bluuebunny/crossref_metadata_2025 huggingface dataset:
On the mechanistic origin of damped oscillations in biochemical reaction systems
Authors: RYDE‐PETTERSSON Ulf
Published: March 2005

A generalized reaction scheme for the kinetic interaction of two reactants in a metabolic pathway has been examined in order to establish what minimal mechanistic patterns are required to support a damped oscillatory transient‐state kinetic behaviour of such a two‐component system when operating near a steady state. All potentially oscillating sub‐systems inherent in this scheme are listed and briefly characterized. The list includes several mechanistic patterns that may be frequently encountered in biological system (e.g. involving feedback inhibition, feed‐forward activation, substrate inhibition or product activation), but also draw attention to some hitherto unforeseen mechanisms by which the kinetic interaction of two metabolites may trigger damped oscillations. The results can be used to identify possible sources of oscillations in metabolic pathways without detailed knowledge about the explicit rate equations that apply.
Primary structure and functional expression of the α‐, β‐, γ‐, δ‐ and ɛ‐subunits of the acetylcholine receptor from rat muscle
Authors: WITZEMANN Veit , STEIN Elke , BARG Brigitte , KONNO Takashi , KOENEN Michael , KUES Wilfried , CRIADO Manuel , HOFMANN Michael , SAKMANN Bert
Published: March 2005

The isolation and characterization of five clones carrying sequences of the α‐, β‐, γ‐, δ‐ and ɛ‐subunit precursors of the rat muscle acetylcholine receptor (AChR) are described. The deduced amino acid sequences indicate that these polypeptides contain 457–519 amino acids and reveal the structural characteristics common to subunits of ligand‐gated ion channels. The pattern of subunit‐specific mRNA levels in rat muscle shows characteristic changes during development and following denervation, suggesting that innervation of muscle reduces the expression of the α‐, β‐ and δ‐subunit mRNAs, suppresses the expression of the γ‐subunit mRNA, and induces expression of ɛ‐subunit mRNA. Subunit‐specific cRNAs generated in vitro were injected into Xenopus laevis oocytes, resulting in the assembly of two functionally different AChR channel subtypes. The AChRγ, composed of the α‐, β‐, γ‐ and δ‐subunits, has functional properties similar to those of the native AChRs in fetal muscle. The AChRɛ, composed of α‐, β‐, δ‐ and ɛ‐subunits, corresponds to the end‐plate channel of the adult muscle. Thus in rat skeletal muscle the motor nerve regulates the expression of two functionally different AChR subtypes with different molecular composition by the differential expression of subunit‐specific mRNAs.
Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity
Authors: GIUDICI‐ORTICONI Marie‐Thérèse , BUC Jean , RICARD Jacques
Published: March 2005

The principles of structural kinetics allow one to define the thermodynamic conditions that are sufficient to generate a certain type of kinetic behavior. If subunits are loosely coupled, that is if no quaternary constraint exists between them, the kinetic behavior of the polymeric enzyme is qualitatively defined by the behavior of an ideal dimer. The nature and the extent of the kinetic cooperativity are defined by the energy of interaction, ΔGQ, between two subunits. This energy of interaction is that of an ideal dimer relative to that of the A2 and B2 states. This thermodynamic formulation of a given type of cooperativity holds whatever the degree of polymerization of the enzyme. Under these conditions of loose coupling between subunits, positive kinetic cooperativity cannot be associated with any sigmoidicity of the rate curve. The range of energy coupling where positive kinetic cooperativity must, of necessity, be observed becomes more and more narrow as the number of subunit interactions is increased. This range, however, is independent of the number of subunits. The same situation is not observed for negative cooperativity which appears to be independent of both the number of subunits and the number of subunit interactions.

If the subunits are tightly coupled, that is if quaternary constraints exist between them, three thermodynamic parameters, ΔG'Q, ΔGλ, ΔGμ, are required to define the nature of kinetic cooperativity. ΔG'Q is the free energy of an ideal strained dimer relative to that of strained A2 and B2 states. ΔGλ and ΔGμ represent the difference of strain energies between conformations A and B and B and B relative to that existing between conformations A and A. One may determine in the parametric space (ΔG'Q, ΔGλ, ΔGμ) the boundaries between the sufficient conditions that generate a certain type of cooperativity and the lack of these conditions.

The kinetic parameters of the rate equation are not all independent. A number of constraint conditions exist between them which depend upon the subunit design of the polymeric enzyme. The existence of these constraint conditions may be diagnostic of a certain type of subunit interactions.
Characterization of human factor VIII and interaction with von Willebrand factor
Authors: HEIJNEN Harry F. G. , KOEDAM Joost. A. , SANDBERG Helena , BEESER‐VISSER Nel H. , SLOT Jan. W. , SIXMA Jan J.
Published: March 2005

Blood coagulation factor VIII is a large glycoprotein that circulates in plasma at relative low concentration (0.1 μg/ml). It consists of a heterogeneous mixture of a series heavy‐chain peptides (90–200 kDa), each associated with a light chain of 80 kDa. To gain insight into the physical properties of the protein, we have characterized purified human factor VIII by electron microscopy and rotary shadowing. Electron microscopy of rotary shadowed factor VIII molecules showed predominantly a single globular domain structure, with a somewhat asymmetric shape, while two‐domain structures were also encountered. The overall dimensions of the globular domains ranged from 4 × 6 nm to 8 × 12 nm. EDTA treatment of factor VIII reduced the overall dimensions (2.5 × 5 nm to 6 × 10 nm) while treatment with thrombin reduced the dimensions to a small extent. In complexes with von Willebrand factor, factor VIII appeared localized at the globular domains of von Willebrand factor multimers. In addition, incubation of factor VIII with Staphylococcus aureus V8 protease fragments SpII and SpIII revealed only binding to the globular domains of SpIII. In this study, the first morphological characterization of human factor VIII is presented, together with its direct localization on von Willebrand factor multimers.
Neurokinin A induces expression of the c‐fos, c‐jun, and c‐myc genes in rat smooth muscle cells
Authors: HULTGÅRDH‐NILSSON Anna , LARSSON Stefan H. , JIN Pei , SEJERSEN Thomas , RINGERTZ Nils R.
Published: March 2005

Neurokinin A, a member of the tachykinin family of neuropeptides, has been identified as a mitogen for cultured smooth muscle cells. Tachykinin‐induced DNA synthesis has previously been shown to be mediated by a receptor‐specific mechanism and to correlate with accumulation of phosphatidylinositol 4,5‐bisphosphate breakdown products. In the present experiments, we have studied intracellular pH and expression of the protooncogenes c‐myc, c‐jun and c‐fos in smooth muscle cells exposed to mitogenic concentrations of neurokinin A. Growth‐arrested smooth muscle cells stimulated with neurokinin A responded with an amiloride‐sensitive intracellular alkalinization, indicating Na+/H+ antiport activation. c‐myc and c‐jun mRNA expression was only slightly elevated by neurokinin A, while c‐fos expression underwent a more pronounced increase. Maximal levels of c‐fos transcripts were found after 15 min and 30 min following neurokinin A stimulation. The results demonstrate that neuropeptides may influence proto‐oncogene expression in smooth muscle cells and suggest a mechanism by which peripheral neurons may modulate differentiation and growth of these cells.
Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes
Authors: LAMPE Paul D. , JOHNSON Ross G.
Published: March 2005

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C‐terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell‐to‐cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP‐dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP‐derived peptides on different HPLC columns and to determine possible cAMP‐dependent protein kinase phosphorylation sites. Lens membranes, which contain 50–60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C‐terminal region of MIP and a major product of 21–22 kDa remained membrane‐associated. Separation of the lysylendopeptidase‐C‐released peptides on C8 reversed‐phase HPLC demonstrated that one of these fragments, corresponding to residues 239–259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP‐dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.
Cloning, nucleotide sequence and regulation of the Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase
Authors: FRICK Michele M. , NEUHARD Jan , KELLN Rod A.
Published: March 2005

The Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase was cloned and sequenced. In total, a sequence of 1286 nucleotide pairs was determined wherein a single open‐reading‐frame of 1011 bp, encoding a polypeptide of 336 amino acids having 95% similarity with the Escherichia coli pyrD gene product, was identified. A region of hyphenated‐dyad symmetry exists within the leader region affording the potential for the formation of a stable secondary structure in the 5′ end of the transcript. Mutations from several regulatory mutants were located within the region of dyad symmetry which would impart changes in the transcript within the putative secondary structure, implicating the secondary structure in regulation. Primer extension analysis revealed multiple transcriptional start sites located six to nine nucleotides downstream from the Pribnow box, with the primary initiation site differing in repressing and derepressing growth conditions. The results are discussed in terms of a translational attenuation model for regulation of pyrD expression.
Avian alcohol dehydrogenase: the chicken liver enzyme
Authors: ESTONIUS Mats , KARLSSON Christina , FOX Edward A. , HÖÖG Jan‐Olov , HOLMQUIST Barton , VALLEE Bert L. , DAVIDSON William S. , JÖRNVALL Hans
Published: March 2005

The major ethanol‐active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full‐length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to γ chains of the human enzyme, a Km for ethanol of 0.5 mM and a Ki for 4‐methyl pyrazole of 2.5 μM. Relationships to the other two classes are non‐identical; residue exchanges towards the human classes increase in the order I < III < II, and human/chicken differences are less than inter‐class differences. Consequently, the origins of the classes are more distant than the avian/mammalian separation. They reflect duplicatory events separated in time, and the lines that lead to present‐day classes I and II deviate early. Integrated with the data for the quail enzyme, the structure of the chicken protein shows that within the avian enzymes the degree of variation is comparable to that within the mammalian class I enzymes, which are more variable than the class III forms. The coenzyme‐binding and substrate‐binding residues of this chicken alcohol dehydrogenase are largely identical to those in the mammalian class I counterparts. However, the subunit‐interacting areas are more variable and suggest some relationships of the avian enzyme with both class I and III mammalian forms. One of the residues, Gly260 (mammalian class I numbering system), previously considered characteristic of all alcohol dehydrogenases, is replaced by Gln.
Limited proteolysis of γII‐crystallin from calf eye lens
Authors: SHARMA Ashok K. , MINKE‐GOGL Verena , GOHL Peter , SIEBENDRITT Renate , JAENICKE Rainer , RUDOLPH Rainer
Published: March 2005

γII‐crystallin from calf eye lens consists of two homologous domains, connected by a six‐residue linker peptide. In order to study the intrinsic properties of the domains and their mutual stabilization, limited proteolysis was applied. Optimum conditions providing a homogeneous 10‐kDa fragment at high yield were pepsin cleavage in 0.1 M NaCl/HCl pH 2.0, in the presence of 3.0 M urea. Determination of the N‐terminus and the C‐terminal sequence showed that cleavage occurred at the Phe88–Arg89 peptide bond, giving rise to the complete N‐terminal domain including the connecting hexapeptide. The C‐terminal part of the polypeptide chain is cleaved to small fragments.

Comparing the spectral properties of the isolated N‐terminal domain and intact γII‐crystallin proved the structure of the fragment to be closely similar to that of the native domain. Small differences in absorbance, fluorescence emission and circular dichroism point to alterations caused by the increase in surface area as a consequence of domain separation. The resistance of the 10‐kDa fragment toward thermal and alkaline denaturation, as well as unfolding in the presence of urea or guanidine · HCl is decreased, due to the lack of domain interactions stabilizing the intact protein.

Unfolding/folding kinetics of the 10‐kDa fragment coincide with the second phase of the bimodal transition of intact γII‐crystallin, in agreement with independent sequential folding and modular assembly of the domains within the native molecule.
Structural characterization of a recombinant CD4‐IgG hybrid molecule
Authors: HARRIS Reed J. , WAGNER Karen L. , SPELLMAN Michael W
Published: March 2005

CD4‐IgG is a homodimer of a hybrid polypeptide consisting of the two amino‐terminal domains (residues 1–180) of human CD4 fused to the hinge region and the second and third constant‐sequence (CH2 and CH3) Fc domains (residues 216–441) of human immunoglobulin G (IgG‐1). This antibody‐like molecule, termed an immunoadhesin, was produced in an effort to combine the binding specificity of CD4 with several potentially desirable properties of IgG molecules [Capon et al. (1989) Nature 337, 525–531]. The structural characteristics of the molecule have been evaluated to demonstrate that CD4‐IgG has the same features as the N‐terminal region of soluble CD4, while retaining those expected for the Fc portion of human IgG. Identification of peptides recovered from the tryptic map confirmed 98.8% of the expected structure of CD4‐IgG. The detection of glucosamine in peptides containing Asn257 and the retention time shift of this tryptic peptide after deglycosylation confirmed the presence of Asn‐linked oligosaccharides at this position. Four pairs of intrachain and two interchain disulfide bonds were also established.
Modelling of substrate binding to 3‐phosphoglycerate kinase with analogues of 3‐phosphoglycerate
Authors: VAS Maria
Published: March 2005

Two short analogues of 3‐phosphoglycerate, −OOC‐CHOH‐CH2‐O‐PO2−3, phosphonolactate, (‐OOCCHOH‐CH2‐PO2−3) and arsonolactate (‐OOC‐CHOH‐CH2‐AsO32−3) have been tested with 3‐phosphoglycerate kinase. None of these served as substrate for the kinase reaction, unlike the previously studied [Orr, G. A. & Knowles, J. R. (1974) Biochem. J. 141, 721–723] analogues ‐OOC‐CHOH‐CH2‐CH2‐PO2−3 and ‐OOC‐CHOHCH2‐CH2‐AsO2−3, which are isosteric with 3‐phosphoglycerate. Thus, a decrease in the substrate size and the accompanying stereochemical changes cannot be tolerated by the catalytic mechanism. Instead, both analogues acted as relatively poor competitive inhibitors with respect to both 3‐phosphoglycerate and MgATP. At pH 8.5 and 20°C, the inhibitory constants (K1) of phosphonolactate and arsnolactate against both substrates are 17 ± 5 mM and 30 ± 7 mM, respectively. Surprisingly, however, both analogues proved to be more effective than either 3‐phosphoglycerate or its isosteric analogues in protecting the enzyme against modification of its fastreacting thiols. This comparison suggests that the shorter analogues bind differently, and that the catalytic mechanism demands a precise fitting of the ‐CH2‐O‐PO2−3 segment of the substrate.
Repeat peptide motifs which contain β‐turns and modulate DNA condensation in chromatin
Authors: ERARD Monique , LAKHDAR‐GHAZAL Faouzi , AMALRIC François
Published: March 2005

In order to understand better the roles of repeating basic peptide motifs in modifying DNA structure, we have synthesized typical repeats found in the C‐terminal domain of histone H1 (KTPKKAKKP)2 and in the N‐terminal domain of nucleolin (ATPAKKAA)2. By using circular dichroism in conjunction with Raman and Fourier‐transform infrared spectroscopies, we demonstrate that the abilities of the two peptides to affect DNA conformation are dramatically different. Whilst the binding of the nucleolin repeat to DNA does not significantly alter its conformation, the binding of H1 repeat induces a very marked DNA condensation, giving rise to a Ψ(‐)‐type circular dichroic spectrum. The H1 repeat thus adopts a more rigid β‐turn‐containing structure which probably binds to the DNA minor groove as assessed by competition with the drug Hoechst 33258. Unexpectedly, the DNA condensation induced by the H1 repeat is enhanced by the nucleolin repeat which by itself does not promote any alteration in DNA conformation.
Human fibroblasts show expression of the leukotriene‐A4‐hydrolase gene, which is increased after simian‐virus‐40 transformation
Authors: MEDINA Juan F. , BARRIOS Carlos , FUNK Colin D. , LARSSON Olle , HAEGGSTRÖM Jesper , RÅDMARK Olof
Published: March 2005

Human fibroblasts in cell culture converted the epoxide intermediate leukotriene A4 into the potent chemotaxin leukotriene B4. The identity of leukotriene B4 was ascertained by its mobility in reverse‐phase high performance liquid chromatography, ultraviolet spectroscopy and gas chromatography/mass spectrometry. The presence of the enzyme responsible for the conversion (i.e. leukotriene A4 hydrolase), as well as the corresponding mRNA, were demonstrated by Western and Northern blot analyses. Leukotriene‐A4‐hydrolase enzyme activity, protein and mRNA were all enhanced (approximately threefold) in human fibroblasts that had been transformed by simian virus 40.
The H+‐induced dissociation of human plasma α2‐macroglobulin
Authors: PAP Sarolta , SJÖBERG Bo , MORTENSEN Kell
Published: March 2005

The dissociation of the tetrameric α2‐macroglobulin molecule into two half‐molecular fragments, which occurs at pH < 4.5, has been investigated using the small‐angle neutron scattering method, and test of trypsin binding activity.

Best fit with the relative forward scattering of neutrons is obtained for a model where the dissociation of the protein is driven by the uptake of H+ on altogether four acid‐base groups, one per monomeric subunit of α2‐macroglobulin. These groups are not (or only slightly) accessible in the native tetramer, but become exposed to the solvent after dissociation of the protein. The H+‐binding constant obtained for these groups, after dissociation of the protein, log K1 in the range 4.2–4.5, suggests that they are most probably carboxylate groups.

From the about 10% increase in the radius of gyration, which occurs when lowering the pH from 4.5 to 2.0, we can conclude that the dissociation is associated with a change in structure of the protein. Tests of trypsin binding show that there is also an irreversible loss in trypsin binding activity, which is directly related to the fraction of dissociated protein.

Thus, at pH <4.5, there is a transition of α2‐macroglobulin which results simultaneously in dissociation, disorganisation of the conformation of the subunits and loss in activity.
α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates
Authors: JOZIASSE David H. , SHAPER Nancy L. , SALYER Linda S. , Van den EIJNDEN Dirk H. , van der SPOEL Aarnoud C. , SHAPER Joel H.
Published: March 2005

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP‐Gal:Galβ1 → 4GlcNAc α→ 3‐galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J. Biol. Chem. 264, 14290–14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high‐level expression of enzymatically active α1 → 3‐galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000‐fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant α1 → 3‐galactosyltransferase could be readily detergent‐solubilized and subsequently purified by affinity chromatography on UDP‐hexanolamine ‐Sepharose. The recombinant α1 → 3‐galactosyltransferase showed the expected preference for the acceptor substrate N‐acetyllactosamine (Galβ1 → 4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity‐purified calf thymus α1 → 3‐galactosyltransferase [Blanken, W. M. & Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927–12934].

In pilot studies, the recombinant enzyme was examined for the ability to synthesize αl → 3‐galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 1H‐NMR, methylation analysis, HPLC. and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Galα1 → 3Galβ1 → 4‐R. Our results demonstrate that catalysis by recombinant α1 → 3‐galactosyltransferase can be used to obtain preparative quantities of various α1 → 3‐galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.
Inactivation of bacteriophage T7 DNA‐dependent RNA polymerase by 5′‐p‐fluorosulfonylbenzoyladenosine
Authors: TUNITSKAYA Vera L. , AKBAROV Akhror Kh. , LUCHIN Sergey V. , MEMELOVA Lyudmila V. , RECHINSKY Vladimir O. , KOCHETKOV Sergey N.
Published: March 2005

Bacteriophage T7 RNA polymerase was covalently modified by 5′‐[4‐fluorosulfonyl)benzoyl]adenosine (4‐FSO2BzAdo). The modified enzyme lacks the ability to catalyze RNA synthesis from the Ø10 promoter of bacteriophage T7; both promoter and GTP binding being markedly decreased. The mild hydrolysis of the ester bond of 4‐FSO2BzAdo within the covalent enzyme‐inhibitor complex restores the RNA synthesis at a lower rate.

Sequence studies show that Lys172 is the target of modification by 4‐FSO2BzAdo. This residue, which is situated in the polypeptide region connecting two domains of RNA polymerase, was shown to be the primary site of the limited proteolysis occurring in vivo [Ikeda, R. A. & Richardson, C. C. (1987) J. Biol. Chem. 262, 3790–3799].

We propose that Lys172 is located outside the active site. Once this residue has reacted with 4‐FSO2BzAdo. the nucleoside moiety of the analog is fixed in the NTP‐binding site of the active centre and prevents binding of the substrates. Here, Lys172 per se is not important for the activity but serves as an ‘anchor’ for binding of the inhibitor.
DNA linking potential generated by gyrase
Authors: KOZYAVKIN Sergei A. , SLESAREV Alexei I. , MALKHOSYAN Sergei R. , PANYUTIN Igor G.
Published: March 2005

Whether or not DNA gyrase can supercoil DNA so that alternative structures will arise in it is the major question of this work. We have shown gyrase to produce in pAO3 DNA a superhelix density sufficient for cruciform formation. However, the transition does not take place because of too slow kinetics. A change of ionic conditions in favour of more intense DNA supercoiling by gyrase shifts the midpoint of the equilibrium transition to the cruciform structure toward more supercoiled topoisomers.

The width of the equilibrium transition to the cruciform as a function of linking number has been revealed to be an order of magnitude larger in buffers containing magnesium and spermidine than in buffers with monovalent cations only. We ascribe this effect to the influence that the counter ions surrounding the DNA molecule have on its elasticity, the coefficient of elasticity being dependent on superhelix density σ. Thus, the free energy of supercoiling (a) depends on the ionic conditions and (b) is not a quadratic function of σ in the physiological range of parameters.

We propose a description of DNA as a system of links that can be either closed or open; we also introduce a new concept of the DNA linking potential akin to the chemical and electric potentials. The linking potential is a suitable parameter for describing the equilibrium distribution of links in heterogeneous DNA, the coexistence of various DNA structures, the equilibrium input and output of DNA links by enzymes, and the nonequilibrium movement of links along DNA chains. Within the framework of this approach DNA gyrase is considered as the source of the DNA linking potential.
Yeast homoserine kinase
Authors: MANNHAUPT Gertrud , POHLENZ Hans‐Dieter , SEEFLUTH Ann Kristin , PILZ Ursula , FELDMANN Horst
Published: March 2005

THR1, the gene from Saccharomyces cerevisiae, encoding homoserine kinase, one of the threonine biosynthetic enzymes, has been cloned by complementation. The nucleotide sequence of a 3.1‐kb region carrying this gene reveals an open reading frame of 356 codons, corresponding to about 40 kDa for the encoded protein. The presence of three canonical GCN4 regulatory sequences in the upstream flanking region suggests that the expression of THR1 is under the general amino acid control. In parallel, the enzyme was purified by four consecutive column chromatographies, monitoring homoserine kinase activity. In SDS gel electrophoresis, homoserine kinase migrates like a 40‐kDa protein; the native enzyme appears to be a homodimer. The sequence of the first 15 NH2‐terminal amino acids, as determined by automated Edman degradation, is in accordance with the amino acid sequence deduced from the nucleotide sequence. Computer‐assisted comparison of the yeast enzyme with the corresponding activities from bacterial sources showed that several segments among these proteins are highly conserved. Further more, the observed homology patterns suggest that the ancestral sequences might have been composed from separate (functional) domains. A block of very similar amino acids is found in the homoserine kinases towards the carboxy terminus that is also present in many other proteins involved in threonine (or serine) metabolism: this motif, therefore, may represent the binding site for the hydroxyamino acids. Limited similarity was detected between a motif conserved among the homoserine kinases and consensus sequences found in other mono‐ or dinucleotide‐binding proteins.
Immune recognition of affinity‐labelled cholecystokinin receptor
Authors: DUFRESNE Marlène , POIROT Sandrine , JIMENEZ Juan , CUBER Jean‐Claude , VAYSSE Nicole , FOURMY Daniel
Published: March 2005

The present study was undertaken to characterize the immune recognition of pancreatic cholecystokinin receptor by an anti‐cholecystokinin antibody. Cholecystokinin receptor from pancreatic plasma membranes was photoaffinity labelled using the specific, cleavable probe 125I‐labelled 2‐(p‐azidosalicylamido)‐1,3‐dithiopro‐pionate‐[Thr28, Ahx31]CCK(25–33) [CCK(25–33) is the C‐terminal nonapeptide of the 33‐amino‐acid form of cholecystokinin]. Labelled receptor was then solubilized and subsequently prepurified on immobilized wheat‐germ agglutinin. The C‐terminal‐directed anti‐cholecystokinin serum (8E) specifically immunoprecipitated a fraction of affinity‐labelled cholecystokinin receptor which was identified at Mr 85000–100000 on SDS/PAGE. The binding affinity of antiserum 8E for covalently labelled cholecystokinin receptor was lower (Kd 0.11 ± 0.02 nM) than for cholecystokinin (Kd 3.65 ± 0.55 pM). The compound L364‐718, an A‐subtype cholecystokinin‐receptor antagonist did not interfere with the immune recognition of cholecystokinin. However, the recognition of affinity‐labelled cholecystokinin receptor was enhanced as a result of an increasing availability of cholecystokinin molecules. Indeed, the amount of immunoprecipitated receptor was doubled in the presence of 10 μM L364–718. This study offers the possibility of using an anti‐cholecystokinin antibody for cholecystokinin‐receptor purification and demonstrates that prepurified affinity‐labelled cholecystokinin receptor retains A‐subtype specificity.
The quaternary structure of Escherichia coli inorganic pyrophosphatase is essential for phosphorylation
Authors: SKLYANKINA Vera A. , AVAEVA Svetlana M.
Published: March 2005

The hexameric inorganic pyrophosphatase (PPase) is irreversibly inactivated by phosphoric acid monoesters. The inactivation kinetics are consistent with the formation of a dissociable complex of the phosphoric acid monoester with the enzyme, followed by phosphorylation of the dicarboxylic amino acid of its active site. PP; and its analogues, binding at the regulatory site, release the inhibitor from the active site and thus restore PPase activity.

Chemically identical subunits in the hexameric PPase interact, promoting their cooperativity in a reaction with phosphoric acid monoesters. The trimeric and monomeric PPase, exhibiting full catalytic activity, form a dissociable complex with the phosphoric acid monoesters but, in contrast to the hexameric PPase, do not form a covalent bond with them. This indicates that the native hexameric structure is essential for the irreversible inactivation of Escherichia coli PPase by phosphoric acid monoesters. Possible nontraditional pathways for activity regulation of PPase are discussed.
Structural investigations and biological activity of inositol sphingophospholipids from Phytophthora capsici
Authors: LHOMME Olivier , BRUNETEAU Maud , COSTELLO Catherine E. , MAS Pierre , MOLOT Paul‐Michel , DELL Anne , TILLER Philip R. , MICHEL Georges
Published: March 2005

Inositol sphingophospholipids that protect pepper (Capsicum annuum c.v. Yolo Wonder) against pathogen have been isolated by chromatographic methods from the mycelium of Phytophthora capsici. The structure of the major compound was determined by chemical methods and mass spectrometry. Phosphodiester bond cleavage of the phospholipid by mild alkaline hydrolysis liberated a ceramide which contained a C16‐sphingosine. This longchain base was identified by gas chromatography and mass spectrometry of its trimethylsilyl derivative. One of the amide‐linked fatty acids was found to be 4‐hydroxy‐2 docosenoic acid. Fast‐atom‐bombardment mass spectrometry and fast‐atom‐bombardment collison‐induced tandem mass spectrometry were used to characterize the ceramide as N(4‐hydroxy‐2‐docosenoyl)C16‐sphingosine. These sphingolipids have a protective effect on cotyledons of young peppers against necrotic lesions induced by the pathogen P. capsici.
Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes*
Authors: KÜHN Hartmut , BELKNER Jutta , WIESNER Rainer
Published: March 2005

Mitochondrial membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,11E)‐(13S)‐13‐hydroxy‐9,11‐octadecadienoic acid, 9S and 9R isomers of (10E,12Z)‐9‐hydroxy‐10,12‐octadecadienoic acid and their all‐E isomers. Furthermore (5Z,8Z,11Z,13E)‐(15S)‐15‐hydroxy‐5,8,11,13‐icosatetraenoic acid, 9‐and 13‐oxooctadecadienoic acid were detected as minor products. The chemical structure of these products has been identified by co‐chromatography with authentic standards, by ultraviolet and infrared spectroscopy, and by gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The oxygenated fatty acids originate most probably from the intracellular action of the erythroid arachidonate 15‐lipoxygenase. In membranes of the mature erythrocyte only small amounts of hydroxy fatty acids were detected. Young peripheral reticulocytes contain more oxygenated polyenoic fatty acids in their membranes than older cells. In mixed cell populations, about 85% of the lipoxygenase products were found esterified to the membrane ester lipids, whereas 15% were associated as free hydroxy fatty acids with the membranes. The hydroxy fatty acid content of the mitochondrial membranes is more than threefold higher than that of the plasma membranes. The pattern of the products isolated from plasma membranes shows a high specificity with (9Z,11E)‐(13S)‐13‐hydroxy‐9,11‐octadecadienoic acid as the main product. In contrast, the pattern found in the mitochondrial membranes was much more unspecific; a complex mixture of all positional and optical isomers was detected.

The data presented indicate that the reticulocyte lipoxygenase in vivo acts on both plasma membranes and mitochondrial membranes. The results are discussed in the light of the involvement of the lipoxygenase in the breakdown of mitochondria and other organelles in reticulocytes during maturation.
Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids
Authors: PLOMP Peter J. A. M. , BOON Louis , CARO L. Heleen P. , van WOERKOM George M. , MEIJER Alfred J.
Published: March 2005

Katz et al. [Katz, J., Golden, S. & Wals, P. A. (1976) Proc. Natl Acad. Sci. USA 73, 3433–3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made.

Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase.

Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents.

3‐Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate.

The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen, K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683–14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase.

It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955–959].
Identification and role of the basal phosphorylation site on hormone‐sensitive lipase
Authors: GARTON Andrew J. , YEAMAN Stephen J.
Published: March 2005

Phosphorylation site 2 on bovine hormone‐sensitive lipase (HSL), which is phosphorylated in vitro by the AMP‐activated protein kinase, has been found also to be phosphorylated in vitro by glycogen synthase kinase‐4. Peptide mapping of HSL phosphorylated in vitro and in isolated adipocytes demonstrates that this site corresponds to the basal phosphorylation site on HSL, which is phosphorylated in intact adipocytes in the absence of lipolytic stimuli. Site 2 has been proposed to have an antilipolytic role in that phosphorylation at this site greatly reduces subsequent phosphorylation (at site 1) and activation of HSL by cyclic‐AMP‐dependent protein kinase. Further evidence for an antilipolytic role of site 2 has been obtained using a synthetic peptide based on the sequence around sites 1 and 2. Phosphorylation of the peptide at site 2 totally prevents the subsequent phosphorylation of site 1 and vice versa.
Comparison of exonucleolytic activities of herpes simplex virus type‐1 DNA polymerase and DNase
Authors: KNOPF Charles W. , WEISSHART Klaus
Published: March 2005

The exonucleolytic activities associated with herpes simplex virus type‐1 (HSV‐1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5′–3′‐exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i.e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double‐stranded DNA in a very processive manner and exclusively in the 5′–3′ direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5′–3′‐exonucleolytic activities of the DNase and the polymerase‐associated 3′–5′ exonuclease.

The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N‐terminal, central and C‐terminal polypeptide domains of HSV‐1 DNA polymerase. From the data presented it can be concluded that the cryptic 3′–5′ exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.
Cytochrome aa3 from Sulfolobus acidocaldarius
Authors: ANEMÜLLER Stefan , SCHÅFER Günter
Published: March 2005

The thermoacidophilic archaebacterium Sulfolobus acidocaladarius (DSM 639) extrudes protons when expending respiratory energy [Moll, R. & Schäfer, G. (1988) FEBS Lett. 232, 359–363]. Cytochromes of the membrane electron‐transport systems are assumed to represent the proton pumps. Only a‐ and b‐type cytochromes can be found; no c‐type cytochromes are present. Of the two terminal oxidases [Anemüller, S. & Schäfer, G. (1989) FEBS Lett. 244, 451–455] one shows an absorption band at 604–605 nm, typical of cytochromes of the aa3 type. This hemoprotein has been solubilized from the membrane and purified to homogeneity. It exhibits distinct differences from known aa3‐type oxidases. (a) It consists of a single polypeptide subunit of 38–40 kDa apparent molecular mass with two heme‐α molecules and two copper ions. (b) In the oxidized state, absorption maxima are found at 421 nm and 597 nm, and in the reduced state at 439 nm and 601 nm; CO difference spectra suggest one heme to be a heme‐a3 centre. (c) The redox potentials of the heme centres are + 220 mV and + 370 mV, respectively. (d)A high‐spin heme signal at g=6 is present in EPR spectra, which is more prominent than the low‐spin heme signal at g=3, the former already being present in the oxidized state. A signal at g=2.1 may be due to one of the copper ions and is superimposed upon a minor free radical signal at g=2. (e) Caldariella quinone was also isolated from the plasma membrane of Sulfolobus. Its redox midpoint potential at pH 6.5 was determined to be + 100 (±5) mV; spectral properties have also been determined. (f) The isolated aa3 preparation does not oxidize cytochrome c; however, it oxidizes N,N,N′,N′‐tetramethyl‐1,4‐phenylenediamine dihydrochloride as an artificial single‐electron donor as well as reduced caldariella quinone, which is assumed to represent the natural substrate. The reaction is cyanide‐sensitive and the product of oxygen reduction is water. (g) On the basis of the results obtained a novel type of cytochrome aa3 is postulated in this paper which oxidizes reduced quinones; its ability to act as a proton pump remains to be shown.
A 19F‐NMR study of 3‐fluoro‐4‐demethoxydaunomycin intercalation complexes with the hexanucleotide d(CTGCAG)2
Authors: HAMMER Brendon C. , RUSSELL Richard A. , WARRENER Ronald N. , COLLINS J. Grant
Published: March 2005

Equilibrium systems containing intercalation complexes formed between the novel anthracycline drug, 3‐fluoro‐4‐demethoxydaunomycin (3FD), and the hexanucleotide duplex d(CTGCAG)2 have been studied by 19FNMR spectroscopy. Solutions containing a 1:1 molar ratio of 3FD/d(CTGCAG)2 gave four 19F signals which have been assigned to each of four possible intercalation isomers for the 1:1 3FD · d(CTGCAG)2 complex, which we denote by [d(CTGCAG)2][3FD]; these were where 3FD bound between the 5′‐CT‐3′,5′‐TG‐3′, 5′‐GC‐3′ or 5′‐CA‐3′ base sequences, with the drug sugar moiety lying in the minor groove and pointed in the 3′ direction in each case. Changes in temperature and NaCl concentration affecting the equilibrium distribution of these isomers were studied and indicated that no overriding binding site preference prevailed under standard biochemical conditions.

Formation of some of the 2:1 3FD · d(CTGCAG)2 complex occurred when a solution of [d(CTGCAG)2][3FD] was exposed to excess 3FD; however, this complex was unstable to gel filtration and no co‐operative binding of the second 3FD molecule was observed.
Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB–cysG region of the Escherichia coli K‐12 chromosome
Authors: PEAKMAN Timothy , CROUZET Joel , MAYAUX Jean Francois , BUSBY Stephen , MOHAN Sudesh , HARBORNE Nerina , WOOTTON John , NICOLSON Rachel , COLE Jeffrey
Published: March 2005

The DNA sequence and derived amino‐acid sequence of a 5618‐base region in the 74‐min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH‐dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG.

Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino‐acid sequence with those of database proteins. A directly repeated sequence, which includes the motif ‐Cys‐Xaa‐Xaa‐Cys‐, is suggested as the binding site for either one [4Fe‐4S] or two [2Fe‐2S] clusters.

The nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino‐acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33‐amino‐acid peptide of unknown function which includes 8 phenylalanyl residues.

The product of the nirC gene is a highly hydrophobic protein with regions of amino‐acid sequence similar to cytochrome oxidase polypeptide 1.
The measurement of the rotational diffusion coefficient of bovine plasma fibronectin by electric birefringence technique
Authors: VUILLARD Laurent , ROUX Bernard , MILLER Andrew
Published: March 2005

Although the conformation of fibronectin has been widely investigated by various techniques, there has not yet been any determination of its rotational diffusion coefficient. We report here this determination by the transient electric birefringence study of solutions of bovine plasma fibronectin at physiological ionic strength.

The solutions showed a positive birefringence. A linear relationship was observed between the intensity of the birefringence at equilibrium and the square of the electric field within the range of fields applied (up to 12.5 kV · cm−1). The field‐independent decay of the induced birefringence was described by a single exponential with a relaxation time of 0.76 (±0.08) μs at 23°C. This establishes fibronectin in solution as a globally rigid structure with a rotational diffusion coefficient, at 20°C, of 202000 s−1.

This result allows the first rigorous determination of the low‐resolution structure of fibronectin. It is important to notice that the analysis combines only results obtained in physiological conditions on native molecules and follows a strict hydrodynamic interpretation. The conclusion of this work is that a hollow sphere of about 20 nm external diameter can be proposed as a model for the three‐dimensional structure of the fibronectin molecule in solution. This new model suggests the fibronectin could have the structure of a carrier protein.
Quinone compounds are able to replace molecular oxygen as terminal electron acceptor in phytoene desaturation in chromoplasts of Narcissus pseudonarcissus L.
Authors: Mayer Matthias P. , Beyer Peter , Kleinig Hans
Published: March 2005

The desaturation of phytoene to ζ‐carotene and of ζ‐carotne to lycopene employs molecular oxygen as the terminal electron acceptor. 2,3,5,6‐Tetramethyl‐1,4‐benzoquinone (duroquinone) and other artificial quinones are able to replace oxygen, which demonstrates that oxygen does not act in a mixed‐function oxygenase‐like mechanism at the desaturase itself, but at a spatially separate site. Evidence for additional redox elements mediating between desaturase and oxygen is presented.
Glycosphingolipids in insects
Authors: WESKE Bianka , DENNIS Roger D. , HELLING Friedhelm , KELLER Martin , NORES Gustavo A. , PETER‐KATALINIC Jasna , EGGE Heinz , DABROWSKI Ursula , WIEGANDT Herbert
Published: March 2005

The two major components of the acidic glycolipid fraction from the pupae of Calliphora vicina were isolated using high‐performance liquid chromatography. The acidic moiety was identified as glucuronic acid by β‐glucuronidase cleavage and gas chromatographic analysis as the pentafluoropropionyl derivative. The structures of the carbohydrate moiety were elucidated by peracetylation, methylation, exoglycosidase cleavage, fast‐atom‐bombardment mass spectrometric and 1H‐nuclear magnetic resonance spectroscopic analysis. The only difference between the two hexasaccharide variants was the presence, in one of them, of a phosphoethanolamine (AeP) sidechain on the third sugar of the sequence, i. e. N‐acetylglucosamine. The composition of the ceramide moiety was dominated by a C20:0 fatty acid (arachidic acid) and a C14:1 sphingoid base (tetradecasphing‐4‐enine). The chemical structures of the two insect acidic glycosphingolipids were determined to be: GlcA(β‐3)Gal‐(β1‐3)GalNAc(β1 ‐4)GlcNAc(β1 ‐3)Man(β1‐4)Glc(β ‐1)Cer; GlcA(β1‐3)Gal(β1‐3)GalNAc(β1 ‐4)[2AeP‐6]‐GlcNAc(β1‐3)Man(β1 ‐4)Glc(β1‐1)Cer.

Such glucuronic‐acid‐containing insect glycosphingolipids have been given the generic name arthrosides, with the implied synonymity to the gangliosides.