bluuebunny/crossref_metadata_2025 huggingface dataset:
The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution
Authors: ARROWSMITH Cheryl , PACHTER Ruth , ALTMAN Russ , JARDETZKY Oleg
Published: March 2005

We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25‐kDa dimer), with and without the co‐repressor l‐tryptophan, from NMR data. This is the largest protein structure thus far determinéd by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van‐der‐Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3–10.

Sequence‐specific 1H‐NMR assignments and the secondary structure of the holopressor (l‐tryptophan‐bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332–6341]. Those for the trp aporepressor (l‐tryptophan‐free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium‐range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long‐range NOE data. The two structures are quite similar in overall topology to one another as well as to published crystal structures; however, the DNA binding region appears to be more disordered in solution than in the crystal structures. We propose that the greater flexibility of the DNA binding region observed by us as well as by others [C. L. Lawson, R.‐G. Zhang, R. W. Schevitz, Z. Otwinowski, A. Joachimiak & P. B. Sigler (1988) Proteins 3, 18] plays an important role in the mechanism of DNA binding.
Purification and biochemical characterization of dog gastric lipase
Authors: CARRIÈRE Frédéric , MOREAU Hervé , RAPHEL Véronique , LAUGIER René , BENICOURT Claude , JUNIEN Jean‐Louis , VERGER Robert
Published: March 2005

A lipase was found to be present in dog stomach which appeared to be more abundant in the fundic than in the pyloric mucosa. Dog gastric lipase was extracted by soaking the gastric tissue and further purified after cation exchange, anion exchange and gel‐filtration using fast protein liquid chromatography. The amino‐acid composition, N‐terminal amino‐acid sequence, substrate specificity, interfacial and kinetic behavior and inactivation by sulfhydryl reagents were determined and compared with those of human and rabbit gastric lipases. We report for the first time that a gastric lipase is 13 times more active on long‐chain than on short‐chain triacylglycerols at pH 4.0, reaching a maximal specific activity of 950 U/mg on Intralipide emulsion.
Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii
Authors: LEYLAND Mark L. , KELLY David J.
Published: March 2005

An isocitrate dehydrogenase able to function with either NADP or NAD as coenzyme was purified to homogeneity from cell‐free extracts of the purple photosynthetic eubacterium Rhodomicrobium vannielii using a rapid two‐step procedure involving dye‐ligand affinity chromatography. The enzyme was obtained in 60% yield with specific activities of 23 U · mg protein−1 (NADP‐linked reaction) and 18.5 U · mg protein−1 (NAD‐linked reaction). The purified enzyme was monomeric and migrated with an approximate Mr of 75000–80000 on both SDS/PAGE and non‐denaturing PAGE. Affinity constants (Km values) of 2.5 μM for NADP and 0.77 mM for NAD and values for Kcat/Km of 981200 min−1· mM−1 (NADP) and 2455 min−1· mM−1 (NAD) indicated a greater specificity for NADP compared to NAD. A number of metabolites were examined for possible differential regulatory effects on the NADP‐ and NAD‐linked reactions, using a dual‐wavelength assay. Oxaloacetate was found to be an effective inhibitor of both reactions and the enzyme was also sensitive to concerted inhibition by glyoxylate and oxaloacetate. The amino‐acid composition and the identity of 39 residues at the N‐terminus were determined and compared to other isocitrate dehydrogenases. The results suggested a relationship between the Rm. vannielii enzyme and the monomeric isocitrate dehydrogenase isoenzyme II from Vibrio ABE‐1.
Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G
Authors: BRINKMANN Thomas , SCHNIERER Susanne , TSCHESCHE Harald
Published: March 2005

The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor.

Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P′2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity.

Recombinant aprotinin variants which act specifically against chymotrypsin‐like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10−8 M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.
Isolation and characterization of a new variant of surfactin, the [Val7]surfactin
Authors: PEYPOUX Françoise , BONMATIN Jean‐Marc , LABBÉ Henri , DAS Bhupesh C. , PTAK Marius , MICHEL Georges
Published: March 2005

Reinvestigation of surfactin, a previously studied peptidolipid surfactant from Bacillus subtilis, by fast‐atom‐bombardment mass spectrometry and 1H‐NMR spectroscopy, as well as by chemical methods, revealed the presence of a closely related second constituent. This new compound, [Val7]surfactin, differs from the known surfactin by the C‐terminal amino acid residue which is valine instead of leucine.
Tracking of proton flow during transition from anaerobiosis to steady state
Authors: LUVISETTO Siro , SCHMEHL Ibolya , COLA Carmen , AZZONE Giovanni Felice
Published: March 2005

1. The kinetics of acidification and realkalinization of the matrix after addition of nigericin to respiring and non‐respiring mitochondria, recorded by intramitochondrial pH indicators such as neutral red and 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF), is complementary to that recorded by extramitochondrial pH indicators. The extent of acidification decreases with the logarithm of the KCl concentration and is inhibited by Pi and ammonium ions.

2. Proton translocation during respiration has been compared with proton extraction from matrix bulk water. During oxygen pulses to EGTA‐untreated mitochondria, BCECF records an extraction of protons from matrix bulk water of about 2–3 nmol H+/mg, reduced to 1–2 nmol H+/mg in EGTA‐treated mitochondria. Since the amount of proton translocation required to achieve steady state is of the order of 6–7 nmol H+/mg, it appears that 75–90% of the protons are not extracted from matrix bulk water. Only a slight response is recorded by neutral red.

3. The effect of permeant cations and of uncouplers on the distribution of proton extraction between membrane and matrix bulk water has been studied in presteady state. During Sr2+ uptake, proton extrusion into cytosolic bulk water, as well as proton extraction from matrix bulk water, corresponds almost to 100% of the protons translocated by the redox proton pumps. In the absence of Sr2+, parallel to the disappearance of the proton extrusion in cytosolic bulk water, the proton extraction from matrix bulk water diminishes to about 20% of the proton translocation.

4. The mechanism by which divalent cation uptake and protonophoric uncouplers affect the distribution of proton extraction between matrix bulk water and membrane domains and the nature of the membrane domains are discussed.
The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense
Authors: VANONI Maria A. , NUZZI Lucia , RESCIGNO Maria , ZANETTI Giuliana , CURTI Bruno
Published: March 2005

The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady‐state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both l‐glutamine‐dependent and ammonia‐dependent reactions of the glutamate synthase from A. brasilense follow an identical two‐site uni‐uni bi‐bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2‐oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron‐sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both l‐glutamine‐dependent and ammonia‐dependent reactions, this hydrogen atom equilibrates with the solvent.

The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia‐dependent reaction of other glutamate synthases. The ammonia‐dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological l‐glutamine‐dependent activity and requiring the enzyme flavins and iron‐sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501–3508] that the small subunit of glutamate synthase is endowed with a glutamate‐dehydrogenase‐like activity.
Sulfur‐containing cyclic ketimines and imino acids
Authors: CAVALLINI Doriano , RICCI Giorgio , DUPRÈ Silvestro , PECCI Laura , COSTA Mara , MATARESE Rosa M. , PENSA Bernardo , ANTONUCCI Antonio , SOLINAS Sandro P. , FONTANA Mario
Published: March 2005

Aminoethylcysteine, lanthionine, cystathionine and cystine are mono‐deaminated either by l‐amino‐acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high‐affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur‐containing cyclic products provided with a possible neurochemical function to be investigated further.
Cloning, sequence and transcriptional analysis of the structural gene for LPD‐3, the third lipoamide dehydrogenase of Pseudomonas putida
Authors: PALMER Judith A. , MADHUSUDHAN Kunapuli T. , HATTER Kenneth , SOKATCH John R.
Published: March 2005

The third lipoamide dehydrogenase structural gene of Pseudomonas putida, lpd3, was isolated from a library of P. putida PpG2 DNA cloned in Escherichia coli TB1. The nucleotide sequence of lpd3 and its flanking regions indicate that lpd3 is not part of an operon, which is unique for a prokaryotic lipoamide dehydrogenase. An open reading frame was found 207 bases upstream from the start of transcription, but is encoded on the strand opposite lpd3. There is no evidence of an open reading frame immediately downstream from lpd3. The coding region of lpd3 consists of 1401 bp, providing for 466 amino acids plus a stop codon with a G/C content of 62.4%. The transcriptional start site was located 33‐bp upstream from the start of translation. The third lipoamide dehydrogenase (LPD‐3) shares amino acid identity with the other two lipoamide dehydrogenases of P. putida, 45% with that of the 2‐oxoglutarate dehydrogenase and pyruvate multienzyme complexes, and 45.9% with the lipoamide dehydrogenase of the branched‐chain oxoacid complex. LPD‐3 is more closely related to eukaryotic lipoamide dehydrogenases since it has 53.6% amino acid sequence identity with pig and human lipoamide dehydrogenases and 51.1% identity with yeast lipoamide dehydrogenase. LPD‐3 was not produced in wild‐type P. putida PpG2 under a variety of growth conditions. However, LPD‐3 was produced in P. putida PpG2 carrying pSP14, a pKT240‐based clone with the entire lpd3 gene plus 104 bases of the leader. The only demonstrated role of LPD‐3 in P. putida is a substitute for lipoamide dehydrogenase of the 2‐oxoglutarate dehydrogenase and pyruvate multienzyme complexes when the latter is inactive or missing.
Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli
Authors: NIKKILA Heli , GENNIS Robert B. , SLIGAR Stephen G.
Published: March 2005

The gene for the soluble cytochrome b562 from Escherichia coli B has been cloned on a SalI fragment. The analysis of the gene reveals the presence of a leader sequence in front of the sequence encoding the mature protein. Expression of cytochrome b562 using the lac‐promoter produced the protein to a level of 3–5% of total protein. This over‐production enables employment of a simple, high‐yield purification protocol to obtain homogeneous cytochrome b562. Spectroscopic and N‐terminal sequence analyses of the purified protein demonstrate that it is identical to the chromosomally expressed cytochrome b562 purified and characterized from E. coli B [Itagaki, E. & Hager, L. P. (1966) J. Biol. Chem. 241, 3687–3695]. It is demonstrated that the genomic sequence codes for a classic N‐terminal signal sequence and that mature cytochrome b562 is translocated to the periplasmic space.
1H NMR studies of echistatin in solution
Authors: DALVIT Claudio , WIDMER Hans , BOVERMANN Günter , BRECKENRIDGE Robin , METTERNICH Rainer
Published: March 2005

Two‐dimensional 1H‐NMR methods have been used to obtain complete proton resonance assignments for the 49‐residue protein echistatin from the viper Echis carinatus. The protein in solution contains only a small amount of regular secondary structure with four very short β‐strands. These β‐strands form two short segments of antiparallel β‐sheet, as evidenced by the observed cross‐strand NOE. The first two strands are connected with a tight reverse turn, whereas the remaining two strands are linked together by an 11‐residue loop forming a so‐called hairpin. The tripeptide unit Arg‐Gly‐Asp, responsible for the binding of echistatin to the fibrinogen receptor glycoprotein GPIIb/IIIa, is located at the tip of this very hydrophilic loop.
Nuclear magnetic resonance studies of the snake toxin echistatin
Authors: COOKE Robert M. , CARTER Brian G. , MARTIN David M. A. , MURRAY‐RUST Peter , WEIR Malcolm P.
Published: March 2005

The 1H‐NMR spectrum of the snake toxin echistatin has been assigned using homonuclear two‐dimensional methods. Consideration of the NOE patterns, coupling constants and putative hydrogen bonds enabled two regular features of secondary structure to be deduced: a β‐sheet/turn between residues 8 and 13 and a small antiparallel β‐sheet and bulge linking residues 16–20 with residues 30–33. The recognition region of the protein containing the residues RGD lies in a loop joining the two strands of the β‐sheet. The β‐bulge and the loop containing the RGD sequence undergo pH‐dependent conformational interconversion, modulated by the side chain of Asp29.
NMR study of the structural characteristics of variants of yeast iso‐1‐cytochrome c in which unvaried aromatic residues have been substituted
Authors: THURGOOD Andrew G. P. , DAVIES Anne M. , GREENWOOD Colin , MAUK A. Grant , SMITH Michael , GUILLEMETTE J. Guy , MOORE Geoffrey R.
Published: March 2005

The structures of variants of yeast iso‐1‐cytochrome c, in which the previously unchanged Tyr48 and Tyr48 + Trp59 have been replaced by Phe, have been characterised by NMR. The NMR data indicated that the structures of the variant cytochromes c are very similar to the wild‐type protein. In particular, the heme environment and interactions of the heme macrocycle were shown to be preserved. The observation of chemical shift differences have allowed for the assessment of conformational changes. The substitution of Trp59 by Phe may have caused a small conformational change, a manifestation of which is the observed chemical shift differences at His39, Val57 and Tyr74.

The structural basis for the reduction in redox potential accompanying the amino acid substitutions is discussed and the proposal made that the changes in potential are a direct consequence of the side chain properties and do not result primarily from conformational changes.
Structural and functional relationships in two families of β‐1,4‐glycanases
Authors: GILKES Neil R. , CLAEYSSENS Marc , AEBERSOLD Ruedi , HENRISSAT Bernard , MEINKE Andreas , MORRISON Hamish D. , KILBURN Douglas G. , WARREN R. Antony J. , MILLER Robert C.
Published: March 2005

CenA and Cex are β‐1,4‐glycanases produced by the cellulolytic bacterium Cellulomonas fimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulosebinding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C. fimi‐type cellulose‐binding domains. CenA and Cex belong to families B and F, respectively, in the classification of β‐1,4‐glucanases and β‐1,4‐xylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active‐site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM‐9 β‐1,4‐glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kcat/Km) for hydrolysis of the heterosidic bond of p‐nitrophenyl‐β‐d‐xylobioside is 14385 min−1· mM−1 at 25°C; the corresponding kcat/Km for p‐nitrophenyl‐β‐d‐cellobioside hydrolysis is 296 min−1· mM−1.
Simulation of the electrochemical behavior of multi‐redox systems
Authors: MORENO Cristina , CAMPOS António , TEIXEIRA Miguel , LeGALL Jean , MONTENEGRO Maria Irene , MOURA Isabel , van DIJK Cees , MOURA José G. J.
Published: March 2005

The direct unmediated electrochemical response of the tetrahemic cytochrome c3 isolated from sulfate reducers Desulfovibrio baculatus (DSM 1743) and D. vulgaris (strain Hildenborough), was evaluated using different electrode systems [graphite (edge cut), gold, semiconductor (InO2) and mercury)] and different electrochemical methods (cyclic voltammetry and differential pulse voltammetry). A computer program was developed for the theoretical simulation of a complete cyclic voltammetry curve, based on the method proposed by Nicholson and Shain [Nicholson, R. S. & Shain, I. (1964) Anal. Chem. 36, 706–723], using the Gauss‐Legendre method for calculation of the integral equations. The experimental data obtained for this multi‐redox center protein was deconvoluted in to the four redox components using theoretically generated cyclic voltammetry curves and the four mid‐point reduction potentials determined. The pH dependence of the four reduction potentials was evaluated using the deconvolution method described.
Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)
Authors: RANSAC Stéphane , GARGOURI Youssef , MOREAU Hervé , VERGER Robert
Published: March 2005

We studied the covalent inhibition of lipases by the monolayer technique. We report the inactivation of porcine pancreatic and human and rabbit gastric lipases, acting on mixed monomolecular films of dicaprin containing tetrahydrolipstatin or new hydrophobic disulfide compounds, which can be described as a ‘poisoned‐interface’ system.

A kinetic model is presented for depicting the covalent inactivation of lipolytic enzymes at a lipid/water interface. The stoichiometry of the interfacial situation can be described as follows: one lipase molecule embedded among 105 substrate molecules will be inactivated to half its initial velocity by the presence of 10 tetrahydrolipstatin molecules. This inactivation was independent of the surface pressure.

When tested in the form of mixed films, all the disulfide compounds investigated specifically reduced the hydrolysis of 1,2‐didecanoyl‐sn‐glycerol films by gastric lipases, but did not affect hydrolysis by pancreatic lipase. With this poisoned‐interface system, tetrahydrolipstatin was found to be the most potent inactivator, whereas disulfide compounds showed a higher degree of selectivity than tetrahydrolipstatin.
The distribution of permeant ions demonstrates the presence of at least two distinct electrical gradients in bloodstream forms of Trypanosoma brucei
Authors: NOLAN Derek P. , VOORHEIS H. Paul
Published: March 2005

The distribution of 86Rb+ and the radiolabelled lipophilic cation [3H]methyltriphenylphosphonium (MePh3P+) was used to investigate the membrane potentials that exist in bloodstream forms of Trypanosoma brucei. Even after correction for binding to cellular constituents, the accumulation of MePh3P+ was approximately tenfold greater than the accumulation of Rb+ under resting conditions. The addition of low concentrations of carbonylcyanide p‐trifluoromethoxyphenylhydrazone or valinomycin reduced the accumulation of MePh3P+ tenfold without perturbing the accumulation of Rb+. Although selective permeabilization of the plasma membrane abolished the accumulation of Rb+ and caused a substantial decrease in the accumulation of MePh3P+, a significant carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone‐sensitive accumulation of MePh3P+ persisted under these conditions. These data were consistent with the presence of at least two distinct membrane potentials (Ψ) in bloodstream forms of T. brucei; a potential across the plasma membrane (Ψp) and an additional Ψ, generated by the electrogenic movement of H+, across the membrane of an intracellular organelle that possesses no electrical permeability to Rb+ or K+.
The solution structure of a monomeric insulin
Authors: KNEGTEL Ronald M. A. , BOELENS Rolf , GANADU Maria Luisa , KAPTEIN Robert
Published: March 2005

The solution conformation of des‐(B26–B30)‐insulin (DPI) has been investigated by 1H‐NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two‐dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance‐bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5‐ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time‐averaged RMD simulation. An average structure was obtained from a trajectory generated during 20‐ps RMD. The final structure was compared with the des‐(B26–B30)‐insulin crystal structure refined by molecular dynamics and the 2‐Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des‐(B26–B30)‐insulin is retained in solution with respect to the crystal structures with a high flexibility at the N‐terminal part of the A chain and at the N‐terminal and C‐terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des‐(B26–B30)‐insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.
Mechanism of action of deoxyribonuclease II from human lymphoblasts
Authors: HAROSH Itzik , BINNINGER David M. , HARRIS Paul V. , MEZZINA Mauro , BOYD James B.
Published: March 2005

Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE‐cellulose and heparin – agarose chromatography followed by fractionation on Mono‐S, Mono‐Q and Superose‐12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single‐strand nicks relative to double‐strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3′ termini to hydrolysis by this enzyme.

Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 μM Fe3+. Likewise we confirmed the presence of 3′‐phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzid by Escherichia coli DNA polymerase I.
Geotrichum candidum produces several lipases with markedly different substrate specificities
Authors: SIDEBOTTOM Christopher M. , CHARTON Emmanuelle , DUNN Paul P. J. , MYCOCK Gary , DAVIES Christine , SUTTON Jane L. , MACRAE Alasdair R. , SLABAS Antoni R.
Published: March 2005

We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and II (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pI. Thus, lipases I and II have native molecular masses of 50.1 kDa and 55.5 kDa, and pI of 4.61 and 4.47, respectively.

Lipases A and B are very similar to lipases I and II with native molecular masses of 53.7 kDa and 48.9 kDa, and pI of 4.71 and 4.50, respectively. Treatment with endo‐β‐N‐acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.
Notechis 11′2, a non‐toxic phospholipase A2 from the venom of Notechis scutatus scutatus
Authors: BOUCHIER Christiane , BOYOT Philippe , TESSON Frédérique , TRÉMEAU Odile , BOUET Françoise , HODGSON Darren , BOULAIN Jean‐Claude , MÉNEZ André
Published: March 2005

Previously, we deduced the amino acid sequence of a novel phospholipase‐A2‐like protein (PLA2) from the nucleotide sequence of a cDNA isolated from a library prepared from the venom gland of the Australian elapid Notechis scutatus scutatus. The corresponding protein has now been identified, purified from the venom and named Notechis 11′2. Its complete amino acid sequence has been determined by automated Edman degradation of both the whole protein and peptides generated by Staphylococcus aureus protease digestion and chemical cleavage at a tryptophan residue. As predicted from its sequence which contains all the residues putatively required for PLA2 activity, Notechis 11′2 exhibits an esterase activity, preferentially against neutral phospholipids. However, despite its sequence homology with other highly toxic PLA2 present in the venom of Notechis scutatus scutatus, notechis 11′2 has no lethal activity. This observation further supports the view that the lethal activity of PLA2 from Notechis scutatus scutatus is not due to the esterasic activity only.
Direct electrochemistry of proteins
Authors: BURROWS Alison L. , GUO Liang‐Hong , HILL H. Allen O. , McLENDON George , SHERMAN Fred
Published: March 2005

Direct electrochemistry of site‐specific mutants of yeast iso‐1‐cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge‐plane pyrolytic graphite (EPG) and bis(4‐pyridyl)‐disulphide‐modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a varietyof electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile–Cys102Thr > Cys102Thr > Asn52Ala–Cys102Thr. Mid‐point potentials follow the order: Arg13Ile (+ 60 ± 5 mV vs. standard calomel electrode) > Cys102Thr (+ 40 ± 5 mV) > Lys27Gln (+ 30 ± 5 mV) > Lys72Asp (+ 30 ± 5 mV) > Asn52Ala–Cys102Thr (+ 15 ± 5 mV) > Asn52Ile ‐Cys102Thr (‐10 ± 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical respose are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single‐site variation could modify or abolish direct electrochemistry in the ternary adduct.
Kinetic characterization of the normal and procaine‐perturbed reaction cycles of the sarcoplasmic reticulum calcium pump
Authors: HENAO Fernando , de FORESTA Béatrice , ORLOWSKI Stéphane , CUENDA Ana , GUTIÉRREZ‐MERINO Carlos , CHAMPEIL Philippe
Published: March 2005

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport‐coupled Ca2+‐ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half‐maximal inhibition occurred with procaine concentrations close to 15–20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast‐kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium‐induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca2+‐dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast‐filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.
The medium chains of the mammalian clathrin‐associated proteins have a homolog in yeast
Authors: NAKAYAMA Yasuhiro , GOEBL Mark , O'BRINE GRECO Betsy , LEMMON Sandy , PINGCHANG CHOW Elizabeth , KIRCHHAUSEN Tomas
Published: March 2005

We have cloned and sequenced mouse brain AP47, the medium chain of the trans‐Golgi network clathrin‐associated protein complex AP‐1. The predicted protein sequence of AP47 is closely related to rat and calf brain AP50, the corresponding medium chain of the plasma‐membrane clathrin‐associated protein complex AP‐2. We have also identified in the yeast genome an open reading frame encoding a protein of previously unknown function. Referred to here as YAP54, its predicted protein sequence displays a striking homology to AP47. We therefore propose that Yap54 is the medium chain subunit of a putative AP‐1 complex in yeast. From the analyses of the optimized sequence alignments of AP47, AP50 and Yap54p, we suggest a model for the domain organization of the medium chains.
Expression and post‐transcriptional regulation of ornithine decarboxylase during early Xenopus development
Authors: OSBORNE H. Beverley , DUVAL Catherine , GHODA Lucy , OMILLI Francis , BASSEZ Therése , COFFINO Philip
Published: March 2005

In this paper we show that large changes in ornithine decarboxylase (ODC) activity accured during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid‐blastula transition (MBT). ODC activity was stable in cycloheximide‐treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post‐MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC‐deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post‐translationally by polyamines, indicating that the post‐MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine‐mediated regulation.
Molecular and cellular aspects of immunologic tolerance
Authors: NOSSAL Gustav J. V.
Published: March 2005

This review seeks to explain the most exciting recent data concerning the nature of self/non‐self discrimination by the immune system in a manner accessible to a biochemical readership. The nature of recognition in the two great lymphocyte families, B cells and T cells, is described with special emphasis on the nature of the ligands recognized by each. The history of the field of immunologic tolerance is surveyed, as are the key experiments on conventional mice which provided a conceptual framework. This suggested that tolerance was essentially due to ‘holes’ in the recognition repertoires of both the T and B cell populations so that lymphocytes competent to react to self antigens were not part of the immunologic dictionary. There were essentially two ways to achieve this situation. On the one hand, self antigens might ‘catch’ developing lymphocytes early in their ontogeny and delete the cell, a process of clonal abortion. On the other hand, self antigens might signal lymphocytes (particularly immature cells) in a negative manner, reducing or abolishing their capacity for later responses, without causing death. This process is referred to as clonal anergy. Evidence for both processes exists.

Special emphasis is placed on a wave of experimentation beginning in 1988 which imaginatively uses transgenic mouse technology to study tolerance. Transgenic manipulations can produce mice which synthesize foreign antigens in a constitutive and/or inducible manner, sometimes only in specific locations; mice which possess T or B lymphocytes almost all expressing a given receptor of known specificity; and mice which are an immunologic time bomb in that the antigen is present and so too are lymphocytes all endowed with receptors for that antigen. These experiments have vindicated the possibility of both clonal abortion and clonal anergy in both T and B cell populations, the choice of which phenomenon occurs depending on a number of operational circumstance. For T cell tolerance, clonal abortion occurs if the self antigenic determinant concerned is present within the thymus; if not, clonal anergy is more likely. For B cell tolerance, the strength of the negative signal and therefore the choice between abortion and anergy depends on the molar concentration of the self antigen, the capacity for multivalent presentation to a B cell, and the affinity of the B cell's receptor for the antigen in question. Some B cells with low affinity for self antigens certainly escape censorship and remain capable of secreting low affinity anti‐self antibodies, which however do no harm. Mechanisms which prevent the emergence of hypermutated, high‐affinity anti‐self B cells are discussed.
Analysis of excitation‐contraction‐coupling components in chronically stimulated canine skeletal muscle
Authors: OHLENDIECK Kay , BRIGGS F. Norman , LEE K. Francis , WECHSLER Andrew W. , CAMPBELL Kevin P.
Published: March 2005

The chronic stimulation of predominantly fast‐twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow‐twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation‐contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains‐all. Consistent with previous experiments, a greatly reduced density was observed for the fast‐twitch isozyme of Ca2+‐ATPase, while the expression of the slow‐twitch Ca2+‐ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K+‐ATPase, dystrophin‐glycoprotein complex) and the free sarcoplasmic reticulum (Ca2+‐binding protein sarcalumenin and a 53‐k Da glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca2+‐release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine‐sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum protiens of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation‐contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.
A simple procedure for isolation of eukaryotic mRNA polyadenylation factors
Authors: ÅSTRÖM Anders , ÅSTRÖM Jonas , VIRTANEN Anders
Published: March 2005

We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeble and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.
Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA
Authors: MASUDA Naoko , KITAMURA Naomi , SAITO Kunihiko
Published: March 2005

Phospholipase B has not yet been well defined. The most important points about this enzyme are its relationships with lysophospholipase and phospholipase A1. As reported [Saito, K., Sugatani, J. & Okumura, T. (1991) Methods Enzymol. 197, 446–456], Penicillium notatum phospholipase B is a glycoprotein with a molecular mass of 95 kDa and intrinsic lysophospholipase and phospholipase B activities; however, by endogenous proteolytic modification, its phospholipase B activity is lost almost completely, whereas its lysophospholipase activity remains unchanged.

A cDNA library of P. notatum was screened by hybridization with two synthetic oligodeoxyribonucleotide probes, which corresponds to two different pentapeptides of the enzyme. A hybridization‐positive clone, pPLB18, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence was quite different from that found previously. Therefore, we rescreened the cDNA library with a Sau3AI fragment derived from pPLB18 and isolated a new clone, pPLB15. Comparison of the nucleotide sequences of pPLB15 and pPLB18 revealed that pPLB18 contained an insertion sequence of 53 bp. Consequently, the reading frame was open downstream for 603 amino acid residues.

From the assigned sequence, it was deduced that the limited proteolysis occurred between Leu175 and Asp176; eight cysteine residues and 16 potential N‐glycosylation sites were also found. No amino acid sequence similarity was found with other proteins, including other phospholipases.
Structural studies on sulfated oligosaccharides derived from the carbohydrate‐protein linkage region of chondroitin sulfate proteoglycans of whale cartilage
Authors: SUGAHARA Kazuyuki , MASUDA Masao , HARADA Tadashi , YAMASHINA Ikuo , de WAARD Pieter , VLIEGENTHART Johannes F. G.
Published: March 2005

From the carbohydrate‐protein linkage region of whale cartilage proteoglycans, which bear predominantly chondroitin 4‐sulfate, one nonsulfated, two monosulfated and one disulfated hexasaccharide alditols were isolated after exhaustive digestions with Actinase E and chondrotinase ABC, and subsequent β‐elimination. Their structures were analyzed by chondroitinase ACII digestion in conjunction with HPLC and by 500‐MHz 1H‐NMR spectroscopy. The nonsulfated compound (A) had the following conventional structure: Δ GlcA(β1–3)‐GalNAc(β1–4)GlcA(β1–3)Gal(β1–3)Gal(β1–4)Xylol, where GlcA, ΔGlcA and GalNAc are glucuronic acid; 4,5‐unsaturated glucuronic acid and 2‐deoxy‐2‐N‐acetylamino‐d‐galactose, respectively. The other compounds were sulfated derivatives of compound A. Two monosulfated compounds (B and C) had an ester sulfate on C4 or C6 of the GalNAc residue, respectively and the disulfated compound (D) had two ester sulfate groups, namely, one on C4 of the GalNAc and the other on C4 of the Gal residue substituted by GlcA. The molar ratio of A/B/C/D was 0.21:0.16:0.36:0.27. The compound containing Gal‐4‐O‐sulfate was previously isolated by us in the form of a sulfated glycosreine [Δ GlcA(β1–3)GalNAc(4‐O‐sufate)(β1–4)GlcA(β1–3)Gal((4‐O‐sulfate)(β1–3)‐Gal(β–4)Xy1β1‐O‐Ser] from the carbohydrate‐protein linkage region of rat chondrosarcoma chondroitin‐4‐sulfate proteoglycans [Sugahara, K., Yamashina, I., DeWaard, P., Van Halbeek, H. & Vliegenthart, J. F. G. (1988) J. Biol. Chem. 263, 10168–10174]. The discovery of this structure in the carbohydrate‐protein linkage region of chondroitin 4‐sulfate proteoglycans from nontumorous cartilage indicates that it is not a tumor‐associated product but rather a physiological biosynthetic product since it represents a significant proportion. The biological significance of this structure is discussed in relation to glycosaminoglycan biosynthesis.