bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Type of Ca2+‐Dependent, Mg2+‐Stimulated ATPase of Rat Liver Plasma Membrane
Authors: Heilmann Claus , Spamer Cornelia , Mössner Wolfgang , Dietz Christine , Reutter Werner , Kreisel Wolfgang
Published: March 2005

Incubation of a glycoprotein fraction obtained from rat liver plasma membrane which has been previously well characterized using [γ‐32P]ATP results in the phosphorylation of a 230‐kDa glycoprotein (pgp230). It is composed of a 120‐kDa subunit (pgp120) and a 110‐kDa subunit (pgp110) linked by interchain disulfide bonds. Peptide maps of pgp120 and pgp110 suggest extensive similarity in their polypeptide chains. Glycan analysis reveals between four and six hybrid‐type oligosaccharide chains for both phosphoproteins. Immunoblotting using monoclonal antibodies and endoglycosidase digestion exclude an identity of pgp120 or pgp110 with the hepatocyte plasma membrane glycoproteins dipeptidylpeptidase IV or the taurocholate transport protein, which co‐purify and comigrate in SDS/PAGE. Protein phosphorylation is ca2+‐dependent (K0.5(Ca2+)= 0.35 μM, in the absence of Mg2+). In the presence of Mg2+, the glycoprotein undergoes rapid cycles of phosphorylation and dephosphorylation, resulting in ATPase activity. Analysis of phosphorylated amino acids identifies phosphothreonine as the major one. Photoaffinity labeling with 8‐azido‐[α‐32P]ATP demonstrates the presence of one or more ATP binding site(s). Preincubation of pgp230 with various purine or pyrimidine nucleotides (ATP, UTP, TTP, ADP, GDP, AMP, CMP) or known P2‐purinoceptor agonists or antagonists (adenosine 5′‐[α,β‐methylene]triphosphate, 2‐methyl‐thio‐adenosine 5′‐triphosphate, suramin) inhibits its phosphorylation by [γ‐32P]ATP.

The biological function of pgp230 is unknown at present. Several findings of the present study are compatible with the idea that pgp230 may be involved in a P2‐purinoceptor function of the hepatocyte. Following this concept, a mechanism is discussed where a cytosolically exposed high‐affinity Ca2+‐binding site of pgp230 would allow for receptor feedback control, via phosphorylation and dephosphorylation, by sensing changes in cytosolic Ca2+ concentration.
Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from Alicyclobacillus Acidocaldarius Atcc 27009
Authors: Schwermann Birgit , Pfau Karsten , Liliensiek Birgit , Schleyer Manfred , Fischer Thomas , Bakker Evert P.
Published: March 2005

The α‐amylase from the thermoacidophilic eubacterium Alicyclobacillus (Bacillus) acidocaldarius strain ATCC 27009 was studied as an example of an acidophilic protein. The enzyme was purified from the culture fluid. On an SDS/polyacrylamide gel, the protein exhibited an apparent molecular mass of 160 kDa, which is approximately 15% higher than that predicted from the nucleotide sequence. The difference is due to the enzyme being a glycoprotein. Deglycosylation or synthesis of the enzyme in Escherichia coli gave a product with the mass expected for the mature protein. The amylase hydrolyzed starch at random and from the inside, and its main hydrolysis products were maltotriose and maltose. It also formed glucose from starch (by hydrolysing the intermediate product maltotetraose to glucose and maltotriose) and exhibited some pullulanase activity. The pH and temperature optima were pH 3 and 75°C, respectively, characterizing the enzyme as being thermoacidophilic. Alignment of the sequence of the enzyme with that of its closests neutrophilic relatives and with that of α‐1,4 or α‐1,6 glycosidic‐bond hydrolyzing enzymes of known three‐dimensional structure showed that the acidophilic α‐amylase contains approximately 30% less charged residues than do its closests relatives, that these residues are replaced by neutral polar residues, and that hot spots for these exchanges are likely to be located at the surface of the protein. Literature data show that similar effects are observed in three other acidophilic proteins. It is proposed that these proteins have adapted to the acidic environment by reducing the density of both positive and negative charges at their surface, that this effect circumvents electrostatic repulsion of charged groups at low pH, and thereby contributes to the acidostability of these proteins.
The Modulation of Enzyme Reaction Rates within Multi‐Enzyme Complexes
Authors: Ricard Jacques , Giudici‐Orticoni Marie‐Thérèse , Gontero Brigitte
Published: March 2005

There is now experimental evidence that association of different enzymes as a multi‐enzyme complex may result in an alteration of the catalytic properties of the enzymes present in this complex. This effect is not related to the channelling of reaction intermediates between different active sites. It appears as a consequence of an information transfer that occurs within the multi‐enzyme complex.

A theory, based on statistical thermodynamics, has been developed which provides an understanding, on a physical basis, for how isologous as well as heterologous interactions between identical, or different, enzymes of the complex may modulate the catalytic properties of an oligomeric enzyme of that complex. The theory predicts three possible types of effects: an alteration, through heterologous interactions, of an already existing cooperativity of the oligomeric enzyme within the complex; a co‐operativity, generated by heterologous interactions in the complex that could not occur if the oligomeric enzyme were isolated from the rest of the complex; a Michaelis‐Menten character of the oligomeric enzyme within the complex, but with altered values of Vm and Km relative to what would have been observed with the naked enzyme.

All these effects appear as a consequence of a transfer of information between different enzymes of the same multi‐protein complex. The following paper in this journal shows how one can demonstrate and characterize experimentally these effects in a multi‐enzyme complex containing ribulose bisphosphate carboxylase‐oxygenase.
Physico‐chemical and Spectroscopic Properties of the Monohemic Cytochrome C552 from Pseudomonas nautica 617
Authors: Saraiva Lígia M. , Fauque Guy , Besson Stéphane , Moura Isabel
Published: March 2005

A c‐type monohemic ferricytochrome c552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2‐terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome c552 has histidine‐methionine as axial ligands, and a pH‐dependent mid‐point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two‐dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two‐dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome c552 is typical of a low‐spin ferric heme.
Secondary Structure and Membrane Interaction of PR‐39, a Pro+Arg‐rich Antibacterial Peptide
Authors: Cabiaux Véronique , Agerberths Birgitta , Johansson Jan , Homblé Fabrice , Goormaghtigh Erik , Ruysschaert Jean‐Marie
Published: March 2005

PR‐39 is a 4719‐Da peptide isolated from pig intestine and belonging to the recently discovered family of Pro + Arg‐rich antibacterial peptides. PR‐39 does not lyse Escherichia coli, instead the lethal action is probably linked to the termination of DNA and protein synthesis [Boman, H. G., Agerberth, B. & Boman, A. (1993) Infect. Immun. 61, 2978–2984].

Circular dichroism and Fourier‐transform infrared spectroscopy have been used to investigate the secondary structure of PR‐39 in the absence or presence of lipids. According to the circular dichroic data, this structure is not altered upon incubation of PR‐39 with negatively charged vesicles, although the infrared spectra suggest that the hydrogen bond pattern is modified upon the peptide—lipid interaction. This is detected by a shift in the maximum wavelength of absorption of PR‐39 from 1636 cm−1 in the absence of lipids to 1645 cm−1 in the presence of lipids. We have further addressed the question of the possible mechanism of interaction of PR‐39 with model membranes (liposomes and planar lipid bilayers) whose lipid compositions mimick that of the E. coli inner membrane. PR‐39 induced a calcein release from large unilamellar vesicles, which is dependent upon the peptide concentration and upon the presence of negatively charged lipid (glycerophosphoglycerol) in the membrane. The binding study of PR‐39 to dioleoylglycerophosphoglycerol vesicles suggests that nearly 100% of the added peptide is membrane‐bound. Addition of PR‐39 to a planar lipid bilayer induced a linear increase in the current but no channel formation was observed since no discrete steps of conductance occurred.
Chemical Modification of Tyr34 and Tyr129 in Rabbit Liver Microsomal Cytochrome b5 Affects Interaction with Cytochrome P‐450 2B4
Authors: Hlavica Peter , Kellermann Josef , Golly Ines , Lehnerer Michael
Published: March 2005

Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 molecule were found to be accessible to the nitrating agent. Co‐modification of tryptophan and histidine residues could be disregarded. CD‐spectral measurements disproved gross changes in cytochrome b5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P‐450 2B4 (P‐450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b5 to shift the spin equilibrium to the high‐spin conformer of P‐4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH‐dependent electron flow to ferric P‐450 2B4. Changes in the redox potential of cytochrome b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4‐nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 in the presence of a protective amount of P‐450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is proposed that Tyr129 might control docking of cytochrome b5 to P‐450 2B4, whereas Tyr34 could be of functional importance in electron transfer.
The Carbon Monoxide Stretching Modes in Camphor‐Bound Cytochrome P‐450cam
Authors: Schulze Heike , Ristau Otto , Jung Christiane
Published: March 2005

The effect of pH, monovalent cations, glycerol, temperature, and pressure on the carbonmonoxy (CO) stretching mode of camphor‐bound cytochrome P‐450cam (CYP 101) was studied. Two effects, band overlap and frequency shift, have been observed. The CO stretch infrared band located at about 1940 cm−1 is asymmetric because of the overlap of three bands at about 1931 cm−1, 1939 cm−1, and 1942 cm−1 with strongly different populations. Reducing the temperature or increasing the pressure leads to splitting the band or switching the asymmetry from the lower energy side to the higher energy side of the infrared band. The overlap of several CO stretch bands indicates conformational substates within the heme pocket. A frequency shift of the predominately populated band is observed by changing all the parameters mentioned. The pH‐induced frequency shift follows an S‐shape with the pK at 6.2, which matches the pK observed for the pH‐induced high‐spin/low‐spin transition. Conformational changes on the proximal heme side are suggested to be the origin. Monovalent cations at saturating concentration induce a small frequency shift depending on the ion radius. The potassium ion is the one that induces a CO stretch frequency with the highest wave‐number while sodium and lithium (smaller radii) and rubidium and caesium ion (larger radii) have diminished values, which is supporting evidence for the special function of the potassium ion within the structure. Glycerol and hydrostatic pressure induce a red shift of the CO stretching frequency. Forced contact of the polar hydroxyl group of Thr252 of the I helix induced by pressure and indirectly by glycerol is suggested to change the CO dipole moment, reflecting in the decreased CO stretching frequency.
Chlorocatechol 1,2‐Dioxygenase from Rhodococcus Erythropolis 1CP
Authors: Maltseva Olga V. , Solyanikova Inna P. , Golovleva Ludmila A.
Published: March 2005

Chlorocatechol 1,2‐dioxygenase from Rhodococcus erythropolis 1CP was purified to homogeneity. In contrast to chlorocatechol 1,2‐dioxygenase from Gram‐negative strains which have a very broad substrate tolerance, the Rhodococcus enzyme was relatively more specific and had a distinct preference for 4‐substituted catechols. Protein and metal analysis indicate an unusual stoichiometry of one atom each of iron and manganese/64‐kDa homodimer. The N‐terminal amino acid sequence (27 residues) of the Rhodococcus chlorocatechol 1,2‐dioxygenase was determined and exhibited 15–22% identity to the published sequences of catechol 1,2‐dioxygenases and other chlorocatechol 1,2‐dioxygenases. Antiserum was raised in rabbits and antibodies against Rhodococcus chlorocatechol 1,2‐dioxygenase were affinity purified. Dot‐blot analysis revealed a very weak reaction between the antibodies and partially purified chlorocatechol 1,2‐dioxygenases from Alcaligenes eutrophus JMP134 and Pseudomonas putida 87. No reaction between these antibodies and above enzymes was observed using Western blotting. Kinetic and immunochemical data as well as comparison of subunit molecular mass and suggest that the Rhodococcus enzyme differs significantly from the known highly similar chlorocatechol 1,2‐dioxygenases of Gram‐negative strains and seems to be only distantly related to them.
Higher Plant Light‐Harvesting Complexes LHCIIa and LHCIIc are Bound by Dicyclohexylcarbodiimide During Inhibition of Energy Dissipation
Authors: Walters Robin G. , Ruban Alexander V. , Horton Peter
Published: March 2005

We have investigated the binding to proteins of the photosynthetic apparatus of the carboxy‐modifying agent dicyclohexylcarbodiimide, (cHxN)2C; this inhibits the protective dissipation of excess absorbed light energy (qE) by the light‐harvesting apparatus of photosystem II (LHCII), suggesting that carboxyl amino‐acid side chains within hydrophobic protein domains may be involved in qE. (cHxN)214C was used to label thylakoids and photosystem II particles, so as to identify proteins which may be involved in the detection of lumen pH during qE induction. Of six thylakoid proteins labelled with (cHxN)2C under conditions where qE is efficiently induced, three are associated with photosystem I, and none with the bulk LHCII. PSII‐associated label is bound to three minor components of LHCII, identified as LHCIIa (two species) and LHCIIc, as shown by protein sequencing of tryptic fragments of purified complexes. pH titration of qE formation and protein labelling in coupled thylakoids showed that both qE and labelling of LHCIIa increased at pH 7–8.
A Novel Class of Adhesion Acidic Glycans in Sea Urchin Embryos
Authors: Papakonstantinou Eleni , Karakiulakis George , Aletras Alexios J. , Misevic Gradimir N.
Published: March 2005

Total glycans were isolated and purified from Lytechinus pictus embryos at early developmental stages by gel‐filtration chromatography after pronase and DNase digestion, and alkali‐borohydride treatment. Fractionation by Superose 6 and HPLC gel‐filtration chromatography revealed three major glycan fractions of 580, 150 and 2 kDa consistently throughout development up to the stage of end gastrula. The 580‐kDa and the 150‐kDa glycan fractions isolated from fertilized eggs up to the stage of end gastrula are highly acidic, whereas the 2‐kDa glycan fractions have no detectable uronic acid residues and charged groups. Chemical analysis of the glycan fractions showed that their content of neutral hexoses, uronic acid, GlcNAc, GalNAc and sulphate changes during development. The resistance of the 580‐kDa and the 150‐kDa glycan fractions to glycosaminoglycan‐degrading enzymes indicates a structure which is different from the glycosaminoglycans. The incorporation of [3H]glucosamine into the 580‐kDa, the 150‐kDa and the 2‐kDa glycan fractions showed that glycan synthesis increases in a linear fashion from the stage of early blastulation to end of gastrulation. Maximal incorporation of the radioligad occurs in the 2‐kDa glycan fractions, with the highest rate observed between the stages of mesenchyme blastula and early gastrulation. Immunological studies, using a monoclonal antibody which inhibits cell aggregation, showed that the total glycans isolated from morula, early blastulation, early gastrulation and the end of gastrulation carry cell‐adhesion epitopes. The number of these epitopes, as indicated by the intensity of the immunostaining, increases from morula formation to end‐gastrulation stages and correlates with the increased rate of morphogenetic movements. These results suggest that controlled expression of the cell‐adhesion glycan epitopes play an important role in sea urchin gastrulation.
In vitro Approaches to Investigation of the Early Steps of Colicin‐Ompf Interaction
Authors: El Kouhen Rachid , Hoenger Andreas , Engel Andreas , Pagès Jean‐Marie
Published: March 2005

The OmpF porin plays a central role during the colicin uptake by sensitive Escherichia coli cells. Lipopolysaccharide‐OmpF complexes (‐1bLPS‐OmpF), which contain one tightly bound and no loosely bound LPS molecules for each porin trimer, is able to recognize and bind to immobilized colicins. This association is specific to colicins A and N, which both use the OmpF porin as receptor, and depends on the presence of the porin‐receptor domain on the bacteriocin molecule. The ‐1bLPS‐OmpF complex protects sensitive cells against colicin A and N activity. The protection level depends on the native conformation, as demonstrated by heat denaturation of the trimeric porin which abolishes the protection. This indicates that the purified OmpF trimer presents an affinity site for the colicin which efficiently mimics the native cellular receptor site. These results are discussed with regard to the conformation of the receptor site and to the early steps of colicin uptake.
The Efficiency of Nuclear Processing of the Tyrosine Aminotransferase mRNA Transcript Increases after Partial Hepatectomy
Authors: Baki Lia , Alexis Michael N.
Published: March 2005

Following a two‐thirds partial hepatectomy, an approximately fivefold increase in the levels of nuclear and total mRNA for tyrosine aminotransferase was observed at 1 h and 1.5 h, respectively, and a return to the levels of the quiescent state, i.e. the levels found in non‐operated livers from adrenalectomized rats, was established 16 h post‐hepatectomy. The increase in mRNA levels was not accounted for by a comparable change in the rate of transcription of the gene which, at 0.5 h post‐hepatectomy, reached a maximum value that amounted to only 1.4‐fold the value for quiescent liver. Subsequent changes in the transcription rate largely accounted for the changes in mRNA levels observed later on. Although tyrosine aminotransferase mRNA levels were equal in quiescent and 16‐h‐regenerating liver, the rate of transcription of the gene in quiescent liver was threefold higher than the rate in 16‐h‐regenerating liver. The maintenance of a higher rate of gene transcription in quiescent liver, as compared to regenerating liver, was shown to depend on ongoing protein synthesis. The possibility that the high rate of gene transcription was due to blockage or pausing during transcript elongation in quiescent liver was excluded. The inference is that the pronounced increase in tyrosine aminotransferase mRNA levels within 1 h of partial hepatectomy is largely due to a rapid increase in the efficiency of nuclear processing of the primary transcript.
A Protein Kinase Isolated from Porcine Brain Microvessels is Similar to a Class of Heat‐Shock Proteins
Authors: Dechert Ute , Weber Peter , König Bernd , Ortwein Claus , Nilson Iris , Linxweiler Winfried , Wollny Eric , Gassen Hans G.
Published: March 2005

To further characterize a protein kinase present in porcine brain microvessels, a cDNA library using porcine microvessel poly(A) RNA was screened with polyclonal antibodies raised against the native protein kinase. Since no full‐length cDNA clone could be obtained, the missing sequence information was completed using two subsequent polymerase chain reactions. The amplified transcripts were cloned and the sequence determined. Additionally, a genomic DNA library from porcine kidney was screened to substantiate the results obtained from the polymerase chain reaction. Earlier hints of a relation to a subclass of the family of heat‐shock proteins (HSPs) based upon a close sequence similarity at its amino‐terminus could be confirmed by comparison of the full‐length cDNA sequences. Common protein kinase consensus sequences, a targeting sequence for proteins of the endoplasmic reticulum at the carboxy‐terminus as well as a hydrophobic leader sequence in the amino‐terminal region of the protein could also be identified. Furthermore, a set of membrane‐associated substrate proteins of this enzyme could be detected in brain capillaries. The results indicate that at least some members of the HSP 90 subfamily undergo autophosphorylation and show protein kinase activity by phosphorylating substrate proteins in vitro.
Equilibrium Unfolding Studies of Horse Muscle Acylphosphatase
Authors: Taddei Niccolo' , Buck Matthias , Broadhurst R. William , Stefani Massimo , Ramponi Giampietro , Dobson Christopher M.
Published: March 2005

The stability and equilibrium unfolding behaviour of horse muscle acylphosphatase have been studied by denaturing the protein under various conditions of temperature, pH, and urea concentration. Far‐ultraviolet circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy indicate that this small monomeric protein unfolds reversibly and cooperatively. Thermodynamic parameters, the Gibbs free energy G and enthalpy H of unfolding, have been estimated for denaturation of the protein from NMR and CD data as 19 kJ mol−1 and 350 kJ mol−1, respectively. CD and 1H‐NMR results suggest the presence of very little persistent residual structure in the denatured states studied under these different conditions. Furthermore, photo‐chemically induced dynamic nuclear polarisation experiments show that in the denatured states aromatic residues are freely accessible to a flavin dye probe.
Calcium‐Induced Changes in Annexin V Behaviour in Solution as Seen by Proton NMR Spectroscopy
Authors: Neumann Jean‐Michel , Sanson Alain , Lewit‐Bentley Anita
Published: March 2005

The behaviour of human annexin V in the presence of calcium was studied by NMR. We observe the formation of well defined dimers, as well as a change in the local dynamics of one His side chain. We assign the observed changes to either His98 or His267 residues and conclude that they could be related either to the hinge‐bending motion reported from crystal structures, or to a local side chain rearrangement within the calcium‐binding loops concerned. Dimerization was also confirmed by a small‐angle neutron‐scattering experiment. Under the experimental conditions used, we do not observe the conformational change involving Trp187 seen in previous studies, which occurs at higher relative calcium concentrations.
Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors
Authors: Berlose Jean‐Philippe , Convert Odile , Brunissen Alié , Chassaing Gérard , Lavielle Solange
Published: March 2005

The S/T‐X1‐X2‐N‐P‐X3‐X4‐Y highly conserved sequence of the seventh transmembrane (TM VII) segment of G‐protein‐coupled receptors is not present in the photon receptor bacteriorhodopsin TM VII domain. Despite this noticeable discrepancy in sequence, the X‐ray structure of bacteriorhodopsin is generally used as the key structure for modelling all G‐protein‐coupled receptors. Thus, a kinked trans ‐Pro helix is usually accepted for the TM VII three‐dimensional structure of G‐protein‐coupled receptors, although Asn‐Pro dipeptide mainly induces a type I/III β‐turn conformation in both model peptides and proteins. NMR studies in various solvents and molecular calculations were undertaken in order to gain insight into the conformational behaviour of a 15‐residue peptide from the tachykinin NK‐1 TM VII domain incorporating this common sequence. The low solubility of this membrane‐embedded peptide precludes methanol or micellar systems mimicking membrane environment; thus only dimethylsulfoxide (Me2SO) or chlorofonn/Me2SO mixture could be used. We also found that perfluoro‐tert ‐butanol, which has not been previously used for NMR studies, constitutes an excellent alternative solvent for the analysis of hydrophobic peptides. The postulated kinked trans ‐Pro helix was only present as a minor conformer in Me2SO and an equilibrium between helical and extended structures existed. From NOE data a type I/III β‐structure, centered around Pro9‐Ile10, probably stabilized by an Asx turn, may be postulated. Addition of chloroform in Me2SO increased the percentage of folded structures but no preferential conformation could be proposed. In perfluoro‐tert ‐butanol/CD3OD (9:1) the N‐ and C‐terminal regions presented an α‐helical structure, and these two domains were linked by a hinge around Asn‐Pro with a γ‐turn for the preceding residue Tyr7 and either a type I/III β‐turn around Pro9–Ile10 or αR orientations for these residues, which are both stabilized by an Asx turn. As determined by energy calculations, these structures were equally as stable as the kinked trans ‐Pro helix and could constitute key structures for analysing the conformational changes and/or the dynamics of TM VII segment induced by the ligand when interacting with the receptor.
Proteolytic Cleavage of the Multienzyme Polypeptide CAD to Release the Mammalian Aspartate Transcarbamoylase
Authors: Hemmens Benjamin , Carrey Elizabeth A.
Published: March 2005

We have demonstrated biochemically that the conformation of the proteolytic fragment (mammalian aspartate transcarbamoylase) from the C‐terminus of the 240‐kDa multienzyme polypeptide carrying the activities carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase (CAD) is similar to that of the catalytic subunits from Escherichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidinium chloride solutions, and have also demonstrated that the protein cross‐reacts with antibodies raised against the E. coli enzyme.

CAD is digested by low concentrations of trypsin in the presence of 0.2 mM UTP to release an active aspartate transcarbamoylase domain and a 195‐kDa ‘nicked CAD’ molecule containing active carbamoyl phosphate synthetase. These two products are easily separated by ion‐exchange chromatography. Similar proteolytic cleavage and trimming by elastase releases a family of aspartate transcarbamoylase fragments. Direct N‐terminal sequencing of the aspartate transcarbamoylase fragments confirms predictions of the most accessible residues in the region linking the aspartate transcarbamoylase and dihydroorotase domains. Only the largest of the four fragments generated by elastase retains phosphorylation site 2. When this largest fragment is phosphorylated, the family of aspartate transcarbamoylase fragments is eluted together from ion‐exchange columns in a different fraction from the completely unphosphorylated preparation, demonstrating the affinity of the domains for each other.
In vitro Synthesis of CDP‐D‐Abequose Using Salmonella Enzymes of Cloned rfb Genes
Authors: Lindqvist Lennart , Schweda K. H. , Reeves Peter R. , Lindberg Alf A.
Published: March 2005

In vitro enzymic synthesis of CDP‐D‐abequose, CDP‐D‐[U‐14C]abequose, CDP‐6‐deoxy‐D‐xylo ‐4‐hexulose and CDP‐3,6‐dideoxy‐D‐xylo ‐4‐hexulose was achieved using enzymes from cell extracts of cultures of Escherichia coli strains harbouring and expressing genes of the rfb gene cluster of Salmonella enterica LT2. From an initial synthesis step, CDP‐6‐deoxy‐D‐xylo ‐4‐hexulose was isolated after 30 min reaction, using CDP‐D‐glucose, NAD and CDP‐glucose 4,6‐dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 90.6%). From that intermediate, CDP‐3,6‐dideoxy‐D‐xylo ‐4‐hexulose was produced in a reaction using NADH and a crude extract containing the required enzymes. CDP‐D‐abequose synthesis was performed either in the presence of excess NADH and NADPH or using an enzymic system which regenerates low concentrations of the coenzymes. In a two‐step reaction, CDP‐D‐glucose was first converted to CDP‐6‐deoxy‐D‐xylo ‐4‐hexulose, then, following addition of the required coenzymes and enzymes, CDP‐D‐abequose was formed from this intermediary product in a 1‐h incubation. Starting from 250 mg CDP‐D‐glucose, the molar yield of CDP‐D‐abequose after protein precipitation and HPLC was 82%, corresponding to more than 200 mg. CDP‐D‐[U‐14C]abequose was synthesised from α‐D‐[U14C]glucose 1‐phosphate and CTP using purified glucose‐1‐phosphate cytidylyltransferase in a reaction preceding the later steps. GC‐MS and NMR revealed that the hexose part of the end product was 3,6‐dideoxy‐D‐galactose (abequose) and that the corresponding intermediates were 4‐keto‐6‐deoxy‐D‐xylo ‐hexose and 4‐keto‐3,6‐dideoxy‐D‐xylo ‐hexose, respectively. The synthesized CDP‐6‐deoxy‐D‐xylo ‐4‐hexulose exhibited the characteristic ultraviolet light absorption at 318 nm but no corresponding absorption was found for CDP‐3,6‐dideoxy‐D‐xylo ‐4‐hexulose. A HPLC technique, where the four CDP‐sugars were baseline separated, was developed and used for enzyme assays and for the analysis of synthesized products.
Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top‐Down Elasticity Analysis
Authors: Kesseler Adolf , Brand Martin D.
Published: March 2005

The aim of this study was to identify the significant sites of action of cadmium on oxidative phosphorylation in potato tuber mitocondria. We simplified the system to three convenient subsystems linked via the production or consumption of a common intermediate, namely protonmotive force. The three subsystems were substrate oxidation, which produces protonmotive force, and the proton leak reactions and the phosphorylation reactions, which consume protonmotive force. By measuring the effect of cadmium on the kinetic response of each subsystem to protonmotive force (top‐down elasticity analysis), we found that cadmium stimulated proton leak reactions and strongly inhibited substrate oxidation, but had no measurable effect on the phosphorylation reactions. Cadmium therefore decreases the amount of ATP produced/oxygen consumed (the effective P/O ratio) not by inhibiting the phosphorylation reactions directly, but by inhibiting the production of protonmotive force and by diverting proton flux from phosphorylation reactions to the proton leak reactions.
Characterisation of D‐Arabinono‐1,4‐Lactone Oxidase from Candida albicans ATCC 10231
Authors: Huh Won‐Ki , Kim Seong‐Tae , Yang Kap‐Seok , Seok Yeong‐Jae , Hah Yung Chil , Kang Sa‐Ouk
Published: March 2005

D‐Erythroascorbic acid was detected from the cell extracts of a dimorphic fungus, Candida albicans. Its concentration in yeast cells grown at 25 °C was estimated to be about 0.45 μmol/ml cell water. D‐Arabinono‐1,4‐lactone oxidase, which catalyses the final step in the biosynthesis of D‐erythroascorbic acid, was purified 639‐fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Triton X‐100 solubilisation, ammonium sulphate precipitation, anion‐exchange, hydrophobic‐interaction, gel‐filtration and dye‐ligand chromatographies. Gel‐filtration chromatography and polyacrylamide‐gradient gel electrophoresis in the presence of deoxycholate gave apparent molecular masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only one protein band corresponding to a molecular mass of 66.7 kDa. Considering the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spectrum of a flavin‐containing enzyme. The flavin was not released by treatment with SDS, CCl3CO2H or boiling, indicating that it may be covalently bound to the enzyme protein. The enzyme was optimally active at 40°C and at pH 6.1. The enzyme was stable in the range pH 7.5–10. An apparent Km, value for D‐arabinono‐1,4‐lactone was 44.1 mM. L‐Galactono‐1,4‐lactone, L‐gulono‐1,4‐lactone and L‐xylono‐1,4‐lactone could also serve as substrates. Competitive inhibition was demonstrated with D‐glucono‐1,5‐lactone, L‐arabinono‐1,4‐lactone, D‐galactono‐1,4‐lactone and D‐gulono‐1,4‐lactone. P‐Chloromercuribenzoate, N ‐ethylmaleimide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd2+, Hg2+, Mn2+ and Zn2+ exhibited inhibitory effects on the enzyme.
Alcohol Dehydrogenase Class III Contrasted to Class I
Authors: Danielsson Olle , Shafqat Jawed , Estonius Mats , Jörnvall Hans
Published: March 2005

Alcohol dehydrogenases of classes I (the classical liver enzyme) and III (formaldehyde dehydrogenase) constitute a pair of moderately related enzymes (63% residue identity between the human forms) that differ fundamentally in many respects. To elucidate the nature of the differences, we have characterized alcohol dehydrogenase from the most primitive vertebrate line (a cyclostome, Atlantic Hagfish), related that to the multiplicity of the human enzyme, and submitted the enzymes to in vitro hybridization for evaluation of subunit interactions. Three findings illustrate important principles of the enzyme system. First, the alcohol dehydrogenase purified from cyclostomes is a class‐III protein, compatible with the facts that cyclostomes constitute the earliest extant vertebrate line and that class III has a distant pre‐vertebrate origin. Second, the hagfish enzyme shows multiplicity, with acidic forms in decreasing yield and with amino acid sequences identical between two major isoforms, both aspects constituting properties similar to those of the corresponding human forms. The chemically different subunits are present as homodimers and heterodimers of unmodified and modified subunits, suggesting that the class‐III multiplicity derives from modification of a type common to lines as divergent as mammals and cyclostomes. Third, the human enzyme can form cross‐species hybrid dimers in vitro with the cod and hagfish or Drosophila class‐III enzymes (positional identity with the human form of 82, 76 and 70%, respectively). Hence, the results provide experimental evidence for little class‐III divergence in the segments of subunit interactions. The extent of conservation of residues directly involved in the formation of the subunit interface also reveals a clearly different pattern between classes I and III. This highlights separation of divergent forms in an enzyme system, with the constant form (class III) resembling house‐keeping enzymes, and exhibiting a correlation between subunit‐interacting and substrate‐interacting segments.
Olfactory Receptor Proteins
Authors: Gat Uri , Nekrasova Elina , Lancet Doron , Natochin Michael
Published: March 2005

A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation with a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N‐terminus of the open reading frame, and a specific polyclonal antibody against a C‐terminal peptide of olp4. Translation in vitro, followed by immunoprecipitation, showed a major olp4‐specific band of 27–29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag‐olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag‐olp4 product on the cell surface of the infected host Sf9 cells, with the N‐terminus and C‐terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.
Structural and Functional Properties of Plasma Membranes from the Filamentous Fungus Penicillium chrysogenum
Authors: Hillenga Dirk J. , Versantvoort Hanneke J. M. , Driessen Arnold J. M. , Konings Wil N.
Published: March 2005

Functional plasma membranes from the filamentous fungus Penicillium chrysogenum have been isolated with the objective of studying transport processes. The isolation procedure consists of three steps, namely homogenization of cells with a Braun MSK homogenizer, followed by Percoll gradient centrifugation and floatation of membranes in a three‐step Nycodenz gradient. This method can be applied to strains which differ significantly in morphology and penicillin‐production capacity. Plasma membranes were fused with liposomes containing the beef heart mitochondria1 cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes maintained a high proton motive force that functions as a driving force for the uptake of the amino acids arginine and valine via distinct transport systems.
Binding Properties of the Coagulation Factor IX/factor X‐binding Protein Isolated from the Venom of Trimeresurus flavoviridis
Authors: Atoda Hideko , Yoshida Naoko , Ishikawa Midori , Morita Takashi
Published: March 2005

The binding properties of the coagulation factor IX/factor X‐binding anticoagulant protein (IW/X‐bp) isolated from the venom of Trimeresurus fluvoviridis (habu snake) were investigated with an enzyme‐linked immunosorbent assay. The half‐maximal binding and maximal binding of IX/X‐bp to both factors IX and X were observed at concentrations of Ca2+ ions of 0.4 mM and 1 mM, respectively. Concentration of IX/X‐bp at half‐maximal binding to solid‐phase bovine factor IX and solid‐phase bovine factor X were 0.4 ± 0.1 nM and 1.1 ± 0.4 nM, respectively, in the presence of 1 mM Ca2+ ions. The kinetics of binding activity of IX/X‐bp to bovine factors IXa and Xa and to human factors IX and X resembled those of the binding to bovine factors IX and X. IX/X‐bp did not bind to solid‐phase coagulation factors other than factor IX/IXa and factor X/Xa, for example, prothrombin, factor VII, protein C, and protein Z, under the conditions of the experiment. To localize the binding sites of IX/X‐bp on the coagulation factors, the ability of IX/X‐bp to bind to various fragments derived from factors IX and X was examined. The binding of IX/X‐bp to solid‐phase factor IX was inhibited by a peptide containing the 4‐carboxyglutamic acid (Gla) domain derived from factor IXaβ′ (residues 1–42) in the liquid phase, but the binding was not inhibited by Gla‐domainless factor IXaβ′. Half‐maximal binding of IX/X‐bp to solid‐phase Gla‐domain peptide of factor IX occurred at 9.2 ± 1.9 nM. Factor X was partially reduced and the S ‐carboxymethylated light and heavy chains of factor X were prepared. IX/X‐bp bound to the S‐carboxymethylated light chain of factor X but not to the heavy chain. The binding of IX/X‐bp to solid‐phase factor X was inhibited by the Gla‐domain peptide of factor X (residues 1–44) but not by Gla‐domainless factor X. IX/X‐bp bound to PCGFX, a recombinant human protein C whose Gla‐domain region (residues 1–43) had been replaced by residues 1–43 of human factor X. The affinity of binding was about one tenth of that to intact human factor X. IX/X‐bp was unable to bind at all to human protein C. These data indicate that IX/X‐bp is a protein that binds to the Gla‐domain regions of factors IX and X in the presence of Ca2+ ions.
Chemical Structure of the Core Region of Escherichia coli J‐5 Lipopolysaccharide
Authors: Müller‐Loennies Sven , Holst Otto , Brade Helmut
Published: March 2005

The lipopolysaccharide of Escherichia coli J‐5 was sequentially de‐O‐acylated, dephosphorylated, reduced, de‐N‐acylated, and N ‐acetylated. The products were separated by high‐performance anion‐exchange chromatography into a nonasaccharide (1), two octasaccharides (2,3), and a heptasaccharide (4). Compositional analysis, methylation analysis, and NMR spectroscopy revealed the structures of the products as:

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in which 1R is l‐α‐d‐Hepp‐(1–5)‐[α‐Kdop‐(2–4)‐]‐α‐Kdop‐(2–6)‐β‐d‐GlcpNAc‐(1–6)‐d‐GlcN‐Acol, and 2R is l‐α‐d‐Hepp‐(1–5)‐α‐Kdop‐(2–6)‐β‐d‐Glcp NAc‐(1–6)‐d‐GlcNAcol (LD‐Hep, l‐glycero‐d‐manno‐heptose; Kdo, 3‐deoxy‐d‐manno‐octulopyranosonic acid; GlcNAcol, 2‐acet‐amido‐2‐deoxy‐glucitol).

Fast‐atom‐bombardment mass spectrometry of de‐O‐acylated and dephosphorylated lipopolysaccharide showed that the isolated oligosaccharides represented the complete carbohydrate moiety of the lipopolysaccharide, and indicated that the non‐reducing terminal d‐GlcN residue in lipopolysaccharide was present as the free base.
Characterization of a Bean (Phaseolus vulgaris L.) Malic‐enzyme Gene
Authors: Walter Michael H. , Grima‐Pettenati Jacqueline , Feuillet Catherine
Published: March 2005

We have isolated a genomic clone encoding a plant NADP+‐dependent malic enzyme (NADP‐ME). This clone, isolated from bean (Phaseolus vulgaris L.), covers the entire gene (exons, introns) and 5′‐flanking regions. DNA sequencing defines 20 exons spanning approximately 4.5 kb, which range over 48–235 bp in size. All 19 introns are fairly small (79–391 bp). The first intron resides in the 5′‐untranslated leader sequence. Introns 10, 11 and 16 are located at positions identical to a rat malic‐enzyme gene. In the promoter region, a TATA box (TATATATA) is easily recognized 41 bp upstream of a single transcription‐initiation site. Two potential cis‐acting elements with homology to elements from plant genes, activated by UV light and fungal elicitors, were identified at positions −153 and −312, respectively. Southern‐blot analysis suggests a single gene copy, but also other distantly related genes, in the bean genome. The deduced NADP‐ME protein of 589 amino acids exhibits features consistent with a cytoplasmic location. We describe the organization of the NADP‐ME protein into functional domains located on separate exons. The evolution of malic‐enzyme genes coding for isoforms in different cellular compartments of plants and animals is discussed.
Structural Phase Transitions Involved in the Interaction of Phospholipid Bilayers with Octyl Glucoside
Authors: De La Maza Alfonso , Parra Jose Luis
Published: March 2005

The transitional stages induced by the interaction of the nonionic surfactant octyl glucoside (OcOse) on phosphatidylcholine liposomes were studied by means of transmission electron microscopy (TEM), light scattering and permeability changes. A linear correlation was observed between the effective surfactant/lipid molar ratio (Re three‐stage model proposed for liposome solubilization) and the OcOse concentration in the initial and final interaction stages, despite showing almost a constant value during bilayer saturation. The bilayer/aqueous phase partition coefficient (K) decreased in the subsolubilizing interaction steps and increased during solubilization. Thus, whereas a preferential distribution of surfactant monomers in the aqueous phase with respect to the lipid bilayers took place in the initial interaction steps, a larger association of OcOse molecules with these lipids in bilayers occured during solubilization. The initial steps of bilayer saturation (50–70% permeability) were attained for a lower free surfactant (Sw) than that for its critical micellar concentration (cmc). When Sw reached the OcOse cmc, solubilization started to occur (Resat). Large unilamellar vesicles began to form as the OcOse exceeded 60 mol/100 mol, exhibiting for 65 mol/100 mol (50% permeability) vesicles of approximately 400 nm. TEM pictures for 100% permeability (72 mol/100 mol) and Resat still showed unilamellar vesicles, albeit that the Resat TEM picture showing traces of smaller structures. Exceeding surfactant amounts led to a decrease in static light scattering; the vesicle‐size curve began to show a bimodal distribution. The TEM picture showed tubular structures together with bilayer fragments. Thereafter, the open structures were gradually affected by the surfactant and the scattered intensity gradually decreased to a constant low value.
Characterization and partial purification of the human receptor for the heat‐stable enterotoxin
Authors: VISWESWARIAH Sandhya S. , RAMACHANDRAN Vasanthi , RAMAMOHAN Surabhi , DAS Goutam , RAMACHANDRAN Janakiraman
Published: March 2005

The receptor for the Escherichia coli heat‐stable enterotoxin has been characterized and partially purified from the T84 human colonic cell line. Using a novel mutant heat‐stable enterotoxin peptide as a radioligand (the C‐terminal tyrosine residue is replaced by phenylalanine in the mutant), a single class of high‐affinity receptor sites was detected in T84 cells, with a Kd of 0.1 nM, similar in affinity to the receptor described in human intestinal tissue. The receptor was solubilised from T84 cell membranes and affinity cross‐linking of the solubilised preparation indicated that a single species of Mr 160000 served as the receptor. Freshly solubilised preparations of the receptor retained heat‐stable enterotoxin‐activable guanylyl cyclase activity. Purification of the receptor was achieved through sequential affinity chromatography on GTP–epoxy‐Sepharose and wheat‐germ‐agglutinin columns resulting in purification of the receptor by 3000 fold. The heat‐stable enterotoxin‐binding characteristics of the receptor were unchanged during the purification and silver staining of the purified receptor preparation indicated a band of Mr 160000, which was specifically cross‐linked to the 125I‐labeled mutant peptide. The purified receptor retained guanylyl cyclase activity, but the activity was not stimulated on addition of human heat‐stable enterotoxin, suggesting that accessory structural factors may be involved in the activation of the guanylyl cyclase/receptor.