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ESCHERICHIA-COLI f1 ATPASE IS REVERSIBLY INHIBITED BY INTRASUBUNIT AND INTERSUBUNIT CROSS-LINKING - AN APPROACH TO ASSESS ROTATIONAL CATALYSIS

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第1期890卷 97-105页

作者： KANDPAL, RP BOYER, PD UNIV CALIF LOS ANGELES DEPT CHEM & BIOCHEM LOS ANGELES CA 90024 USA UNIV CALIF LOS ANGELES INST MOLEC BIOL LOS ANGELES CA 90024 USA

Reaction of the multisubunit f1 ATPase from Escherichia coli (Ef1) with a bifunctional cleavable crosslinker, 3,3''-dithiobis(succinimidylpropionate) (DSP), has been used to explore the possibility that during catalysis a rotational movement of catalytic subunits relative to noncatalytic subunits occurs. The premise is that such rotational catalysis is tenable if intersubunit crosslinking of a major subunit with one of the minor subunits inhibits the enzyme activity and if upon cleavage of the crosslinks, the enzyme regains activity. The results presented in this paper show that crosslinking of about 5-6 reactive groups on Ef1 with DSP is accompanied by a loss of 2/3 of the enzyme activity. Both intra- and intersubunit crosslinks are formed. The most prominent intersubunit crosslinks are those of .gamma. and .delta. subunits with the .alpha. subunit. Nearly complete recovery of activity can be attained by cleaving the disulfide bond in the crosslinker with dithiothreitol. Because the chemical modification of enzyme groups remains after the crosslinker is cleaved, the loss in activity before cleavage can be ascribed to conformational restraints. The results show that catalysis by the Ef1 ATPase is highly sensitive to the restrictions of crosslinking, and are consistent with the view that catalysis is accompanied by appreciable movements of the major subunits with respect to the minor subunits, as suggested for rotational catalysis.

关键词： f 1 -ATPase ATPase crosslinking Rotational catalysis ATPase conformation ( E. coli ) f 1 coupling factor one ATPase Ef 1 ATPase f 1 ATPase from Escherichia coli Mops 4-morpholinepropanesulfonic acid DSP 3,3′-dithiobis(succinimidylpropionate) SDS sodium dodecyl sulfate DMSO dimethylsulfoxide P i inorganic phosphate DTT dithiothreitol

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SUBUNIT COMPOSITION AND COLD STABILITY Of THE PEA COTYLEDON MITOCHONDRIAL f1-ATPASE

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期893卷 190-196页

作者： HORAK, A HORAK, H PACKER, M Department of Plant Science The University of Alberta Edmonton Alberta Canada

The f1-ATPase isolated from pea cotyledon sumitochondrial particles contained six types of subunit with molecular weights of 57000 (.alpha.), 55000 (.beta.), 36500 (.gamma.), 26500 (.delta.), 22500 (.delta.'') and 8000 (.epsilon.). The same polypeptide composition was observed even when the purification was carried out at ***.C in the presence of proteolytic inhibitors, suggesting that the sixth subunit was not a proteolytic product formed during the isolation procedure. The six-subunit f1-ATPase exhibited considerable cold stability: it retained 65% of its activity after 24 h of incubation at ***.C and was more than 90% active after 48 h of incubation at ***.C. The 26.5 kDa protein could be dissociated from the remaining f1-ATPase by centrifugation in a linear sucrose gradient containing (NH4)2SO4 and deoxycholate. The resulting five-subunit f1-ATPase was considerably less stable at ***.C than the six-subunit enzyme. Several features suggest the possibility of a functional and structural relationship between the 26.5 kDa a protein of the pea cotyledon mitochondria and the mammalian oligomycin-sensitivity-conferring protein.

关键词： Mitochondrion Mitochondrial f 1 -ATPase Coupling factor f 1 -subunit Cold stability (Pea) f 1 mitochondrial coupling factor PMSf phenylmethylsulfonylfluoride Tricine N -[2-hydroxy-11-bis(hydroxymethyl)ethyl]glycine P i inorganic phosphate

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BETA-SUBUNIT Of ESCHERICHIA-COLI f1-ATPASE - AN AMINO-ACID REPLACEMENT WITHIN A CONSERVED SEQUENCE (G-X-X-X-X-G-K-T/S) Of NUCLEOTIDE-BINDING PROTEINS

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fEBS LETTERS 1987年第2期218卷 222-226页

作者： HSU, SY NOUMI, T TAKEYAMA, M MAEDA, M ISHIBASHI, S fUTAI, M OSAKA UNIV INST SCI & IND RESDEPT ORGAN CHEM & BIOCHEMIBARAKIOSAKA 567JAPAN

A mutant strain Kf87 of E. coli with a defective beta-subunit (Ala-151----Val) of f1-ATPase was isolated. The mutation is within the conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins. The mutant f1-ATPase had a much higher rate of uni-site hydrolysis of ATP than the wild type, and about 6% of the wild-type multi-site activity. The mutant enzyme showed defective transmission of conformational change(s) between the ligand- and aurovertin-binding sites.

关键词： f 1 -ATPase Nucleotide-binding protein Uni-site catalysis Oncogene

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METABOLISM Of 2-AMINO-3-METHYLIMIDAZO[4,5-f]QUINOLINE (IQ) AND 2-AMINO-3,4-DIMETHYLIMIDAZO[4,5-f]QUINOLINE (MEIQ) IN SUSPENSIONS Of ISOLATED RAT-LIVER CELLS

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TOXICOLOGY IN VITRO 1987年第4期1卷 175-181页

作者： HOLME, JA ALEXANDER, J BECHER, G TRYGG, B Department of Toxicology National Institute of Public Health Geitmyrsveien 75 N-0462 Oslo 4 Norway.

Suspensions of rat-liver cells metabolized (14)C-labelled 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to metabolites that were ethyl acetate extractable, water soluble or covalently bound to macromolecules. The major ethyl acetate extractable metabolite(s) had a retention time on the high-performance liquid chromatograph (HPLC), an ultraviolet spectrum and an R(f) value on the thin-layer chromatograph that corresponded to those of the N-acetylated derivative. Relatively more of this metabolite was formed from MeIQ than from IQ. In contrast, IQ was more easily converted to water-soluble metabolites than was MeIQ. The amount of covalently bound metabolite(s) found with MeIQ as test substance was larger than that found with IQ. No major increase in the ethyl acetate-extractable metabolites was obtained after incubation of the aqueous phase with beta-glucuronidase or aryl sulphatase in comparison with untreated controls. HPLC analysis showed that after acid hydrolysis of the water-soluble metabolites, about 55 and 20% of the hydrolysed metabolites had retention times that were the same as those of IQ and MeIQ, respectively. The ratio between covalently bound (activated) metabolites and water-soluble (detoxified) metabolites was larger for MeIQ than for IQ. from these data, one would expect MeIQ to be more potent than IQ as a liver carcinogen in male rats.

关键词： AcIQ N -acetyl-2-amino-3-methylimidazo[4,5- f ]quinoline AcMeIQ N -acetyl-2-amino-3,4-dimethylimidazo[4,5- f ]quinoline DMSO dimethylsulphoxide EGTA ethylene glycol bis-(β-aminoethyl ether) N , N , N ′, N ′-tetraacetic acid HPLC high-pressure liquid chromatography IQ 2-amino-3-methylimidazo[4,5- f ]quinoline MeIQ 2-amino-3,4-dimethylimidazo[4,5- f ]quinoline

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THE PURIfICATION Of CYTOCHROME-f AND PLASTOCYANIN USING AffINITY-CHROMATOGRAPHY

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期894卷 327-331页

作者： MOLNAR, SA ANDERSON, GP GROSS, EL Department of Biochemistry The Ohio State University Columbus OH U.S.A.

Both plastocyanin and cytochrome f were purified using a combination of affinity chromatography together with established methods. Plastocyanin was partially purified using the method of Davis and San Pietro (Anal. Biochem. 95 (1979) 254–259), after which it was further purified using a column of cytochrome c covalently attached to Sepharose 4B. The affinity column was prepared using the method of Godinot and Gautheron (Methods Enzymol. 54 (1979) 112–114). The final purity index ratio ( A 278 A 597 ) was less than 1.2, which is equal to that obtained using the more expensive fPLC procedure (Anderson, G.P., Sanderson, D.G., Lee, C.H., Durell, S., Anderson, L.B. and Gross, E.L. (1987) Biochim. Biophys. Acta 894, issue 3). Cytochrome f was partially purified using a modification of the method of Matazaki et al. (Plant Cell. Physiol. 16 (1975) 237–246) and bound to an affinity column of plastocyanin covalently attached to Sepharose 4B. Cytochrome f purified using this procedure had a purity index ratio ( A 554.5 A 277 ) of 1.2. Both proteins are tyrosine proteins containing no tryptophan residues. After the affinity chromatography step, the fluorescence emission spectrum of either plastocyanin or cytochrome f was typical of a tyrosine protein free from tryptophan contamination.

关键词： Cytochrome f Plastocyanin Cytochrome c Protein-protein interaction Affinity chromatography CM-Sephadex carboxymethyl-Sephadex fPLC fast protein liquid chromatography PC-Sepharose plastocyanin covalently attached to Sepharose 4B

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THERMAL INACTIVATION Of ELECTRON-TRANSPORT fUNCTIONS AND f0f1-ATPASE ACTIVITIES

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第1期894卷 16-28页

作者： TOMITA, M KNOX, BE TSONG, TY JOHNS HOPKINS UNIV SCH MED DEPT BIOL CHEM BALTIMORE MD 21205 USA

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at ***.C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 .+-. ***.C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, f0f1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 .+-. ***.C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 .+-. ***.C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of f0f1 persists to 61.0 .+-. ***.C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 .+-. ***.C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic and release a total of approx. 105 J/g protein. Solubilized and reconstituted f0f1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 .+-. ***.C, but the latter at 58.0 .+-. ***.C. Differential scanning calorimetry also detects biphasic transitions of f0f1, with similar temperatures of denaturation (49.0 and ***.C). from these and other results presented in this communication, the following is concluded. (1) A selective in

关键词： ATPase, f 0 f 1 - Electron transport chain Thermal denaturation (Bovine heart mitochondrion) Hepes 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid Tricine N -[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine P i inorganic phosphate DCCD N , N ′-dicyclohexylcarbodiimide DSC differential scanning microcalorimetry fCCP carbonylcyanide p -trifluoromethoxyphenylhydrazone

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MEASUREMENT Of CYTOCHROME-f IN THYLAKOIDS Of HIGHER-PLANTS USING QUANTITATIVE GEL-ELECTROPHORESIS

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ANALYTICAL BIOCHEMISTRY 1987年第1期164卷 31-34页

作者： HOLLOWAY, PJ MACLEAN, DJ SCOTT, KJ UNIV QUEENSLAND DEPT BIOCHEM ST LUCIA QLD 4067 AUSTRALIA

A quantitative method was developed to estimate the concentration of cytochrome (cyt) f in isolated thylakoids, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with a heme-specific reagent containing 3,3'',5,5''-tetramethylbenzidine and hydrogen peroxide. This densitometric technique was at least as sensitive as difference spectroscopy. Analysis of thylakoid preparations by densitometry of stained bands using cyt c as standard gave molar ratios of cyt/chlorophyll which were identical to ratios obtained by difference spectroscopy. Densitometric assays demonstrated that the molar ratio of cyt t/chlorophyll decreased during leaf aging in seven higher plants;however, there was a marked difference in the rate at which cyt f was lost from the leaves of different species.

关键词： cytochrome f gel electrophoresis photosynthesis thylakoids heme-dependent peroxidase tetramethylbenzidine

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ON THE RATE Of f1-ATPASE TURNOVER DURING ATP HYDROLYSIS BY THE SINGLE CATALYTIC SITE - EVIDENCE THAT HYDROLYSIS WITH A SLOW RATE Of PRODUCT RELEASE DOES NOT OCCUR AT THE ALTERNATING ACTIVE-SITE

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fEBS LETTERS 1987年第1期222卷 32-36页

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

Under conditions of molar excess of enzyme, isolated f1-ATPase from beef heart mitochondria catalyses ATP hydrolysis biphasically. The rate constants for product release are approximately 10(-1) and 10(-4)-10(-3) s-1, respectively. The slow phase of ATP hydrolysis is insensitive to EDTA. [gamma-32P]ATP splitting in the slow phase cannot be chased from the enzyme during several catalytic turnovers. It follows from these results that the slow single-site hydrolysis of ATP (kcat approximately 10(-4) s-1), initially observed by Grubmeyer et al. [(1982) J. Biol. Chem. 257, 12092-12100], is not carried out by the normal catalytic site. In contrast, the phase of rapid ATP hydrolysis (kcat approximately 10(-1) s-1) is completely prevented by EDTA and is believed to be the normal function of the normal catalytic site of f1-ATPase.

关键词： f 1 -ATPase Single-site catalysis Turnover rate

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STEADY-STATE RATE Of f1-ATPASE TURNOVER DURING ATP HYDROLYSIS BY THE SINGLE CATALYTIC SITE

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fEBS LETTERS 1987年第1期212卷 63-67页

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

Under the conditions of ATP regeneration and molar excess of nucleotide-depleted f1-ATPase the enzyme catalyses steady-state ATP hydrolysis by the single catalytic site. Values of Km = 10(-8) M and Vm = 0.05 s-1 for the single-site catalysis have been determined. ADP release limits single-site ATP hydrolysis under steady-state conditions. The equilibrium constant for ATP hydrolysis at the f1-ATPase catalytic site is less than or equal to 0.7.

关键词： f 1 -ATPase Single-site catalysis Active site Equilibrium constant

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ANAEROBIC METABOLISM Of 2-AMINO-3-METHYL-3H-IMIDAZO[4,5-f]QUINOLINE (IQ) BY HUMAN fECAL fLORA

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MUTATION RESEARCH 1987年第3期190卷 187-190页

作者： BASHIR, M KINGSTON, DGI CARMAN, RJ VANTASSELL, RL WILKINS, TD VIRGINIA POLYTECH INST & STATE UNIV DEPT ANAEROB MICROBIOL BLACKSBURG VA 24061 USA

Incubation of the heterocyclic cooked food mutagen IQ with mixed human fecal microflora under anaerobic conditions yielded 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (2) as the major detectable meta... 详细信息

关键词： 2-Amino-3-methyl-3 H -imidazo[4,5- f ]quinoline IQ (Human fecal microflora) Cooked food mutagen IQ, 2-amino-3 H -imidazo[4,5- f ]quinoline, 76180-96-6 N 6 -methyl-5,6-quinolinediamine, 14204-98-4 PTLC, preparative thin-layer chromatography BHI, brain heart infusion DMSO, dimethyl sulfoxide EI, electron impact

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