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THE BINDING AND RELEASE Of THE INHIBITOR PROTEIN ARE GOVERNED INDEPENDENTLY BY ATP AND MEMBRANE-POTENTIAL IN OX-HEART SUBMITOCHONDRIAL VESICLES

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第1期933卷 12-21页

作者： LIPPE, G SORGATO, MC HARRIS, DA UNIV PADUA INST BIOCHEM I-35100 PADUA ITALY UNIV PADUA CNR STUDY MITOCHONDRIAL PHYSIOL UNIT I-35100 PADUA ITALY UNIV OXFORD DEPT BIOCHEM OXFORD ENGLAND

(1) The effects of membrane potential (.DELTA..psi.) and nucleotides on the interaction between the f1-ATP synthase and its natural inhibitor protein (If1) are studied in ox-heart submitochondrial vesicles. (2) Membrane potential causes displacement of If1 from submitochondrial vesicles, as shown by measuring both .DELTA..psi.-dependent stimulation of ATPase capacity and release of 125I-labelled If1 from the vesicles. These effects are abolished if ATP is included in the incubation. (3) There is a linear increase in the steady-state ATPase capacity of oxidising vesicles as .DELTA..psi. is increased from 100 mV to 135 mV. Increasing .DELTA..psi. above 140 mV leads to no further change. (4) At a constant membrane potential, ATP suppresses the increase in ATPase capacity, with a concentration for half maximal effect of 140 .mu.M. This value is close to the Km for ATP hydrolysis by membrane-bound f1. This suppression is related to ATP concentrations rather than to .*** or ATP/ADP ratio. (5) The unidirectional on- and off-rates of If1 were measured separately. The off-rate of If1 is increased by membrane potential but unaffected by ATP. The on-rate, conversely, is increased by ATP. Thus, the suppression of the potential-dependent net release of If1 from submitochondrial vesicles by ATP results from an increase of the If1 on-rate above the off-rate.

关键词： Inhibitor protein ATP synthase ATP synthase inhibitor protein interaction ATP Membrane potential Bovine heart submitochondrial vesicle If 1 naturally occurring inhibitor protein of mitochondrial ATPase [ 125 I]If 1 radioiodinated inhibitor protein MgATP SMP submitochondrial vesicles prepared in the presence of ATP and magnesium [ 125 I]If 1 SMP submitochondrial vesicles supplemented with [ 125 I]If 1 Δp protonmotive force = Δ∼μ H f = Δψ + (2.303 RT f ) ΔpH , where Δψ is the electrical component and ΔpH the chemical component of the transmembrane difference of the electrochemical potential of protons ( Δ ∼ μ H ) P i inorganic phosphate ΔG P = ΔG O′ + RT In ( [ATP] [ADP][P i ] ) Ap 5 A, P 1 ,P 5 -di(adenosine-5′-)pentaphosphate fCCP carbonylcyanide p -trifluoromethoxyphenylhydrazone CCCP carbonylcyanide m -chlorophenylhydrazone

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SITE-DIRECTED MUTAGENESIS Of STABLE ADENOSINE-TRIPHOSPHATE SYNTHASE

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第1期933卷 156-164页

作者： YOHDA, M OHTA, S HISABORI, T KAGAWA, Y JICHI MED SCH DEPT BIOCHEM MINAMIKAWACHI TOCHIGI 32904 JAPAN WASEDA UNIV SCH EDUC DEPT BIOL SHINJUKU KU TOKYO 160 JAPAN

Evidence was obtained that four ionizable residues in the .alpha. and .beta. subunits of thermophilic ATP synthase (Tf0f1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. Tf0f1 was used because it is the only ATP synthase whose .alpha.-, .beta.-and .gamma.-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its .beta.-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting .beta.I164 and .beta.N252 subunits were both noncatalytic after re-assembly into the .alpha..beta..gamma.-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the .alpha.-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting .alpha.I175 subunit reassembled weakly into an oligomer, while the .alpha.N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an .alpha..beta..gamma.-complex that showed no inter-subunit cooperativity.

关键词： Site directed mutagenesis ATP synthase MgATP binding segment Subunit cooperativity f 0 f 1 f 0 f 1 ATP synthase Tf 0 f 1 thermophilic f 0 f 1 f 1 a catalytic portion of ATP synthase Tf 1 thermophilic f 1 Ef 1 Escherichia coli f 1 , 2 × TY medium, 16 g bactotryptone, 10 g yeast extract and 5 g NaCl per 1 UV ultraviolet

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LONG-WAVELENGTH OPTICAL LATTICE-VIBRATIONS IN MNXCD1-Xf2 fLUORIDES

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JOURNAL Of PHYSICS AND CHEMISTRY Of SOLIDS 1988年第10期49卷 1197-1203页

作者： KROL, A SWIATEK, K UNIV WARSAW INST EXPTL PHYSPL-00681 WARSAWPOLAND POLISH ACAD SCI INST PHYSPL-02668 WARSAWPOLAND

The far-infrared reflectivity spectra of Mn x Cd 1− x f 2 mixed fluorides (0 ⩽ x ⩽0.26) were measured at 80 and 300 K over the spectral range 100–600 cm −1 . A single reststrahlen band with some fine structure was observed. The position of this band shifts with the composition x . The MREI model was adapted to the investigated system. The long-wavelength optical phonons' frequencies were obtained and compared with model computations.

关键词： Infrared spectra optical phonons Mn x Cd 1− x f 2 mixed fluorides

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CORRELATION BETWEEN HEAT TOLERANCE AND DROUGHT TOLERANCE IN CEREALS DEMONSTRATED BY RAPID CHLOROPHYLL fLUORESCENCE TESTS

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JOURNAL Of PLANT PHYSIOLOGY 1988年第5期133卷 555-560页

作者： HAVAUX, M ERNEZ, M LANNOYE, R Laboratoire de Physiologie végétale Université Libre de Bruxelles CP 169 28 avenue Paul Heger B-1050 Brussels Belgium

Drought and heat tolerances were measured in intact leaves of a large number of genotypes of durum wheat (Triticum durum Desf.), barley (Hordeum vulgare L.) and triticale (Triticum durum L. .times. Secale cereale L.) using rapid in vivo chlorophyll fluorescence tests. The high temperature r(Tp) corresponding to maximal fo-fluorescence in leaf samples heated at a rate of ***. C min-1 was used to estimate the relative heat tolerance. On the other hand, drought tolerance was measured by the reduction of the photochemical quenching of chlorophyll fluorescence (qQ) after a short desiccation treatment. The data indicate a very wide range of genotypic adaptation to drought and heat in cereals. The two types of tolerance were, however, closely related: the most heat tolerant cereal varieties were also the most drought tolerant ones. The results presented in this paper also provide a good example of the usefulness and the simplicity of modulated chlorophyll fluorescence measurements as rapid screening tests for stress tolerance in crop plants.

关键词： Triticum durum Hordeum vulgare triticale chlorophyll fluorescence drought heat tolerance f o dark (or initial) level of chlorophyll fluorescence f v variable fluorescence level f m maximal fluorescence level q Q photochemical quenching of chlorophyll fluorescence PS II and 1 photosystems II and I Q A , primary electron acceptor of PS II T c critical temperature T p , temperature of peak fluorescence LHCP Light Harvesting Chlorophyll Protein Complexes

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fLUORESCENCE ANALYSIS Of SIZE DISTRIBUTION AND MODE Of DYE RELEASE fROM CARBOXYfLUORESCEIN-LOADED VESICLES - APPLICATION TO THE STUDY Of COMPLEMENT-MEMBRANE INTERACTIONS

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第2期945卷 253-262页

作者： LIU, ZY SOLOW, R HU, VW GEORGE WASHINGTON UNIV SCH MED & HLTH SCI DEPT BIOCHEM 2300 EYE ST NW WASHINGTON DC 20037 USA UNIFORMED SERV UNIV HLTH SCI DEPT BIOCHEM BETHESDA MD 20814 USA

We wish to report a novel method for visualizing large unilamellar vesicles loaded with a fluorescent dye and for monitoring changes in the size distribution as well as state of aggregation of such dye-loaded liposomes. In addition, we demonstrate that this method can be used to distinguish between all-or-none release of dye and graded release of dye from individual vesicles. Using this technique, we have characterized complement-mediated release of carboxyfluorescein from large unilamellar vesicles and have found that C5-8 complexes mediate a graded release of dye while C5-9 complexes cause an all-or-none release. furthermore, complement appears to preferentially attack the medium to larger-sized vesicles in our population of large unilamellar vesicles while smaller vesicles appear to be selectively spared.

关键词： Car☐yfluorescein-loaded vesicle Size distribution Vesicle aggregation Complement-induced permeability Permeability Dye release LUV large unilamellar vesicle Cf 6-car☐yfluorescein PBS phosphate-buffered saline EGTA ethylene glycol bis(β-aminoethyl ether)- N,N,N ′,N′-tetraacetic acid CRT cathode ray tube PMT photomultiplier tube f o fluorescence of Cf-loaded vesicles f t fluorescence after solubilization of vesicles with Triton X-100 C5 complement proteins comprising the membrane attack complex C6 complement proteins comprising the membrane attack complex C7 complement proteins comprising the membrane attack complex C8 complement proteins comprising the membrane attack complex C9 complement proteins comprising the membrane attack complex

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INfLUENCE Of ELECTROSTATIC SCREENING BY CATIONS ON ENERGY COUPLING BETWEEN PHOTOSYSTEM-II REACTION CENTERS AND THE LIGHT-HARVESTING CHLOROPHYLL-A/B PROTEIN COMPLEX-II

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第1期934卷 144-150页

作者： ZUCCHELLI, G GARLASCHI, fM JENNINGS, RC Centro C.N.R. Biologia Cellulare e Molecolare delle Piante Dipartimento di Biologia Università di Milano Milano Italy

In the present paper the increased f 0 fluorescence emission encountered upon lowering the cation concentration below saturating levels is studied. Comparison of the f 0 fluorescence excitation spectra at different cation concentration with that of chlorina barley mutant which lacks LHCP II and also with phosphorylated spinach chloroplasts indicates that the increased f 0 fluorescence originates from LHCP II which becomes partially uncoupled from the Photosystem II reaction centre at low cation concentration. Studies with cations of different valency suggest that this phenomenon is controlled by membrane surface electrostatic charges in a similar way to thylakoid stacking and the ‘spill-over’-type interactions between the two photosystems. Cation titration experiments of phosphorylated chloroplasts suggest that both the ‘mobile’ and the ‘non-mobile’ LHCP II display a cation-sensitive energy transfer to Photosystem II reaction centres.

关键词： Light harvesting chlorophyll a b complex Cation Photosystem II Energy transfer Chlorophyll fluorescence (Spinach chloroplast) f 0 nonvariable fluorescence f M maximal fluorescence PS I Photosystem I PS II Photosystem II LHCP II light-harvesting chlorophyll a b protein complex II DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea Tricine N -[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine TEC tris-(ethylenediamine)cobalt(III)chloride

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TIME-RESOLVED CHLOROPHYLL fLUORESCENCE STUDIES ON PIGMENT-PROTEIN COMPLEXES fROM PHOTOSYNTHETIC MEMBRANES

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第1期935卷 41-52页

作者： HODGES, M MOYA, I CNRS INST PHYSIOL VEGETALE F-91198 GIF SUR YVETTE FRANCE UNIV PARIS 11 F-91405 ORSAY FRANCE

Room-temperature single-photon timing measurements at low excitation energies have been carried out on Photosystem II (PS II) pigment-protein complexes containing 40-70 chlorophylls per reaction centre (RC) as a function of PS II trap closure, Chl fluorescence emission spectra and detergent concentration. The lifetime and amplitude values obtained, using a four exponential model, have been compared with the values exhibited by the isolated light harvesting Chl a/b complex of PS II (LHC II) and a Photosystem I (PS I) complex containing 100 Chl/RC. The results suggest that the two rapid decays seen in the PS II preparations arise from the 47 kDa (.tau. = 54 ps) and 43 kDa (.tau. = 180 ps) Chl containing polypeptides but that in vivo these two proteins give rise to a single variable decay. The differences are discussed in terms of a perturbation in excitation energy migration by detergent molecules, although the presence of high detergent levels does not alter the kinetic and amplitude characteristics of these decays. The two long-lived decays seen in the PS II-core complexes have the same lifetimes and characteristics with respect to detergent addition as the isolated LHC II, suggesting that they arise from LHC II contamination. Unlike the two fast decays, the two long-lived components are extremely sensitive to detergent concentration. The requirement of two decay components in LHC II suggests the presence of a heterogeneous population. Isolated PS I also gives rise to four decay components with 99% of the excitation energy located in pigment-proteins which exhibit lifetimes of 110 ps and 27 ps. The lifetime and amplitude values have been used to calculate estimations for the single-step transfer time between interacting Chl molecules, the number of visits required before trapping takes place and the quantum yield of photochemistry.

关键词： Chlorophyll fluorescence Excitation transfer Photosystem I Photon counting Pigment-protein complex Photosystem II Chl chlorophyll PS Photosystem LHC light harvesting Chl a b complex PS Photosystem RC reaction centre Q A primary stable electron acceptor of PS II f M maximum Chl fluorescence f 0 minimum Chl fluorescence A exp preexponential factor (amplitude) Mes 4-morpholine-ethanesulphonic acid Hepes N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic acid OGP octylglucopyranoside JT transfer time between chlorophylls NV number of visits before trapping fPT first passage time QY quantum yield

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CONVERSION Of COUPLING fACTOR-I Of RHODOSPIRILLUM-RUBRUM fROM A CA2+-ATPASE INTO A MG2+-ATPASE

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第2期935卷 123-129页

作者： NORLING, B STRID, A NYREN, P Department of Biochemistry Arrhenius Laboratory University of Stockholm Stockholm Sweden

Isolation of f1-ATPase from Rhodospirillum rubrum by chloroform extraction of chromatophores, followed by purification on a glycerol gradient, results in a very pure enzyme preparation containing five subunits with high Ca2+-ATPase activity (15 .*** per min per mg protein). furthermore, conditions are reported under which the purified f1 exhibits Mg2+-dependent ATPase activity of about 35 .*** per min per mg protein. NaHCO3 stimulates the Mg2+-activity from 1.5 .*** per min per mg protein to 5 .*** per min per mg protein giving a maximal activity at a concentration of about 60 mM NaHCO3. Lauryl dimethylamine oxide (LAO), octyl glucoside and noananoyl N-methylglucamide enhance the Mg2+-ATPase activity from 1.5 to 14, 22 and 35 .*** per min per mg protein, respectively, in the absence of NaHCO3, and from 5 to 34, 30 and 37 .*** per min per mg protein, respectively, in the presence of 50 mM NaHCO3. The Vmax is increased, but the Km for ATP remains the same, about 0.22 mM, both in the absence of activators and in the presence of NaHCO3, LDAO or NaHCO3 plus LDAO. Ca2+-dependent ATPase activity is slightly stimulated by NaHCO3 but strongly inhibited by octyl glucoside.

关键词： f 1 -ATPase Detergent activation Cf 1 ATPase, Ca 2+ ATPase, Mg 2+ ( R. rubrum ) Rrf 1 f 1 -ATPase isolated from Rhodospirillum rubrum chromatophores OG n -octyl glucoside LDAO lauryl dimethylamine oxide MEGA-9 nonanoyl N -methylglucomide cmc critical micellar concentration

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UNCOUPLING PROTEIN IN EMBRYONIC BROWN ADIPOSE-TISSUE - EXISTENCE Of NONTHERMOGENIC AND THERMOGENIC MITOCHONDRIA

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BIOCHIMICA ET BIOPHYSICA ACTA 1988年第1期935卷 19-25页

作者： HOUSTEK, J KOPECKY, J RYCHTER, Z SOUKUP, T Institute of Physiology Czechoslovak Academy of Sciences 142 20 Prague Czechoslovakia

Embryonic development of mouse and rat brown adipose tissue was characterized by electron microscopy and by quantifying the mitochondrial oxidative, phosphorylating and thermogenic capacities immunochemically, using antibodies against cytochrome oxidase, f1-ATPase and uncoupling protein, respectively. Mitochondria and cytochrome oxidase were detected from the 15-16th day of pregnacny and their amounts continuously increased toward birth. f1-ATPase was also found on the 15th day but it reached a maximum level already on the 19th day when the uncoupling protein appeared and rapidly increased during further maturation of brown adipose tissue. It thus appears that mitochondria in early prenatal brown adipose tissue lack completely uncoupling protein and are nonthermogenic. They transform into typical thermogenic mitochondria abruptly only 2 days before birth.

关键词： Uncoupling protein Brown adipose tissue Embryogenesis Thermogenesis Mitochondrion SDS sodium dodecyl sulfate f 1 catalytic part of mitochondrial H + -translocating ATP synthase

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INHIBITION Of RAT-BRAIN PYRUVATE-DEHYDROGENASE BY THIAMINE ANALOGS

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BIOCHEMICAL PHARMACOLOGY 1988年第22期37卷 4375-4380页

作者： KANDIKO, CT SMITH, D YAMAMOTO, BK NE OHIO UNIV COLL MED DEPT PHARMACOL ROOTSTOWN OH 44272 USA

The effects of thiamine thiazolone (TT) and thiamine thiazolone pyrophosphate (TTPP) on the in vitro and in vivo inhibition of pyruvate dehydrogenase complex (PDHC) from rat cortex and hippocampus were characterized. TTPP decreased PDHC activity in vitro but had no effect in vivo following its direct chronic administration via osmotic mini-pumps into the brains of behaving rats. In contrast, TT had no direct effect in vitro following a differential centrifugation purification of the mitochondrial PDHC fraction, but decreased PDHC activity in vivo. Additional experiments demonstrated that the cytosolic fraction converted TT to TTPP which, in turn inhibited PDHC in vitro. A mechanism is proposed to explain these effects that is consistent with a non-competitive inhibition of brain PDHC by TTPP.

关键词： PDHC pyruvate dehydrogenase complex R f distance traveled from baseline by compound/ distance traveled from baseline by solvent PMSf phenylmethanesulfonyl fluoride V m maximum velocity obtained when PDHC was saturated with pyruvate s pyruvate concentrations in mM v specific activity (μmol product/ min/mg protein) TT thiamine thiazolone TTPP thiamine thiazolone pyrophosphate

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