bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for fast and discriminatory electron transfer of proteins at modified gold electrodes
Authors: BOND Alan M. , HILL H. Allen O. , PAGE David J. , PSALTI Ioanna S. M. , WALTON Nicholas J.
Published: March 2005

The electrochemistry of the redox proteins, cytochrome c, cytochrome b5, plastocyanin and ferredoxin at modified gold electrodes has been examined on the basis that electron transfer takes place at electroactive sites which are microscopic in size. Using this model, it is now proposed that electrochemistry of these proteins occurs at suitably modified sites with fast rates at potentials near the standard redox potential. The microscopic model implies that redox proteins and enzymes take part in fast electron transfer at specific sites on the electrode, other sites being completely ineffective. This form of molecular recognition, i.e. the ability to discriminate between the different sites on an electrode surface, mimics homogeneous redox reactions wherein redox active proteins ‘recognize’ their biological partners in a very specific sense. Previously, protein electrochemistry has been interpreted via use of a macroscopic model in which the proteins are transported to the electrode surface by linear diffusion followed by quasi‐reversible or irreversible electron transfer to the electrode surface. The microscopic model, which assumes that the movement of the protein occurs predominantly by radial diffusion to very small sites, would appear to explain the data more satisfactorily and be consistent with biologically important, homogeneous redox reactions which are known to be fast.
Subcellular distribution of energy metabolites in the pre‐ischaemic and post‐ischaemic perfused working rat heart
Authors: HUMPHREY Stuart M. , BUCKMAN Jane E. , HOLLISS Des G.
Published: March 2005

Isolated working rat hearts were subjected to 20 min of global ischaemia and either 5 min or 15 min of reperfusion. The subcellular distribution of ATP, ADP, AMP, phosphocreatine and Pi were determined before and after ischaemia by the method of non‐aqueous tissue fractionation. Ventricular function and the cytosolic, mitochondrial and ATPase‐associated compartmentation of metabolites were measured. After 5 min of reperfusion, only 13±9% of the pre‐ischaemic contractile function was restored compared to 67±8% after 15 min reperfusion. ATP was reduced in all cellular compartments after 5 min of reperfusion but was only decreased from pre‐ischaemic values in the cytosolic compartment after 15 min of reperfusion (17.1±3.9 nmol/mg vs. 4.3±1.5 nmol/mg total protein; P<0.05). The mitochondrial [ATP]/[ADP] was reduced from a normal value of 4.36 to 1.79 after 5 min but recovered to 4.62 after 15 min of reperfusion. Most of the Pi was located in the mitochondria or with the ATPase fraction of the cell, with only 16% of the total Pi free in the cytosol. This study indicates that the capacity of the heart to recover function may be compromised during early reperfusion by a 59% increase in mitochondrial phosphate content and during late reperfusion by a reduced cytosolic/mitochondrial concentration ratio of both ATP (from 0.85 to 0.19) and phosphocreatine (from 3.9 to 1.24).
Complete amino‐acid sequence of glyceraldehyde‐3‐phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima
Authors: SCHULTES Verena , DEUTZMANN Rainer , JAENICKE Rainer
Published: March 2005

The complete amino‐acid sequence of D‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) from the extreme thermophilic eubacterium Thermotoga maritima has been determined by classical automated sequence analysis of peptides derived by chemical fragmentation with cyanogen bromide and enzymatic cleavages with specific proteases.

The protein contains 332 amino acids per subunit. Its sequence is as follows: A R V A I N G F G R10 I G R L V Y R I I Y20 E R K N P D I E V V30 A I N D L T D T K T40 L A H L L K Y D S V50 H K K F P G K V E Y60 T E N S L I V D G K70 E I K V F A E P D P80 S K L P W K D L G V90 D F V I E S T G V F100 R N R E K A E L H L110 Q A G A K K V I I T120 A P A K G E D I T V130 V I G C N E D Q L K140 P E H T I I S C A S150 C T T N S I A P I V160 K V L H E K F G I V170 S G M L T T V H S Y180 T N D Q R V L D L P190 H K D L R R A R A A200 A V N I I P T T T G210 A A K A V A L V V P220 E V K G K L D G M A230 I R V P T P D G S I240 T D L T V L V E K E250 T T V E E V V A V M260 K E A T E G R K G270 I I G Y N D E P I V280 S S D I I G T T F S290 G I F D A T I T N V300 I G G K L V K V A S310 W Y D N E Y G Y S N320 R V V D T L E L L L330 K M.

Comparing the given sequence with those of the enzymes from the moderate and extreme thermophilic bacteria Bacillus stearothermophilus and Thermus aquaticus, 63% and 59% identity are observed. Alignment of the sequences of GAPDHs from a variety of sources yields one deletion (one amino acid) and one insertion (two amino acids).

Thermal stability is caused by minute adjustments of the local three‐dimensional structure. Previous ‘strategies of thermal adaptation’ in terms of preferred amino‐acid exchanges are not in accordance with the present sequence data.
Serine/threonine‐specific protein kinase activity associated with viral pp60src protein
Authors: DAVID‐PFEUTY Thérèse , NOUVIAN‐DOOGHE Yolande
Published: March 2005

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60‐kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild‐type Rous sarcoma virus (SRD‐FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v‐src, the mAb327 [Lipsich, L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352–360] and to be phosphorylated in vitro at serine/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c‐src protein immunoprecipitated with mAb327 from extracts of NIH c‐src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor‐bearing rabbit (TBR) serum and reported in an earlier work [David‐Pfeuty, T. & Hovanessian, A. (1984) Eur. J. Biochem. 140, 325–342], together with those reported here, suggest that the TBR‐immunoprecipitated pp60v‐src coprecipitates with a cellular protein related to the 60‐kDa subunit of the Ca2+/calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v‐src, but not of pp60c‐src, are shown to contain a Ca2+/calmodulin‐dependent protein kinase activity that phosphorylates a 52‐kDa protein substrate.
N‐Acetyl‐L‐galactosaminuronic acid as an epitope common to the O‐polysaccharides of Pseudomonas aeruginosa serotype A and H (Homma) recognized by a protective human monoclonal antibody
Authors: YOKOTA Shin‐Ichi , OCHI Hiroshi , UEZUMI Ikuko , OHTSUKA Hiroshi , IRIE Kenji , NOGUCHI Hiroshi
Published: March 2005

We have established a human–mouse heterohybridoma cell line producing a human monoclonal antibody TS‐3G2 (IgGγ1,κ). This monoclonal antibody specifically bound to O‐polysaccharides belonging to plural Pseudomonas aeruginosa Homma serotypes, A and H, in contrast to serotype‐specific monoclonal antibody which exclusively bound to strains belonging to a single specific serotype. The binding affinity for serotype A strains was higher than that for serotype H strains. Competitive enzyme immunoassay experiments with O‐polysaccharide preparations derived from IID 1001, NCTC 8505 (serotype A) and IID 1009 (serotype H) and their derivatives demonstrated that the N‐acetyl‐L‐galactosaminuronic acid residue in O‐polysaccharide was essentially involved in the epitope for TS‐3G2. Furthermore, a 6‐deoxy‐hexosamine residue neighboring the reducing terminal of N‐acetyl‐L‐galactosaminuronic acid residues was also concerned with the epitope to some extent. In the experimental infection model of normal mice, the monoclonal antibody TS‐3G2 showed a protective activity against both strains of serotype A and H.
Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105
Authors: SIZMANN Dorothea , KEILMANN Corinna , BÖCK August
Published: March 2005

The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post‐translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C‐terminus were absolutely necessary for the maturation process. Internal deletions within the β‐subunit domain also prevented post‐translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the α subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post‐translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.
Citrate lyase from Klebsiella pneumoniae
Authors: HUPPERICH Martin , HENSCHEN Agnes , EGGERER Hermann
Published: March 2005

The primary structure of the β‐subunit (acyl lyase subunit) of citrate lyase from Klebsiella pneumoniae (ATCC 13882) was determined with protein chemical methods. The polypeptide chain consists of 289 amino acid residues and has a molecular mass of 31 352 Da. The two half‐cystine residues of the subunit are present as cysteines and not involved in disulfide bridges. The sequence shows no homology to known sequences of proteins or nucleic acids and reads

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A vitamin‐K2‐binding factor secreted from Bacillus subtilis
Authors: IKEDA Hiro , DOI Yukio
Published: March 2005

The synthesis of vitamin K2 by Bacillus subtilis was found to increase in parallel with the number of cells. In the exponential growth phase, most vitamin K2 was found in the bacterial cells. At the end of the growth phase, however, the soluble form of vitamin K2 appeared in the incubation media and amounted to 40% of the total vitamin K2 at the stationary phase. DEAE ion‐exchange chromatography of the incubation medium showed a single elution peak corresponding to vitamin K2, suggesting the secretion of vitamin K2 in the form of a soluble complex with a specific acidic binding factor. This factor was partially purified by consecutive DEAE chromatography, and examined by polyacrylamide disc gel electrophoresis. A band stainable with acridine orange, a cationic dye, was found to co‐migrate with vitamin K2. This band was also visualized by periodic acid Schiff staining. Gel‐permeation chromatography showed that the binding factor in its active form has an apparent molecular mass of 100 kDa and is capable of binding exogenous menaquinone but not menadione. Chemical analysis indicated that this binding factor contained about 20% carbohydrate and 80% peptide which consisted mainly of Leu, Glu, Asp and Val. The new acidic glycoconjugate was designated as the vitamin‐K2‐binding factor.
A 1H‐NMR study of electronic structure of the active site of Galeorhinus japonicus metmyoglobin
Authors: YAMAMOTO Yasuhiko , ŌSAWA Akemi , INOUE Yoshio , CHÛJÔ Riichirô , SUZUKI Tomohiko
Published: March 2005

The ferric high‐spin form of the myoglobin from the shark Galeorhinus japonicus, which possesses a Gln residue at the distal site instead of the usual His residue, has been studied by 1H‐NMR spectroscopy. Using the heme meso‐proton (C5H, C10H, C15H and C20H) resonance shift as a diagnostic probe for identifying the coordination system of the iron center in ferric high‐spin form of hemoprotein, it has been shown that G. japonicus metmyoglobin (metMb) possesses the pentacoordinated active site. The pH‐dependence study of NMR spectra of G. japonicus metMb revealed the appearance of the hydroxyl form of metMb at high pH, indicating that the protein undergoes the transition between the acidic and alkaline forms. The pK value and the rate for this acid‐alkaline transition in G. japonicus metMb were found to be ∼ 10 and ≪ 4 × 102 s−1, respectively. Since the pK value of the acidalkaline transition for the pentacoordinated heme in Aplysia limacina metMb is 7.8 [Giacometti, G. M., Das Ros, A., Antonini, E. & Brunori, M. (1975) Biochemistry 14, 1584–1588] and that of the hexacoordinated heme in sperm whale metMb is 9.1 [Brunori, M., Antonini, E., Fasella, P., Wyman, J. & Rossi‐Fanelli, A. (1968) J. Mol. Biol. 34, 497.–504], the OH− affinity of the ferric heme iron does not appear to depend on its coordination system. The acid‐alkaline transition rate in A. limacina metMb was reported to be ≪ 1.5 × 102 s−1 [Pande, U., La Mar, G. N., Lecomte, J. T. J., Ascoli, F., Brunori, M., Smith, K. M., Pandey, R. K., Parish, D. W. & Thanabal, V. (1986) Biochemistry 25, 5638–5646] and therefore a slow transition rate may be unique to the pentacoordinated active site of Mb.
Novel proteins mediate an interaction between clathrin‐coated vesicles and polymerizing actin filaments
Authors: KOHTZ D. Stave , HANSON Veneta , PUSZKIN Saul
Published: March 2005

A monoclonal antibody, A‐7C11, was generated which reacts with two polypeptides of 40 kDa and 80 kDa associated with the coat proteins of purified brain clathirn‐coated vesicles. The 40‐kDa antigen was purified and found to display actin‐binding properties. Negative‐staining electron microscopy showed that one of the antigens reactive with A‐7C11 appears to mediate the association of isolated clathrin‐coated vesicles with assembling actin filaments in vitro. Immunofluorescence microscopy of cultured fibroblasts with A‐7C11 revealed the antigens aligned with both actin filaments and as punctate structures near the plasma membrane. The data suggest that the interaction between clathrin‐coated vesicles and the actin cytoskeleton is mediated by antigens identified by monoclonal antibody A‐7C11.
Transcription factor TFIIIA stimulates DNA supercoiling promoted by a fractionated cell‐free extract from Xenopus laevis
Authors: SEKIGUCHI Jo Ann M. , COLE Allyson D. , GARDNER Kevin H. , KMIEC Eric B.
Published: March 2005

An activity than can introduce negative supercoils into relaxed covalently closed DNA molecules has been isolated from a Xenopus laevis cell‐free extract (S‐150) and purified over 200‐fold. The exogenous addition of ATP, other ribonucleotides and deoxyribonucleotides, as well as nonhydrolyzable analogs, stimulate DNA supercoiling which may occur by a pathway involving multiple protein components. DNA supercoiling occurs in topological single steps and is inhibited by camptothecin and berenil, but not novobiocin or VM‐26, suggesting a catalytic role for topoisomerase I in the reaction. Transcription factor TFIIIA stimulates DNA supercoiling catalyzed by the isolated fraction at low factor to DNA ratios. Taken together, these results suggest that a isolated DNA supercoiling activity from the X. laevis S‐150 cell‐free extract can be stimulated by transcription factor TFIIIA.
Sequence‐specific 1H‐NMR assignment and conformation of proteolytic fragment 163–231 of bacterioopsin
Authors: BARSUKOV Igor L. , ABDULAEVA Galina V. , ARSENIEV Alexander S. , BYSTROV Vladimir F.
Published: March 2005

Proteolytic fragment 163–231 of bacterioopsin was isolated from Halobacterium halobium purple membrane treated with NaBH4 and papain under nondenaturing conditions. Two‐dimensional 1H‐NMR spectra of (163–231)‐bacterioopsin solubilized in chloroform/methanol (1:1), 0.1 M LiClO4 indicated the existence of one predominant conformation. Most of the resonances in the 1H‐NMR spectra of (163–231)‐bacterioopsin were assigned by two‐dimensional techniques. Two extended right‐handed α‐helical regions Ala168–Ile191 and Asn202–Arg227 were identified on the basis of NOE connectivities and deuterium exchange rates. The N‐terminal part of the peptide is flexible and the region of Gly192–Leu201 adopts a specific conformation. The protons of OH groups of Thr178, Ser183 and Ser214 slowly exchange with solvent, and side‐chain conformations of these residues, as evaluated by NOE connectivities of OH protons, are optimal for the formation of hydrogen bonds between OH and backbone carbonyl groups.
An anti‐inflammatory protein secreted from the rat seminal vesicle epithelium inhibits the synthesis of platelet‐activating factor and the release of arachidonic acid and prostacyclin
Authors: CAMUSSI Giovanni , TETTA Ciro , BUSSOLINO Federico , METAFORA Salvatore , PELUSO Gianfranco , ESPOSITO Carla , PORTA Raffaele
Published: March 2005

Platele‐activating factor (PAF), a 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine, is a mediator of inflammation and endotoxic shock produced by a variety of stimulated cells. Since the main biosynthetic pathway of PAF involves acetylation of 1‐O‐alkyl‐sn‐glycero‐3‐phosphocholine (lyso‐PAF) generated from 1‐O‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphocholine by phospholipase A2, we suggest a general physiological role played by steroid‐induced anti‐(phospholipase A2) proteins in the modulation of PAF synthesis. The results of the present study support this hypothesis since an androgen‐induced anti‐inflammatory protein, SV‐IV, secreted from rat seminal vesicles, inhibits PAF synthesis in stimulated polymorphonuclear neutrophils, macrophages and endothelial cells. SV‐IV impairs PAF synthesis by inhibiting the activation of phospholipase A2, that also results in the inhibition of arachidonic acid and prostacyclin release, and of acetyl‐CoA:lyso‐PAF acetyltransferase.
Regulation of heme oxygenase gene expression by cobalt in rat liver and kidney
Authors: LIN Jane H.‐C. , VILLALON Patricio , MARTASEK Pavel , ABRAHAM Nader G.
Published: March 2005

The effect of heavy metals such as cobalt chloride and the corresponding metalloporphyrin on the transcription of the heme oxygenase gene in tissues was examined using cDNA for rat heme oxygenase as the probe. An increase in heme oxygenase mRNA level was observed in response to cobalt chloride and cobalt protoporphyrin treatment in both liver and kidney. Quantitative evaluation of the heme oxygenase transcript was obtained by determining the intensity of mRNA bands by scanning densitometry, and indicated that cobalt chloride increased heme oxygenase mRNA by 40–60‐fold after 2 h of metal exposure. Accumulation of heme oxygenase mRNA after cobalt chloride administration was prevented by co‐administration of actinomycin D or cycloheximide. These results indicate that the increased expression of heme oxygenase by cobalt chloride required de novo protein synthesis and was regulated at the transcriptional level. The time course of heme oxygenase transcript accumulation following administration of cobalt protoporphyrin was different from that of cobalt chloride. There was a sharp increase in heme oxygenase mRNA after cobalt chloride administration at 2 h and cobalt protoporphyrin at 10 h. Heme, to which cobalt protoporphyrin is structurally analogous. acted as a potent inducer of heme oxygenase transcripts in both liver and kidney. Variation in heme oxygenase mRNA levels resulting from enhanced transcription of the heme oxygenase gene was evaluated by nuclear runoff assay using isolated rat liver nuclei after cobalt chloride administration. Quantification of specific nuclear RNAs labeled during the in vitro transcription revealed active heme oxygenase gene transcription in liver nuclei from cobalt‐chloride‐treated rats. Transcription of heme oxygenase is greatly increased within 1 h of administration of cobalt chloride in rat liver. as evidenced by the level of [α‐32P]UTP incorporation into nuclear RNA. The transcription was increased by 40‐fold after 3 h of cobalt chloride administration. The activation of the heme oxygenase gene by metal ions is the most rapid transcriptional response to heavy metals yet described and highlights the regulatory role of heme oxygenase in heme degradation during deviating environmental conditions. On the other hand, cobalt protoporphyrin and heme arginate increase transcription of the heme oxygenase gene in a similar pattern but at a slower rate than that of the heavy metal, suggesting that the heme oxygenase promotor region may contain additional elements conferring the inducing effect of these two agents.
In vivo activation of a microtubule‐associated protein kinase during meiotic maturation of the Xenopus oocyte
Authors: HACCARD Olivier , JESSUS Catherine , CAYLA Xavier , GORIS Jozef , MERLEVEDE Wilfried , OZON René
Published: March 2005

We have characterized a serine/threonine protein kinase from Xenopus metaphase‐II‐blocked oocytes, which phosphorylates in vitro the microtubule‐associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone‐induced meiotic maturation (G2‐M transition of the cell cycle). p‐Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactiyated by the low‐Mr polycation‐stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine‐specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necssary to convert pre‐M‐phase‐promoting factor (pre‐MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.
The formation of protein complexes between ferricytochrome b5 and ferricytochrome c studied using high‐resolution 1H‐NMR spectroscopy
Authors: WHITFORD David , CONCAR David W. , VEITCH Nigel C. , WILLIAMS Robert J. P.
Published: March 2005

The association of the tryptic fragment of bovine microsomal cytochrome b5 with cytochrome c has been studied by one‐ and two‐dimensional 1H‐NMR spectroscopy. The association of cytochromes to form protein complexes is apparent from the increase in linewidths for resonances of ferricytochrome b5 as well as small perturbations in their chemical shifts that occur upon increasing the cytochrome c/b5 molar ratio. The changes in the chemical shifts of hyperfine shifted resonances of ferricytochrome b5 with increasing ratios of ferricytochrome c indicate the formation of binary 1:1 complexes and ternary 1:2 complexes. Similarly, titrations of the linewidth of resolved resonances of ferricytochrome b5 are consistent with stoichiometries of 1:1 and 1:2 for complexes formed between cytochromes b5 and c. Surprisingly, in the 1:1 complex, mobility is shown to be a function of ionic strength. Two‐dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY) of the binary complex formed between ferricytochrome b5 and c indicate that the positions of many resonances attributable to amino acids are unaltered by protein association, although distinctive chemical shift changes are detected in the α‐CH of the haem C17 propionate. The protein complex detected by NMR is discussed with respect to the model for the binary complex proposed by Salemme and possible mechanisms of electron transfer.
NADP‐dependent malate dehydrogenase (decarboxylating) from sugar cane leaves
Authors: IGLESIAS Alberto A. , ANDREO Carlos S.
Published: March 2005

NADP‐dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel‐filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme‐dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer > dimer > monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP‐dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.
The role of fructose 2,6‐bisphosphate in glycolytic/oscillations in extracts and cells of Saccharomyces cerevisiae
Authors: YUAN Zhi , MEDINA Miguel A. , BOITEUX Arnold , MÜLLER Stefan C. , HESS Benno
Published: March 2005

Fructose 2,6‐bisphosphate is physiologically one of the most potent activators of yeast 6‐phosphofructo‐1‐kinase. The glycolytic oscillation observed in cell‐free cytoplasmic extracts of the yeast Saccharomyces cerevisiae responds to the addition of fructose 2,6‐bisphosphate in micromolar concentrations by showing a pronounced decrease of both the amplitude and the period. The oscillations can be suppressed completely by 10 μM and above of this activator but recovers almost fully (95%) to the unperturbed state after 3 h. Fructose 2,6‐bisphosphate shifts the phases of the oscillations by a maximal ± 60°. Oscillations in concentration of endogenous fructose 2,6‐bisphosphate in the extract were also observed. Fructose 2,6‐bisphosphate alters the dynamic properties of 6‐phosphofructo‐1‐kinase which are vital for its role as the ‘oscillophore’. However, the minute amount (∼ 0.3 μM) of endogenous fructose 2,6‐bisphosphate and the phase relationship of its oscillations compared with other metabolites indicate that this activator is not an essential component of the oscillatory mechanism. Further support for this conclusion is the observation of sustained oscillations in both the extracts and a population of intact cells of a mutant strain (YFA) of S. cerevisiae with no detectable fructose 2,6‐bisphosphate (< 5 nM).
A comparison of the effect of vanadate on the binding of myosin‐subfragment‐1 · ADP to actin and on actomyosin subfragment 1 ATPase activity
Authors: SMITH Susan J. , EISENBERG Evan
Published: March 2005

Equilibrium binding studies were used to determine the binding constant of vanadate ion (Vi), to the complex of actomyosin subfragment 1 (S1) with ADP and Vi and of actin to the myosin S1 · ADP · Vi complex. The proteins used were obtained from rabbit skeletal muscle. Pre‐steady‐state measurements were also performed to determine the rates of Vi association and dissociation from the actomyosin S1 · ADP · Vi complex. Using these measured values in a simple model, the steady‐state actomyosin S1 ATPase activity was predicted over a range of Vi concentrations. This model predicted that Vi would have little effect on the actomyosin S1 ATPase activity. In agreement with this prediction, the measured ATPase activity of actomyosin S1 was not greatly inhibited by Vi, except at high concentrations at which polymeric species of Vi may occur (> 900 μM).
Carbonyl reductases from rat testis and vas deferens
Authors: IWATA Nobuhisa , INAZU Norihisa , TAKEO Satoshi , SATOH Tetsuo
Published: March 2005

Three enzyme forms (T1, T2, T3) from rat testis and two from rat vas deferens (V1, V2) of carbonyl reductase have been highly purified to apparent homogeneity. These carbonyl reductases from rat reproductive organs have several similarities in terms of molecular mass (32 – 33 kDa), isoelectric point (pI 5.9 – 6.4), immunochemical properties, cofactor requirement (NADPH dependency) and sensitivity to sulfhydryl reagents. The isoenzymes from the vas deferens (V1, V2) have similar catalytic activities, whereas those from the testis (T1, T2, T3) showed different catalytic activities from each other. All enzymes, however, reduced quinones, aromatic aldehydes and ketones, while T3, V1 and V2 were characterized as possessing high affinity towards prostaglandins. An immunoinhibition study using a specific antibody indicated that these enzymes were solely responsible for the overall catalytic activities of 13,14‐dihydro‐15‐oxo‐prostaglandin F2α, 4‐benzoylpyridine, and 4‐nitroacetophenone reduction and prostaglandin F2α oxidation in both testis and vas deferens cytosol. The immunohistochemical staining revealed a positive immunoreactivity to antibody only in the Leydig cells of the testis, but neither the germ cells nor Sertoli cells in the seminiferous tubule. The staining also showed that the enzymes in the vas deferens were primarily localized in mucosal epithelium cells.
Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei
Authors: JACKMAN Simon A. , HOUGH David W. , DANSON Michael J. , STEVENSON Kenneth J. , OPPERDOES Fred R.
Published: March 2005

In the long‐slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2‐oxo‐acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism [Danson, M. J., Conroy, K., McQuattie, A. & Stevenson, K. J. (1987) Biochem. J. 243, 661–665]. In the present paper, a complete subcellular fractionation of T. brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane. In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction. A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane.

The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T. brucei. Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid‐hydrolysed extracts of T. brucei contain 1.7 (± 0.2) ng of the cofactor/mg protein. The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry.
Species‐dependent binding of serum albumins to the streptococcal receptor protein G
Authors: NYGREN Per‐Åke , LJUNGQUIST Charlotta , TRØMBORG Hanne , NUSTAD Kjell , UHLÉN Mathias
Published: March 2005

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37°C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.
Muscle‐specific response to thyroid hormone of myosin isoform transitions during rat postnatal development
Authors: d'ALBIS Anne , CHANOINE Christophe , JANMOT Chantal , MIRA Jean‐Claude , COUTEAUX René
Published: March 2005

Transitions from embryonic and neonatal to adult‐type‐II isomyosins are known to be related to the increase in the thyroid hormone plasma concentration during postnatal development. These transitions have been shown, however, to occur at different times, depending on the muscle, suggesting that each muscle responds differently to the thyroid hormone. We have investigated quantitatively the effects of experimental hypothyroidism and hyperthyroidism on isomyosin transitions from birth until the 45th postnatal day in eight rat muscles: diaphragm, intercostals, gastrocnemius medialis, soleus, plantar muscles of the foot, tongue muscle, levator ani and bulbocavernosus complex, and masseter.

Hypothyroidism delayed the isomyosin transitions in all the muscles examined, particularly in the sexually dimorphic muscles (levator ani and bulbocavernosus complex and masseter). However, it did not eventually inhibit isomyosin transitions, indicating that the thyroid hormone was not an absolute requirement for these to occur.

Hyperthyroidism had only a slight effect on isomyosin transition in the diaphragm, and accelerated such transitions in the other muscles. The transition curves of all the muscles investigated, except those of the sexually dimorphic muscles, became similar to that of the diaphragm, demonstrating that the various muscles did not display the same sensitivity to the thyroid hormone but were regulated by it in the same way. The isomyosin transitions in the sexually dimorphic muscles remained late in comparison to that in the diaphragm, which suggests a more complex regulation. The effect of hyperthyroidism was not permanent and could be reversed, by interruption of the treatment, to a greater or lesser extent depending on the muscle.

In all muscles containing slow‐type‐I isomyosin, hypothyroidism had no effect on this isomyosin synthesis, whereas hyperthyroidism inhibited it. This inhibition ceased rapidly after the interruption of the treatment.
Hormone binding to natural mutants of human serum albumin
Authors: KRAGH‐HANSEN Ulrich , MINCHIOTTI Lorenzo , BRENNAN Stephen O. , SUGITA Osamu
Published: March 2005

High‐affinity binding of progesterone, testosterone, prostaglandin F2α and l‐thyroxine to five genetic variants of human serum albumin with defined point mutations was investigated by equilibrium dialysis (pH 7.4). Endogenous albumin A (Alb A) from each individual and commercial human serum albumin were used as controls in each case. The association constant for binding of progesterone to Alb Canterbury (Lys313 → Asn) was 1.5 times that calculated for binding to the corresponding, endogenous Alb A. In contrast, the variants Alb Niigata (Asp269 → Gly), Alb Roma (Glu321 → Lys), Alb Parklands (Asp365 → His) and Alb Verona (Glu570 → Lys) all had normal progesterone binding properties. Specificity with respect to the type of mutation was also found for the binding of testosterone and prostaglandin F2α. Testosterone binding to Alb Roma was only 0.7 of that determined for endogenous Alb A, whereas prostaglandin F2α binding to Alb Niigata was increased by a factor 2.4. In the case of l‐thyroxine normal binding properties were found for all the variants. Steric effects and/or conformational changes of the protein, introduced by the amino acid substitutions, probably account for the altered hormone binding. However, in the case of the increased binding of prostaglandin F2α to Alb Niigata electrostatic effects could also be involved. The experimental findings suggest different high‐affinity sites for the four hormones. Progesterone, testosterone and prostaglandin F2α are apparently bound within the middle third (domain II) of the protein molecule. The possible position of the primary l‐thyroxine site is discussed.
A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis)
Authors: BALESTRIERI Ciro , CASTALDO Domenico , GIOVANE Alfonso , QUAGLIUOLO Lucio , SERVILLO Luigi
Published: March 2005

The finding of a powerful inhibitor of pectin methylesterase in ripe kiwi fruit is reported. The inhibitor was revealed to be a glycoprotein. It was purified to homogeneity and found to have a molecular mass of about 28 kDa, as estimated by gel filtration chromatography, SDS/PAGE and analytical ultracentrifugation. The sugar portion is composed of galactose, arabinose and rhamnose, the latter being much less represented. The amino acid composition showed a very high content of acidic residues compared to basic ones, which is the reason for the very low isoelectric point of the protein (less than 3.5). The kind of inhibition on kiwi pectin methylesterase was found to be competitive with an apparent Ki of 0.22 μM, using citrus pectin as a substrate. Moreover, the inhibitor is effective in inhibiting pectin methylesterase in the pH range 3.5–7.5. Kiwi inhibitor appears to be specific for pectin methylesterase, inasmuch as it was found to be ineffective against other polysaccharide‐degrading enzymes, such as polygalacturonase and amylase. Conversely, it appears to be completely aspecific as far as the pectin methylesterase source is concerned. In fact, it was found to inhibit this enzyme effectively from all the sources we assayed, i. e. orange, tomato, apple, banana, potato.
Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg)
Authors: HEDDERICH Reiner , BERKESSEL Albrecht , THAUER Rudolf K.
Published: March 2005

The reduction of the heterodisulfide of coenzyme M (H‐S‐CoM) and 7‐mercaptoheptanoyl‐l‐threonine phosphate (H‐S‐HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80‐fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 ± 1 nmol FAD, 130 ± 10 nmol non‐heme iron and 130 ± 10 nmol acid‐labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM‐S‐S‐HTP (app. Km= 0.1 mM) with reduced benzylviologen at a specific rate of 30 μmol · min−1· mg protein−1 (kcat= 68 s−1) and the reduction of methylene blue with H‐S‐CoM (app. Km= 0.2 mM) plus H‐S‐HTP (app. Km < 0.05 mM) at a specific rate of 15 μmol · min−1· mg−1. The enzyme was highly specific for CoM‐S‐S‐HTP and H‐S‐CoM plus H‐S‐HTP. The physiological electron donor/acceptor remains to be identified.
A 31P‐NMR study of bovine adrenocortical mitochondrial metabolic activities
Authors: PERRIN Anne , DEFAYE Geneviève , GUIDICELLI Claudette , CHAMBAZ Edmond , ALBRAND Jean‐Pierre , ROBY Claude
Published: March 2005

High‐field 31P‐NMR spectroscopy has been used to study the metabolic activities of coupled bovine adrenocortical mitochondria in vitro. These differentiated organelles use oxygen as a substrate to support both oxidative phosphorylation and specific steroid hydroxylation reactions.

The NMR technique allowed the resolution of two inorganic phosphate signals, attributed to the matrix and external medium phosphate pools, at low and high field, respectively. These signals were used to calculate the respective Pi concentrations and to obtain the pH of the two corresponding compartments. In addition, the NMR spectra displayed resonance signals corresponding to ADP added to the medium and to ATP synthesized during oxidative phosphorylation. NMR analysis of the mitochondrial perchloric acid extracts identified the major phosphate‐containing metabolites, namely NADP+, NAD+, phosphocholine, phosphoethanolamine, sn‐glycero‐(3)phosphocholine, AMP, ADP, ATP and Pi.

Upon addition of ADP and malate to the oxygenated suspension, the kinetics of mitochondrial external Pi consumption and of ATP synthesis, along with the intra‐ and extraorganelle pH variations could be monitored over time periods of approximately 30 min, in the absence and presence of different steroid hydroxylation substrates. A major observation was that oxidative phosphorylation, which takes place in the absence of steroid, was markedly inhibited as soon as steroid hydroxylation was operating. These observations show the potential of 31P‐NMR spectroscopy in the study of metabolic activities of isolated intact mitochondrial organelles. Such an approach appears promising for further determination of the underlying mechanisms in the balance between vital oxidative phosphorylation and differentiated steroid hydroxylation which are under hormonal control in adrenocortical mitochondria as well as in other steroidogenic cell systems.
Expression of three plant glutamine synthetase cDNA in Escherichia coli
Authors: BENNETT Malcolm , CULLIMORE Julie
Published: March 2005

Three cDNA clones encoding the closely related glutamine synthetase (GS) α, β and γ polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant α, β and γ polypeptides which then assembled into catalytically active homo‐octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion‐exchange and gel‐filtration chromatography. Furthermore, the α and γ isoenzymes complemented a GS(glnA)‐deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo‐octameric GS isoenzymes.
Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury
Authors: OKAJIMA Ai , MIYAZAWA Keiji , KITAMURA Naomi
Published: March 2005

Overlapping cDNA clones for rat hepatocyte growth factor (rHGF) were isolated by cross‐hybridization with the cloned cDNA for human hepatocyte growth factor (hHGF) and the nucleotide sequence of the cDNA was determined. The entire primary structure of rHGF was deduced from the sequence. Comparison of the amino acid sequences between rat and human HGFs revealed that the two sequences are highly conserved throughout the protein structures, suggesting that rat and human HGFs may be functionally similar.

Responses of the rHGF mRNA during liver regeneration in rats were examined by Northern blot hybridization analysis with the aid of the cDNA probe for rHGF. The mRNA levels increased in the liver and spleen but not in the kidney after administration of carbon tetrachloride. At the maximum level of induction, the rHGF mRNA increased in the liver about 4.5‐fold over its normal level. The mRNA levels also increased in the liver and spleen after administration of d‐galactosamine. On the other hand, no obvious increase of the mRNA was observed in the liver and spleen after partial hepatectomy. These observations suggest that HGF may function as a regulator of liver regeneration following hepatic injury caused by hepatotoxins.
Analysis of the binding of glyceraldehyde‐3‐phosphate dehydrogenase to microtubules, the mechanism of bundle formation and the linkage effect
Authors: SOMERS Marijke , ENGELBORGHS Yves , BAERT Johan
Published: March 2005

The binding of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) to microtubules was analysed by the determination of the concentration of the free enzyme in equilibrium with the complex. At low ionic strength (0.03 M) the binding data are best described by a sum of high (Kd= 0.28 μM) and low affinity (Kd= 7.14 μM) sites, showing positive cooperativity. Addition of 1 mM adenosine 5′‐[β,γ‐imido]triphosphate (p[NH]ppA) or increasing the ionic strength to 0.1 M reduces the binding constant of the high‐affinity sites considerably. Adding microtubule‐associated proteins (at I= 0.1 M) does not appreciably influence the affinities. Total stoichiometries vary over 2.1 – 1.2 tubulin dimers involved in a binding site for GAPDH.

Bundling is reduced concomitantly with the reduction of the affinities and the increase of the stoichiometry to close to 1 mol GAPDH/mol tubulin dimer. The critical concentration of tubulin is practically not influenced by the binding of the enzyme. This behaviour is discussed in terms of the concept of linked functions.

p[NH]ppA dissociates the bundles very rapidly. Analytical sedimentation studies showed that the dissociation of the bundles by p[NH]ppA is not due to the dissociation of the tetrameric enzyme. Bundling slows down association and dissociation of microtubules. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration.
Carbohydrate epitope structural elucidation by 1H‐NMR spectroscopy of a new Mycobacterium kansasii phenolic glycolipid antigen
Authors: GILLERON Martine , VENISSE Anne , RIVIÈRE Michel , SERVIN Philippe , PUZO Germain
Published: March 2005

The complete primary structure of the carbohydrate moiety of a new phenolic glycolipid antigen namely PheGl K‐IV from Mycobacterium kansasii was successfully established from only one‐ and two‐dimensional 1H‐NMR data. Among the scalar two‐dimensional techniques, correlated spectroscopy with a 45° mixing pulse and phase‐sensitive double‐quantum‐filtered correlated spectroscopy were selected, combined with two‐dimensional dipolar techniques (nuclear Overhauser effect). These techniques using milligram of quantities native PheGl K‐IV allowed the following monoacetylated tetrasaccharide to be proposed for its carbohydrate part:

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The PheGl K‐IV shares, with the other phenolic glycolipids isolated from M. kansasii (K‐I, K‐II), a common core assigned to the lipid aglycone glycosylated by the monoacetylated trisaccharide part. It differs in the structure of the distal monosaccharide residue.
The inositol‐phospholipid‐accelerated activation of prekallikrein by activated factor XII at physiological ionic strength requires zinc ions and high‐Mr kininogen
Authors: SCHOUSBOE Inger
Published: March 2005

In a system consisting of purified proteins inositol‐phospholipid‐accelerated activation of prekallikrein by α‐factor XIIa was determined by measuring the appearance of kallikrein amidolytic activity towards the chromogenic substrate, H‐d‐Pro‐Phe‐Arg‐NH‐PhNO2 (PhNO2,4‐nitrophenyl). The activation reaction was ionic‐strength dependent. In the absence of high‐Mr kininogen optimal activity was recorded at I= 50 mM. Searching for conditions, which could change this optimum towards physiological values, high‐Mr kininogen was added. This resulted in an inhibitition of the activity, with no change in ionic strength optimum. If, however, Zn2+ were added concomitant with high‐Mr kininogen, the inhibition was abolished and optimal activity recorded at physiological ionic strength. The optimal Zn2+ concentration was found to be 0.1 mM. Kinetic analysis of the reaction demonstrated that the kcat/Km was 1.2 × 105 M−1 s−1 in the absence and 1.1 × 106 M−1 s−1 in the presence of Zn2+. Zn2+ were also required for inositol‐phospholipid‐accelerated initiation of the contact activation in whole plasma.