bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen‐activated protein (MAP) kinase and MAP‐kinase‐activated kinases 1 and 2
Authors: SUTHERLAND Calum , ALTERIO Janine , CAMPBELL David G. , LE BOURDELLÈS Béatrice , MALLET Jacques , HAAVIK Jan , COHEN Philip
Published: March 2005

Mitogen‐activated protein‐kinase (MAP) kinase‐activated protein kinases 1 and 2 (MAPKAP kinase‐1, MAPKAP kinase‐2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in vitro at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases‐1 and ‐2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase‐1) and Ser19 and Ser40 (MAPKAP kinase‐2). In contrast to calmodulin‐dependent protein kinase‐II, which phosphorylates Ser19 faster than Ser40, MAPKAP kinase‐2 phosphorylated Ser40 about twice as fast as Ser19. The maximal activation of tyrosine hydroxylase by MAPKAP kinase‐1 or‐2 was about 3‐fold, and activation by MAPKAP kinases‐1 and ‐2 or calmodulin‐dependent protein kinase‐II correlated with the extent of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase, but at markedly different rates (3=4 > 1 » 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase‐1, MAPKAP kinase‐2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.
Putative dehydrogenase tms1 suppresses growth arrest induced by a p53 tumour mutant in fission yeast
Authors: WAGNER Peter , GRIMALDI Margaret , JENKINS John R.
Published: March 2005

Expression of a human tumour‐derived p53 His 273 cDNA induced growth arrest in fission yeast Schizosaccharomyces pombe. Based on the p53‐induced growth arrest, we cloned an extragenic suppressor, termed tms1, by complementation. The open reading frame of the tms1 gene corresponded to a protein of 347 amino acids with a calculated mass of 37 380 Da. The transcriptional start site of the tms1 gene was mapped and, in addition, the corresponding cDNA was isolated and expressed in Escherichia coli. Recombinant tms1 protein served as an antigen to produce specific polyclonal antibodies to aid identification of the tms1‐gene‐product in total yeast lysates. Comparison of the deduced amino acid sequence of tms1 with available databases revealed significant similarity to dehydrogenases, suggesting that the tms1 protein itself might posses dehydrogenase activity.
Comparison of X‐ray powder‐diffraction data of various bacterial lipopolysaccharide structures with theoretical model conformations
Authors: KASTOWSKY Manfred , GUTBERLET Thomas , BRADACZEK Hans
Published: March 2005

X‐ray powder‐diffraction experiments have been performed on dry samples of lipid A and various rough‐mutant lipopolysaccharides (LPS) of Salmonella minnesota, Salmonella typhimurium and Escherichia coli. The diffraction patterns obtained indicated exclusively lamellar, bilayered arrangements in all samples. The periodicities were found to be in the range 4.5 nm for lipid A to 8.8 nm for Ra‐LPS. Upon treatment with water‐saturated air, swelling of the lamellar structures was achieved, as indicated by shifts of reflections. The increase in bilayer dimensions normally was about 0.3 nm.

X‐ray intensities were used for the determination of the inner bilayer structure, i.e. for calculation of the one‐dimensional electron‐density distribution across the bilayer. For lipid A and several Re‐LPS, Rd2‐LPS, Rd1‐LPS and Rc‐LPS samples, a striking coincidence of the electron‐density distributions in the lipid‐A domain was found, suggesting that in all these structures the lipid‐A portion is similarly arranged. For Rb1 and Ra‐LPS, the lipid‐A domain could not be resolved due to the limited number of observed reflections. For other Re‐mutant lipopolysaccharide samples, quite different X‐ray patterns were obtained. Some samples yielded diffraction patterns indicating a very high state of order in the lipid‐A domain, whereas, in others, a significantly reduced order in the lipid‐A domain was infered.

Comparison of the X‐ray data with features of a calculated three‐dimensional molecular model of lipopolysaccharide revealed reasonable agreement in molecular dimensions and bilayer structure.
The promoter region of the human type‐I‐DNA‐topoisomerase gene
Authors: HEILAND Susanne , KNIPPERS Rolf , KUNZE Norbert
Published: March 2005

We examined the promoter of the human type‐I‐DNA topoisomerase gene (hTOP1) for regions protected against DNase I digestion by nuclear proteins from HeLa or from adenovirus‐transformed 293 cells. We identified ten protected DNA sequences within 580 bp of DNA upstream of the transcriptional‐start sites and one additional site, which is located between the two clusters of transcriptional‐start sites. Several of these protein‐binding sites have significant similarities to recognition sequences of known transcription factors including factors Sp1, octamer transcription factor, cAMP‐responsive‐element‐binding protein (CREB/ATF), NF‐kB and members of the Myc‐related family of basic/helix‐loop‐helix/leucine‐zipper proteins. Other protein‐binding sites show less or no similarities to known consensus sequences. We investigated the physiological significance of these protein‐binding sites using a set of deletion and nucleotide‐exchange mutants. We conclude that the expression of the hTOP1 gene is regulated by a complex network of negatively and positively acting transcription factors.
Reconstruction of translation
Authors: GANOZA M. Clelia , AOKI Hiroyuki , KOZIERADZKI Ivona , SCHWARTZ Ira
Published: March 2005

The in vitro reversal of conditionally lethal mutations has greatly aided the study of translation. N4316 is a mutant of Escherichia coli that has a temperature‐sensitive defect in a protein called the rescue protein. Without the rescue protein, translation in vivo and in vitro is drastically reduced and frameshift errors, as well as increased read‐through of nonsense codons, occurs. Using reversal of temperature‐sensitivity as an assay, the rescue protein was purified from a ribosomal eluate of the parental (D10) strain.

Composite polyacrylamide/agarose gel electrophoresis and sedimentation on sucrose density gradients were employed to examine the distribution of 70S ribosomes and ribosomal subunits in the mutant (N4316) and the parental (D10) extracts at restrictive (43°C) and non‐restrictive (35°C) temperatures. Fewer polysomes and a larger proportion of 70S ribosomes relative to subunits were observed at 43°C with N4316, but not with D10 extracts. Addition of the rescue protein had no effect at 35°C with either strain, but restored the polysome pattern of N4316 at 43°C.

The purified rescue protein labelied by methylation retained activity and bound preferentially to 30S subunits. Rescue bound to 30S particles prevented the action of IF‐3 fostering formation of 70S ribosomes. Thus the rescue protein enables formation of 70S ribosomes from 30S and 50S subunits. 70S ribosomes which contain the rescue protein are active in translation and resist dissociation induced by high centrifugal fields. We propose that the rescue protein alters the conformation of 70S ribosomes resulting in a tighter association of subunits which, in turn, fosters both higher rates and increased accuracy of translation.
Conformations and conformational changes of four Phe→ Trp variants of the DNA‐binding histone‐like protein, HBsu, from Bacillus subtilis studied by circular dichroism and fluorescence spectroscopy
Authors: WELFLE Heinz , MISSEL WITZ Rolf , WELFLE Karin , SCHINDELIN Hermann , SCHOLTZ Andreas S. , HEINEMANN Udo
Published: March 2005

Circular dichroic spectra in the region 180–260 nm of the DNA‐binding histone‐like protein, HBsu, from Bacillus subtilis and of four mutants with a Phe residue replaced by Trp, i.e. [F29W]HBsu, [F47W]HBsu, [F50W]HBsu and [F79W]HBsu, show minor differences only and demonstrate the general similarity of the conformations of these proteins. Fluorescence maxima at 315–320 nm and 330–335 nm indicate a more hydrophobic environment or a more effective stacking of Trp residues in mutants [F29W]HBsu and [F50W]HBsu in comparison to [F47W]HBsu and [F79W]HBsu, respectively. Unfolding of the mutants in high‐ionic‐strength buffers by increasing concentrations of urea results in a red shift of the fluorescence emission maxima to about 350 nm; the fluorescence intensities decrease strongly for [F29W]HBsu and [F50W]HBsu but show a small increase for [F47W]HBsu and [F79W]HBsu. The data suggest complex unfolding patterns with subtle differences between the single mutants. The circular dichroic spectra in the region 250–320 nm are dominated by the effects of the Trp residues and signal position‐dependent differences in the environment of the Trp residues. The conformations of the mutant proteins depend on the ionic strength of the buffer and become more stable against unfolding by denaturants or increasing temperatures at higher ionic strength. At low ionic strength a pronounced protein‐concentration dependence of the conformation of the mutants is seen.
Cloning of a new antenna gene cluster and expression analysis of the antenna gene family of Rhodopseudomonas palustris
Authors: TADROS Monier H. , KATSIOU Eleni , HOON Mark A. , YURKOVA Natalie , RAMJI Dipak P.
Published: March 2005

The genome of Rhodopseudomonas palustris contains five antenna gene clusters, αβa, αβb, αβc, αβd and αβe, which encode the light‐harvesting peripheral antenna complex II polypeptides. The isolation and characterisation of the gene which encodes the αe and βe polypeptides are reported. The primary structure of the βe polypeptide is identical to that of βb whilst the structure of αe is different from the other α subunits so far characterised. All five of the gene clusters were transcribed under high‐light conditions while under low‐light conditions only three were transcribed (αβb, αβd and αβe). Furthermore, Northern‐blot analysis showed that the gene clusters encode RNA transcripts of either 500 or 650 nucleotides. Individual members of the gene family showed a differential response in terms of the regulation of abundance of mRNA upon growth under either high‐light or low‐light intensities. Possible promoter sequences and operator sites upstream of the αβb, αβd and αβe genes were located. Furthermore using puc‐lacZ fusions in trans in R. palustris, we were able to examine the positions of the promoter of the gene clusters. The significance of these observations with respect to the regulation, organization and role of the peripheral antenna is discussed.
Isolation, structural characterization and biological function of a lysine‐conopressin in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata
Authors: SALZET Michel , BULET Philippe , VAN DORSSELAER Alain , MALECHA Jean
Published: March 2005

Several neuropeptides are suspected to act on the control of hydric balance in leeches. One of these peptides, a peptide immunoreactive to an antibody against oxytocin, was previously characterized from the central nervous system of the leech Erpobdella octoculata [Salzet, M., Wattez, C., Verger‐Bocquet, M., Beauvillain, J.‐C. & Malecha, J. (1993) Brain Res. 601, 173–184].

This paper reports the isolation from the central nervous system of E. octoculata of another peptide of the oxytocin/vasopressin family; a lysine‐vasopressin‐like molecule. Its purification was performed by reverse‐phase high‐performance liquid chromatography combined with both dot immunobinding assay and enzyme‐linked immunosorbent assay for lysine‐vasopressin. The amino acid sequence was established by Edman degradation and confirmed by electrospray‐mass‐spectrometry measurement. The nonapeptide obtained corresponded to the lysine‐conopressin previously isolated from the venom of the mollusc Conus geographus [Cruz, L. L., de Santos, V., Zafaralla, G. C., Ramilo, C. A., Zeikus, R., Gray, W. R. & Olivera, B. M. (1987) J. Biol. Chem. 262, 15821–15824]. In leeches, synthetic lysine‐conopressin exerts a diuretic effect which can be compared to that of the arginine‐vasopressin‐like peptide isolated in the Insect Locusta migratoria [Proux, J., Miller, C. A., Li, J. P., Carney, R. L., Girardie, A., Delaage, M. & Schooley, D. A. (1987) Biochem. Biophys. Res. Commun. 149, 180–186].
Expression of human interferon‐α2 in Sf9 cells
Authors: SUGYIAMA Kenji , AHORN Horst , MAURER‐FOGY Ingrid , VOSS Tilman
Published: March 2005

Human interferon α2 (IFN‐α2) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. The protein purified by immunoaffinity chromatography exhibited biological activity identical to that of leukocyte‐derived ‘natural’ IFN‐α2. However, the protein was found to be heterogeneously glycosylated, partially truncated by proteolysis and partially lacking a disulfide bridge. The major product was shown to be O‐glycosylated at the same position as natural human IFN‐α2. Enzymatic cleavage, reverse‐phase HPLC peptide mapping and plasma‐desorption mass spectroscopy analysis revealed the presence of two types of O‐linked carbohydrates. The major O‐linked carbohydrate was found to be the disaccharide galactosyl(β1–3)‐N‐acetylgalactosamine, the minor component the monosaccharide N‐acetylgalactosamine. No evidence for sialylation was found. The non‐glycosylated species representing about 40% of the total material were shown to partially lack the C‐terminal three amino acids. In addition an unglycosylated, reduction‐sensitive dimer was observed. This was formed due to the lack of the N‐terminal cysteine normally forming an intramolecular disulfide bridge. Furthermore, a minor species was identified which contains Cys1 and Cys98 in a modified form, thereby hindering the formation of a disulfide bridge between these two residues.
Site‐directed mutagenesis of β‐lactamase TEM‐1
Authors: LENFANT Françise , PETIT Agnès , LABIA Roger , MAVEYRAUD Laurent , SAMAMA Jean‐Pierre , MASSON Jean‐Michel
Published: March 2005

From sequence alignments, two groups can be defined for the carbenicillin‐hydrolysing β‐lactamases (CARB enzymes). One group includes the Pseudomonas‐specific enzymes PSE‐1, PSE‐4, CARB‐3, CARB‐4 and also the Proteus mirabilis GN79, for which the well‐conserved residue Lys 234 in all class‐A β‐lactamases is changed to an arginine residue. The second group includes the enzymes PSE‐3 and AER‐1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM‐1 β‐lactamase to study their potential role in defining the substrate profile of the CARB enzymes.

The mutations K234R and E240G in TEM‐1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second‐generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to α‐carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third‐generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM‐1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three‐dimensional structures of the active site of the TEM‐1 enzyme and PSE‐3, PSE‐4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM‐1 protein variants.
Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases
Authors: SCHOU Charlotte , RASMUSSEN Grethe , KALTOFT Maj‐Britt , HENRISSAT Bernard , SCHÜLEIN Martin
Published: March 2005

The catalytic activity of nine enzymes (endoglucanases I–III, V, VI and cellobiohydrolases I and II from Humicola insolens; endoglucanases A and C from Bacillus lautus), representative of cellulase families A–C, H, J and K, has been investigated using a series of reduced cellooligosaccharides (cellotriitol to cellohexaitol) as substrates. For each enzyme, the specificity of cleavage was determined by analytical HPLC while the kinetic constants were obtained from a kinetic assay involving a cellobiose dehydrogenase purified from H. insolens as a coupled enzyme using 2,6‐dichloroindophenol as the electron acceptor. These data were used to estimate the number of subsites in the enzymes. The stereochemical course of hydrolysis by seven enzymes, representing the six different families, was assessed using 1H‐NMR. The enzymes belonging to families which had already been investigated (A–C), showed results in agreement with previous studies. The three other families (H, J and K), for which no mechanistic data was previously available, gave results which indicated that enzymes in group H had retaining‐type activity and enzymes in groups J and K had inverting‐type activity. The retaining endoglucanases I and III displayed a high glycosyl‐transferase activity under the conditions used during the NMR experiments resulting in precipitates of higher oligomers.
Dihydrorhodamine 123: a fluorescent probe for superoxide generation?
Authors: HENDERSON Lydia M. , CHAPPELL J. Brian
Published: March 2005

Imaging techniques, such as confocal microscopy and fluorescent activated cells scan are facilitating the study of responses at the single‐cell level. Superoxide is reported to oxidise the non‐fluorescent dihydrorhodamine 123 (DHR) to rhodamine 123. The generation of rhodamine 123 by human neutrophils, stimulated by the phorbol ester phorbol 12‐myristate 13‐acetate was inhibited slowly by diphenylene iodonium and rapidly by azide, but not by superoxide dismutase. In the absence of enzymes H2O2 (but not ) oxidised DHR slowly but the rate was greatly enhanced by peroxidases. The rhodamine 123 generated by phorbol‐ester‐stimulated neutrophils was observed to be located within the cell despite the fact that neutrophils failed to accumulate external rhodamine 123. This stimulated rise in cellular fluorescence was eliminated by excess extracellular catalase. It appears that H2O2, released on the outside, crosses the plasma membrane where oxidation of DHR is catalysed by cellular peroxidases. Since in a mixed population DHR failed to distinguish between ‐producing and non‐producing HL60 cells it is not a suitable probe for single‐cell observations. We conclude that DHR oxidation reports only the presence of H2O2 and intracellular peroxidases, and not the generation of by any one cell. Only peroxidase‐containing cells fluoresce.
Voltammetric studies of the catalytic electron‐transfer process between the Desulfovibrio gigas hydrogenase and small proteins isolated from the same genus
Authors: MORENO Cristina , FRANCO Ricardo , MOURA Isabel , LE GALL Jean , MOURA José J. G.
Published: March 2005

The kinetics of electron transfer between the Desulfovibrio gigas hydrogenase and several electron‐transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, square‐wave voltammetry and chronoamperometry. The cytochrome c3 from Desulfovibrio vulgaris (Hil‐denborough), Desulfovibrio desulfuricans (Norway 4), Desulfovibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron‐transfer proteins such as ferredoxin I, ferredoxin II and rubredoxin from D. gigas were tentatively used as redox carriers. Only ferredoxin II was effective in mediating electron exchange between hydrogenase and the working electrode. The second‐order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mechanism [Moreno, C., Costa, C., Moura, I., Le Gall, J., Liu, M. Y., Payne, W. J., van Dijk, C. & Moura, J. J. G. (1993) Eur. J. Biochem. 212, 79–86] and the results obtained by cyclic voltammetry were compared with those obtained by chronoamperometry. Values for k of 105–106 M−1 s−1 (cytochrome c3 as electron carrier) and 104 M−1 s−1 (ferredoxin II as the electron carrier) were determined. The rate‐constant values are discussed in terms of the existence of an electrostatic interaction between the electrode surface and the redox carrier and between the redox carrier and a positively charged part of the enzyme.
Comparison of the biochemical properties of unprocessed and processed forms of the small GTP‐binding protein, rab6p
Authors: YANG Chunzhi , MOLLAT Patrick , CHAFFOTTE Alain , McCAFFREY Mary , CABANIÉ Lucien , GOUD Bruno
Published: March 2005

The rab6 protein (rab6p) belongs to a large family of ras‐like low‐molecular‐mass GTP‐binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. When expressed in the baculovirus/insect cell system, two major forms of rab6p are obtained; a 24‐kDa cytosolic unprocessed form and a 23‐kDa membrane‐bound form which represents the processed lipid‐modified protein. Here, we have purified both forms to homogeneity and we have studied and compared their biochemical properties. Unprocessed and processed rab6p display similar binding‐rate constants (kon) for GDP and GTP (1–1.9 μM−1 min−1). However, significant differences exist in the dissociation constants of bound guanine nucleotides. Processed rab6p in low and high magnesium solutions displays similar koff values for GTP and GDP. However, unprocessed rab6p has a koff value higher for GDP than for GTP in both low and high magnesium solutions. Their intrinsic GTPase activities also differ; unprocessed rab6p has an almost undetectable GTPase activity, whereas that of processed rab6p is in the same range as that reported for other ras and ras‐like GTP‐binding proteins (0.012 ± 0.002 min−1). These results suggest that post‐translational modifications of rab6p might induce subtle changes in the three‐dimensional structure of the protein which affect the guanine‐nucleotide‐binding/hydrolysis activity.
Structure/activity relationship of leukotriene B4 and its structural analogues in chemotactic, lysosomal‐enzyme release and receptor‐binding assays
Authors: SOYOMBO Olukayode , SPUR Bernd W. , SOH Cecilia , LEE Tak H.
Published: March 2005

The biological activities of chemically synthesized leukotriene B4 and eight structural analogues have been studied using chemotaxis, lysosomal‐enzyme release and receptor‐binding assays on human neutrophils. The results show that increasing the number of double bonds between C14 and C20, having triple bonds at C6 or C14, substitution of the primary carboxyl group at C1, changing the geometry of the double bond at C6 from the cis to trans configuration and changing the chirality of the hydroxyl group at C12 from the R to the S configuration result in substantial loss of both biological activity and the capacity to bind to the LTB4 recognition site in parallel. We suggest that the functional epitopes of 5S, 12R‐dihydroxy‐6, 14‐cis‐8, 10‐trans‐icosatetraenoic acid (LTB4) are either the same, or reside in the same domain as the binding site for the LTB4 receptor. Development of LTB4 antagonists to the high‐affinity LTB4 receptor, based on the structure of LTB4, is unlikely to be successful.
Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23‐nm resolution and localization of the γ subunit
Authors: FICNER Ralf , HUBER Robert
Published: March 2005

The three‐dimensional structure of the light‐harvesting pigment‐protein b‐phycoerythrin from the red alga Porphyridium cruentum has been determined at 0.23‐nm resolution. The b‐phycoerythrin structure is very similar to the structure of B‐phycoerythrin from Porphyridium sordidum. Besides three non‐identical residues there are only small differences between b‐phycoerythrin and B‐phycoerythrin α and β subunits, respectively. In the crystals b‐phycoerythrin forms an (αβ)6 hexamer (molecular mass: 236 kDa), whereas B‐phycoerythrin additionally contains a 30‐kDa γ subunit. The comparison of the b‐phycoerythrin and B‐phycoerythrin electron‐density maps clearly reveals, that the γ subunit is located inside the (αβ)6 aggregate.
Characterization of the post‐translational modifications in tubulin from the marginal band of avian erythrocytes
Authors: RÜDIGER Manfred , WEBER Klaus
Published: March 2005

Tubulin purified from turkey erythrocytes was characterized by partial protein sequence data, high‐resolution IEF and by its reaction with antibodies specific for certain post‐translational modifications. The tubulin from the marginal band contains a single α and β isotype, i.e. α1 and β6. Partial protein sequences and immunoblotting with antibody 6–11B‐1 show that erythrocyte α1 tubulin is not acetylated at Lys40. The acidic carboxy‐terminal peptides purified by Mono Q chromatography and reverse‐phase HPLC were characterized by sequence analysis and mass spectrometry. Although erythrocyte α tubulin is almost completely detyrosinated it retains the penultimate glutamic acid residue, which is partially lost in brain tubulin. Thus erythrocyte tubulin is an excellent substrate for extensive in vitro tyrosination by tubulin‐tyrosine ligase. Erythrocyte α and β tubulin lack the side‐chain polyglutamylation found in all major tubulins from adult brain. Finally we show that about 10% of the β tubulin is phosphorylated at Ser441. Thus erythrocyte tubulin is an unusual homogeneous preparation. It contains the minimum possible number of tubulin isotypes and the only post‐translational modifications detected (detyrosination and phosphorylation) are reversible.
Mouse gelatinase B
Authors: MASURE Stefan , NYS Guy , FITEN Pierre , VAN DAMME Jo , OPDENAKKER Ghislain
Published: March 2005

Gelatinase B is a regulated matrix metalloproteinase with an important role in the remodelling of extracellular matrices and of basement membranes. To study the structure and function of gelatinase B in the mouse, the cDNA was cloned from a macrophage cell line (WEHI‐3). Using this cDNA, a cosmid clone with the mouse gene was isolated. The complete gene (8 kbp) was sequenced and compared with the human gene structure. There was 78% similarity at the cDNA level and the exon/intron structure of the murine gene was similar to the human counterpart. At the 5′ untranslated side, 1200 bp of the promoter/enhancer region were sequenced and found to contain several trans‐acting‐factor‐binding sites. The mRNA transcription‐initiation site was determined by non‐isotopic primer‐extension analysis. Polymerase‐chain‐reaction amplification of cDNAs yielded indirect evidence for a reverse‐transcription stop in WEHI‐3 cell mRNA. The DNA‐derived mouse‐protein structure exhibited 82% similarity with the human one. This similarity was functionally reflected by cross‐reactivity of the mouse protein with an antiserum against human gelatinase B. The production of murine gelatinase B was studied at the protein level by zymography and at the mRNA level by Northern blot analysis. In WEHI‐3 cells the gelatinase B protein is induced by bacterial lipopolysaccharide, phorbol ester, double‐stranded RNA and the cytokine interleukin‐1. Regulation of activity and structural heterogeneity of gelatinase B in WEHI‐3 cells were shown to occur at the gene regulatory level, by expression of the matrix metalloproteinase inhibitor TIMP‐1, and by glycosylation of the secreted protein.
Identification of O‐linked oligosaccharide chains in the activation peptides of blood coagulation factor X
Authors: INOUE Keisuke , MORITA Takashi
Published: March 2005

Conversion of factor X to factor Xa results in release of a heavily glycosylated activation peptide. Analysis of protease‐digested glycopeptides derived from the activation peptides of bovine and human blood coagulation factor X allowed the identification of sites of the O‐linked oligosaccharide chains in these peptides. Glycopeptides were prepared from the activation peptides by digestion with chymotrypsin or Staphylococcus aureus V8 protease. By combined analysis of amino acid sequence and sialic acid content, we found that bovine factor X had an O‐linked oligosaccharide chain linked to Thr26, and human factor X had four carbohydrate‐attachment sites, namely, O‐glycosidic linkages to Thr17 and Thr29, respectively, and N‐glycosidic linkages to Asn39 and Asn49, respectively, in their activation peptides. The O‐linked carbohydrate‐attachment sites were identified since the yields of phenylthiohydantoin derivatives of amino acids that corresponded to their residues were increased during amino acid sequencing after deglycosylation of the glycopeptides with sialidase and O‐glycanase. The effect of deglycosylation of bovine factor X1 was investigated with factor‐X‐activating enzyme from Russell's viper venom or extrinsic Xase (factor VIIa/tissue factor/phosholipid) by examining the activation rates of derivatives of factor X prepared using O‐glycanase, sialidase, and/or N‐glycanase. The removal of O‐linked carbohydrate resulted in a decrease in the rate of activation. It appears that carbohydrate residues in factor X play an important role in the activation of the zymogen.
Structure and dynamics of the acidic compact state of apomyoglobin by frequency‐domain fluorometry
Authors: BISMUTO Ettore , GRATTON Enrico , SIRANGELO Ivana , IRACE Gaetano
Published: March 2005

The conformational dynamic properties of tuna apomyoglobin, a single tryptophan‐containing protein, in the acidic compact state, as well as in the native and in the fully unfolded state, have been explored by frequency‐domain fluorometry. Apomyoglobin at acidic pH in the presence of high salt concentration displays bimodal tryptophanyl lifetime distributions which may be related to the simultaneous presence of different populations of structural states (compact and fully unfolded states). The tryptophanyl anisotropy decay indicated that the acidic compact state displays at least two rotational correlational times, suggesting that this state possesses a complex geometrical organization. 1‐Anilino‐8‐naphthalene sulfonate (ANS), bound both to native and compact protein forms, shows broad unimodal lifetime distributions. The small time dependence of the ANS emission spectra indicated that the solvent dipolar reorganization are either absent or they occur on a time scale much shorter than the lifetime of the excited ANS molecule bound to apomyoglobin. The anisotropy decay data relative to the extrinsic fluorophore (ANS) are consistent with the presence of a single rotational correlation time for both native (12.1 ns) and compact (6.2 ns) states.
Gly387 of murine ornithine decarboxylase is essential for the formation of stable homodimers
Authors: TOBIAS Karin E. , MAMROUD‐KIDRON Emanuelle , KAHANA Chaim
Published: March 2005

In its active form mammalian ornithine decarboxylase (ODC) is a homodimer composed of two 53‐kDa subunits while the monomer retains no enzymic activity. In the present study we demonstrate that Gly387 of mouse ODC plays an important role in enabling dimer formation. Gly387 of mouse ODC, an evolutionary conserved residue, was converted to all possible 19 amino acids using site‐directed mutagenesis. With the exception of alanine, all other substitutions of Gly387 completely abolished enzymic activity. Cross‐linking analysis and fractionation through a Superose‐12 sizing column have demonstrated that mutant subunits are detected only in their monomeric form. These results strongly suggest that the primary lesion of substitution at position 387 of mouse ODC is the inability of mutant subunits to associate with each other to form the active homodimers. In agreement with this conclusion, G387 A, the only mutant that retained partial activity, displayed reduced dimerization. The degradation rate of ODC mutants in which Gly387 was substituted by aspartic acid or alanine was enhanced compared to the wild‐type enzyme, suggesting that monomers may be more susceptible to degradation.
Structure/activity relationships in porphobilinogen oxygenase and horseradish peroxidase
Authors: FERNANDEZ Marcelo , FRYDMAN Rosalía B. , HURST Jorge , BULDAIN Graciela
Published: March 2005

The apo‐enzymes of porphobilinogen oxygenase and horseradish peroxidase were reconstituted with hemin IX, deuterohemin IX, 2,4‐diacetyldeuterohemin IX, 2‐vinyl‐4‐deuterohemin IX and hemin I. The apoproteins did not reconstitute with the dimethyl or diethyl esters of hemin IX. The native enzymes and the synthetic hemoproteins showed similar oxygenase activities toward porphobilinogen in the presence of dithionite and oxygen. They also showed peroxidase activity in the presence of H2O2, which was affected by the side‐chain substitution pattern of the hemes. Oxygenase activities, however, were not affected by the heme structure. Iron chelators completely inhibited the oxygenase, but not the peroxidase activities. The EPR spectra of the native and synthetic porphobilinogen oxygenase showed that dithionite reduction produced a rapid disappearance of the high‐spin heme‐iron signal at g= 6.0. It reappeared 1 min later but the enzyme retained its catalytic activity. The changes in the EPR spectra could be correlated with the biphasic kinetics of the oxygenase reaction which was very fast during the first minute and then decreased to a half‐value rate. The oxygenase reaction was inhibited by addition of superoxide dismutase during the fast rate phase, but not during the slower phase. These results could be explained by the formation of a superoxide anion during the first minute of the oxygenase reaction, after which a protein‐stabilized radical (g= 2.0) is generated (very likely a tyrosyl radical). The latter then oxidizes the substrate porphobilinogen and facilitates its reaction with O2 to give oxopyrrolenines.
The separate roles of plant cis and trans prenyl transferases in cis‐1,4‐polyisoprene biosynthesis
Authors: CORNISH Katrina
Published: March 2005

In plants, the elongation of cis‐1,4‐polyisoprene (natural rubber, Mr > 106) requires a small trans‐allylic diphosphate (≦C20) initiator. The trans‐allylic diphosphates are hydrophilic cytosolic compounds, whereas cis‐1,4‐polyisoprene is hydrophobic and compartmentalised in subcellular rubber particles.

In this paper, it is demonstrated that soluble trans‐prenyl transferase from latex of Hevea brasiliensis functions solely as farnesyl diphosphate synthase, and plays no direct role in cis‐1,4‐polyisoprene elongation. The cis‐1,4‐prenyl transferase is firmly associated with the H. brasiliensis rubber particle, as is also the case in other rubber‐producing species [Archer, B. L., Audley, B. G., Cockbain, E. G. & McSweeney, G. P. (1963) Biochem. J. 89, 565–574; Madhavan, S., Greenblatt, G. A., Foster, M. A. & Benedict, C. R. (1989) Plant Physiol. 89, 506–511; Siler, D. J. & Cornish, K. (1993) Phytochemistry 32, 1097–1102]. The experimental data explain and refute previous reports in which soluble trans‐prenyl transferase isolated from H. brasiliensis latex was attributed both trans‐prenyl transferase and cis‐prenyl transferase activities [Light, D. R. & Dennis, M. S. (1989) J. Biol. Chem. 264, 18589–18597; Light, D. R., Lazarus, R. A. & Dennis, M. S. (1989) J. Biol. Chem. 264, 18598–18607].

Thus, it appears that plant prenyl transferases are comparable to animal enzyme systems in which trans‐prenyl transferases are soluble enzymes whilst cis‐prenyl transferases are membranebound [Ericcson, J., Runquist, M., Thelin, A., Andersson, M., Chojnacki, T. & Dallner, G. (1992) J. Biol Chem. 268, 832–838].
Biosynthetic 15N and 13C isotope labelling of glutathione in the mixed disulfide with Escherichia coli glutaredoxin documented by sequence‐specific NMR assignments
Authors: BUSHWELLER John H. , HOLMGREN Arne , WÜTHRICH Kurt
Published: March 2005

A biosynthetic procedure for obtaining 13C‐15N doubly labelled glutathione from readily available precursor molecules is described. Isolation of the mutant Escherichia coli [C14S]glutaredoxin from E. coli cultures grown on 15N‐13C doubly labelled media in the absence of reducing agents yields the mixed disulfide labelled in both the protein and the glutathione. 15N NMR assignments for glutathione obtained from two‐dimensional [15N,1H]‐correlation spectroscopy (COSY), and 13C NMR assignments for the entire mixed disulfide obtained from combined use of three‐dimensional ct‐HA[CAN]HN experiments and HCCH‐total correlation spectroscopy ([HCCH]‐TOCSY) demonstrated unequivocally that the glutathione is uniformly labelled with both 15N and 13C. This result also supports earlier suggestions that the intracellular glutaredoxin activity is sensitive to the glutathione redox status of the cell. Complete sets of 1H, 13C and 15N chemical shifts of both components in the mixed disulfide of [C14S]glutaredoxin and glutathione were obtained from the sequence‐specific NMR assignments.
19F‐NMR study on the pH‐dependent regioselectivity and rate of the ortho‐hydroxylation of 3‐fluorophenol by phenol hydroxylase from Trichosporon cutaneum
Authors: PEELEN Sjaak , RIETJENS Ivonne M. C. M. , VAN BERKEL Willem J. H. , VAN WORKUM Wilbert A. T. , VERVOORT Jacques
Published: March 2005

The regioselectivity and rate of the ortho‐hydroxylation of 3‐fluorophenol by phenol hydroxylase from Trichosporon cutaneum (EC 1.14.13.7) was studied using 19F‐NMR. The regioselective hydroxylation as well as the rate of ortho‐hydroxylation are pH dependent with a pKa of 6.5. At pH values below 6.5, 3‐fluorophenol preferentially becomes hydroxylated at the C6 ortho position, resulting in a maximum C6/C2 hydroxylation ratio of 6.7. Upon increasing the pH, the total rate of conversion increases. Also, the C2 ortho‐hydroxylation increases relatively to the C6 ortho‐hydroxylation and yields a minimum C6/C2 hydroxylation ratio of 2.2 at pH values above 7.5.

Based on data from 19F‐NMR binding studies and molecular orbital calculations, a hypothesis is put forward which explains the pH‐dependent effects observed. A mechanism is proposed involving an active‐site amino acid residue acting as a base in the reduced form of the protein. Deprotonation of this residue results in hydrogen bond formation with the hydroxyl moiety of the phenolic substrate, leading to (partial) deprotonation of the substrate. Molecular orbital calculations demonstrate that such a (partial) deprotonation increases (a) the overall reactivity of 3‐fluorophenol for an electrophilic attack and (b) the reactivity of C2 relative to the C6 position. The hypothesis may explain the decrease in the C6/C2 hydroxylation ratio. Furthermore the increased amount of ortho‐hydroxylated products formed with increasing pH can also be explained by this hypothesis.
Inactivation of phosphorylated rat tyrosine hydroxylase by ascorbate in vitro
Authors: ROSKOSKI Robert , GAHN Laura G. , ROSKOSKI Laura M.
Published: March 2005

Tyrosine hydroxylase activity is reversibly controlled by the actions of several protein kinases. Previous studies showed that, following phosphorylation by protein kinase A, physiological concentrations of ascorbate irreversibly inactivate tyrosine hydroxylase. Several studies were performed to establish the mechanism of inactivation. We found that inactivation occurred under oxygen‐free conditions. The results of this and other experiments suggest that oxygenated species such as super‐oxide or hydrogen peroxide were not required for inactivation by ascorbate. Inhibition of tyrosine hydroxylase by low concentrations of ascorbate raised the question concerning the mechanism for maintaining enzyme activity under physiological conditions. We report that tyrosine, Nα‐methyl tyrosine, 3‐iodotyrosine, and phenylalanine protected the phosphorylated enzyme against ascorbate inactivation. Catecholamines (dopamine, norepinephrine, and some of their analogues) also protected the enzyme against ascorbate inactivation. We performed studies to assess conformational changes of tyrosine hydroxylase by measuring the extrinsic fluorescence using 8‐anilino‐1‐naphthalenesulfonic acid as a reporter group. Phosphorylation of tyrosine hydroxylase by protein kinase A decreased the extrinsic fluorescence. Treatment of tyrosine hydroxylase with ascorbate produced a further decrease in fluorescence. These results provide evidence for conformational changes following these treatments. In contrast to extrinsic fluorescence, the circular dichroic spectrum of tyrosine hydroxylase failed to change following phosphorylation by protein kinase A or inhibition by ascorbate. The spectrum was consistent with a secondary structure of tyrosine hydroxylase with 55%α helix, 20%β sheet, 2%β turn, and 23% random coil.
Purification of ATP synthase from beef heart mitochondria (FoF1) and co‐reconstitution with monomeric bacteriorhodopsin into liposomes capable of light‐driven ATP synthesis
Authors: DEISINGER Barbara , NAWROTH Thomas , ZWICKER Klaus , MATUSCHKA Simone , JOHN Gabriele , ZIMMER Guido , FREISLEBEN Hans‐Joachim
Published: March 2005

ATP synthase was isolated from beef heart mitochondria by extraction with N,N‐bis‐(3‐D‐gluconamidopropyl)deoxycholamide or by traditional cholate extraction. The enzyme was purified subsequently by ion‐exchange and gel‐permeation chromatographies in the presence of glycerol and the protease inhibitor diisopropylfluorophosphate. The ATP synthase consisted of 12–14 subunits and contained three tightly bound nucleotides. The co‐reconstitution of crude or purified ATP synthase with monomeric bacteriorhodopsin by the method of detergent incubation of liposomes yielded proteoliposomes capable of light‐driven ATP synthesis, as detected with a luciferase system for at least 30 min. The reaction was suppressed by the inhibitors oligomycin (>90%) and dicyclohexylcarbodiimide (85%) and by the uncoupler carbonylcyanide‐p‐trifluormethoxyphenylhydrazone (>95%). The purified ATP synthase was apparently free of cytochrome impurities and of adenylate kinase activity, i.e. the enzyme exhibited light‐driven ATP synthesis without the dark reaction. For the first time, this is demonstrated with purified ATP synthase from beef heart mitochondria.
NAD turnover and utilisation of metabolites for RNA synthesis in a reaction sensing the redox state of the cytochrome b6f complex in isolated chloroplasts
Authors: PEARSON Colin K. , WILSON Stanley B. , SCHAFFER Robert , ROSS Alexander W.
Published: March 2005

NAD is normally regarded as a redox molecule or as the substrate for ADP‐ribosylation reactions. In this study, we describe the rapid metabolism of NAD by Percoll‐gradient‐purified lettuce chloroplasts and show that the adenine moiety can be incorporated into RNA in a dark‐activated reaction that senses the redox state of the cytochrome b6f complex. Isolated chloroplasts rapidly metabolised radiolabelled NAD+ to 5′‐AMP (within seconds) and adenosine during a 60‐min incubation in vitro; the products were analysed by high‐performance liquid chromatography. No radiolabelled ADP‐ribose was detected. Radioactivity was incorporated into trichloroacetic‐acid‐insoluble material during this period, with approximately 2–4‐fold more incorporation occurring in the dark. Most of this radiolabel was rendered acid‐soluble by dilute alkaline digestion at 37°C, yielding an approximately equal mixture of 2′‐AMP and 3′‐AMP, and by RNase digestion, identifying the acid‐insoluble radioactive material as RNA. Protein‐bound ADP‐ribose would have yielded 5′‐AMP and/ or oligomeric/polymeric ADP‐ribose after alkali digestion. The utilisation of NAD metabolites for RNA synthesis was restricted to the thylakoid compartment of the chloroplast.

The use of a variety of electron‐transport inhibitors such as 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone, bromanil (tetrabromo‐1,4‐benzoquinone), electron donors (dithiothreitol), electron acceptors (ferricyanide) and an uncoupler showed that the incorporation of radiolabel from NAD into acid‐insoluble material was favoured when the cytochrome b6f complex was in the oxidised state (as pertaining to incubations in the dark).
Pressure‐induced dissociation of ribosomes and elongation cycle intermediates
Authors: GROSS Michael , LEHLE Karla , JAENICKE Rainer , NIERHAUS Knud H.
Published: March 2005

Pressure‐induced dissociation of ribosomes has been considered a major reason for the inhibition of protein biosynthesis and, hence, bacterial growth at high hydrostatic pressure [Jaenicke, R. (1981) Annu. Rev. Biophys. Bioeng. 10, 1–67]. We reexamined the issue, using a buffer system with polyamines that has been optimized to reproduce in‐vivo‐like performance of protein biosynthesis in vitro. By slightly modifying this buffer, we were able to find conditions that stabilize functional ribosomal complexes against the dissociating effect of pressure up to 100 MPa and uncharged tight couples up to 60 MPa. Approaching the physiological conditions by reducing the Mg2+ concentration down to 4 mM, one finds a significant destabilization of the post‐translocational complex, which represents the most pressure‐sensitive intermediate of the elongation cycle and is possibly the limiting factor for the pressure‐induced block of protein biosynthesis and bacterial growth.
Ion‐exchange reactions of proteins during DNA binding
Authors: FRIED Michael G. , STICKLE Douglas F.
Published: March 2005

The equilibrium association constant observed for many DNA/protein interactions in vitro (Kobs) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that displacement of cations from the DNA is the predominant form of the involvement of ions in the binding reaction. With this assumption, the graph of log Kobs versus log [MX] is predicted to have a constant slope proportional to the number of ions displaced from the DNA upon protein binding [Record, M. T., Lohman, T. M. & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145–158]. Experimental data often deviate from linearity, however, at lower salt concentrations. Such deviations can be due to differential cation binding, anion binding or changes in macromolecular hydration, or differential screening effects of the electrolyte on protein and/or DNA charges. Here the theoretical effects on Kobs of a simple form of ion–protein interaction are examined. A model for binding interactions is used that includes a mass balance of ions bound to both protein and DNA as the protein is transferred from the salt concentration of bulk solvent to the typically higher cation and lower anion concentrations characteristic of the volume adjacent to the DNA. We show that models in which the cation and anion stoichiometries of a protein change as it associates with DNA are consistent with the curvature of plots of log Kobs versus log [MX]. Such mechanisms could reduce the sensitivity of gene‐regulatory interactions to changes in environmental salt concentration.