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fORMATION Of MUTAGENS, 2-AMINO-3,8-DIMETHYLIMIDAZO[4,5-f]QUINOXALINE (MEIQX) AND 2-AMINO-3,4,8-TRIMETHYLIMIDAZO[4,5-f]QUINOXALINE (4,8-DIMEIQX), IN HEATED fISH MEATS

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MUTATION RESEARCH 1987年第1期179卷 5-14页

作者： KIKUGAWA, K KATO, T Tokyo College of Pharmacy 1432-1 Horinouchi Hachioji Tokyo 192-03 Japan

fish meats were heated under conditions close to those used for cooking and processing. The mutagenic activity of the heated fish meats was estimated toward Salmonella typhimurium TA98 with metabolic activation after extraction with boiling water and adsorption to blue cotton. The numbers of His+ revertant colonies/5 g of the meat heat-dried without charring at ***. C for 15 min were about 3000 for bonito, about 1000 for tunny, less than 500 for mackerel, salmon, swordfish, sardine, horse mackerel and cod, and 0 for cuttlefish. The mutagens in the heat-dried bonito meat were purified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). They were identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) by comparison with the authentic specimen with respect to Rf values in TLC, retention times in HPLC, ultraviolet absorption spectra and mass spectra. The contents of MeIQx and 4,8-DiMeIQx in the bonito meat were estimated to be 5.2 and 5.4 ng/g, respectively. The major mutagens produced in the bonito, tunny and mackerel meats heated without charring at ***. C for 48 h and at ***. C for 15 min were found to be MeIQx and 4,8-DiMeIQx. It is interesting to note that the bonito and sardine meats grilled with charring for 15 min contained MeIQx and 4,8-DiMeIQx but higher mutagenicity was observed in the fraction that may contain 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and/or 2-aminodipyrido[1,2-a:3'',2''-d]imidazole (Glu-P-2).

关键词： fish meats, heated MeIQx 4,8-DiMeIQX Trp-P-1 3-amino-1,4-dimethyl-5 H -pyrido[4,3- b ]indole Trp-P-2 3-amino-1-methyl-5 H -pyrido[4,3- b ]indole Glu-P-1 2-amino-6-methyldipyrido[1,2- a : 3′,2′-d ]imidazole Glu-P-2 2-aminodipyrido[1,2- a : 3′,2′-d] imidazole AαC 2-amino-9 H -pyrido[2,3- b ]indole MeAαC, 2-amino-3-methyl-9 H -pyrido[2,3- b ]indole IQ 2-amino-3-methylimidazo[4,5- f ]quinoline MeIQ 2-amino-3,4-dimethylimidazo[4,5- f ]quinoline MeIQx 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline 4,8-DiMeIQx 2-amino-3,4,8-trimethylimidazo[4,5- f ]quinoxaline 7,8-DiMeIQx 2-amino-3,7,8-trimethylimidazo[4,5- f ]quinoxaline

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MONOCLONAL IMMUNOASSAYS fOR MAJOR DUST MITE (DERMATOPHAGOIDES) ALLERGENS, DER-P-I AND DER-f-I, AND QUANTITATIVE-ANALYSIS Of THE ALLERGEN CONTENT Of MITE AND HOUSE DUST EXTRACTS

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JOURNAL Of ALLERGY AND CLINICAL IMMUNOLOGY 1987年第2期80卷 184-194页

作者： CHAPMAN, MD HEYMANN, PW WILKINS, SR BROWN, MJ PLATTSMILLS, TAE UNIV VIRGINIA MED CTR DEPT MED DIV ALLERGY & CLIN IMMUNOL BOX 225 CHARLOTTESVILLE VA 22908 USA

Monoclonal, two-site radioimmunoassay (RIAs) were developed to measure allergen Der p I of Dermatophagoides pteronyssinus or Der f I of D. farinae. Microtiter plates coated with monoclonal antibody (Mab) were incubated with mite extract, and bound allergen was detected with a second 125I-labeled Mab of different epitope specificity. The Mab RIAs were very sensitive (nanogram range) and highly specific. D. pteronyssinus extracts with different concentrations of Der p I demonstrated parallel binding curves, whereas a potent D. farinae extract demonstrated < 5% of the Der p I binding in the same assay. Similar parallel curves were obtained with several D. farinae extracts in the Der f I assay, whereas D. pteronyssinus extract demonstrated little or no binding. The Mab RIAs were compared with an inhibition RIA that measured cross-reacting determinants on both Der p I and Der f I (antigen P1 equivalent [AgP1Eq]). The results demonstrated good quantitative agreement between these assays in commercial mite and house dust extracts (mean difference 1.57 .+-. 0.5-fold). Thirty house dust samples with known mite counts. Der p I, and AgP1Eq content were also compared. The summed Mab RIA values for Der p I and Der f I demonstrated a very good correlations with AgP1Eq values (r = 0.86;p < 0.001) and with assessments of total mite-allergen content by RAST inhibition (n = 21, r = 0.77, p < 0.001). furthermore, in samples with more than 10 mites per 100 mg of dust, the Der p I: Der f I ratio closely correlated with the ratio of the two mites counted by microscopy (n = 15, r = 0.89;p < 0.001). The Mab RIAs can measure allergen levels in mite or dust extracts without the need for purified allergen or affinity-purified antibodies and can readily be standardized. These assays will be useful in epidemiologic studies of allergic asthma, to assess patients'' exposure to mite allergens, and the effects of avoidance regimens. Because of the long-term stability and reproducibility of the reag

关键词： Mab Monoclonal antibody RIA Radioimmunoassay Der p I A major 24,000 MW allergen of Dermatophagoides pteronyssinus Der f I A major 24,000 MW allergen of D. farinae WHO/IUIS World Health Organization/Intemational Union of Immunological Societies NIBSC National Institute for Biological Standards and Control UVA University of Virginia SAS Saturated ammonium sulfate PBS-T Phosphate-buffered saline, pH 7.2 containing 0.05% Tween 20 BBS Borate-buffered saline, pH 8.0 BSA Bovine serum albumin AgP 1 Eq Antigen P 1 equivalent 10B9 Anti- Der p I monoclonal antibody 10B9 f6 A12 5H8 Anti- Der p I monoclonal antibody 5H8 C12 D9 6A8 Anti- Der f I monoclonal antibody 6A8 BIO D12 4C1 Monoclonal antibody 4Cl B8 3f8 that recognizes both Der p I and Der f I

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INHIBITION Of YEAST MITOCHONDRIAL f1-ATPASE,f0f1-ATPASE AND SUBMITOCHONDRIAL PARTICLES BY RHODAMINES AND ETHIDIUM-BROMIDE

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第1期892卷 108-117页

作者： WIEKER, HJ KUSCHMITZ, D HESS, B Max-Planck-Institut fur Ernahrungsphysiologie D-4600 Dortmund Germany

ATP hydrolysis by f1-ATPase is strongly inhibited by cationic rhodamines;neutral rhodamines are very poor inhibitors. Rhodamine 6G is a noncompetitive inhibitor of purified f0f1-ATPase and submitochondrial particles, however, an uncompetitive inhibitor of f1-ATPase (KI .apprxeq. 2.4 .mu.M for all three enzyme forms). Ethidium bromide is a noncompetitive inhibitor of f0f1-ATPase, submitochondrial particles and also f1-ATPase (KI .apprxeq. 270 .mu.M). Neither of the inhibitors affects the negative cooperativity (nH .apprxeq. 0.7). The non-identical binding sites for rhodamine 6G and ethidium bromide are located on the f1-moiety and are topologically distinct from the catalytic site. Binding of the inhibitors prevents the conformational changes essential for energy transduction. It is concluded that the inhibitor binding sites are involved in proton translocation. In f1-ATPase, binding of MgATP at a catalytic site causes conformational changes, which allosterically induced the correct structure of the rhodamine 6G binding site. In f0f1-ATPase, this conformation of the f1-moiety exists a priori, due to allosteric interactions with f0-subunits. The binding site for ethidium bromide on f1-ATPase does not require substrate binding at the catalytic site and is not affected by f0f1-subunit interactions.

关键词： ATPase inhibition, H + Rhodamine 6G Ethidium bromide (Yeast mitochondria) P i inorganic phosphate f 1 the ATPase component of ATP synthase f 0 the proton-conducting component (membrane sector) of ATP synthase Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid Rho6G rhodamine 6G

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AMINO-ACID SUBSTITUTIONS IN THE KAPPA-SUBUNIT Of THE f1f0-ATPASE Of ESCHERICHIA-COLI

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期890卷 195-204页

作者： COX, GB HATCH, L WEBB, D fIMMEL, AL LIN, ZH SENIOR, AE GIBSON, f Department of Biochemistry John Curtin School of Medical Research Australian National University Canberra City Australia

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature .epsilon.-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton tanslocation and lowered oxidative phosphorylation, but did not affect proton translocation through the f0. Purified f1-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal f1-ATPase. Partial revertant strains were isolated in which Pro-47 of the .epsilon.-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II .beta.-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III .beta.-turn. Space-filling models of the .beta.-turn (residues 46-49) in the normal, mutant and partial revertant .epsilon.-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the .epsilon.-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the f1-ATPase.

关键词： f 1 f 0 -ATPase Inhibition ε-Subunit Amino acid substitution Proton translocation

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NOVEL APPROACHES TOWARDS CHARACTERIZATION Of THE HIGH-AffINITY NUCLEOTIDE BINDING-SITES ON MITOCHONDRIAL f1-ATPASE BY THE fLUORESCENCE PROBES 3'-O-(1-NAPHTHOYL)ADENOSINE DIPHOSPHATE AND TRIPHOSPHATE

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第1期892卷 30-41页

作者： WEBER, J ROGNER, M SCHAfER, G TECH UNIV BERLIN MAX VOLMER INST BIOPHYS & PHYS CHEM D-1000 BERLIN 12 GERMANY

The fluorescence properties of 3''-O-(1-napththoyl)adenosine di- and triphosphates (termed N-ADP and N-ATP, respectively) were investigated in detail. Of special importance for the use of these analogues as environmental probes is their high quantum yield (0.58 in water) and the polarity dependence of shape and wavelength position of the emission spectrum. Upon binding of N-ADP and N-ATP to mitochondrial f1-ATPase, the fluorescence intensity is markedly decreased, due to polarity changes and ''ground-state''quenching. Using this signal for equilibrium binding studies, three (at least a priori) equivalent nucleotide-binding sites were detected on the enzyme. The perspective intrinsic dissociation constants are as follows: N-ADP/Mg2+ 120 nM;N-ATP/Mg2+ 160 nM;N-ADP/EDTA 560 nM;N-ATP/EDTA 3500 nM. for bound ligand the environment was found to be rather unpolar;the rotational mobility of the fluorophore is restricted, its accessibility for iodide anions (as a quencher) is hindered. These facts show a location of the binding sites quite deeply embedded in the protein. The conformation of the binding domains is strongly dependent on the absence or presence of Mg2+, as can be seen from the relative efficiencies of the singlet-singlet energy transfer from tyrosine residues in the protein to bound naphthoyl moieties. Investigation of the binding kinetics revealed this process as biphasic (in presence of Mg2+). After the first fast step (kon > 1 .cntdot. 106 M-1 .cntdot. s-1), in which the analogue is bound to the enzyme, a slow local conformational rearrangement occurs.

关键词： fluorescence probe Nucleotide analog Nucleotide binding ATPase, f 1 - High-affinity binding site f 1 -ATPase soluble part of the proton-translocating adenosine triphosphatase complex from bovine heart mitochondria (EC 3.6.1.34) pyruvate kinase (EC 2.7.1.40) lactate dehydrogenase (EC 1.1.1.27) photinus inciferin 4-monooxygenase (ATP-hydrolyzing) or luciferase (EC 1.13.12.7) N-AD(M,T)P 3′- O -(1-naphthoyl)adenosine di(mono, tri)phosphate lin -benzo-ADP 8- amino -3-(β- d - ribofuranosyl)imidazo [4,5-g] quinazoline 5′- diphosphate

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ASSOCIATION Of A SPERM-SPECIfIC PROTEIN WITH THE MITOCHONDRIAL f1f0-ATPASE IN HELIOTHIS - IMPLICATIONS fOR STERILITY Of HELIOTHIS-VIRESCENS X HELIOTHIS-SUBfLEXA BACKCROSS HYBRIDS

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INSECT BIOCHEMISTRY 1987年第3期17卷 417-+页

作者： MILLER, SG Insect Attractants Behavior and Basic Biology Research Laboratory Agricultural Research Service U.S. Department of Agriculture Gainesville FL 32604 U.S.A.

Evidence is presented that a mitochondrial protein that displays a species-specific net charge in the Heliothis spp complex is associated with subunits of the f1-ATPase. The expression of this 63 kd polypeptide, p63, was restricted to sperm and its developmental pattern of synthesis and accumulation paralleled that of the putative .beta.-subunit of the f1-ATPase complex. Comparisons of the enzyme from fertile Heliothis virescens and sterile (H. virescens .times. H. subflexa) backcross hybrid males revealed two differences. first, the specific activity of the f1-ATPase isolated from sterile males was half that of preparations from fertile males;and second, the p63 protein was bound less tightly to the complex in backcross sperm. The implications of these findings in relation to both the identification of the cause of backcross male sterility and prospects for future research are discussed.

关键词： Sperm mitochondria f 1 -ATPase male sterility

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AVAILABILITY TO MONOCLONAL-ANTIBODIES Of ANTIGENIC SITES Of THE ALPHA-SUBUNIT AND BETA-SUBUNIT IN ACTIVE, DENATURED OR MEMBRANE-BOUND MITOCHONDRIAL f1-ATPASE

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第1期890卷 55-65页

作者： MORADIAMELI, M GODINOT, C UNIV LYON 1 CNRS BIOL & TECHNOL MEMBRANES LAB 43 BLVD 11 NOVEMBRE 1918 F-69622 VILLEURBANNE FRANCE

The binding of five monoclonal antibodies to mitochondrial f1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sits. The accessibility of the epitopes has been compared for f1 integrated in the mitochondrial membrane, for purified .beta.-subunit and for purified f1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-.beta. monoclonal antibodies bound more easily to the .beta.-subunit than to active f1, and recognized equally active f1 and f1 intergrated in the membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound f1 and better exposed in the isolated .beta.-subunit. In addition, unfolding f1 by urea srongly increased the binding of one anti-.beta. monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the .beta.-subunit. One anti-.alpha. monoclonal antibody (20 D6) bound poorly to f1 integrated in the membrane, while the other (7 B3) had a higher affinity for f1 integrated in the membrane than for soluble f1. Therefore, 20 D6 recognizes an epitope of the .alpha.-subunit buried inside f1 integrated in the membrane, while 7 B3 binds to a domain of the .alpha.-subunit well exposed at the surface of the inner face of the mitochondrial membrane.

关键词： Monoclonal antibody Protein conformation ATPase f 1 (Pig heart mitochondria f 1 mitochondrial f 1 -ATPase mAb monoclonal antibody ELISA enzyme-linked immunosorbent assay RIA radioimmunoassay

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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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OXIDATION REDUCTION KINETICS Of CYTOCHROME-f IN WHOLE CELLS

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JOURNAL Of PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 1987年第1期1卷 45-60页

作者： MAROTI, P MENDE, D WIESSNER, W UNIV GOTTINGEN INST PFLANZENPHYSIOLEXPTL PHYKOL ABTD-3400 GOTTINGENFED REP GER

The kinetic aspects of the cytochrome f function and its relation to the non-cyclic electron transport were investigated in photosynthetizing green algae under physiological conditions. Easy-to-use and low cost single- and double-beam spectrophotometers were constructed which enabled the measurement of light-induced absorption changes of cytochromes of highly scattering samples under different light—dark preconditioning regimes. The complexity of the light-induced absorption spectra in the 540 – 570 nm spectral range is discussed. The lack of separation of the components based on their kinetic behaviour indicated that different cytochrome f forms contributed to the spectra. By comparison with the time course of the variable part of the fluorescence at 744 nm at room temperature, it was demonstrated that the oxidation of cytochrome f is a pure photochemical process if the half-risetime is greater than 50 ms. The dark-reduction kinetics is monophasic after a single-flash excitation of dark-adapted algae ( t 1 2 = 2 ms) or after producing a photostationary state by means of a long (1 s) and relatively weak (half-risetime of the oxidation greater than 50 ms) flash ( t 1 2 = 500 ms, highly dependent on the systematic position and the states of the algae). The biphasicity (the existence of a mixture of fast and slow components) is evident in the reduction kinetics after excitation by a long and intense flash. The results are discussed within the framework of a simple linear pathway: the electrons from a pool of reduced plastoquinone are transferred to cytochrome f , to plastocyanin and then to the reaction centre of photosystem I. We distinguish basically two states of electron pressure in the plastoquinone pool: high if the half-time of dark reduction is less than 15 ms, and low if it is greater than 100 ms. The states of the pool can interconvert by preconditioning (dark—light regime) and excitation (oxidation). The majority of the data presented in this paper were obta

关键词： Green algae photosynthesis electron transport cytochrome f light—dark redox reactions.

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DISSOCIATION-RECONSTITUTION EXPERIMENTS SUPPORT THE PRESENCE Of 2 CATALYTIC BETA-SUBUNITS IN MITOCHONDRIAL f1

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期894卷 277-283页

作者： NIEBOER, P HARTOG, Af BERDEN, JA UNIV AMSTERDAM BCP JANSEN INST BIOCHEM LABPLANTAGE MUIDERGRACHT 121018 TV AMSTERDAMNETHERLANDS

Mitochondrial f 1 , inactivated to various extents with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), was dissociated with LiCl and reconstituted after removal of the salt. This procedure resulted in a reactivation that corresponded with a reactivation theoretically expected on the basis of the assumption that the reassociation of β-subunits into native f 1 molecules is random and that two out of the three β-subunits are directly involved in catalysis. Repeated inactivation of such reactivated f 1 , followed by the same dissociation-association procedure, resulted in similar data. After inactivation of f 1 by covalent binding of 2-N-AT(D)P to one catalytic site, no reactivation upon dissociation-reassociation was obtained due to the fact that such modified f 1 did not dissociate under the experimental conditions used.

关键词： ATPase, f 1 - Quaternary structure Chemical modification β-Subunit Catalytic site (Bovine heart mitochondria) NBD-Cl 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (also named 4-chloro-7-nitrobenzofurazan) f 1 mitochondrial ATPase DCCD dicyclohexylcarbodiimide

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