrial ATPase f 1 -ATPase Redox titration

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AN ATP DEPENDENCE Of MITOCHONDRIAL f1-ATPASE INACTIVATION BY THE NATURAL INHIBITOR PROTEIN AGREES WITH THE ALTERNATING-SITE BINDING-CHANGE MECHANISM

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fEBS LETTERS 1989年第1-2期246卷 202-206页

作者： MILGROM, YM A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry Moscow State University 119899 Moscow USSR

The rate of inactivation of f 1 -ATPase, isolated from beef heart mitochondria, by the active acidic form of the natural inhibitor protein depends on the ATP concentration. An increase in concentration of ATP to ∼ 20 μM leads to a decrease in that of the inhibitor protein inducing 50% inhibition of the f 1 -ATPase during 5 s preincubation ( C 50 ); further increase in ATP concentration to 1 mM causes little, if any, change in C 50 . However, the C 50 values show a rise at ATP concentrations higher than 1 mM. This ATP dependence of the inhibitor action may be in agreement with a version of the alternating-site binding-change mechanism, which assumes that the two-site catalytic cycle intermediates possessing (i) the products (ADP + P i ) bound in the low-affinity state at one of the active sites and (ii) an ATP molecule at the other active site are the targets for the acidic form of the inhibitor protein.

关键词： ATPase, f 1 - Catalytic intermediate Inhibitor protein f 1 , mitochondrial f 1 -ATPase IP, natural inhibitor protein of mitochondrial f 1 -ATPase

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EffECT Of LEAD ON INTRACELLULAR fREE CALCIUM-ION CONCENTRATION IN A PRESYNAPTIC NEURONAL MODEL - f-19-NMR STUDY Of NG108-15 CELLS

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BRAIN RESEARCH 1989年第2期503卷 308-311页

作者： SCHANNE, fAX MOSKAL, JR GUPTA, RK MONTEFIORE MED CTR ALBERT EINSTEIN COLL MED DEPT PEDIAT 111 E 210TH ST BRONX NY 10467 USA MONTEFIORE MED CTR ALBERT EINSTEIN COLL MED DEPT PATHOL BRONX NY 10467 USA MONTEFIORE MED CTR ALBERT EINSTEIN COLL MED DEPT NEUROSURG BRONX NY 10467 USA MONTEFIORE MED CTR ALBERT EINSTEIN COLL MED DEPT NEUROSCI BRONX NY 10467 USA YESHIVA UNIV ALBERT EINSTEIN COLL MED DEPT PHYSIOL & BIOPHYS BRONX NY 10461 USA YESHIVA UNIV ALBERT EINSTEIN COLL MED DEPT BIOCHEM BRONX NY 10461 USA

The intracellular free calcium ion concentration ([Ca2+]i) of the neuroblastoma .times. glioma hybrid cell line, NG108-15, was measured using the 19f-nuclear magnetic resonance divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N'',N''-tetra-acetic acid (5f-BAPTA). The basal [Ca2+]i was measured to be 106 .+-. 14 nM. Treatment with 5 .mu.M lead (Pb) for 2 h produced a 2-fold increase in [Ca2+]i to 200 .+-. 24 nM and a measurable free Pb2+ concentration ([Pb2+]i) of 30 .+-. 10 pM. Intracellular free Zn2+ concentrations ([Zn2+]i) were also observed in the presence of Pb. This represents the first direct demonstration that Pb elevates the [Ca2+]i in neurons, thus providing evidence for a role of [Ca2+]i in mediating the neurotoxicity of Pb.

关键词： 19 f calcium lead neuron NG108-15 nuclear magnetic resonance spectroscopy

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THE INHIBITION Of QUINOL OXIDATION BY STIGMATELLIN IS SIMILAR IN CYTOCHROME BC1 AND B6f COMPLEXES

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BIOCHIMICA ET BIOPHYSICA ACTA 1989年第2期974卷 223-226页

作者： NITSCHKE, W HAUSKA, G RUTHERfORD, AW UNIV REGENSBURG INST ZELLBIOL & PFLANZENPHYSIOL UNIVERSITATSTR 31 D-8400 REGENSBURG FED REP GER CENS DEPT BIOL SERV BIOPHYS F-91190 GIF SUR YVETTE FRANCE

Stigmatellin is shown to raise the midpoint potential of the Rieske feS-center of isolated cytochrome b6 f complex from 320 to about 460 mV. This increase in potential is accompanied by a modified EPR spectrum, i.e., a shift of the gy line and a sharpening of the gz peak. An equivalent effect on the mitochondrial cytochrome bc1 complex has been observed earlier. Stigmatellin therefore inhibits both types of cytochrome complexes in the same way, by altering the feS center to an unfavorable reductant for cytochrome f or c1. The decreased reduction of cytochrome f by quinol in the presence of stigmatellin is accompanied by an increased transient reduction of cytochrome b6. This can be explained by an increased formation of semiquinone during quinol oxidation by a stronger oxidizing feS center.

关键词： Cytochrome bc 1 , complex Cytochrome b 6 f complex Stigmatellin Quinol oxidation Inhibition ( S. aurantiaca ) Mes 4-morpholineethanesulfonic acid Mops 4-morpholinepropanesulfonic acid UHDBT 5- N -undecyl-6-hydroxy-4,7-dioxobenzothiazole

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HETEROGENEITY Of ATP-HYDROLYZING SITES ON RECONSTITUTED Cf0f1

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fEBS LETTERS 1989年第1期259卷 33-36页

作者： fROMME, P GRABER, P UNIV STUTTGART INST BIOLW-7000 STUTTGARTGERMANY

The proton translocating ATP-synthase from chloroplasts, Cf 0 f 1 , was isolated and reconstituted into asolectin liposomes. [γ- 32 P]ATP hydrolysis was measured under uni-site conditions. When 1 mM unlabeled ATP was added so that all ATP-binding sites were occupied, [γ- 32 P]ATP bound to the first site, was hydrolyzed with a rate of 0.5 ATP/(Cf 0 f 1 s). In a second experiment, first cold ATP was hydrolyzed under uni-site conditions and then 1 mM [γ- 32 P]ATP was added. This allows under otherwise identical conditions the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. It resulted in a rate of 80 ATP/(Cf 0 f 1 s). It is concluded that the catalytic nucleotide binding sites are heterogeneous: there exists one nucleotide binding site which hydrolyzes ATP with a maximal turnover of 0.5/s and another one (or two) which hydrolyze ATP with a turnover of 80/s. The latter one is the catalytic site for maximal turnover.

关键词： Chloroplast ATPase, H + - Cf 0 f 1 Reconstitution Nucleotide binding site Uni-site catalysis Cf 0 f 1 H + -translocating ATPase (ATP-synthase) from chloroplasts

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