bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha
Authors: BERKS Ben C. , RICHARDSON David J. , ROBINSON Carol , REILLY Ann , APLIN Robin T. , FERGUSON Stuart J.
Published: March 2005

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M‐6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93‐kDa polypeptide and a 16‐kDa nitrate‐oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane‐bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93‐kDa subunit exhibits immunological cross‐reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo‐cytochrome c552 and apo‐cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately −15 mV and +80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.
Posttranslational modification of nitrogenase
Authors: DURNER Jörg , BÖHM Ines , HILZ Helmuth , BÖGER Peter
Published: March 2005

In the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulatus post‐translational regulation of nitrogenase is due to ADP‐ribosylation of the Fe‐protein, the dinitrogenase reductase [Burris, R. H. (1991) J. Biol. Chem. 266, 9339–9342]. This mechanism has been assumed to be responsible for nitrogenase modification in a variety of organisms. In the present study, we examined whether ADP‐ribosylation holds true for the filamentous cyanobacterium Anabaena variabilis. Genes coding for the nitrogenase‐modifying enzymes dinitrogenase reductase‐activating glycohydrolase (DRAG) and dinitrogenase reductase ADP‐ribosyl transferase (DRAT) from R. rubrum have been subcloned and overexpressed in Escherichia coli. After isolation under anaerobic conditions, both proteins were functional as determined by in‐vitro assays using nitrogenase from R. rubrum as substrate. In contrast to the R. rubrum enzyme, nitrogenase from A. variabilis was not affected by DRAG or DRAT. Neither could inactive nitrogenase be restored by DRAG, nor nitrogenase activity suppressed by DRAT. Using specific antibodies against arginine‐bound ADP‐ribose [Meyer, T. & Hilz, H. (1986) Eur. J. Biochem. 155, 157–165], immunoblotting of the inactive, modified form of the Fe‐protein from R. rubrum but not that from A. variabilis showed a strong cross reaction. Furthermore, differently to R. rubrum no ADP‐ribosylated proteins could be detected at all, indicating the absence of this posttranslational modification in A. variabilis.
Effects of pH on carboxypeptidase‐Y‐catalyzed hydrolysis and aminolysis reactions
Authors: CHRISTENSEN Ulla
Published: March 2005

The pH dependencies of serine carboxypeptidase‐Y‐catalysed hydrolysis and aminolysis reactions using L‐amino acids and L‐amino acid amides as nucleophiles, have been studied and analyzed. The results reveal two catalytically important ionizing groups of the enzyme with rather similar pK values (5–6), the active site His397 and a possibly Glu residue, which is not only important in interactions with carboxylic groups of substrates and nucleophiles [Liao, D.‐I., Breddam, K., Sweet, R. M., Bullock, T. & Remmington, S. J. (1992) Biochemistry 31, 9796–9812], but also indirectly play a role in catalysis. This explains the pH behaviour of hydrolysis of both peptide and ester substrates and further, that L‐amino acid amides are better nucleophiles in aminolysis reactions than L‐amino acids.
The role of electrostatic charge in the membrane insertion of colicin A
Authors: LAKEY Jeremy H. , PARKER Michael W. , GONZÁLEZ‐MAÑAS Juan M. , DUCHÉ Denis , VRIEND Gert , BATY Daniel , PATTUS Franc
Published: March 2005

The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface‐bound toxin must subsequently, however, become an acidic ‘molten globule’ before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite‐difference Poisson‐Boltzmann method of the Delphi programme and the known X‐ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pI 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild‐type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.
Involvement of a lysine residue in the N‐terminal Ni2+ and Cu2+ binding site of serum albumins
Authors: SADLER Peter J. , TUCKER Alan , VILES John H.
Published: March 2005

We report one‐dimensional and two‐dimensional 1H‐NMR studies of the binding of Ni2+, Cu2+, Co2+, Cd2+ and Al3+ to defatted bovine and human serum albumins. The diamagnetic shifts induced by Ni2+, and paramagnetic effects due to Cu2+, were consistent with strong binding to a square‐planar site formed by the three N‐terminal amino acid residues (Asp‐Thr‐His for bovine, and Asp‐Ala‐His for human albumin). In contrast to previous studies on isolated 1–24 N‐terminal peptide, a Lys residue also appeared to be involved in the binding site, and is assigned as Lys4. A second His residue is also close to the Cu2+/Ni2+ binding site in bovine serum albumin and is assigned to His59 (not present in human albumin).

Co2+ caused specific perturbation of the resonances for the three N‐terminal residues as well as those for Lys4. This is the first evidence for Co2+ binding to the N‐terminal metal site of serum albumin. Neither Al3+ nor Cd2+ perturbed resonances for the N‐terminal amino acids, but bind elsewhere in the protein.
Purification and characterisation of a water‐soluble ferrochelatase from Bacillus subtilis
Authors: HANSSON Mats , HEDERSTEDT Lars
Published: March 2005

Bacillus subtilis ferrochelatase is encoded by the hemH gene of the hemEHY gene cluster and catalyses the incorporation of Fe2+ into protoporphyrin IX. B. subtilis ferrochelatase produced in Escherichia coli was purified. It was found to be a monomeric, water‐soluble enzyme of molecular mass 35 kDa which in addition to Fe2+ can incorporate Zn2+ and Cu2+ into protoporphyrin IX. Chemical modification experiments indicated that the single cysteine residue in the ferrochelatase is required for enzyme activity although it is not a conserved residue compared to other ferrochelatases. In growing B. subtilis, the ferrochelatase constitutes approximately 0.05% (by mass) of the total cell protein, which corresponds to some 600 ferrochelatase molecules/cell. The turnover number of isolated ferrochelatase, 18–29 min−1, was found to be consistent with the rate of haem synthesis in exponentially growing cells (0.2 mol haem formed/min/mol enzyme). It is concluded that the B. subtilis ferrochelatase has enzymic properties which are similar to those of other characterised ferrochelatases of known primary structure, i.e. ferrochelatases of the mitochondrial inner membrane of yeast and mammalian cells. However, in contrast to these enzymes the B. subtilis enzyme is a water‐soluble protein and should be more amenable to structural analysis.
Conservation analysis and structure prediction of the protein serine/threonine phosphatases
Authors: BARTON Geoffrey J. , COHEN Patricia T. W. , BARFORD David
Published: March 2005

A multiple sequence alignment of 44 serine/threonine‐specific protein phosphatases has been performed. This reveals the position of a common conserved catalytic core, the location of invariant residues, insertions and deletions. The multiple alignment has been used to guide and improve a consensus secondary‐structure prediction for the common catalytic core. The location of insertions and deletions has aided in defining the positions of surface loops and turns. The prediction suggests that the core protein phosphatase structure comprises two domains: the first has a single, β sheet flanked by α helices, while the second is predominantly α helical. Knowledge of the core secondary structures provides a guide for the design of site‐directed‐mutagenesis experiments that will not disrupt the native phosphatase fold. A sequence similarity between eukaryotic serine/threonine protein phosphatases and the Escherichia coli diadenosine tetraphosphatase has been identified. This extends over the N‐terminal 100 residues of bacteriophage phosphatases and E. coli diadenosine tetraphosphatase. Residues which are invariant amongst these classes are likely to be important in catalysis and protein folding. These include Arg92, Asn138, Asp59, Asp88, Gly58, Gly62, Gly87, Gly93, Gly137, His61, His139 and Val90 and fall into three clusters with the consensus sequences GD(IVTL)HG, GD(LYF)V(DA)RG and GNH, where brackers surround alternative amino acids. The first two consensus sequences are predicted to fall in the β‐α and β‐β loops of a β‐α‐β‐β secondary‐structure motif. This places the predicted phosphate‐binding site at the N‐terminus of the α helix, where phosphate binding may be stabilised by the α‐helix dipole.
Recombinant human casein kinase II
Authors: BODENBACH Ludger , FAUSS Jürgen , ROBITZKI Andrea , KREHAN Andreas , LORENZ Peter , LOZEMAN Frederick J. , PYERIN Walter
Published: March 2005

Human casein kinase II (CKII) is a ubiquitous and multipotential Ser/Thr kinase involved in the regulation of cell growth and differentiation. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits (α and/or α′) and two regulatory subunits (β); second, the autophosphorylation of the holoenzyme at the N‐terminus of CKIIβ, suspected to be involved in tuning of the kinase activity. Whether CKIIα and CKIIα′ reconstitute comparably with CKIIβ to form holoenzyme is unclear. For a systematic investigation, the complete set of recombinant CKII subunits and of autophosphorylation mutants of CKIIβ were expressed in Escherichia coli and comparative reconstitutions carried out. At 1:1 molar ratio, CKIIβ stimulated both catalytic subunits roughly fivefold with phosvitin as a substrate. The level of activity reached with both of the reconstituted CKII isoforms was of the same order of magnitude as that of holoenzyme isolated from human placenta. It was also similar to a recombinant α2β2 holoenzyme whose expression had been attained in E. coli with a bicistronic construct containing the coding regions of CKIIβ and CKIIα in a tandem arrangement. Both Ser2 and Ser3 were identified as the autophosphorylation sites; replacement of one of these with Ala by oligonucleotide‐mediated site‐directed mutagenesis influenced only the extent of CKIIβ autophosphorylation, replacement of both resulted in a loss of autophosphorylation. Despite these differences, the stimulatory effect of all the CKIIβ mutants was comparable both to each other and to that of wild‐type CKIIβ. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream‐binding factor (UBF). However, the degree of stimulation was substrate specific and ranged from 2–5‐fold with no major differences between CKIIα and CKIIα′ stimulation. Calmodulin phosphorylation by both CKIIα and CKIIα′ was decreased similarly by CKIIβ and the CKIIβ mutants. Proteins such as cAMP‐responsive‐element‐binding protein (CREB), HPV16 E7 or Jun were not phosphorylated by either catalytic subunit but became substrates of both in the presence of CKIIβ or CKIIβ mutants. The data suggest that CKIIα and CKIIα′ form similar CKII holoenzymes and that the tuning of holoenzyme activity is independent of the autophosphorylation status of CKIIβ.
Hydrophobic interaction at the subunit interface contributes to the thermostability of 3‐isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus
Authors: KIRINO Hiromi , AOKI Makoto , AOSHIMA Miho , HAYASHI Yumiko , OHBA Masayuki , YAMAGISHI Akihiko , WAKAGI Takayoshi , OSHIMA Tairo
Published: March 2005

We cloned and sequenced the leuB gene encoding 3‐isopropylmalate dehydrogenase from Escherichia coli K‐12 (JM103). Errors (33 residues) were found and corrected in the sequence previously reported for the leuB gene of Thermus thermophilus. The three‐dimensional structure of the thermophile enzyme and the amino acid sequence comparison suggested that a part of the high stability of the T. thermophilus enzyme is conferred by increased hydrophobic interaction at the subunit‐subunit interface. Two residues at the interface of the T. thermophilus enzyme, Leu246 and Val249, are substituted with less hydrophobic residues, Glu and Met, respectively, in the E. coli enzyme, whereas other residues in this region are highly conserved. The mutated T. thermophilus enzyme [L246E, V249M]IPMDH had reduced stability to heat. Two residues of the E. coli dehydrogenase, Glu256 and Met259, were replaced with the corresponding residues from the thermophile sequence. The resulted mutant enzyme was more resistant to heat than the wild‐type enzyme.
The carboxy‐terminal peptide of detyrosinated α tubulin provides a minimal system to study the substrate specificity of tubulin–tyrosine ligase
Authors: RÜDIGER Manfred , WEHLAND Jürgen , WEBER Klaus
Published: March 2005

The ATP‐dependent tubulin–tyrosine ligase (TTL) restores the carboxy‐terminal tyrosine of α tubulin in αβ tubulin that has been previously detyrosinated. Here we show that the carboxy‐terminal tetradecapeptide of detyrosinated α tubulin is used by TTL as a substrate, albeit at 50‐fold lower efficiency than αβ tubulin. The minimal system provided by the TTL/peptide combination mirrors the TTL/tubulin system in all aspects tested, and shows a pronounced substrate inhibition. Synthetic peptides varying in length and/or containing single amino acid replacements were used to analyze the TTL specificity for the carboxy‐terminal sequence of detyrosinated α tubulin. Peptides ending like α tubulin with the sequence Gly‐Glu‐Glu are optimally tyrosinated once a peptide length of 12 residues is reached. Position ‐1 of this recognition sequence, to which the tyrosine is added, must be glutamic acid. Position ‐2 accepts only an acidic amino acid but glutamic acid is by far preferred over aspartic acid. These results explain why a subpopulation of brain α tubulin, which ends with the sequence Gly‐Glu, is not tyrosinated by TTL. The carboxy‐terminal dodecapeptide of brain α tubulin with its polyglutamyl side‐chain on position ‐6 shows the same substrate activity as the corresponding synthetic peptide lacking the side‐chain. We discuss the substrate specificity of TTL for different α tubulins and speculate why tubulin is a better substrate than the optimal peptide covering the carboxy‐terminal of detyrosinated α tubulin.
Enhancement by lipoprotein‐free plasma of the inhibitory effect of endotoxin on endocytotic catabolism of low density lipoproteins in Hep G2 cells
Authors: LIAO Wei , FLORÉN Claes‐Henrik
Published: March 2005

We previously showed that the presence of microgram levels of endotoxin inhibited low‐density lipoprotein (LDL) uptake and degradation in Hep G2 cells. We also showed that both the polysaccharide and lipid A parts of endotoxins are needed for the inhibitory effects of endotoxins on cellular LDL uptake. The current study was carried out by inclusion of lipoprotein‐free plasma (LFP) in tissue culture medium to observe the modulatory infleucen of non‐lipoprotein factor(s) on endotoxin‐induced inhibition of endocytotic catabolism of LDL in Hep G2 cells. We found that LFP dramatically promotes the inhibitory effect of endotoxins with a complete polysaccharide, but has no influence on the effect of the Re mutant endotoxin (from S. minnesota Re595), which lacks polysaccharide. By using gel‐filtration chromatography, agarose electrophoresis and agarose isoelectric focusing, we further showed that in the presence of LFP, both the endotoxins with a complete polysaccharide and the Re mutant endotoxin complex with and anionize LDL, while in the absence of LFP, these endotoxins poorly interact with LDL. Thus, endotoxin inhibits cellular endocytotic catabolism of LDL by forming LDL‐endotoxin complexes, and LFP enhances endotoxin‐induced inhibition of endocytotic catabolism of LDL by promoting the interaction between endotoxin and LDL. In addition, our finding that the Re mutant endotoxin also interacts with LDL to form LDL‐endotoxin complexes, but has no significant effect on LDL uptake and degradation, further supports the notion that both the polysaccharide and lipid A parts of endotoxins are needed for the inhibitory effects of endotoxins on cellular LDL uptake.
Sequence comparison of a segment of the gene for 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase in zygomycetes
Authors: BURMESTER Anke , CZEMPINSKI Katrin
Published: March 2005

In this paper we compare the sequences of a segment of the 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase gene, isolated from eleven different strains belonging to four species of the fungal order Mucorales, Parasitella parasitica, Absidia glauca, Mucor mucedo (Mucoraceae) and Blakeslea trispora (Choanephoraceae). The segment was obtained by polynucleotide‐chain‐reaction amplification with primers derived from conservative regions of the gene. For the species M. mucedo and P. parasitica we have obtained evidence for two different types of HMG‐CoA reductase genes by hybridization of genomic DNA with the amplified fragment and by cloning and sequencing of two different fragments. The different genes from one species show a sequence similarity of around 80% at the protein sequence level, whereas sequences of the same type from different species show similarity ranging between 91–96%. The highest similarity was found between the genes of type 1 from B. trispora and M. mucedo, although these species belong to different families. Southern‐blot analysis of A. glauca DNA and B. trispora DNA revealed a second copy of the genes.
X‐ray crystallographic and calorimetric studies of the effects of the mutation Trp59→ Tyr in ribonuclease T1
Authors: SCHUBERT Wolf‐Dieter , SCHLUCKEBIER Gerd , BACKMANN Jan , GRANZIN Joachim , KISKER Caroline , CHOE Hui‐Woog , HAHN Ulrich , PFEIL Wolfgang , SAENGER Wolfram
Published: March 2005

Two mutants of ribonuclease T1 (RNaseT1), [59‐tyrosine]ribonuclease T1 (W59Y) and [45‐tryptophan, 59‐tyrosine]ribonuclease T1 (Y45W/W59Y) possess between 150% and 190% wild‐type activity. They have been crystallised as complexes of the inhibitor 2′‐guanylic acid and analysed by X‐ray diffraction at resolutions of 0.23 nm and 0.24 nm, respectively. The space group for both is monoclinic, P21, with two molecules/asymmetric unit, W59Y: a= 4.934 nm, b= 4.820 nm, c= 4.025 nm, β= 90.29°. Y45W/W59Y: a= 4.915 nm, b= 4.815 nm, c= 4.015 nm, β= 90.35°. Compared to wild‐type RNaseT1 in complex with 2′‐guanylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04‐nm inward shift of the single α‐helix and to significant differences in the active‐site geometry, inhibitor conformation and inhibitor binding. Calorimetric studies of a range of mutants [24‐tryptophan]ribonuclease T1 (Y24W), [42‐tryptophan]ribonuclease T1 (Y42W), [45‐tryptophan]ribonuclease T1 (Y45W), [92‐alanine]ribonuclease T1 (H92A) and [92‐threonine]ribonuclease T1 (H92T) with and without the further mutation Trp59→Tyr showed that mutant proteins for which Trp59 is replaced by Tyr exhibit slightly decreased thermal stability.
Evidence that the recently discovered high‐energy nucleotide derivative, oligo(phosphoglyceroyl‐ATP) forms the end chains of purinogen, a complex polymeric major stroage form of adenine nucleotide and phosphate in heart
Authors: PATEL Brinda , SARCINA Mary , MOWBRAY John
Published: March 2005

Previous work in our laboratory has shown that free adenine nucleotides in heart, liver and kidney exchange rapidly with a major acid‐insoluble species which we have characterised as oligo[3‐phospho‐glyceroyl‐γ‐triphospho(5′)adenosine(3′)], abbreviated to (PG‐ATP)n. This has been found in liver and in heart to be complexed to a specific 3′‐phosphodiesterase which releases PG‐ATP monomers and is located in mitochondrial inter‐membrane space. In [14C]adenosine‐perfused hearts, (PG‐ATP)n has been shown to reach specific radioactivity equilibrium with free ATP within 30 min. Attempts to estimate the quantities of nucleotide in (PG‐ATP)n and free nucleotides in response to a variety of stimuli using previously labelled hearts showed by contrast that the free and sequestered nucleotide pools were not at equilibrium. Re‐examination of the rapidly labelled acid‐insoluble species led to the recognition of radioactive inhomogeneity and of three additional nucleotide components. Perfusion of hearts with phosphate‐free medium increased the proportion of 14C incorporated into the sequestered form by 70% and caused the net transfer of 0.9 μmol · g−1 of purine from free adenine nucleotides to the sequestered form. The findings point to the existence of a more complex polymer whose rapidly exchanging end chains we had previously isolated and characterised as (PG‐ATP)n; we suggest the name purinogen for this polymer and show that it can contain between 25% and 55% of the tissue nucleotide pool in rat heart.
Regulation of transferrin receptor mRNA expression
Authors: CHAN Roxanne Y. Y. , SEISER Christian , SCHULMAN Herbert M. , KÜHN Lukas C. , PONKA Prem
Published: March 2005

In proliferating non‐erythroid cells, the expression of transferrin receptors (TfR) is negatively regulated by the amount of intracellular iron. Fe‐dependent regulation of TfR occurs post‐transcriptionally and is mediated by iron‐responsive elements (IRE) located in the 3′ untranslated region of the TfR mRNA. IREs are recognized by a specific cytoplasmic binding protein (IRE‐BP) that, in the absence of Fe, binds with high affinity to TfR mRNA, preventing its degradation. While TfR numbers are positively correlated with proliferation in non‐erythroid cells, in hemoglobin‐synthesizing cells, their numbers increase during differentiation and are, therefore, negatively correlated with proliferation. This suggests a distinct regulation of erythroid TfR expression and evidence, as follows, for this was found in the present study. (a) With nuclear run‐on assays, our experiments show increased TfR mRNA transcription following induction of erythroid differentiation of murine erythroleukemia (MEL) with Me2SO. (b) Me2SO treatment of MEL cells does not increase IRE‐BP activity which is, however, increased in uninduced MEL cells by Fe chelators. (c) Following induction of MEL cells, there is an increase in the stability of TfR mRNA, whose level is only slightly affected by iron excess. (d) Heme‐synthesis inhibitors, such as succinylacetone and isonicotinic acid hydrazide, which inhibit numerous aspects of erythroid differentiation, also inhibit TfR mRNA expression in induced MEL cells. However, heme‐synthesis inhibition does not lead to a decrease in TfR mRNA levels in uninduced MEL cells. Thus, these studies indicate that TfR gene expression is regulated differently in hemoglobin synthesizing as compared to uninduced MEL cells.
Isolation and expression of a gene specifying a cdc2‐like protein kinase from the human malaria parasite Plasmodium falciparum
Authors: ROSS‐MACDONALD Petra B. , GRAESER Ralph , KAPPES Barbara , FRANKLIN Richard , WILLIAMSON Donald H.
Published: March 2005

A partially redundant oligonucleotide based on conserved protein sequences of cdk and cdc2‐like proteins was used to isolate from genomic libraries of Plasmodium falciparum fragments of chromosome XIII carrying a 288‐residue open‐reading frame encoding a protein kinase sharing 57–58% identity with yeast p34cdc2. Based on sequence data, base composition and the striking similarity with other cdk and related proteins, four intervening sequences were identified. Their removal in vitro allowed expression of the gene, designated PfPK5, in Escherichia coli, the resulting product having kinase activity against casein and histone H1. Western blotting using a polyclonal antibody raised against the expressed protein showed that the kinase was located in the parasite's cytosol and was present in approximately constant amounts throughout the intra‐erythrocytic asexual reproductive stage of the life cycle. The PSTAIRE region of the PfPK5 protein differs at three sites from that of p34cdc2, and the gene failed to complement cdc2/28 yeast mutants. However, Western blotting showed that the gene was not expressed in yeast, so this does not eliminate the possibility that it is the malarial version of cdc2.
Human tumor necrosis factor mutants with preferential binding to and activity on either the R55 or R75 receptor
Authors: VAN OSTADE Xaveer , VANDENABEELE Peter , TAVERNIER Jan , FIERS Walter
Published: March 2005

Previously, we reported that the cytotoxic activity of human (h) tumor necrosis factor (TNF) on murine (m) L929 cells requires the integrity of three loops (positions 30–36, 84–88 and 138–150) which cluster around the interface between each two subunits of the trimeric hTNF structure. The collection of hTNF mutants was further characterized by their activity on various human cell systems as well as by their binding to the two types of hTNF receptor (R), R55 and R75. It turned out that two amino acids (Leu29 and Arg32) were specifically involved in hR75 binding, as Leu29→Ser (L29S) and Arg32→Trp (R32W) mutant molecules had largely lost binding to hR75, but not to hR55. In order to screen for more highly R55‐specific mutants, nine other amino acids were inserted at these two positions; only the substitutions L29G and L29Y showed an increased differential binding as compared to L29S, while no further improvement was found with mutations at position 32 compared to R32W. Biological assays mediated either by hR55 or hR75 confirmed the results obtained by physical binding to purified receptors. A similar substitution in mTNF, Arg32→Tyr, also resulted in a preferential loss of binding to hR75 and a large decrease in mR75‐mediated bioactivity. Except for the double mutant L29S‐R32W, all other tested amino acid substitutions in the loops at positions 30–36 or 84–88 of hTNF led to a substantial loss of affinity for both receptors and a concomitant reduction of biological activity. In the loop at positions 138–150, the non‐conservative replacement of Glu by Lys at position 146 (E146K) resulted in an even lower binding to R75 as compared to R32W, while binding on and bioactivity through R55 was only slightly reduced. Remarkably, a reversed differential binding was observed after substitution at position 143 in hTNF; replacing Asp by non‐conservative residues such as Tyr, Phe or Asn resulted in a much larger decrease in binding to R55 than to R75. In conclusion, receptor‐specific mutants such as R32W, E146K and D143N can be used to study the function either of R55 or R75 on different human cell types. In vivo, we presume that the R55‐specific mutants will retain antitumor activity in the absence of R75‐dependent, severe side effects.
Isolation of transcription factor IIIC from Dictyostelium discoideum
Authors: BUKENBERGER Monika , DINGERMANN Theodor , MEISSNER Wolfgang , SEIFART Klaus H. , WINCKLER Thomas
Published: March 2005

Transcription factor IIIC (TFIIIC) binds in a sequence‐specific manner to RNA‐polymerase‐III‐transcribed genes (e.g. tRNA genes). It sequesters other transcription factors into the preformed complex, thereby activating transcription by RNA polymerase III. The Dictyostelium discoideum homologue of TFIIIC was highly purified by affinity chromatography based on its tDNA‐binding activity. This TFIIIC homologue is a multicomponent factor (molecular mass 380 kDa), which binds to the B‐box element of the internal tRNA gene promoter without significant A‐box interaction. Partially purified D. discoideum TFIIIC is able to functionally complement a human RNA polymerase III in vitro transcription system depleted of human TFIIIC. We provide evidence that partially purified D. discoideum TFIIIC interacts in vitro with gene‐external B‐box elements present downstream of many D. discoideum tRNA genes.
Characterization of a 100‐kDa heat‐stable microtubule‐associated protein from higher plants
Authors: VANTARD Marylin , PETER Christine , FELLOUS Arlette , SCHELLENBAUM Paul , LAMBERT Anne‐Marie
Published: March 2005

In higher‐plant cells, the different cell‐cycle‐dependent microtubule arrays are involved in a wide range of activities including chromosome segregation, cell‐plate formation and cellulose microfibril distribution and orientation. A wealth of data, obtained using animal cells, has indicated that the differential stability and function of microtubules during cell‐cycle and/or differentiation could be primarily regulated by selective microtubule‐associated proteins (MAP). Compared to animal MAP, our knowledge of plant MAP is so far very limited. In this study, we have identified a maize heat‐stable protein with apparent molecular mass 100 kDa (P‐100) which binds to taxol‐stabilized neurotubules and copolymerizes in vitro with purified neural tubulin. Moreover, P‐100 cross‐reacts with affinity‐purified · antibodies like a maize 83‐kDa putative MAP described previously [Vantard, M., Schellenbaum, P., Fellous, A. & Lambert, A. M. (1991) Biochemistry 30, 9334–9340]. Polyclonal antibodies directed against P‐100 were obtained and indicated that this protein is found in diverse higher‐plant cultured cells suggesting the ubiquitous nature of this protein. P‐100 can be phosphorylated in vitro by protein kinases present in a maize cytosol extract. Together, our data suggest that P‐100 could be a higher plant MAP.
Control of cellular iron homeostasis by iron‐responsive elements in vivo
Authors: WARD Roberta J. , KÜHN Lukas C. , KALDY Pierre , FLORENCE Anne , PETERS Timothy J. , CRICHTON Robert R.
Published: March 2005

It has recently been proposed that cellular iron homeostasis in mammalian cells is regulated at the post‐transcriptional level by the reciprocal control of transferrin receptor and ferritin mRNA expression via an iron‐regulatory factor. This iron‐regulatory factor has been shown to be a cytoplasmic aconitase which can bind to iron‐responsive elements in the corresponding mRNAs with greater or lesser affinity as a function of the iron status of the cell. In the present study, we show that in vivo the affinity of iron‐regulatory factor for iron‐responsive elements in liver reflects the long‐term iron status of the tissue in animal models for iron overloading and iron deficiency, when combined with altered transferrin saturation and serum iron levels. In contrast hepatic iron overload achieved without altering such haematopoeitic indices, had a less pronounced effect. In both spleen and heart, the affinities of iron‐regulatory factor changed in parallel with both altered iron status and haematological markers. In brain and duodenum, there were no consistent changes in iron‐regulatory‐factor activity with iron loading or depletion. Iron‐regulatory‐factor activity in kidney responded in an as yet unexplained manner.
Analysis of genes encoding highly conserved lysine‐rich proteins in Aplysia californica and Saccharomyces cerevisiae
Authors: AUCLAIR Daniel , LANG B. Franz , FOREST Pierre , DESGROSEILLERS Luc
Published: March 2005

To isolate a gene that can be used as an internal control in studies on gene expression in Aplysia californica neurons, we have characterized a cDNA clone (pKRP‐A) isolated on the basis of its high expression in A. californica neurons. This cDNA is of 850 nucleotides and codes for a putative 29‐kDa lysine‐rich protein. Blotting experiments revealed that the gene is expressed in all tested A. californica tissues, and in individually identified neurons of the abdominal ganglion, suggesting that this gene can be efficiently used as internal control in studies of gene expression. We have also isolated one cDNA and two different genomic clones from yeast libraries that show 59% identity with pKRP‐A. Sequence comparison of genomic clones, as well as PCR and Southern blotting experiments, revealed that at least two homologous genes are present in yeast. Northern blotting experiments revealed that the expression of the gene is strongly repressed at 39°C.
Receptor‐stimulated guanine‐nucleotide‐triphosphate binding to guanine‐nucleotide‐binding regulatory proteins
Authors: KALDENBERG‐STASCH Sylvia , BADEN Michael , FESSELER Barbara , JAKOBS Karl H. , WIELAND Thomas
Published: March 2005

In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine‐nucleotide‐binding regulatory proteins, binding of the GTP analogues, guanosine 5′‐[γ‐thio]triphosphate, GTP[S], and guanosine 5′‐[β,γ‐imino]triphosphate, p[NH]ppG, and the regulation of binding by the formly‐peptide‐receptor agonist, fMet‐Leu‐Phe, were studied in membranes of differentiated HL‐60 cells. For fMet‐Leu‐Phe‐stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of fMet‐Leu‐Phe‐stimulated binding of GTP[S] and p[NH]ppG were similar for up to approximately 30 s. Thereafter, receptor‐stimulated binding of p[NH]ppG rapidly reached equilibrium, whereas the binding of GTP[S] proceeded further. At equipotent concentrations of p[NH]ppG and GTP[S], maximal fMet‐Leu‐Phe‐stimulated binding of GTP[S] was approximately twofold higher than that of p[NH]ppG. Finally, for half‐maximal receptor‐stimulated binding of GTP[S], approximately fivefold higher concentrations of both Mg2+ and GDP were required than for p[NH]ppG binding. With p[NH]ppG as the competing nucleotide, the extent of receptor‐stimulated binding of GTP[S] as well as its Mg2+ requirement and time course were similar to the receptor‐stimulated p[NH]ppG binding observed in the presence of GDP. However, with GTP[S] as the competing nucleotide, fMet‐Leu‐Phe reduced the binding of p[NH]ppG, a reaction further enhanced when GDP was additionally present. Under similar conditions as used in the binding studies, GTP[S] thiophosphorylated a 35‐kDa protein, which is most likely a guanine‐nucleotide‐binding regulatory protein β subunit [Wieland, T., Nürnberg, B., Ulibarri, I., Kaldenberg‐Stasch, S., Schultz, G. & Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111–18118]. The thiophosphorylation state of this protein was regulated by guanine nucleotides, Mg2+ and, most importantly, by activated formlypeptide receptors. The data thus provide evidence for an essential difference between GTP[S] and p[NH]ppG binding to guanine‐nucleotide‐binding regulatory proteins and suggest that, in addition to the nucleotide‐exchange reaction, a (thio)phosphate‐group‐transfer process via guanine‐nucleotide‐binding regulatory protein β subunits is involved in the receptor‐stimulated binding of guanine nucleotide triphosphates to guanine‐nucleotide‐binding regulatory proteins.
Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting
Authors: LUMB Michael J. , PURDUE P. Edward , DANPURE Christopher J.
Published: March 2005

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species‐specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group).

Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern‐blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45508 Da. The N‐terminal 22 amino acids comprised the putative mitochondrial‐targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre‐mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase‐protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial‐targeting sequence is excluded from the open reading frame). In vitro studies showed that the 45 kDa polypeptide was imported into rat liver mitochondria and processed to a mature protein of ∼43 kDa, compatible with the cleavage of the N‐terminal 22 amino acids, as is also the case in rat and marmoset. A polypeptide in which the N‐terminal 22 amino acids was absent could not be imported into mitochondria in vitro.
Cloning, sequencing and expression of the gene encoding the coenzyme B12‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli
Authors: BEATRIX Birgitta , ZELDER Oskar , LINDER Dietmar , BUCKEL Wolfgang
Published: March 2005

The coenzyme B12 (adenosylcobalamin)‐dependent 2‐methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2‐methyleneglutarate to (R)‐3‐methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri.

(a) The mgm gene encoding 2‐methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8‐kDa protein of 614 amino acids. It shows significant similarity in its C‐terminal part to that of other cobamide‐dependent enzymes. Probably, this is the coenzyme‐binding region.

(b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/PAGE and Western‐blot analysis with rabbit antiserum directed against the native mutase.

(c) Cell‐free extracts from E. coli carrying the mgm gene showed 2‐methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B12. Experiments are presented which suggest that the expression product is an apoenzyme.
Functional properties of a hybrid vacuolar H+‐ATPase in Saccharomyces cells expressing the Nephrops 16‐kDa proteolipid
Authors: HARRISON Michael A. , JONES Philip C. , KIM Yong‐In , FINBOW Malcolm E. , FINDLAY John B. C.
Published: March 2005

The hydrophobic 16‐kDa polypeptide which forms gap‐junction‐like structures in the crustacean Nephrops norvegicus is a member of a highly conserved family of proteolipids involved in a variety of membrane transport functions in eukaryotic cells. This family also includes the product of the Saccharomyces cerevisiae VMA3 gene which encodes an integral membrane component of the vacuolar membrane H+‐ATPase. The cDNA for the Nephrops proteolipid complements a mutation in the yeast VMA3 gene, resulting in assembly of a hybrid H+‐ATPase comprising yeast catalytic subunits and Nephrops integral membrane components. The hybrid vacuolar ATPase was capable of ATP hydrolysis which was coupled to proton translocation and showed inhibitor binding and enzymological properties similar to those of wild‐type V‐ATPases (Km for ATP, 0.4 mM), suggesting that both yeast and crustacean proteolipids share conserved structure at regions of protein interaction.

To facilitate isolation of the Nephrops proteolipid by affinity chromatography on a Ni2+‐binding support, six C‐terminal histidine residues were added to the proteolipid. This modification did not prohibit assembly into the hybrid H+‐ATPase, although the resultant enzyme did have a markedly elevated Km (1.8 mM). The membrane‐bound Vo sector of the ATPase was isolated by the affinity‐chromatography procedure and reconstituted into synthetic vesicles. This complex was found to be impermeable to small cations in the absence of catalytic ATPase subunits either in situ in the vacuolar membrane or in the reconstituted system. The functional significance of this impermeability and the structure/function relationships between proteolipids from different sources are discussed.
Recombinant anti‐sialidase single‐chain variable fragment antibody
Authors: KORTT Alexander A. , MALBY Robyn L. , CALDWELL J. Bruce , GRUEN L. Clem , IVANCIC Neva , LAWRENCE Michael C. , HOWLETT Geoffrey J. , WEBSTER Robert G. , HUDSON Peter J. , COLMAN Peter M.
Published: March 2005

The single‐chain antibody variable fragment (scFv), with a 15‐residue polypeptide linker (Gly4Ser)3, of monoclonal antibody NC10 was expressed in Escherichia coli and purified to homogeneity. This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4° and 20°C. At higher protein concentrations (∼ 5 mg/ml) dimer and higher‐molecular‐mass multimers were formed and freezing enhanced this aggregation. The dimer was not stable and dissociated to monomer at 20°C with a half‐life of approximately 8 days. The higher‐molecular‐mass multimers and dimer dissociated to monomer in 60% ethylene glycol. Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules bound/sialidase tetramer and that the dimer was bivalent and cross‐linked two sialidase tetramers. Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NC10 antigen‐binding fragment (Fab) molecule. A complex between tern N9 sialidase and NC10 scFv was crystallized and the structure of the complex was solved at 0.3‐nm resolution by X‐ray diffraction. Comparison of this scFv/sialidase structure with the parent Fab/sialidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar. There was no discernible electron density for the peptide linker joining the variable heavy (VH) and variable light (VL) chains. A close interaction between two symmetry‐related scFv suggests that they may have crystallized as dimers.
The structure of the O‐specific chain of Legionella pneumophila serogroup 1 lipopolysaccharide
Authors: KNIREL Yuriy A. , RIETSCHEL Ernst Th. , MARRE Reinhard , ZÄHRINGER Ulrich
Published: March 2005

The O‐polysaccharide chain of Legionella pneumophila Philadelphia strain 1 (serogroup 1) lipopolysaccharide was investigated by means of 1H‐ and 13C‐NMR spectroscopy, and chemical analysis. It was found to consist of an α‐(2→4) interlinked homopolymer of a 5‐acetamidino‐7‐acetamido‐8‐O‐acetyl‐3,5,7,9‐tetradeoxy‐nonulosonic acid possessing most likely the d‐glycero‐l‐galacto configuration, representing the first example of an acidic homopolymer of a higher sugar of this class. The ladder‐like banding pattern exhibiting small distances between individual bands in the SDS/PAGE is compatible with a monosaccharide repeating unit.
Position‐independent, high‐level, and correct regional expression of the rat aldolase C gene in the central nervous system of transgenic mice
Authors: ARAI Yuji , KAJIHARA Susumu , MASUDA Junichi , OHISHI Sachiko , ZEN Katsuhiro , OGATA Jun , MUKAI Tsunehiro
Published: March 2005

Aldolase C is mainly expressed in the central nervous system (CNS). To clarify the regulatory mechanisms for the CNS‐specific expression, transgenic mice were created using two constructs of the rat aldolase C gene. A fusion gene comprising the 5′ regulatory region of the aldolase C gene was expressed in a CNS‐specific manner. However, the expression levels and the cellular localization of the gene varied among transgenic mice. The other construct, including both 5′ and 3′ regulatory regions of the gene, showed position‐independent and high‐level expression as well as the correct regional distribution in the CNS. These results indicate that the 13‐kb sequence of the rat aldolase C gene contains sufficient information for faithful expression of the gene.
Mechanism of inhibition of trypanothione reductase and glutathione reductase by trivalent organic arsenicals
Authors: CUNNINGHAM Mark L. , ZVELEBIL Marketa J. J. M. , FAIRLAMB Alan H.
Published: March 2005

The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione in mammalian systems, has been shown to interact with anti‐trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p‐(4,6‐diamino‐s‐triazin‐2‐yl)aminophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo‐first‐order kinetics with kinact of 14.3×10−4 s−1 and 1.06×10−4 s−1 and Ki of 17.2 μM and 9.6 μM for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time‐dependent loss of the characteristic charge‐transfer absorbance band at 530 nm is observed upon addition of arsenical to pre‐reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH4 form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme‐inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur‐arsenic bond leads to the binding of the arsenical to the charge‐transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide‐binding sites.

Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.