bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli
Authors: AIRAS R. Kalervo
Published: March 2005

A total rate equation was used to calculate the discrimination of valine by the isoleucyl‐tRNA synthetas from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E ·aa‐AMP possible. If the E ·IIe‐AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E ·IIe‐AMP with E ·IIe and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E ·IIe‐AMP and the corrected *EtRNAIIe‐AMP. If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be benefitial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.
Zinc‐specific activation of a HeLa cell nuclear protein which interacts with a metal responsive element of the human metallothionein‐IIA gene
Authors: KOIZUMI Shinji , YAMADA Hirotomo , SUZUKI Kaoru , OTSUKA Fuminori
Published: March 2005

Transcription of metallothionein genes is activated by heavy metals such as zinc and cadmium, and a DNA element called metal responsive element (MRE) is essential for this process. By mobility‐shift assay, we identified a HeLa‐cell nuclear protein which specifically binds to MREa of human metallothionein‐IIA gene. This protein, named ZRF (zinc‐regulatory factor), is present in the cells untreated with heavy metals. Zinc is essential for, and increases in a dose‐dependent manner, the binding of ZRF to MREa. Other heavy metals which can also induce metallothioneins, including cadmium, copper and mercury, do not activate ZRF. A MREa‐containing oligonucleotide that can bind ZRF confers heavy metal‐inducibility to a heterologous promoter, suggesting that ZRF is a zinc‐dependent transcriptional activator. In addition to the MRE core sequence, the surrounding sequences are also important for both ZRF binding in vitro, and zinc‐dependent transcriptional activation in vivo. MREa by itself responds not only to zinc but also to other metallothionein‐inducing heavy metals, indicating that the ZRF protein, not the MREa sequence, is responsible for the zinc specificity.
Non‐lysosomal degradation of misfolded human lysozymes with and without an asparagine‐linked glycosylation site
Authors: OMURA Fumihiko , OTSU Mieko , YOSHIMORI Tamotsu , TASHIRO Yutaka , KIKUCHI Masakazu
Published: March 2005

Human lysozyme is a monomeric secretory protein composed of 130 amino acid residues, with four intramolecular disulfide bonds and no oligosaccharides. In this study, a mutant protein, [Ala 128] lysozyme, Which cannot fold because it lacks a disulfide bond, Cys6‐Cys128, was expressed in mouse fibroblasts and was found to be mostly degraded in the cells, whereas the control wild‐type lysozyme was quantitatively secreted into the media. The degradation of [Ala 128]lysozyme was independent of the transport from the endoplasmic reticulum to the Golgi apparatus. The degradation was greatly inhibited by incubation of cells at 15°C, but was minimally affected by treatment of cells with the lysosomotropic agent, chloroquine, implying a non‐lysosomal process. Additional mutations (Gly48 → Ser or Met29 → Thr) were created to make asparagine‐linked (N‐linked) glycosylation site in the [Ala128]lysozyme, and the resultant double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were analyzed with respect to their intracellular degradation. These mutant proteins were susceptible to N‐linked glycosylation, and were degraded in a similar manner to that of [Ala128] lysozyme, except that the onset of degradation of [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, but not of [Ala128]lysozyme, was preceded by a lag period of up to 60 min. Furthermore, the degradative double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were glycosylated post‐translationally as well as co‐translationally. These observations suggest that there is some interaction between the mechanisms of glycosylation and degradation.
Studies on the interaction of α subunits of GTP‐binding proteins with βγ dimers
Authors: GRAF Rolf , MATTERA Rafael , CODINA Juan , EVANS Tony , HO Yee‐Kin , ESTES Mary K. , BIRNBAUMER Lutz
Published: March 2005

The interaction of several preparations of purified βγ dimers with two types of guanosinenucleotide‐binding‐regulatory‐(G)‐protein α subunits, a recombinant bvαi3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and αs, either purified from human erythrocyte Gs‐type GTP‐binding protein, and activated by NaF/AICI3, or unpurified as found in a natural membrane, were studied. The βγ dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of β subunits that, upon electrophoresis, migrated as two bands with approximate Mr of 35 000 and 36 000, as well as distinct complements of at least two γ subunits each. When tested for their ability to recombine at submaximal concentrations with bvαi3, ROS, brain, hRBC and placental βγ dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS βγ dimers were tested for their ability to promote deactivation of Gs, brain and placental βγ dimers were equipotent and at least 10‐fold more potent than that of ROS βγ dimers; likewise, brain βγ and placental dimers were equipotent in inhibiting GTP‐activated and GTP‐plus‐isoproterenol‐activated adenylyl cyclase, while ROS βγ dimers were less potent when assayed at the same concentration. The possibility that different α subunits may distinguish subsets of βγ dimers from a single cell was investigated by analyzing the βγ composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogenity from a single cell type, the human erythrocyte. No evidence for an α‐subunit‐specific difference in βγ composition was found. These findings suggests that, in most cells, α subunits interact indistinctly with a common pool of βγ dimers. However, since at least, one βγ preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of βγ dimers may exhibit selectivity for the α subunits they interact with.
Hydration of the Gly2 and Gly3 peptide oxygens of [Leu5]‐enkephalin in aqueous solution as revealed by the combined use of 17O‐NMR and Fourier‐transform infrared spectroscopy
Authors: GEROTHANASSIS Joannis P. , BIRLIRAKIS Nick , KARAYANNIS Theodoros , SAKARELLOS‐DAITSIOTIS Maria , SAKARELLOS Constantinos , VITOUX Bernard , MARRAUD Michel
Published: March 2005

Solvent‐induced and temperature‐induced 17O chemical shifts of[17O‐Gly2, Leu5]‐enkephalin and [17O‐Gly3, Leu5]‐enkephalin and solvent‐induced spectral modifications of the amide‐I' stretching vibrations of [1‐13C‐Gly2, Leu5]‐enkephalin and [1‐13C‐Gly2, Leu5]‐enkephalin are reported and correlated with the spectroscopic characteristics of model amides. It is demonstrated that both Gly2 and Gly3 peptide oxygens are motionally equivalent and form solvation species which are essentially monohydrated in aqueous solution, contrary to several simple amides and model peptides in which water largely forms dihydrates. It is shown that the combined use of 17O‐NMR and Fourier transform infrared is a unique methodology for studying the hydration state of specific peptide oxygens in peptide hormones.
A weighting method for predicting protein structural class from amino acid composition
Authors: GENFA Zhou , XINHUA Xu , CHUN‐TING Zhang
Published: March 2005

A protein is generally classified into one of the following four structural classes: all α, all β, α+β and α/β. In this paper, based on the weighting to the 20 constituent amino acids, a new method is proposed for predicting the structural class of a protein according to its amino acid composition. The 20 weighting parameters, which reflect the different properties of the 20 constituent amino acids, have been obtained from a training set of proteins through the linear‐programming approach. The rate of correct prediction for a training set of proteins by means of the new method was 100%, whereas the highest rate of previous methods was 82.8%. Furthermore, the results showed that the more numerous training proteins, the more effective the new method.
Characterisation of the control of respiration in potato tuber mitochondria using the top‐down approach of metabolic control analysis
Authors: KESSELER Adolf , DIOLEZ Philippe , BRINKMANN Klaus , BRAND Martin D.
Published: March 2005

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top‐down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton‐leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8 – 1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and – 0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between –0.2 and + 0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.
Biochemical characterization of single‐chain chimeric plasminogen activators consisting of a single‐chain Fv fragment of a fibrin‐specific antibody and single‐chain urokinase
Authors: HOLVOET Paul , LAROCHE Yves , LIJNEN Henri Roger , VAN HOEF Berthe , BROUWERS Els , DE COCK Frans , LAUWEREYS Marc , GANSEMANS Yannick , COLLEN Désiré
Published: March 2005

K12G0S32 is a 57‐kDa recombinant single‐chain chimeric plasminogen activator consisting of scFv‐K12G0, a single‐chain variable‐region antigen‐binding fragment (Fv) of the monoclonal anti‐body MA‐15C5, which is specific for fragment D‐dimer of human cross‐linked fibrin, and a low‐molecular‐mass (33 kDa) urokinase‐type plasminogen activator (u‐PA‐33k) containing amino acids Ala132‐Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717–19724). In addition, the Arg156‐Phe157 thrombin‐cleavage site in the u‐PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I‐fibrin‐labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single‐chain u‐Pa (rscu‐PA), but 17‐fold higher than that of rscu‐PA(M), a variant of rscu‐PA in which the thrombin‐cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin‐cleavage site, was 6–15‐fold higher than that of rscu‐PA. Conversion of 1 μM single‐chain K12G0S32 or rscu‐PA(M) into their two‐chain derivatives with plasmin occurred at a rate of 1.0 ± 0.15 nmol · min−1· nmol plasmin−1 and 0.85 ± 0.074 nmol · min−1· nmol plasmin−1, compared to 14 ± 2.3 nmol · min−1· nmol plasmin−1 and 18 ± 2.6 nM · min−1· nmol plasmin−1 for K12G0S32T and rscu‐PA, respectively. Purified fragment D‐dimer of human cross‐linked fibrin inhibited the fibrinolytic potency of single‐chain K12G0S32T, but not of two‐chain K12G0S32T, in a dose‐dependent manner. Furthermore, the fibrinolytic potencies of two‐chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two‐chain u‐PA (rtcu‐PA) or of rtcu‐PA(M). These findings suggest that the 50‐fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu‐PA(M), is due both to targeting of the activator to the clot via the single‐chain Fv fragment (sixfold increase) and to a more efficient conversion of single‐chain K12G0S32T to its two‐chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin‐specific antibodies results in a significant increase of the fibrinolytic potency of single‐chain but not of two‐chain u‐PA. K12G0S45 which contains the scu‐PA kringle domain, and K12G0S54 which contains intact scu‐PA, had a similar fibrinolytic potency as K12G0S32, indicating that the fibrin‐targeting in the Fv domain of K12G0S32 is not hampered by spatial constraints in the molecule.
Overproduction of prismane protein in Desulfovibrio vulgaris (Hildenborough): evidence for a second S= 1/2‐spin system in the one‐electron reduced state
Authors: STOKKERMANS Jack P. W. G. , HOUBA Pieter H. J. , PIERIK Antonio J. , HAGEN Wilfred R. , van DONGEN Walter M. A. M. , VEEGER Cees
Published: March 2005

The gene encoding the prismane protein from Desulfovibrio vulgaris (Hildenborough) was inserted into broad‐host‐range vector pSUP104. The recombinant plasmid, pJSP104, was transferred to D. vulgaris by conjugal plasmid transfer. In the transconjugant D. vulgaris cells the prismane protein was 25‐fold overproduced. The overproduced prismane protein was characterized by molecular mass, isoelectric point, iron content and spectroscopical properties. Both the iron content and the ultraviolet/visible spectrum are identical to the wild‐type protein indicating that iron incorporation in the overproduced protein is complete. EPR spectra of the dithionite‐reduced form of the overproduced protein indicated that the Fe‐S cluster might occur in a similar structure as found in inorganic model compounds containing a [6Fe‐6S] prismane core. The as‐isolated overproduced protein showed the presence of a second S= 1/2 spin system that was also detected in the corresponding prismane protein from D desulfuricans (ATCC 27774), but not in the protein from wild‐type D. vulgaris. This additional signal was irreversibly transformed to the ‘wild‐type’ high‐spin and low‐spin systems upon two reduction/re‐oxidation cycles. It is shown that the EPR spectroscopy of the overproduced prismane protein is very similar to that of the D. desulfuricans enzyme and, with the exception of the second S= 1/2 spin system, to that of the prismane protein from wild‐type D. vulgaris. Contrary to claims for the D. desulfuricans protein, it is shown here that all data can be fully explained assuming a single [6Fe‐6S] cluster, that might be titrated into four different redox states and occurs in up to three different spin systems in the one‐electron reduced state.
The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium
Authors: MARAS Bruno , CONSALVI Valerio , CHIARALUCE Roberta , POLITI Laura , DE ROSA Mario , BOSSA Francesco , SCANDURRA Roberto , BARRA Donatella
Published: March 2005

The complete amino acid sequence of glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage by trypsin, cyanogen bromide, Staphylococcus aureus V8 protease and pepsin. The enzyme subunit is composed of 421 amino acid residues yielding a molecular mass of 46.078 kDa. The presence of N‐Δ‐methyllysine in six positions of the sequence was observed. Comparison of the sequence of glutamate dehydrogenase from S. solfataricus with the other known primary structures of the corresponding enzyme from different sources, gives an overall identity of 9.2% and shows a symmetrical evolutionary distance of this archaebacterial protein from the two groups of vertebrate on one side and eubacterial and low eucaryote enzymes on the other side. The occurrence of specific substitutions and a possible role for N‐δ‐methylation of lysine residues are discussed in view of current hypotheses on the molecular basis of thermal adaptation of proteins.
Purification and properties of NADP‐isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803
Authors: MURO‐PASTOR Maria Isabel , FLORENCIO F. Javier
Published: March 2005

NADP‐dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive‐Red‐120‐agarose affinity chromatography and phenyl‐Sepharose chromatography as main steps. The enzyme was purified about 600‐fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 μM for Mg2+, d,l‐isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 μM for Mn2+, d,l‐isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non‐competitively by ADP (Ki, 6.4 mM) and 2‐oxoglutarate, (Ki, 6mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular‐dichroism spectrum showed a protein with a secondary structure consisting of about 30%α‐helix and 36%β‐pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2‐terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well‐known E. coli enzyme.
Inhibition of factor X, factor V and prothrombin activation by the bis(lactobionic acid amide) LW10082
Authors: OFOSU Frederick A. , FAREED Jawed , SMITH Lindsay M. , ANVARI Noorildan , HOPPENSTEADT Debra , BLAJCHMAN Morris A.
Published: March 2005

The minimum concentrations of heparin, dermatan sulfate, hirudin, and D‐Phe‐Pro‐ArgCH2Cl required to delay the onset of prothrombin activation in contact‐activated plasma also prolong the lag phases associated with both factor X and factor V activation. Heparin and dermatan sulfate prolong the lag phases associated with the activation of the three proteins by catalyzing the inhibition of endogenously generated thrombin. Thrombin usually activates factor V and factor VIII during coagulation. The smallest fragment of heparin able to catalyze thrombin inhibition by antithrombin III is an octadecasaccharide with high affinity for antithrombin III. In contrast, a dermatan sulfate hexasaccharide with high affinity for heparin cofactor II can catalyze thrombin inhibition by heparin cofactor II. A highly sulfated bis(lactobionic acid amide), LW10082 (Mr 2288), which catalyzes thrombin inhibition by heparin cofactor II and has both antithrombotic and anticoagulant activities, has been synthesized. In this study, we determined how the minimum concentration of LW10082 required to delay the conset of intrinsic prothrombin activation achieved this effect. We demonstrate that, like heparin and dermatan sulfate, LW10082 delays the onset of intrinsic prothrombin activation by prolonging the lag phase associated with both factor X and factor V activation. In addition, LW10082 is approximately 25% as effective as heparin and 10 times as effective as dermatan sulfate in its ability to delay the onset of prothrombin activation. The strong anticoagulant action of LW10082 is consistent with previous reports which show that the degree of sulfation is an important parameter for the catalytic effectiveness of sulfated polysaccharides on thrombin inhibition.
Nucleic acid binding properties of the 92‐kDa replicase subunit of alfalfa mosaic virus produced in yeast
Authors: ERNY Claude , SCHOUMACHER Fabrice , GODEFROY‐COLBURN Thérèse , STUSSI‐GARAUD Christiane
Published: March 2005

The 92‐kDa non‐structural protein of alfalfa mosaic virus (one of the replicase subunits) was synthesized by Saccharomyces cerevisiae transformed with a recombinant expression vector. The yeast‐expressed protein had the immunological and size characteristics of the naturally made viral protein. It was partially purified and its nucleic acid binding properties were tested by gel‐retardation electrophoresis and nitrocellulose adsorption. The protein interacted with single‐standed RNA, double‐standed RNA and double‐stranded DNA in a salt‐dependent manner, with a slight preference for RNA. These properties may be related to its putative function as a core RNA polymerase.
Subunits VIIa,b,c of human cytochrome c oxidase
Authors: VAN KUILENBURG André B. P. , VAN BEEUMEN Jozef J. , VAN DER MEER Niek M. , MUIJSERS Anton O.
Published: March 2005

The N‐terminal amino acid sequences and the electrophoretic mobilities of the subunits VIIa, VIIb and VIIc of cytochrome c oxidase purified from human heart were investigated and compared with those from human skeletal muscle and from bovine heart. In purified human heart cytochrome c oxidase, both so‐called ‘heart‐type’ and ‘liver‐type’ isoforms of subunit VIIa were found. The first 30 residues of the N‐terminal amino acid sequences of these ‘heart‐type’ and ‘liver‐type’ subunits VIIa showed nine differences. The two isoforms of subunit VIIa in human heart were present in almost equal amounts, in contrast to the situation in skeletal muscle, where the ‘heart‐type’ subunit VIIa was predominant. Therefore, our results imply that in human heart a cytochrome c oxidase isoform pattern is present that differs from that found in skeletal muscle.

Subunits VIIb and VIIc purified from human heart oxidase proved to be very similar to their bovine heart counterparts. Our direct demonstration of the presence of subunit VIIb, the sequence of which has only recently been identified in the bovine heart enzyme, suggests that human cytochrome c oxidase also contains 13 subunits. We found no evidence for the presence of different isoforms of subunit VIIc in cytochrome c oxidase from human heart and skeletal muscle.

We observed clear differencs in the electrophoretic mobility of the subunits VIIa,b,c between bovine and human cytochrome c oxidase. On Tricine/glycerol/SDS/polyacrylamide gels the ‘heart‐type’ and ‘liver‐type’ subunits VIIa present in human heart cytochrome c oxidase migrated with almost the same electrophoretic mobility. Subunit VIIb migrated only slightly faster than subunit VIIa, whereas VIIc proved to have the highest electrophoretic mobility on Tricine/SDS/glycerol/polyacrylamide gels. Our findings may have implications for the elucidation of certair tissue‐specific cytochrome c oxidase deficiencies in man.
The identification of cation‐binding domains on the surface of microsomal cytochrome b5 using 1H‐NMR paramagnetic difference spectroscopy
Authors: WHITFORD David
Published: March 2005

One‐dimensional and two‐dimensional 1H‐NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3+3] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T= 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3+3 and cytochrome b5 using two‐dimensional double‐quantumfiltered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one‐dimensional experiments. In a binary complex with ferricytochrome c, Cr(en)3+3 broadens many cytochrome b5 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line‐broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.
Photoaffinity labeling of the nucleotide‐binding site of the uncoupling protein from hamster brown adipose tissue
Authors: WINKLER Edith , KLINGENBERG Martin
Published: March 2005

The nucleotide binding center of the uncoupling protein from brown adipose tissue (UCP) was probed by photoaffinity labeling with 8‐azido‐ATP. The isolated dimeric UCP in non‐ionic detergent was used. 8‐azido‐ATP binds to UCP with a Kd= 3 μM, i.e. with an only threefold lower affinity than ATP and a maximum number of binding sites of about 12 μmol/g protein corresponding to about 1 mol/mol dimer UCP. UCP is rapidly degraded by ultraviolet radiation, and therefore only near ultraviolet and visible light can be used for photoaffinity labeling. The total covalent incorporation is shown to be dependent on the concentration of azido‐ATP and on competing phospholipids. The specific, i.e. ATP‐sensitive incorporation only to the binding site depends on the presence of cysteine. With CNBr cleavage the 8‐azido‐[γ‐32P]ATP insertion within the primary structure was located by identifying ATP‐sensitive labeled peptides in SDS/PAGE. A major specific 8‐azido‐ATP incorporation was found by autoradiography in the smallest CNBr fragments. Identification of the radioactive peptides was difficult since 8‐azido‐ATP insertion causes a distinct shift in the gels from the stained peptides. Identification was possible by specific disulfide formation at the C‐terminal within the UCP dimer which only removed the CB7 (CB, CNBr fragment) portion of the low‐molecular‐mass peptides but did not move the radioactive band. This excludes the C‐terminal CB7 and identifies the labeled peptide as CB6. Also, limited tryptic cleavage of intact UCP at Lys293 did not remove the radioactivity. Cleavage of tryptophanes support localization of 8‐azido‐ATP between residues 173–280 which includes CB6. Solid‐phase sequencing of the labeled CB6 both after serine lactone and carboxyl coupling suggest incorporation into Thr260. These results indicate that the adenine‐binding site is within the third domain of the tripartite UCP structure at a putative hydrophilic channel which can be assessed both from the cytosol and matrix of mitochondria.
Dephosphorylation of phosphorylated atrial natriuretic peptide by protein phosphatase 2A
Authors: DAUTZENBERG Frank M. , MÜLLER Dieter , RICHTER Dietmar
Published: March 2005

A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the α subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric from of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km= 42 μM, Vmax= 10.3 μmol × min−1× mg−1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km= 2.5 μM, Vmax= 0.008 μmol × min−1× mg−1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A‐like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP‐dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification.
Isolation and structural characterization of novel sialylated oligosaccharide‐alditols from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis
Authors: KLEIN André , CARNOY Christophe , LAMBLIN Geneviève , ROUSSEL Philippe , van KUIK J. Albert , VLIEGENTHART Johannes F. G.
Published: March 2005

The carbohydrate chains of the respiratory‐mucus glycoproteins of a patient (blood group O) suffering from bronchiectasis due to Kartagener's syndrome, were released by alkaline borohydride treatment of a pronase digest. The structures of 82 neutral and low‐molecular‐mass sialylated oligosaccharides have been described previously [van Kuik A., de Waard P., Vliegenthart J. F. G., Klein A., Carnoy C., Lamblin G. Roussel P. (1991) Eur. J. Biochem. 198, 169–182]. In the present work, medium‐size sialylated oligosaccharides were obtained after ion‐exchange chromatography and were subsequently separated into 36 fractions utilizing gel filtration, HPLC on normal‐phase alkylaminebonded silica and reverse‐phase HPLC. From these fractions, the following six sialylated hepta‐ and octa‐saccharide‐alditols have been characterized by employing 500‐MHz 1H‐NMR spectroscopy, in conjunction with fast‐atom‐bombardment mass spectroscopy and methylation analysis.

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Evaluation of 13C and 1H Fermi contact shifts in horse cytochrome c The origin of the anti‐Curie effect
Authors: TURNER David L.
Published: March 2005

Many ferricytochromes c exhibit a peculiar effect in which the 1H chemical shifts of the haem methyl groups appear in pairs and, although the paramagnetic shifts of the two groups with the larger shifts decrease with temperature, those of the pair with the smaller shifts actually increase. Recent NMR studies [Santos, H. and Turner, D. L. (1992) Eur. J. Biochem. 206, 721–728] gave 1H and 13C assignments for most of the haem substituents and the axial ligands in horse cytochrome c at 30°C and 50°C in both oxidation states. These data are used together with an empirically determined magnetic susceptibility tensor to evaluate the Fermi contact contribution to the paramagnetic shift and hence map the delocalisation of the unpaired electron. The anti‐Curie effect is explained by a Boltzmann distribution between partially filled porphyrin 3e(π) molecular orbitals with an energy difference of 3 kJ/mol. The fact that the energy gap is small with respect to the energy of binding to the electron transfer partners calls into question the significance of the asymmetry of the electron distribution in the electron transfer process.
Inhibition of proton‐translocating transhydrogenase from photosynthetic bacteria by N,N′‐dicyclohexylcarbodiimide
Authors: PALMER Tracy , WILLIAMS Ross , COTTON Nick P. J. , THOMAS Christopher M. , JACKSON J. Baz
Published: March 2005

The effects of N,N′‐dicyclohexylcarbodiimide [(cHxN)2C] on the proton‐translocating enzyme, NAD(P) H+‐transhydrogenase (H+‐Thase), from two species of phototrophic bacteria have been investigated. The polypeptides of H+‐Thase from Rhodobacter capsulatus are membrane‐associated, requiring detergent to maintain solubility. The enzyme from Rhodospirillum rubrum, however, has a water soluble polypeptide (Ths) and a membrane‐associated component (Thm) which, separately, have no activity but which can be fully reconstituted to give functional complex.

Two observations suggest that (cHxN)2C inhibited H+‐Thase from both species by modification either close to or at the NADP(H)‐binding site on the enzyme: (a) the presence of NADP+ or NADPH caused increased inhibition by (cHxN)2C and (b) after treatment of the purified enzyme from Rb. capsulatus with (cHxN)2C, the release of NADP+ became rate‐limiting, as evidenced by a stimulated rate of NADPH‐dependent reduction of acetylpyridine adenine dinucleotide by NADH. Experiments in which Ths and Thm from R. rubrum were separately treated with (cHxN)2C then reconstituted with the complementary, untreated component revealed that the NADP(H)‐enhanced modification by (cHxN)2C was confined to Thm.

In contrast to some experiments with mitochondrial H+‐Thase [Wakabayashi, S. & Hatefi, Y. (1987) Biochem. Int. 15, 667–675], there was no protective effect of either NAD+ or NADH on the inhibition by (cHxN)2C of enzyme from photosynthetic bacteria. However, amino acid sequence analysis of proteolytic fragments of Ths revealed that the NAD(H)‐protectable, (cHxN)2C‐reactive glutamate residue in mitochondrial H+‐Thase might be replaced by glutamine in R. rubrum.
The role of inositol phospholipids in the association of band 4.1 with the human erythrocyte membrane
Authors: GASCARD Philippe , PAWELCZYK Tadeusz , LOWENSTEIN John M. , COHEN Carl M.
Published: March 2005

Band 4.1 is a major protein of the erythrocyte membrane skeleton. It promotes the binding of spectrin to F‐actin and may anchor the skeletal network to the plasma membrane via its association with integral membrane proteins. Here, we have investigated the involvement of inositol phospholipids in the binding of band 4.1 to erythrocyte membranes using membrane vesicles stripped of all peripheral proteins at alkallne pH. Trypsinization of these vesicles allows the discrimination of two classes of band 4.1 binding sites: trypsin‐sensitive sites (60–65% of the total), largely or exclusively on band 3, and trypsin‐resistant sites (35–40% of the total), composed, at least in part, of the glycophorins. ATP depletion or activation of erythrocyte phosphoinositol phospholipase C led to a reduction in membrane phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] content by 20–70% in different experiments. The resulting decrease of band 4.1 binding to vesicles by was variable, but averaged about 15–20%. The same treatments led to an average decrease in the band 4.1 binding capacity of trypsinized vesicles of 55%. Since this is equivalent to a 20% decrease in the binding capacity of non‐trypsinized vesicles (consistent with the above result), it indicates that PtdIns(4,5)P2 regulates the binding of band 4.1 only to trypsin‐resistant binding sites (and to only a subset of these) accounting for about 15–20% of total band 4.1 binding sites on membranes. We found that hydrolysis of > 95% of PtdIns(4,5)P2 with exogenous phospholipase C‐δ (PLCδ) resulted in no further decrease in band 4.1 binding to vesicles than did hydrolysis of 65–70% of PtdIns(4,5)P2 which is accessible to erythrocyte phosphoinositol phospholipase C. This suggests that only 65–70% of total membrane PtdIns(4,5)P2 is involved in regulating band 4.1 binding. Significantly, the pool of PtdIns(4,5)P2 involved is the same pool which can be hydrolysed by erythrocyte phosphoinositol phospholipase C, and which has been shown to be metabolically labile in erythrocytes. The membrane binding capacity for band 4.1 found in this study (averaging 1000 μg/mg vesicle protein) is considerably higher than that found in previous studies. The results are consistent with the existence of a binding site for band 4.1 on each copy of the major transmembrane proteins (band 3 and the glycophorins). These results provide new insights into the involvement of membrane inositol phospholipids in cytoskeletal–membrane interactions.
Unidirectional arginine transport in reconstituted plasma‐membrane vesicles from yeast overexpressing CAN1
Authors: OPEKAROVÁ Miroslava , CASPARI Thomas , TANNER Widmar
Published: March 2005

Amino acids are accumulated in Saccharomyces cerevisiae by strictly unidirectional influx systems. To see whether cellular compartmentation causes this unusual amino‐acid‐transport behaviour, arginine transport was studied in plasma‐membrane vesicles. The arginine permease gene CANI was overexpressed in S. cerevisiae RH218a and in a permease‐deficient mutant RS453 (canl). Reconstituted piasma‐membrane vesicles from these transformants, energized by incorporated cytochrome‐c oxidase, showed 3–4‐fold increased rates of arginine uptake compared to vesicles from wild‐type cells. The KT values were 32.5 μM in vesicles from wild‐type and 28.6 μM in vesicles from transformed cells; the corresponding in vivo values were 17.5 μM and 11.4 μM, respectively. It could be demonstrated that unidirectional arginine transport and accumulation also exist in vesicles; thus, unidirectional influx is not related to cellular compartmentation.
Interaction of myelin basic protein with artificial membranes
Authors: ter BEEST Martin B. A. , HOEKSTRA Dick
Published: March 2005

The interaction of myelin basic protein (MBP) with large unilamellar vesicles, composed of phosphatidylserine (PtdSer), phosphatidylserine/phosphatidylcholine (PtdSer/Ole2GroPCho) and phosphatidylcholine/cholesterol (Ole2GroPCho/cholesterol) was examined. Binding of MBP to the bilayers as well as the kinetics of this process were determined by a resonance energy transfer procedure. The ability of the protein to aggregate the vesicles subsequently was monitored continuously by absorbance measurements. The interaction was further characterized by determining the ability of MBP to induce membrane perturbations, as reflected by release of aqueous vesicle contents, and lipid mixing.

The results demonstrate that Ole2GroPCho inhibits, while PtdSer and cholesterol strongly facilitate MBP‐induced membrane aggregation. Furthermore, binding of MBP to vesicles and the subsequent aggregation event are separate processes, i. e. the extent of binding does not necessarily retlect the aggregation susceptibility. Overall, aggregation appears to the rate‐limiting step. Interaction of MBP with PtdSer bilayers results in a limited degree of lipid mixing, which is accompanied by extensive release of vesicle contents. For all other compositions, no lipid mixing occurs, while cholesterol effectively prevents release of vesicle contents. pH‐dependent experiments indicate distinct mechanisms to be operative in MBP‐induced aggregation of PtdSer and Ole2GroPCho/cholesterol bilayers. At neutral pH, protein‐protein interactions appear relevant, while at acidic pH intervesicular bridges, established by monomers that may cause aggregation of PtdSer vesicles, but not of Ole2GroPCho/cholesterol vesicles. The observation that divalent cations reverse MBP‐induced vesicle aggregation may have physiological relevance.
Overexpression of trypanosomal triosephosphate isomerase in Escherichia coli and characterisation of a dimer‐interface mutant
Authors: BORCHERT Torben V. , PRATT Kathryn , ZEELEN Johan Ph. , CALLENS Mia , NOBLE Martin E. M. , OPPERDOES Fred R. , MICHELS Paul A. M. , WIERENGA Rik K.
Published: March 2005

In this paper, the successful expression of trypanosomal triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Escherichia coli, using a T7‐polymerase‐based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure.

A TIM mutant was made in which a unique dimer‐interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could be expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild‐type TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild‐type trypanosomal TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution‐dependent inactivation is reversile. His47 is involved in a water‐mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water‐mediated hydrogen bond is important for the stability of the TIM dimer.
Specificity of the binding domain of glucoamylase 1
Authors: BELSHAW Nigel J. , WILLIAMSON Gary
Published: March 2005

Glucoamylase 1 from Aspergillus niger hydrolyses granular starch at an increased rate due to the presence of a C‐terminal starch‐binding domain. This domain was isolated and shown to bind to the malto‐oligosaccharides Glc2 to Glc11 with a stoichiometry of 1 mol ligand/mol protein. The affinity for these ligands increased with increasing degree of polymerisation until Glc9, above which no further increase was observed. We suggest that this indicates that for maximum affinity the substrate should be able to form a helical conformation, which mimics the conformation of amylose in granular starch. We propose a model of how the complex between the malto‐oligosaccharides and the binding domain is formed and indicate how this affects the differences in binding modes for soluble and insoluble substrates.

Glucono‐1,5‐lactone interacts with the binding domain at a different site to the malto‐oligosaccharides allowing the formation of a ternary complex between the binding domain, a malto‐oligosaccharide and glucono‐1,5‐lactone. The binding domain also binds to linear α‐1,6‐linked glucose digosaccharides (dextran), but with much lower affinity than for α‐1,4‐linked glucose. This ligand appears to interact with the binding domain at both binding sites, i. e. at the site to which the malto‐oligosaccharides bind and also at the site to which glucono‐1,5‐lactone binds. The relevance of the results to the mechanism of action of other polysaccharide‐hydrolysing enzymes containing both a catalytic and a binding domain is discussed.
Localization of the tightly bound divalent‐cation‐dependent and nucleotide‐dependent conformation changes in G‐actin using limited proteolytic digestion
Authors: STRZELECKA‐GOŁASZEWSKA Hanna , MORACZEWSKA Joanna , KHAITLINA Sofia Y. , MOSSAKOWSKA Malgorzata
Published: March 2005

Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca2+ by Mg2+ in ATP‐actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C‐terminal segment. Conversion of ATP‐actin to ADP‐actin to (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39–51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39–51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.
Involvement of the Arg179 in the active site of human IL‐6
Authors: FONTAINE Véronique , SAVINO Rocco , ARCONE Rosaria , De WIT Lucas , BRAKENHOFF Just P. J. , CONTENT Jean , CILIBERTO Gennaro
Published: March 2005

Three internal‐amino acid deletions of amino acids 171–179 of human interleukin 6 (IL‐6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation‐specific anti‐(IL‐6) monoclonal antibodies showed that only mutant 177–179 does not present major alterations in folding. This finding, together with the observation that 177–179 is not able to compete with IL‐6 for binding to the soluble human IL‐6 receptor, suggested that some or all of these three residues participate to the composition of the receptor‐binding site of human IL‐6. A large number of single‐amino‐acid‐substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg 179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive change in position 179 for the interaction of human IL‐6 with its receptor.