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PURIfICATION AND PROPERTIES Of SUCROSE - SUCROSE 1f-BETA-D-fRUCTOSYLTRANSfERASE IN ONION SEEDS

PHYTOCHEMISTRY 1985年第4期24卷 695-698页

作者： SHIOMI, N KIDO, H KIRIYAMA, S Department of Agricultural Chemistry Faculty of Agriculture Hokkaido University Sapporo 060 Japan

A sucrose:sucrose 1f-.beta.-D-fructosyltransferase (EC 2.4.1.9) has been purified from onion seeds by fractionation with ammonium sulfate and then by chromatography on DEAE-cellulose. CM-cellulose, octyl-Sepharose and Sephadex G-200. The purified enzyme which showed a single protein band on polyacrylamide gel electrophoresis was free from the other fructosyltransferases, catalyzed fructosyltransfer from sucrose to another sucrose to form 1-kestose and glucose, and also in some degree transferred a fructosyl residue from sucrose to raffinose and stachyose but did not to 1-kestose and nystose. The enzyme had a MW of .apprx. 68,000, an optimum pH of 5.4 and Km of 0.083 M, was stable at ***.-***. for 10 min, and was inhibited by Hg2+, Ag+, Mn2+ and p-chloromercuribenzoate.

关键词： Allium cepa , Liliaceae onion sucrose:sucrose-1 f -β- d -fructosyltransferase fructosyltransferase.

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THE f1f0-ATPASE Of ESCHERICHIA-COLI - THE SUBSTITUTION Of ALANINE BY THREONINE AT POSITION-25 IN THE C-SUBUNIT AffECTS fUNCTION BUT NOT ASSEMBLY

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BIOCHIMICA ET BIOPHYSICA ACTA 1985年第2期808卷 252-258页

作者： fIMMEL, AL JANS, DA HATCH, L JAMES, LB GIBSON, f COX, GB Department of Biochemistry John Curtin School of Medical Research Canberra ACT 2601 Australia

A mutant strain of E. coli carrying a mutation in the uncE gene which codes for the c-subunit of the f1f0-ATPase was isolated and examined. The mutant allele, designated uncE513, results in alanine at position 25 of the c-subunit being replaced by threonine. The mutant f1f0-ATPase appears to be fully assembled and is partially functional with respect to oxidative phosphorylation. The ATPase activity of membranes from the mutant strain is resistant to the inhibitor dicyclohexylcarbodiimide, but this is due to the f1-ATPase being lost from the membranes in the presence of the inhibitor. Mutant membranes from which the f1-ATPase was removed have a greatly reduced proton permeability compared with similarly treated normal membranes. The results are discussed in relation to a previously proposed mechanism of oxidative phosphorylation.

关键词： f 1 f 0 -ATPase Amino acid substitution Oxidative phosphorylation ( E. coli ) ATPase Mg 2+ -stimulated adenosine triphosphatase (EC 3.6.1.3) DCCD dicyclohexylcarbodiimide NTG N -methyl- N ′-nitro- N -nitrosoguanidine P i inorganic phosphate

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INVOLVEMENT Of PLASTOQUINONE AND LIPIDS IN ELECTRON-TRANSPORT REACTIONS MEDIATED BY THE CYTOCHROME B6-f COMPLEX ISOLATED fROM SPINACH

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fEBS LETTERS 1985年第2期180卷 321-325页

作者： CHAIN, RK Division of Molecular Plant Biology University of California Berkeley CA 94720 USA

The isolation of a cytochrome b6-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.

关键词： Cytochrome b 6 -f complex Plastoquinone Lipid Reconstitution PC, phosphatidylcholine PG, phosphatidylglycerol MGDG monogalactosyldiacylglycerol DGDG, digalactosyldiacylglycerol SQDG, sulfoquinovosyldiacylglycerol TLC, thin-layer chromatography DNP-INT, 2,4-dinitrophenyl ether of iodonitrothymol, PQ, plastoquinone b 6 , cytochrome b 6 f , cytochrome f

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THE INHIBITOR PEPTIDE Of THE MITOCHONDRIAL f1.f0-ATPASE INTERACTS WITH CALMODULIN AND STIMULATES THE CALMODULIN-DEPENDENT CA2+-ATPASE Of ERYTHROCYTES

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BIOCHIMICA ET BIOPHYSICA ACTA 1985年第1期816卷 63-67页

作者： SCHWERZMANN, K MULLER, M CARAfOLI, E UNIV BERN INST MED CHEM CH-3000 BERN 9 SWITZERLAND SWISS FED INST TECHNOL BIOCHEM LAB CH-8092 ZURICH SWITZERLAND

The binding of calmodulin to the [rat] mitochondrial f1 .cntdot. f0-ATPase was studied. [125I]Iodoazidocalmodulin binds to the .epsilon.-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes was also analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the 2 mechanisms of activation are different. These in vitro effects of the 2 regulatory proteins may reflect a common origin of the 2 ATPases considered and/or of the regulatory proteins.

关键词： ATPase inhibitor f 1 · f 0 -ATPase Calmodulin Ca 2+ -ATPase Erythrocyte

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ALPHA,BETA-BIDENTATE CRADP ABOLISHES THE NEGATIVE COOPERATIVITY Of YEAST MITOCHONDRIAL f1-ATPASE

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BIOCHIMICA ET BIOPHYSICA ACTA 1985年第1期806卷 35-41页

作者： WIEKER, HJ HESS, B Max-Planck-Institut für Ernährungsphysiologie Rheinlanddamm 201 D-4600 Dortmund 1 F.R.G.

The hydrolysis of MgATP and MgITP by mitochondrial f1-ATPase from S. cerevisiae was competitively inhibited by .alpha.,b-CrADP, .alpha.,.beta.,.gamma.-CrATP and .beta.,.gamma.-CrATP. The apparent K''i values of the 3 complexes are in the range of the 1/2-saturating MgATP concentration. The negative cooperativity (nH = 0.7) of MgATP hydrolysis was totally abolished by .alpha.,.beta.-CrADP (nH = 1.0), while it was not affected by the CrATP. It is concluded that .alpha.,.beta.-CrADP binds exclusively at the regulatory site and that CrATP binds exclusively to the catalytic site.

关键词： f 1 -ATPase kinetics Chromium-nucleotide complex (Yeast mitochondria) Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Mes 4-morpholineethanesulfonic acid CD circular dichroism

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THE NUCLEOTIDE BINDING-SITE Of f1-ATPASE WHICH CARRIES OUT UNI-SITE CATALYSIS IS ONE Of THE ALTERNATING ACTIVE-SITES Of THE ENZYME

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fEBS LETTERS 1985年第2期189卷 286-290页

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

Nucleotide-depleted mitochondrial f1-ATPase binds 3'-(2')-O-(2-nitro-4-azidobenzoyl)-derivatives of ATP (NAB-ATP) and GTP (NAB-GTP) when these nucleotide analogues are added to the enzyme in equimolar quantities in the presence of Mg2+ (uni-site catalysis conditions). The binding of NAB-ATP is accompanied by its hydrolysis and inorganic phosphate dissociation from the enzyme; NAB-ADP remains bound to f1-ATPase. The f1-ATPase X NAB-ADP complex has no ATPase activity and its reactivation in the presence of an excess of ATP is accompanied by NAB-ADP release. The illumination of the f1-ATPase complexes with NAB-ADP or NAB-GDP leads to the covalent binding of one nucleotide analogue molecule to the enzyme and to the irreversible inactivation of f1-ATPase. It follows from the results obtained that the modification of just one of the f1-ATPase catalytic sites is sufficient to complete the inhibition of ATPase activity.

关键词： f 1 -ATPase Uni-site catalysis Photoactive nucleotide analog NAB-AD(T)P, 3'(2')- O -(2-nitro-4-azidobenzoyl) adenosine-5'-di(tri)phosphate NAB-GD(T)P, 3'(2')- O -(2-nitro-4-azidobenzoyl)guanosine-5'-di(tri)phosphate Mops, 4-morpholinepropanesulfonic acid

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ANALYSIS Of MUTAGENIC IMIDAZO[4,5-f]QUINOLINES AND IMIDAZO[4,5-f]QUINOXALINES (IQ COMPOUNDS) - COMPARISON Of ELECTROCHEMICAL AND ULTRAVIOLET DETECTION

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MUTATION RESEARCH 1985年第1-2期142卷 5-8页

作者： GRIVAS, S NYHAMMAR, T Department of Chemistry and Molecular Biology Swedish University of Agricultural Sciences S-75007 Uppsala (Sweden)

Two synthetic imidazoquinolin-2-amines (IQ and MeIQ) and two imidazoquinoxalin-2-amines (MeIQx and 4,8-DiMeIQx), all known potent mutagens, have been separated by reversed-phase HPLC and detected by two methods — UV detection and electrochemical (EC) detection. The limits of detection were found to be 2.5 pmoles for UV detection and 0.5–1.5 pmoles for electrochemical detection.

关键词： IQ 3-methyl-3 H -imidazo[4,5- f ]quinolin-2-amine, 76180-96-6 MeIQ 3,4-dimethyl-3 H -imidazo[4,5- f ]-quinolin-2-amine, 77094-11-2 MeIQx 3,8-dimethyl-3 H -imidazo[4,5- f ]quinoxalin-2-amine, 77500-04-0 4,8-DiMeIQx 3,4,8-trimethyl-3 H -imidazo[4,5- f ]quinoxalin-2-amine HPLC high-performance liquid chromatography

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DISTRIBUTION-fREE COMPUTER METHODS fOR ANALYZING LIGAND-BINDING AND ENZYME MECHANISMS

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COMPUTERS IN BIOLOGY AND MEDICINE 1985年第3期15卷 111-121页

作者： CRABBE, MJC Nuffield Laboratory of Ophthalmology University of Oxford Walton Street Oxford OX2 6AW U.K.

BASIC computer programs were designed to calculate enzyme kinetic and ligand binding parameters using distribution-free methods. Statistical estimates for the parameters can be calculated, and models distinguished by ... 详细信息

关键词： Enzyme kinetics Ligand binding Distribution-free Integrated rate equations t RNA Opiates Receptors Computers BASIC f -test

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INHIBITION Of PANCREATIC COLIPASE BY ANTIBODIES AND fAB fRAGMENTS - SELECTIVE EffECTS Of 2 fRACTIONS Of ANTIBODIES ON THE fUNCTIONAL SITES Of THE COfACTOR

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BIOCHIMICA ET BIOPHYSICA ACTA 1985年第2期827卷 109-118页

作者： BOSCBIERNE, I PERROT, C SARDA, L RATHELOT, J FAC ST CHARLES BIOCHIM LAB PL V HUGO F-13003 MARSEILLE FRANCE

Rabbit antiserum was raised against porcine pancreatic colipase and fab fragments were prepared by papain digestion of purified antibodies followed by purification on protein A-Sepharose. fab fragments showed inactivation toward porcine colipase activity similar to that of antiserum and purified antibodies. from inactivation studies carried out by incubating porcine colipase and lipase with fab fragments in the absence of lipid or in the presence of triolein and sodium deoxycholate, it could be concluded that polyclonal antiporcine colipase antibodies contain fractions that bind specifically to epitopes at or near the functional regions of the porcine cofactor. Studies with an enzyme-linked immunosorbent assay [ELISA] showed that cross-reactivity of horse or chicken colipase with antiporcine colipase antiserum was lower than that of the human or porcine protein. Results of immunoactivation kinetic studies performed with the same proteins, fully confirmed these observations. Partial cross-reactivity between porcine and chicken colipases allowed antibodies to be fractionated by immunoaffinity chromatography on immobilized chicken colipase. fraction I contains antibodies absorbed on porcine colipase not accessible when the cofactor is bound to lipid. Antibodies of fraction II, nonadsorbed on chicken colipase, inactivate porcine colipase preincubated with triolein/deoxycholate. Lipase had a protective effect against inactivation. Antibodies of fraction II bind likely to epitopes close to the specific region of colipase interacting with lipase. These conclusions are in good agreement with analysis of the sequence of porcine, equine and human colipases by calculating local hydrophilicity indices.

关键词： Colipase f ab fragment Immunoinactivation Immunological cross-reactivity Antigenic determinant (Porcine pancreas)

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CORRELATIONS BETWEEN fLUORESCENCE AND PHOSPHORYLATION CHANGES IN THYLAKOID MEMBRANES Of CHLAMYDOMONAS-REINHARDTII INVIVO - A KINETIC-ANALYSIS

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BIOCHIMICA ET BIOPHYSICA ACTA 1985年第2期809卷 277-283页

作者： DELEPELAIRE, P WOLLMAN, fA INST BIOL PHYSICO-CHIM 13 RUE P & M CURIE F-75005 PARIS FRANCE

The reorganization of the light-harvesting antenna in the thylakoid membranes upon phosphorylation of some of its apoproteins was further characterized in vivo using the green algae Chlamydomonas reinhardtii. To this end we have studied light-to-dark transitions on intact cells placed in the anaerobic state using the f34 mutant strain which lacks PS II centers. We show that the 50% decrese in fluorescence yeld in such transitions is accompanied by a 50% increase in PS I antenna size. The half-times of the kinetics of the fluorescence changes in the dark-to-light and light-to-dark transitions are of 320 and 120 s, respectively. The rate-limiting steps in these transitions are attributed to the dephosphorylation and phosphorylation processes themselves rather than to the activation of the kinase or to the diffusion of the phosphorylated complexes in the thylakoid membrane. Accordingly, the chanes in phosphorylation of three of the main phosphopolypeptides occur with the same kinetics as those of the fluorescence changes. Different phosphorylation kinetics are observed for two phospohpolypeptides which are, however, also part of the light-harvesting complexes. Possible heterogeneities in the kinse enzymatic activities are discussed. The peculiar status of the phosphopolypeptide D2, associated with the PS II center, is described.

关键词： Light-harvesting complex Protein phosphorylation Thylakoid protein Photosystem I fluorescence ( reinhardtii ) LHC light-harvesting complexes PQ plastoquinone DCMU 3,4-dichlorophenyl-1,1-dimethylurea f m , maximum level of fluorescence PS I, Photosystem I, PS II, Photosystem II Chl chlorophyll A , absorbance

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