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fAST ELECTRON-TRANSfER fROM LOW-POTENTIAL TO HIGH-POTENTIAL CYTOCHROME-B6 IN ISOLATED CYTOCHROME-B6f COMPLEX

fEBS LETTERS 1988年第1期232卷 204-208页

作者： NITSCHKE, W HAUSKA, G CROfTS, AR UNIV REGENSBURG INST BOTUNIV STR 31D-8400 REGENSBURGFED REP GER UNIV ILLINOIS DEPT PHYSIOL & BIOPHYSURBANAIL 61801

The two hemes of cytochrome b 6 in the cytochrome b 6 f complex isolated from chloroplasts exhibit slight spectral differences in the α-band at room temperature, sufficient to deconvolute redox kinetics of cytochrome b 6 into the contributions of the two components. The deconvoluted changes observed can be explained by fast electron transfer from low- to high-potential cytochrome b 6 .

关键词： Cytochrome b 6 f complex Cytochrome b Oxidant-induced reduction Q-cycle Spectral deconvolution PQH 2 , plastoquinol b h , high-potential cytochrome b b 1 , low-potential cytochrome b f , cytochrome f b 6 f , cytochrome b 6 f complex bc 1 , cytochrome bc 1 complex

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KINETIC-ANALYSIS Of PROTON TRANSLOCATION CATALYZED BY f0f1 ATPASE

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fEBS LETTERS 1988年第2期234卷 455-458页

作者： MUNEYUKI, E HIRATA, H JICHI MED SCH DEPT BIOCHEMMINAMIKAWACHITOCHIGI 32904JAPAN UNIV TOKYO FAC SCIDEPT BIOCHEM & BIOPHYSBUNKYO KUTOKYO 113JAPAN

Kinetic analysis of both proton translocating and steady-state ATP hydrolytic activities catalyzed by f0f1 ATPase in submitochondrial particles were carried out over an ATP concentration range of 1-2000 microM. The results were examined in relation to the prediction based on the alternate binding change model proposed by Gresser et al. [(1982) J. Biol. Chem. 257, 12030-12038] in which energy transduction occurs only at the tri-site catalytic cycle. The present results essentially contrast with the model and rather indicate that if the alternate binding mechanism holds for the ATP hydrolytic reaction, the proton translocation should be coupled to at least both bi-site and tri-site cycles.

关键词： f 0 f 1 -ATPase Proton translocation Alternate binding change model Negative cooperativity (Submitochondrial particle) SMP, submitochondrial particle ANS, 8-anilino-naphthalene-1-sulfonate E D , an enzyme which binds ADP and P i E DT , an enzyme which binds ATP, ADP and P i E DTD , an enzyme which binds two ADPs, P i s and ATP E DTD , the energized form of E DTD

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H-1-NMR STUDIES ON NUCLEOTIDE BINDING TO THE CATALYTIC SITES Of BOVINE MITOCHONDRIAL f1-ATPASE

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fEBS LETTERS 1988年第1期242卷 178-182页

作者： GARIN, J VIGNAIS, PV GRONENBORN, AM CLORE, GM GAO, Z BAEUERLEIN, E NIDDKD CHEM PHYS LABBETHESDAMD 20892 MAX PLANCK INST BIOCHEM MEMBRANBIOCHEM ABTD-8033 MARTINSRIEDFED REP GER

The conformation of adenine nucleotides bound to bovine mitochondrial f 1 -ATPase was investigated using transfer nuclear Overhauser enhancement measurements. It is shown that all nucleotides investigated adopt a predominantly anti conformation when bound to the catalytic sites. furthermore, the experiment suggests that 8-azido-ADP and 8-azido-ATP, which are predominantly in the syn conformation in solution, are in the anti conformation when bound to f 1 catalytic sites.

关键词： f 1 -ATPase Nucleotide Bound conformation NOE transferred AdoPP(NH)P, adenosine 5′-[β,γ-imido]triphosphate NOE, nuclear Overhauser enhancement TRNOE, transferred nuclear Overhauser enhancement

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THE COMPLEX Of MITOCHONDRIAL f1-ATPASE WITH THE NATURAL INHIBITOR PROTEIN IS UNABLE TO CATALYZE SINGLE-SITE ATP HYDROLYSIS

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fEBS LETTERS 1988年第1-2期230卷 163-166页

作者： KALASHNIKOVA, TY MILGROM, YM POSTANOGOVA, NV MOSCOW STATE UNIV AN BELOZERSKY LAB MOLEC BIOL & BIOORGAN CHEMMOSCOW 119899USSR

Interaction of mitochondrial f 1 -ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [ 14 C]nucleotides in f 1 -ATPase during steady-state [ 14 C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein.

关键词： f 1 -ATPase Inhibitor protein Single-site catalysis IP, natural inhibitor protein of mitochondrial f 1 -ATPase BSA, bovine serum albumin

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STUDIES ON KINASE-CONTROLLED STATE TRANSITIONS IN PHOTOSYSTEM-II AND B6f MUTANTS fROM CHLAMYDOMONAS-REINHARDTII WHICH LACK QUINONE-BINDING PROTEINS

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第1期933卷 85-94页

作者： WOLLMAN, fA LEMAIRE, C Service de Photosynthèse Institut de Biologie Physico-Chimique ParisFrance

We have studied kinase-dependent state transitions in vivo using photosyntheic mutants from the green alga Chlamydomonas reinhardtii lacking in quinone-binding proteins. The aim of our study was to identify proteins involved in the plastoquinone-dependent activation of the LHC-kinase. Whereas mutants totally devoid of the quinone-binding subunits D1 and D2 of Photosystem II showed unaltered state transitions, mutants lacking the b6f complexes were incapable of state transitions. These mutants were blocked in State 1, which is indicative of either the absence of the LHC-kinase responsible for the regulation, or of the loss of a component responsible for the activation of this enzyme. These two hypotheses are discussed in light of (i) the patterns of phosphorylation of the thylakoid membrane proteins observed in the b6f mutants and (ii) the characteristics of the kinase activities recovered from their thylakoids.

关键词： LHC-kinase b 6 f complex State 1-State 2 transition Plastoquinone pool, oxidized/reduced ( C. reinhardtii ) LHC light-harvesting chlorophyll ab protein complex PS Photosystem Cf coupling factor PQ plastoquinone f max maximal fluorescence level PC plastocyanin fe-S iron-sulfur centre TMBZ 3,3,5,5′-tetramethylbenzidine DBMIB 2,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone PMSf phenylmethylsulphonyl fluoride NQNO 2- n -nonyl-4-hydroxyquinoline N -oxide DNP-INT dinitrophenyl ether iodonitrothymol Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid

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IDENTIfICATION Of ALPHA-SUBUNIT LYS201 AND BETA-SUBUNIT LYS155 AT THE ATP-BINDING SITES IN ESCHERICHIA-COLI f1-ATPASE

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fEBS LETTERS 1988年第2期233卷 347-351页

作者： TAGAYA, M NOUMI, T NAKANO, K fUTAI, M fUKUI, T Institute of Scientific and Industrial Research Osaka University Japan.

Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli f1-ATPase resulted in the nearly complete inactivation of the enzyme [(1987) J. Biol. Chem. 262, 7686-7692]. About two thirds of the label was bound to the alpha-subunit, and the rest to the beta-subunit. The present study revealed that Lys201 in the alpha-subunit and Lys155 in the glycine-rich region of the beta-subunit are the major sites labeled with this reagent. Thus, these two residues might be located close to the gamma-phosphate of the bound ATP.

关键词： f 1 -ATPase Nucleotide-binding site Affinity label Adenosine polyphosphopyridoxal Glycine-rich region AP 3 -PL adenosine triphosphopyridoxal fSBA 5′- p -fluorosulfonylbenzoyl adenosine fSBI 5′- p -fluorosulfonylbenzoyl inosine HPLC high performance liquid chromatography

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THE MITOCHONDRIAL ATP SYNTHASE INHIBITOR PROTEIN BINDS NEAR THE C-TERMINUS Of THE f1 BETA-SUBUNIT

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fEBS LETTERS 1988年第1期229卷 224-228页

作者： JACKSON, PJ HARRIS, DA UNIV OXFORD DEPT BIOCHEMS PARKS RDOXFORD OX1 3QUENGLAND

The specific, mitochondrial ATP synthase protein (If1) was covalently cross-linked to its binding site on the catalytic sector of the enzyme (f1-ATPase). The cross-linked complex was selectively cleaved, leaving If1 intact to facilitate the subsequent purification of the f1 fragment to which If1 was cross-linked. This fragment was identified by sequence analysis as comprising residues 394-459 on the f1 beta-subunit, near the C-terminus. This finding is discussed in the light of secondary structure predictions for both If1 and the f1 beta-subunit, and sequence homologies between mitochondrial and other ATP synthases.

关键词： Mitochondria ATP synthase f 1 -ATPase ATPase inhibitor protein (Bovine heart) EEDQ N -ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline HPLC high-performance liquid chromatography If 1 mitochondrial ATP synthase inhibitor protein PTH phenylthiohydantoin SDS-PAGE SDS-polyacrylamide gel electrophoresis

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OXIDATIVE-PHOSPHORYLATION IN A HYBRID SYSTEM CONTAINING BOVINE HEART MEMBRANES AND PEA MITOCHONDRIAL f1-ATPASE

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第2期933卷 389-392页

作者： HORAK, H PACKER, M HORAK, A UNIV ALBERTA DEPT PLANT SCI EDMONTON T6G 2E1 ALBERTA CANADA

Purified pea mitochondrial f1-ATPase reconstituted oxidative phosphorylation in both partially and completely f1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP synthesis when combined with bovine heart membranes, suggesting great evolutionary conservation of the ATP synthase complex in mitochondria.

关键词： Oxidative phosphorylation Plant f 1 -ATPase Reconstitution Mitochondrion f 1 mitochondrial coupling factor ASU-particles bovine heart submitochondrial particles treated with ammonia, Sephadex and urea OSCP oligomycin sensitivity-conferring protein

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THE ROTATION Of THE ALPHA-SUBUNIT Of f1 RELATIVE TO MINOR SUBUNITS IS NOT INVOLVED IN ATP SYNTHESIS - EVIDENCE GIVEN BY USING AN ANTI-ALPHA SUBUNIT MONOCLONAL-ANTIBODY

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第2期934卷 269-273页

作者： MORADIAMELI, M GODINOT, C Laboratoire de Biologie et Technologie des Membranes de Centre National de la Recherche Scientifique (CNRS) 69622 Villeurbanne Cedex France

To test whether ATP synthesis could occur via a mechanism of rotational catalysis in which the .alpha. and .beta. subunits of f1 would rotate with respect to the minor subunits, we have measured the rate of ATP synthesis after binding various masses of antibodies to f1. If the rotation was an essential feature of the mechanism, the rate of ATP synthesis should be inhibited either completely or proportionately to the load carried by f1. Bivalent immunoglobulins (IgG) or monovalent fab fragments of an anti-.alpha. monoclonal antibody (7B3) were bound to f1 present in electron-transport particles in a ratio of 2 fab or 2 IgG per f1. This binding similarly inhibited the rate of ATP synthesis by a maximum of about 50%. When anti-mouse immunoglobulins were added to the f1-7B3 (IgG) complex, no significant change in the rate of inhibition was observed. In conclusion, the rate of ATP synthesis was the same when f1 was loaded with 100 kDa (2 fab), 300 kDa (2 IGG, 7B3) or 900 kDa (2 IgG + 4 anti-mouse IgG). It is concluded that the rotation of the .alpha. subunits is extremely unlikely to play an essential role in the mechanism of ATP synthesis.

关键词： ATP synthesis f 1 Rotational catalysis α-Subunit rotation Monoclonal antibody P i inorganic phosphate PBS phosphate-buffered saline

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ESCHERICHIA-COLI f1-ATPASE - SITE-DIRECTED MUTAGENESIS Of THE BETA-SUBUNIT

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fEBS LETTERS 1988年第1期232卷 111-114页

作者： PARSONAGE, D WILKEMOUNTS, S SENIOR, AE Dept of Biochemistry Box 607 University of Rochester Medical Center Rochester NY 14642 USA

Residues βGlu-181 and βGlu-192 of E. coli f 1 -ATPase (the DCCD-reactive residues) were mutated to Gln. Purified βGln-181 f 1 showed 7-fold impairment of ‘unisite’ P i formation from ATP and a large decrease in affinity for ATP. Thus the β-181 carboxyl group in normal f 1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 × 10 4 - to 548-fold in βGln-181 f 1 . In contrast, purified βGln-192 f 1 showed only 6-fold reduction in ‘multisite’ ATPase activity. Residues βGly-149 and βGly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative ‘flexible loop’ in β-subunit. Impairment of purified f 1 -ATPase ranged from 5 to 61%, with the double mutant f 1 less impaired than either single mutant. f 1 preparations containing βIle-154 showed 2-fold activation after release from membranes, suggesting association with f 0 restrained turnover on f 1 in these mutants.

关键词： f 1 -ATPase Mutagenesis DCCD-reactive glutamate Conserved glycine residue DCCD, dicyclohexylcarbodiimide residue numbers are rationalized to the E. coli β-subunit sequence throughout

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