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REGULATORY ROLE Of THE ATPASE INHIBITOR PROTEIN ON PROTON CONDUCTION BY MITOCHONDRIAL H+-ATPASE COMPLEX

fEBS LETTERS 1987年第1期213卷 67-72页

作者： GUERRIERI, f SCARfO, R ZANOTTI, f YAN, WC PAPA, S UNIV BARI CNR CTR STUDY MITOCHONDRIA & ENERGY METAB I-70124 BARI ITALY

This study shows that the natural inhibitor protein of mitochondrial H+-ATPase complex (If1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by If1 is less effective in the purified f1 than in submitochondrial particles where f1 is bound to f0. No inhibition of H+ conductivity by f0 is observed in f1-depleted particles.

关键词： H + -ATPase Inhibitor protein Proton conductivity fo, membrane integral sector of the mitochondrial membrane f 1 , catalytic part of mitochondrial H + -ATPase If 1 , natural inhibitor protein of mitochondrial H + -ATPase ESMP, submitochondrial particles prepared in the presence of EDTA Sephadex-ESMP, particles depleted of If 1 by passing ESMP through a Sephadex column USMP, particles depleted of f 1 by urea treatment of Sephadex-ESMP

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ON THE MECHANISM Of LINOLENIC ACID INHIBITION IN PHOTOSYSTEM-II

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期890卷 215-223页

作者： WARDEN, JT CSATORDAY, K Department of Chemistry Rensselaer Polytechnic Institute Troy NY U.S.A.

Recent studies in our laboratory have reexamined the interaction of the unsaturated fatty acid, linolenic acid, with Photosystem II and have documented two principal regions of inhibition: one associated with the donor complex (Signal 2f or D1) to the reaction center, and the other located on the reducing side between pheophytin and Qa (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263-271). A further characterization of fatty acid inhibition of secondary electron transport in Photosystem II at room and cryogenic temperatures is presented in this paper. These studies demonstrate that linolenic acid, and related fatty acid analogs, (1) eliminate the transient absorption increase at 320 nm, attributed to QA-;(2) abolish the production, either chemically or photochemically, of the ESR signal (Q-fe) associated with the bound quinone acceptor, QA-;and (3) prevent the photooxidation of Signal 21t(D1) at cryogenic temperature. Linolenic-acid-treated samples are characterized by a high initial fluorescence yield (fi) equivalent to the maximum level of fluorescence (fmax);however, the spin-polarized triplet, associated with the reaction-center electron donor, P-680, is observed only in inhibited samples that have been prereduced with sodium dithionite. These results suggest the presence of an additional acceptor intermediate between pheophytin and Qa. The donor-assisted photoaccumulation of pheophytin anion in Photosystem II particles, as monitored by the decline of fluorescence yield, is inhibited by linolenic acid. Redox titrations of the fluorescence yield in control and inhibited preparations demonstrate that the midpoint potential for the primary acceptor for Photosystem II is insensitive to the fatty acid (Em .apprxeq. -583 mV) and thus indicate that primary photochemistry is functional during linolenic-acid inhibition. These data are consistent with the hypothesis that unsaturated fatty acids inhibit secondary electron transport in Photosystem II v

关键词： Linolenic acid Photosystem II Quinone ESR (Spinach chloroplast) Chl chlorophyll D 1 the primary quinoidal donor to P-680 + D-10 digitonin PS II particles DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea f i initial fluorescence yield f max maximum fluorescence yield f v variable fluorescence yield Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid P-680 primary electron donor of Photosystem II Pheo pheophytin PS I Photosystem I PS II Photosystem II Q a primary quinone acceptor Q b secondary quinone acceptor Q − fe iron-perturbed quinone ESR resonance associated with Q − a TSf-2 triton Photosystem II particles

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WESTERN-BLOTTING METHOD fOR DETECTING ANTIBODIES AGAINST HUMAN-MUSCLE CONTRACTILE PROTEINS IN MYOSITIS

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JOURNAL Of IMMUNOLOGICAL METHODS 1987年第1期105卷 15-21页

作者： KOGA, K ABE, S HASHIMOTO, H YAMAGUCHI, M JUNTENDO UNIV SCH MED DEPT MED 2-1-1 HONGO BUNKYO KU TOKYO 113 JAPAN TOHOKU UNIV SCH MED INST REHABIL MED SENDAI MIYAGI 980 JAPAN JUNTENDO UNIV SCH HLTH & PHYS EDUC DEPT BIOCHEM & NUTR CHIBA 275 JAPAN

Many investigators report anti-muscle antibodies using various kinds of methods. The Western-blotting method, however, has not previously been used for this purpose. We have detected antibodies to muscle contractile proteins in sera from patients with collagen disease and muscular dystrophy by this method. The antigens detected included myosin heavy and light chains, tropomyosin and troponin complex. Our method is a quick and sensitive way to determine which are the antigenic muscle contractile proteins.

关键词： Polymyositis Dermatomyositis Autoantibody Western-blotting method Glycerinated muscle, human Muscle contractile protein MG myasthenia gravis ELISA enzyme-linked immunosorbent assay PM polymyositis DM dermatomyositis MD muscular dystrophy RA rheumatoid arthritis SLE systemic lupus erythematosus PSS progressive systemic sclerosic SDS sodium dodecyl sulfate TBS Tris-buffered saline HC myosin heavy chains TN-T, I and C troponin-T, I and C TM-β and -α tropomyosin-β and -α LC myosin light chains f fast isoforms S slow isoforms CPK creatine phosphokinase

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fORMALDEHYDE REACTIONS AND THE BURDEN Of PROOf

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JOURNAL Of ALLERGY AND CLINICAL IMMUNOLOGY 1987年第5期79卷 705-706页

作者： PATTERSON, R DYKEWICZ, MS GRAMMER, LC PRUZANSKY, JJ ZEISS, CR HARRIS, KE Northwestern University Medical School Section of Allergy-Immunology 303 E. Chicago Ave. Chicago IL 60611 U.S.A.

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PHOTOSYSTEM-I CHARGE SEPARATION IN THE ABSENCE Of CENTER-A AND CENTER-B .3. BIOCHEMICAL-CHARACTERIZATION Of A REACTION CENTER PARTICLE CONTAINING P-700 AND fX

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期893卷 149-160页

作者： GOLBECK, JH PARRETT, KG MCDERMOTT, AE UNIV CALIF BERKELEY CHEM BIODYNAM LAB BERKELEY CA 94720 USA

The Photosystem I reaction center is a membrane-bound, multiprotein complex containing a primary electron donor (P-700), a primary electron acceptor (A0), and intermediate electron acceptor (A1) and three membrane-bound iron-sulfur centers (fX, fB, and fA). We reported in part I of this series (Golbeck, J.H. and Cornelius, J.M. (1986) Biochim. Biophys. Acta 849, 16-24) that in the presence of 1% lithium dodecyl sulfate (LDS), the reaction center becomes dissociated, resulting in charge separation and recombination between P-700 and fX without the need for prereduction of fA and fB. In this paper, we report (i) the LDS-induced onset of the 1.2-ms ''fast'' phase of the P-700 absorption transient is time-dependent, attaining a maximum 3:1 ratio of ''fast'' to ''slow'' kinetic phases;(ii) the ''fast'' kinetic phase, corresponding to the P-700+ fX- backreaction, is stabilized indefinitely by dilution of the LDS-treated particle followed by ultrafiltration over a YM-100 membrane;(iii) without stabilization, the P-700+ fX- reaction deteriorates, leading to the rise of the long-lived P-700 triplet formed from the P-700+ AO-backreaction: (iv) the ''slow'' kinetic phase correlates with the redox and ESR properties of fA and/or fB, which indicates that in a minority of particles the terminal iron-sulfur protein remains attached to the reaction center core;(v) the ultrafiltered reaction center is severely deficient in all of the low molecular-weight polypeptides, particularly the 19-kDa, 18-kDa and 12-kDa polypeptides relative to the 64-kDa polypeptide(s);(vi) the stabilized particle contains 5.8 mol labile sulfide per mol photoactive P-700, reflecting largely the iron-sulfur content of fX, but also residual fA and fB, on the reaction center;and (vii) the apoproteins of fA and fB are physically removed from the reaction center particle as indicated by the presence of protein-bound zero-valence sulfur in the YM-100 filtrate. These results are interpreted in terms of a model for

关键词： Photosystem I P-700 Reaction center Charge separation EXAfS PS I Photosystem I Chl chlorophyll ESR electron spin resonance f X synonym for center X (ESR nomenclature) and A 2 (optical nomenclature) f B synonym for center B (ESR nomenclature) f A synonym for center A (ESR nomenclature) and P-430 (optical nomenclature) CHAPS 3-[(3-cholamidopropyl)dimethylamino]-1-propane-sulfonate DCIP 2,6-dichlorophenolindophenol EXAfS extended X-ray absorption fine structure

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PHENYLGLYOXAL MODIfICATION Of THE PHOTOSYSTEM-II REACTION CENTER

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期890卷 224-232页

作者： CSATORDAY, K KUMAR, S WARDEN, JT RENSSELAER POLYTECH INST DEPT CHEM TROY NY 12180 USA

Time-resolved spectroscopic techniques, including optical flash photolysis and eletron spin resonance (ESR), have been used in conjunction with fluorescence-induction and dye-reduction assays to monitor electron transport in Photosystem II (PS II) subchloroplast particles incubated with the covalent modifier, phenylglyoxal. Phenylglyoxal-modified digitonin (D-10) particles from spinach are characterized by (1) a high initial fluorescence yield (fi) and an abolition of the variable component of fluorescence (fv);(2) and inhibition of PS-II-mediated reduction of dichlorophenol indophenol (DPIP) by sym-diphenylcarbazide;(3) an abolition of flash-induced absorption transients (t1/2 > 2 .mu.s) at 820 nm attributed to the primary electron donor, P-680+;(4) the inhibition of photoreduction of the acceptor Qa;and (5) the elimination of the ESR Signal 2s and Signal 2f. These observations suggest the critical participation of specific arginine residues on both the oxidizing and reducing sides of Photosystem II and also implicate phenylglyoxal as a quinone-binding site inhibitor (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263-271).

关键词： Photosystem II ESR Phenylglyoxal P-680 Chl chlorophyll DPIP 2,6-dichlorophenol indophenol Mes 4-morpholinoethanesulfonic acid Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid D-10 digitonin Photosystem II particles TSf-2 triton Photosystem II particles PS I Photosystem I PS II Photosystem II f i initial fluorescence yield f max maximum fluorescence yield f v variable fluorescence yield P-680 primary electron donor of Photosystem II Q a primary quinone electron acceptor Q b secondary quinone electron acceptor Pheo pheophytin

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INHIBITORY EffECT Of MYOGLOBIN AND HEMOGLOBIN ON THE DIRECT-ACTING MUTAGENICITY Of PROTEIN PYROLYSATE HETEROCYCLIC AMINE DERIVATIVES

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MUTATION RESEARCH 1987年第4期192卷 253-258页

作者： ARIMOTO, S OHARA, Y HIRAMOTO, K HAYATSU, H OKAYAMA UNIV FAC PHARMACEUT SCI OKAYAMA 700 JAPAN

We have shown in our earlier reports (Arimoto et al., 1980a, b) that hemin and some other porphyrins can inhibit the mutagenicity in Salmonella of heterocyclic amines derived from cooking of proteins. A direct interaction btween hemin and the mutagens was implicated on the basis of the observation that some of the mutagens were inactivated when they had been metabolically converted into direct-acting mutagens before the treatment with hemin. Hemin is a bound constitutent of various proteins including hemoglobin and myoglobin, which are abundantly present in the blood and muscles, respectively. An interesting question is whether or not hemoglobin and myoglobin can inhibit the activities of the mutagenic heterocyclic amines.

关键词： Myoglobin Hemoglobin Cytochrome c Heterocyclic amines N-OH-Trp-P-2 3-hydroxyamino-1-methyl-5 H -pyrido[4,3- b ]indole 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 MeAαC 2-amino-3-methyl-9 H -pyrido[2,3- b ]indole AαC 2-amino-9 H -pyrido[2,3- b ]indole MeIQ 2-amino-3,4-dimethylimidazo[4,5- f ]quinoline MeIQx 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline IQ 2-amino-3-methylimidazo[4,5- f ]quinoline N-OH-Glu-P-1 2-hydroxyamino-6-methylimidazo[4,5- a ]3′,2′]imidazole NADPH nicotinamide-adenine dinucleotide phosphate reduced form

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A RABBIT MODEL Of HYPERSENSITIVITY TO PLICATIC ACID, THE AGENT RESPONSIBLE fOR RED CEDAR ASTHMA

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JOURNAL Of ALLERGY AND CLINICAL IMMUNOLOGY 1987年第5期79卷 762-767页

作者： CHAN, H TSE, KS VANOOSTDAM, J MORENO, R PARE, PD CHANYEUNG, M UNIV BRITISH COLUMBIA VANCOUVER GEN HOSP DIV RESP 2775 HEATHER ST VANCOUVER V5Z 3J5 BC CANADA

We describe a rabbit model for the study of the immunogenicity and allergenicity of plicatic acid (PA), the small molecular weight compound in western red cedar responsible for occupational asthma in exposed workers. Specific anti-PA IgE as well as IgG antibodies could be raised, depending on the method of immunization. The sensitized rabbits reacted to antigenic challenge with PA-protein conjugates intravenously, with increases in respiratory frequency and pulmonary resistance. This animal model may be used for the further elucidation of the mechanism of occupational asthma induced by small molecular weight chemical compounds.

关键词： PA Plicatic acid HSA Human serum albumin PBS Phosphate-buffered saline OA Ovalbumin f Respiratory frequency V T Tidal volume R L Pulmonary resistance PCA Passive cutaneous anaphylaxis TDI Toluene diisocyanate A1(OH) 3 Aluminum hydroxide

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ENHANCEMENT Of COMPLEMENT-MEDIATED LYSIS Of DITHIOTHREITOL-TREATED ERYTHROCYTES INVOLVES INCREASED C9 INSERTION AND POLYMERIZATION

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MOLECULAR IMMUNOLOGY 1987年第8期24卷 887-896页

作者： HU, VW MAZOROW, DL NICHOLSONWELLER, A SHIN, ML HARVARD UNIV SCH MEDDEPT MEDBOSTONMA 02115 BETH ISRAEL HOSP BOSTONMA 02215 UNIV MARYLAND SCH MEDDEPT PATHOLBALTIMOREMD 21201

Treatment of human erythrocytes with dithiothreitol (DTT) increases the sensitivity of normal cells to complement (C)-mediated lysis. We have investigated the mechanism through which DTT increases cell susceptibility to complement by comparing the interactions of complement proteins with DTT-treated erythrocytes and with normal cells. In addition, we have studied the eflfect of DTT on the physical state of the erythrocyte membrane. Results indicated that the DTT primarily affects the interactions of the late components of complement with the cell membrane. In particular, the insertion efficiency of C9 and its ability to form tubular poly-C9 are enhanced on DTT-treated cells. Electron spin resonance (ESR) spectroscopic analyses of the treated and untreated membranes showed essentially no correlation between bulk membrane fluidity and the DTT-induced change in lytic susceptibility, suggesting no gross disruption of the membrane lipid structure by DTT. In view of the fact that DTT-treated erythrocytes have been proposed as a possible model for the abnormally complement-sensitive erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) which are deficient in a 75,000 mol. wt membrane protein called decay accelerating factor (DAf), we explored the possibility that DAf might be affected by DTT. Studies with anti-DAf f(ab’)₂ antibodies indicated that DAf activity is protected from DTT-treatment. These results are reinforced by the observation that DTT-treatment of DAf-deficient Type III PNH-E also led to enhanced lysis of PNH-E, implying that DTT affects membrane structures other than DAf. Thus, we conclude: (1) that DTT increases the lytic susceptibility of human erythrocytes to late components of human complement by modifying membrane structures to facilitate C9 insertion and polymerization, and (2) that DTT-treated erythrocytes are not a suitable model for PNH erythrocytes.

关键词： PNH paroxysmal nocturnal hemoglobinuria C complement E normal human erythrocytes PNH-E PNH erythrocytes DTT dithiothreitol DTT-E dithiothreitol-treated erythrocytes EA antibody-sensitized erythrocytes NHS normal human serum DAf decay accelerating factor f ( ab' ) 2 divalent antibody fragment without fc portion SDS-PAGE sodium dodecyi sulfate-polyacrylamide gel electrophoresis 12-APS-GlcN 12-(4-azido-2-nitrophenoxy) stearoyl[1− 14 C]-glucosamine ESR electron spin resonance 5-DS 5-doxyl stearate S membrane order parameter as determined by ESR spectroscopic methods C components are designated as C1, C2, etc and fragments of C components are expressed as C2a, C2b, etc C5b-9 the membrane attack complex of C poly-C9 the SDS-resistant polymerized form of C9 (tubular poly-C9)

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DIETHYLSTILBESTROL IS A POTENT INHIBITOR Of THE H+-PPASE BUT NOT Of THE H+-ATPASE Of RHODOSPIRILLUM-RUBRUM CHROMATOPHORES

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BIOCHIMICA ET BIOPHYSICA ACTA 1987年第2期892卷 236-244页

作者： STRID, A NORE, Bf NYREN, P BALTSCHEffSKY, M UNIV STOCKHOLM ARRHENIUS LAB DEPT BIOCHEM S-10691 STOCKHOLM SWEDEN

In Rhodospirillum rubrum chromatophores, diethylstilbestrol inhibits the photoinduced synthesis of ATP and PPi by the membrane-bound H+-ATPase and H+-PPase, respectively. 50% inhibition of ATP synthesis is obtained at 8 .mu.M diethylstilbestrol in the presence of 0.13 .mu.M BChl, while I50 for PPi formation is 20 .mu.M diethylstilbestrol at the same chromatophore concentration. Diethylstilbestrol also inhibits the hydrolytic activity of H+-PPase, both in the membrane-bound and in the solubilized and purified state. Inhibition to 50 is already attained at 3 .mu.M diethylstilbestrol in chromatophores when 1 .mu.M fCCP is present and the BChl-concentration is 0.62 .mu.M. The hydrolysis by the solubilized enzyme has an I50 of 5 .mu.M when 5 .mu.g protein/ml is used. In contrast to the PPi-hydrolysis, the ATPase activity of the chromatophores shows a small activation at low diethylstillbestrol concentration and becomes inhibited at higher concentrations. Also, solublized f0f1-ATPase is activated to a small extent by diethylstilbestrol at the concentrations tested. At low concentrations of BChl, the inhibitory action of diethylstilbestrol on ATP and PPi synthesis can be reversed by addition of bovine serum albumin. The time dependence and inhibition dependence on the energy state of the membrane and on the BChl concentration are examined for the ATP synthesis. The mechanism of inhibition by diethylstilbestrol is discussed.

关键词： Photophosphorylation Inhibition ATPase,H + - Pyrophosphatase,H + − Diethylstilbestrol ( R. rubrum ) H + -ATPase membrane-bound proton translocating ATP synthase f 0 f 1 -ATPase solubilized ATP synthase H + -PPase membrane-bound proton translocating PP i -synthase P i inorganic phosphate PP i inorganic pyrophosphate DCCD N , N ′-dicyclohexylcarbodiimide BChl bacteriochlorophyll MEGA-9 nonanoyl N -methylglucamide fCCP carbonyl cyanide p -trifluoromethoxyhydrazone

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