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ANALYSIS Of ELECTROPHORETIC BEHAVIOR Of CYTOCHROME-C OXIDASE SUBUNITS - RETARDATION COEffICIENT VERSUS MOLECULAR-WEIGHT IN DODECYL-SULfATE UREA GELS

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第1期576卷 109-117页

作者： TRACY, RP CHAN, SHP SYRACUSE UNIV DEPT BIOL BIOL RES LABS SYRACUSE NY 13210 USA

Standard proteins were examined by electrophoresis in highly cross-linked polyacrylamide gels containing sodium dodecyl sulfate and urea. Their behavior was analyzed at a single gel concentration (by MW vs. relative mobility) and at several gel concentrations (MW vs. retardation coefficient). The validity of the latter method of analysis was established for this gel system. Beef heart cytochrome c oxidase [EC 1.9.3.1] was subjected to analysis by this method. Compared to standards, subunits I and III showed increased free electrophoretic mobility, while that of subunit V was slightly decreased. The MW values derived were: I, 44,600;II, 22,700;III, 23,500;IV 16,900;V, 9400;VI, 7600;VII, 4300. The SE were all less than .+-. 7%. Isolated V and VI were analyzed by 2 dimensional dodecyl sulfate electrophoresis, in which the 2nd dimension also contained urea. In contrast to their behavior when the holo-enzyme was examined, these isolated subunits V and VI no longer exchange migrating positions relative to each other during the 2 dimensional analysis. The MW values of the isolated subunits agree with those of the holo-enzyme.

关键词： Cytochrome c oxidase subunit Subunit molecular weight Retardation coefficient R f relative mobility f r retardation coefficient M o free mobility (extrapolated Y intercept of the ferguson plot)

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ANTIBODY INDUCED fLUORESCENCE ENHANCEMENT Of AN N-(3PYRENE)MALEIMIDE CONJUGATE Of RABBIT ANTI-HUMAN IMMUNOGLOBULIN-G - QUANTITATION Of HUMAN-IGG

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JOURNAL Of IMMUNOLOGICAL METHODS 1979年第3-4期28卷 233-242页

作者： LIBURDY, RP USAF SCH AEROSP MED DIV RADIAT SCI BROOKS AFB TX 78235 USA

PM [N-(3-pyrene)maleimide] is a non-fluorescent dye that covalently binds to freely reactive amino or sulfhydryl groups to produce highly fluorescent protein conjugates. An application of PM to the study of antigen-antibody interactions is reported. PM was reacted with the Ig[immunoglobulin]G fraction of rabbit anti-human IgG (PM-RaHIgG) and the fluorescent adduct was isolated and its fluorescence properties characterized. Introduction of HIgG to PM-RaHIgG produced a marked enhancement of PM fluorescence with no spectral shift;enhancement increased monotonically for increasing amounts of added HIgG. This enhancement did not occur in the presence of serum treated with Sephadex G-200 RaHIgG immunosorbent, indicating that the presence of HIgG was required for fluorescence enhancement. Trypsin cleavage of the fc region of PM-RaHIgG resulted in a 30% reduction in PM fluorescence, indicating that PM had covalently labeled freely reactive .epsilon.-amino groups on lysine residues throughout the RaHIgG molecule. PM fluorescence increases markedly as the pH of the microenvironment is increased from 7.0 to 8.0;a 10 nm spectral shift occurs at pH values greater than 8.0. HIgG binding may cause an increase in microenvironmental pH that does not exceed pH 8.0 at or near PM molecules on PM-RaHIgG. fluorescence sensitivity of PM-RaHIgG to HIgG made quantitation of serum HIgG possible by direct addition of test serum to PM-RaHIgG. This rapid, simple and inexpensive technique is a potentially useful complement to existing methodologies for the detection of serum IgG.

关键词： PM N-(3-pyrene)maleimide HIgG human immunoglobulin G RaHIgG rabbit IgG directed against HIgG PM-RaHIgG PM conjugated RaHIgG PMBME PM conjugate of β-mercaptoethanol f ab single antibody binding site fragment f c crystallizable complement binding site fragment BSA bovine serum albumin LYS lysozyme PHA phytohemagglutinin-M HEPES N-2-hydroxylthylpiperazine-N′-2-ethanesulfonic acid

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CYTOCHROME fUNCTION IN THE CYCLIC ELECTRON-TRANSPORT PATHWAY Of CHLOROPLASTS

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第1期547卷 138-148页

作者： SLOVACEK, RE CROWTHER, D HIND, G BROOKHAVEN NATL LAB DEPT BIOL UPTON NY 11973 USA

flash excitation of isolated intact [spinach] chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the .alpha.-bands of cytochrome b6 and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b6 and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 .mu.M 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH4Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b6 and f by factors of 10 or more. These observations indicate that cytochrome b6, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40-120 .*** ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO2 fixation.

关键词： Electron transport Cytochrome b 6 Cytochrome f Photophosphorylation Antimycin DBMIB 2,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea CCCP carbonylcyanide- m -chlorophenylhydrazone P -700 reaction center pigment for Photosystem I

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CHEMICAL-REACTIVITY Of METALLOPROTEINS IN CONfORMATIONALLY OUT-Of-EQUILIBRIUM STATES

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第3-4期549卷 255-280页

作者： BLUMENfELD, LA DAVIDOV, RM Institute of Chemical Physics Academy of Science of the U.S.S.R. Moscow U.S.S.R.

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ISOLATION, CHARACTERIZATION AND RADIOIMMUNOASSAY Of RAT ALPHA-MACROfETOPROTEIN (ACUTE PHASE-A2 MACROGLOBULIN)

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MOLECULAR IMMUNOLOGY 1979年第8期16卷 547-554页

作者： HUDIG, D SELL, S UNIV CALIF SAN DIEGO DEPT PATHOL LA JOLLA CA 92093 USA

Rat alpha-macrofetoprotein (AMf)§ has been isolated and purified by antibody affinity chromatography and quantitated using a double antibody radioimmunoassay (RIA) with a sensitivity of 20 ng. Two forms of AMf, differing in reference mobilities ( R ) in 5% polyacrylamide gels and in isoelectric focusing points (pI), are present in serum. The R f 0.10 AMf has a pI of 4.35 and R f 0.25 AMf a pI of 4.65. R f 0.25 AMf competes 2.2 times better than R f 0.10 AMf in an RIA with 125 I R f 0.25 AMf as the predominant antigen. The two forms do not differ substantially in mol. wt. AMf has a mol. wt of 700,000 ± 70,000 determined by SDS polyacrylamide gel electrophoresis. Dithiothreitol reduction of AMf resulted in six different chains, the largest with mol. wt 175,000. It is suggested that the five smaller chains are the result of interaction of AMf with endoproteases. The E 1% 280 of AMf is 9.16 ± 0.3 by the biuret method using bovine serum albumin as a standard and 10.6 ± 0.3 by microKjeldahl N analysis. Amino-acid analysis of AMf is compared with the analyses recently reported by others. Normal adult rat serum AMf concentrations determined for two groups of rats are 17 ± 2 and 32 ± 6 μg/ml.

关键词： AMf, alpha-macrofetoprotein AAAMf antibody affinity purified macrofetoprotein DEAE diethylaminoethyl cellulose E 1% 280 absorbance at 280 nm of a 1% wt/volume solution IgG immunoglobulin G IgM immunoglobulin M HA 2 M human alpha 2 -macroglobulin NAM normal alpha-macroglobulin pI isoelectric point s.e.m. standard error of the mean R f reference mobility RIA radioimmunoassay SDS sodium dodecyl sulfate

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HISTIDYL AND TYROSYL RESIDUE IONIZATION STUDIES Of SUBTILISIN NOVO

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第2期578卷 261-268页

作者： OMAR, S BROWN, Mf SILVER, P SCHLEICH, T UNIV CALIF SANTA CRUZ DIV NAT SCI SANTA CRUZ CA 95064 USA

The low field portion of the 360 MHz 1H NMR spectrum of phenylmethanesulfonyl-subtilisin Novo (EC 3.4.21.14) was studied as a function of pH. Analysis of the pH-induced chemcial shift changes occurring between 6-7 ppm revealed 5 classes of ionizable residues with pK values (uncorrected) of 10.3, 10.7, 10.7, 10.8 and 11.0. With a single exception, the titration curves can be fit by assuming a simple proton ionization equilibrium. four classes of low intensity broad resonances, assigned to the histidyl residues, are observed between 8 and 9 ppm. Uncorrected pK values of 5.4, 5.7, 6.0 and 6.4 were determined for the residues comprising each of these classes. The spectral data are consistent with protonation of one or more histidyl residues upon acid induced denaturation of the protein. These results are compared with those of analogues studies performed by the use of other techniques.

关键词： Subtilisin Novo Serine protease Histidyl residue Tyrosyl residue Titration curve PhCH 2 SO 2 f phenylmethanesulfonyl fluoride PhCh 2 SO 2 - phenylmethanesulfonyl NMR nuclear magnetic resonance ppm parts per million TSP sodium 3-trimethylsilylpropanecarboxylate

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EffECTS Of PHOSPHOLIPID ACYL CHAIN STRUCTURE ON PHYSICAL-PROPERTIES .1. ISOBRANCHED PHOSPHATIDYLCHOLINES

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CHEMISTRY AND PHYSICS Of LIPIDS 1979年第3期24卷 287-296页

作者： SILVIUS, JR MCELHANEY, RN Department of Biochemistry University of Alberta Edmonton Alberta T6G 2H7 Canada

All isobranched fatty acids of 12-18 C were synthesized in gram quantities by a convenient acetylene coupling reaction followed by catalytic hydrogenation. The corresponding phosphatidylcholines (PC) were synthesized and their thermotropic phase behavior investigated by differential thermal analysis. The isobranched acyl phosphatidylcholines show gel-to-liquid-crystalline phase transition temperatures (Tcs) some ***. C below those of the corresponding straight-chain PC and appear to exhibit 2 slowly interconverting low-temperature phases below Tc. The observed strong alternation of Tc between isobranched PC with odd- and even-C number acyl chains contrasts with the behavior of the straight-chain PC and suggests that the acyl chains of the branched-chain PC are strongly tilted with respect to the bilayer normal below and/or above Tc while those of the straight-chain PC are not. Significant differences apparently exist in the overall packing of branched- and straight-chain PC in the gel and possibly the liquid-crystalline state.

关键词： DMSO dimethyl sulfoxide DTA differential thermal analysis GLC gas-liquid chromatography PC phosphatidylcholine T c temperatures of major heating endotherm for a PC T f temperatures of major cooling exotherm for a PC TLC thin-layer chromatography T m fatty acid melting temperature

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BIOLOGICAL ENERGY-PRODUCTION IN THE APPARENT ABSENCE Of ELECTRON-TRANSPORT AND SUBSTRATE LEVEL PHOSPHORYLATION

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fEBS LETTERS 1979年第1期107卷 1-3页

作者： ZEHNDER, AJB BROCK, TD Department of Bacteriology University of Wisconsin Madison WI 53706 USA

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TERNARY COMPLEX-fORMATION Of PIG-HEART LACTATE-DEHYDROGENASE WITH SPIN-LABELED COENZYMES AND INHIBITORS AS STUDIED BY ELECTRON-SPIN RESONANCE

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第2期568卷 287-296页

作者： WENZEL, HR TROMMER, WE RUHR UNIV BOCHUM LEHRSTUHL BIOCHEM CHEM ABT D-4630 BOCHUM FED REP GER

The formation of ternary inhibitor and dead end complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) was studied by means of 2 NAD derivatives, spin-labeled at N6 and C-8 of the adenine ring. Dissociation constants calculated for the inhibitors oxamate and oxalate from their corresponding ternary complexes are in excellent agreement with data from literature derived from sedimentation experiments. However, the recently postulated enzyme-NADH-sulfite complex was not observed. The mobility of the spin-label, i.e., the protein conformation near the adenine binding pocket in various ternary complexes, depends on the type of inhibition or substrate employed.

关键词： Lactate dehydrogenase Spin label Ternary complex ESR NAD N 6 SL- or 8SL-preceding NAD denote a nitroxyl spin-label attached to the N 6 - or 8-position of the adenine moiety as shown in fig. 2, SL signifies N 6 SL or 8SL. The subscripts t , b, f with concentration data mean total bound and free respectively

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ENERGY-CONVERSION IN THE fUNCTIONAL MEMBRANE Of PHOTOSYNTHESIS - ANALYSIS BY LIGHT-PULSE AND ELECTRIC PULSE METHODS - CENTRAL ROLE Of THE ELECTRIC-fIELD

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第3-4期505卷 355-427页

作者： WITT, HT Max-Volmer-Institut für Physikalische Chemie und Molekularbiologie Technische Universität Berlin Strasse des 17. Juni 135 1 Berlin 12 Germany

The pulse methods are applied to study of the physicochemical foundations of photosynthesis of green plants. Some of the topics in this review are: photoreactions of the chlorophylls;electron transfer;proton transloca... 详细信息

关键词： ΔA field-indicating absorption change and electrochromic shift BChl bacteriochlorophyll Car carotenoid CCCP carbonylcyanide m -chloropheylhydrazone Cf 1 coupling factor of the ATPase Ch1 chlorophyll Chl- a chlorophyll a Chl- b chlorophyll b Chl- a I ( P -700) chlorophyl a I (reaction center of antennae system I) Chl- a II ( P -680) chlorophyl a II (reaction center of antennae system II) Cyte cytochrome DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea DCIP 2,6-dichlorophenoll indophenol Δφ electrical potential difference Δψ surface potential difference f electric filed strength G free energy i ion current i H + H + current i K + K + current j current density PC plastocyanine PMS N -methylphenazonium sulfate PQ (1) plastoquinone primary electron-acceptro of Chl- a II (≡ X -320) PQ (2) plastoquinone, connector between PQ (1) and PQ pool PQ plastoquinone, member of the PQ pool τ half-life and half-rise time X -320 plastoquinone primary electron-acceptro of Chl- a II (≡PQ (1) 1 (…) single turnover conditions ss (…) steady state in saturating permanent light S enzyme system for the cleavage of H 2 O

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