bluuebunny/crossref_metadata_2025 huggingface dataset:
Secondary Structure of the Single‐stranded DNA Binding Protein Encoded by Filamentous Phage Pf3 as Determined by NMR
Authors: Folmer Rutger H. A. , Folkers Paul J. M. , Kaan Anita , Jonker Aafke J. , Aelen Jan M. A. , Konings Ruud N. H. , Hilbers Cornelis W.
Published: March 2005

Nuclear magnetic resonance spectroscopy was employed to study the single‐stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3. The protein is 78 amino acids long and occurs in solution predominantly as a homodimer with a molecular mass of 18 kDa. Sequence‐specific 1H and 15N resonance assignments have been obtained using homo‐ and heteronuclear two‐ and three‐dimensional experiments. The secondary structure of the protein monomer was determined from a qualitative interpretation of nuclear Overhauser enhancement spectra and amide exchange data. It consists of a five‐stranded antiparallel β‐sheet and three β‐hairpins. Problems caused by the protein's tendency to aggregate at concentrations needed for NMR spectroscopy were largely overcome by designing a mutant (Phe36→His) which exhibits significantly improved solubility characteristics over the wild‐type protein. It is shown that this mutation only locally affects the structure of the protein; the chemical shifts of the wild‐type and mutant species differ only for a few residues near the site of the mutation, and the secondary structures of the proteins are identical. The secondary structure of the Pf3 single‐stranded DNA binding protein is compared to that of the Ff gene V protein, the only single‐stranded DNA binding protein for which the complete three‐dimensional structure is known to date [Folkers, P. J. M., Nilges, M., Folmer, R. H. A., Konings, R. N. H. & Hilbers, C. W. (1994) J. Mol. Biol. 236, 229–246; Skinner, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y., Bellamy, H., Sweet, R. M., Gray, C. W., Konings, R. N. H., Wang, A. H.‐J. & Terwilliger, T. C. (1994) Proc. Natl Acad. Sci. USA 91, 2071–2075]. It is found that the secondary structures of the two proteins are very similar which supports the hypothesis that a five‐stranded antiparallel β‐sheet with protruding β‐hairpins is a common motif in a certain class of single‐stranded DNA binding proteins. In addition, the sequence and folding predicted earlier for the DNA binding wing in the single‐stranded DNA binding protein of phage Pf3 [de Jong, E. A. M., van Duynhoven, J. P. M., Harmsen, B. J. M., Tesser, G. I., Konings, R. N. H. & Hilbers, C. W. (1989) J. Mol. Biol. 206, 133–156] is borne out by the present study. It closely resembles that in the single‐stranded DNA binding protein of phage Ff, which may indicate that such a wing is a recurrent motif as well.
Mechanisms of phospholipase D stimulation by m3 muscarinic acetylcholine receptors
Authors: Schmidt Martina , HÜWe Sabine M. , Fasselt Birgit , Homann Doris , RÜMenapp Ulrich , Sandmann Joachim , Jakobs Karl H.
Published: March 2005

In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTP‐binding proteins by AlF‐4 and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium‐dependent events were studied. Prevention of an agonist‐induced increase in cytoplasmic calcium concentration did not alter the mAChR‐induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12‐myristate 13‐acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down‐regulation of protein kinase C by prolonged exposure to phorbol 12‐myristate 13‐acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP‐binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol‐induced and AlF‐4 induced phospholipase D activation but had no effect on enzyme activation by phorbol 12‐myristate 13‐acetate. The data indicate that phospholipase D in m3 mAChR‐expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP‐binding proteins, protein kinase C‐dependent and calcium‐dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phospholipase D activation by the m3 mAChR and heterotrimeric GTP‐binding proteins.
Identification and characterization of DBK, a novel putative serine/threonine protein kinase from human endothelial cells
Authors: Chu Wei , Presky David H. , Danho Waleed , Swerlick Robert A. , Burns Daniel K.
Published: March 2005

Protein kinases are involved in signal transduction pathways and play important roles in the regulation of cell functions. cDNA clones encoding a novel serine/threonine protein kinase sequence, designated as DBK, were isolated from cDNA libraries made from human endothelial cells. The compiled nucleotide sequence is 1636 base pairs long, consisting of an open reading frame encoding a 479‐amino‐acid protein with a calculated molecular mass of 53 kDa. The deduced amino acid sequence contains a protein kinase catalytic domain of 263 residues which includes all the characteristic features of a serine/threonine protein kinase. The invariant amino acid residues scattered throughout the catalytic domain of almost all known protein kinases are also found in DBK. Sequence comparison of DBK catalytic domain shows approximately 51% sequence identities to that of human protein kinase C family members. DBK shares the highest sequence identity, 53%, to that of Drosophila PKC. Northern blot analysis of various human tissues and cultured cell lines with a DBK gene‐specific cDNA probe demonstrated a single band of 2.0 kb that is expressed in all tissues and cell lines examined. Although the expression of DBK kinase was detected in all human tissues analyzed, the levels of expression varied significantly, with the highest expression detected in lung and heart, and the lowest expression found in brain and liver. Anti‐DBK peptide‐specific rabbit antisera were prepared, and were capable of immunoprecipitating DBK protein from COS cells transfected with DBK cDNA. The DBK gene is a single‐copy gene, and is highly conserved across species from human to yeast. Using somatic cell hybrids, the DBK gene has been localized to human chromosome 14. The ubiquitous expression and high degree of conservation of DBK across species suggest that DBK may play an important role in cell functions.
Sequence‐specific assignment of the 1H and 15N nuclear magnetic resonance spectra of the reduced recombinant high‐potential iron‐sulfur protein I from Ectothiorhodospira halophila
Authors: Bertini Ivano , Felli Isabella Caterina , Kastrau Dieter H. W. , Luchinat Claudio , Piccioli Mario , Viezzoli Maria Silvia
Published: March 2005

A 1H and 15N NMR investigation through two‐dimensional and three‐dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high‐potential iron‐sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are paramagnetic with a relatively low μeff of about 0.8 μB/iron ion, but the paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closest to the paramagnetic center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X‐ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a paramagnetic protein to date.

Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter‐residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X‐ray crystallographic analysis of the wild‐type protein at 250‐pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild‐type protein. These data are meaningful for the detection of structural differences among mutants in future studies.
The three‐dimensional structure in solution of the paramagnetic high‐potential iron‐sulfur protein I from Ectothiorhodospira halophila through nuclear magnetic resonance
Authors: Banci Lucia , Bertini Ivano , Eltis Lindsay D. , Felli Isabella Caterina , Kastrau Dieter H. W. , Luchinat Claudio , Piccioli Mario , Pierattelli Roberta , Smith Michael
Published: March 2005

The three‐dimensional structure in solution of reduced recombinant high‐potential iron‐sulfur protein iso‐I from Ectothiorhodospira halophila was determined using 948 relevant interproton NOEs out of the 1246 observed NOEs. The determination was accomplished using the XEASY program for spectral analysis and the distance geometry (DG) program DIANA for generation of the structure as described by Wüthrich [Wüthrich, K. (1989) Acc. Chem. Res. 22, 36–44]. The FeS cluster was simulated using an amino acid residue constructed for the present work from a cysteinyl residue with an iron and a sulfur atom attached to the terminal thiol. The family of structures obtained from distance geometry were subjected to energy minimization and molecular dynamics simulations using previously defined force field parameters. The quality of these structures at each stage of the refinement process is discussed with respect to the dihedral angle order parameter and the root‐mean‐square deviation of the atomic coordinates. The latter values for the backbone atoms vary from 67 pm for the distance‐geometry structures to 60 pm for the energy‐minimized structures to 51 pm for the structures subjected to restrained molecular dynamics. Finally, the structure in best agreement with the NOE constraints has been further treated with extensive restrained molecular dynamics in water. The solution structure is well defined and is very similar to the available X‐ray structure. We do not know of any previous determination of the structure of a paramagnetic protein in solution by NMR. The effect of paramagnetism on the quality of the structure determination is discussed.
A gelsolin‐related actin‐severing protein with fully reversible actin‐binding properties from the tail muscle of crayfish, Astacus leptodactylus
Authors: Bock Detlef , Hinssen Horst , D'Haese Jochen
Published: March 2005

A Ca2+‐dependent actin‐severing protein was purified from the tail muscle of the crayfish Astacus leptodactylus. The isolation procedure involved extraction at low ionic strength in the presence of EGTA, followed by ammonium sulfate fractionation, ion‐exchange chromatography and gel filtration. The purified crayfish actin modulator appeared as a single band with a molecular mass of 105 kDa on SDS/PAGE. The crustacean actin modulator revealed basic functional properties in common with vertebrate gelsolin, like the Ca2+‐activated severing of F‐actin and the nucleation of actin polymerization. However, both proteins differed in major aspects: Ca2+ activation of crayfish actin modulator started at lower threshold concentrations (0.1 μM). The effect of the modulator on shortening the nucleation phase of actin polymerization was significantly weaker at lower modulator/actin ratios. The modulator formed three distinct stoichiometric complexes with G‐actin, identified as binary, ternary and quaternary. Binding of G‐actin occurred in a low cooperative manner and was completely reversible by EGTA. Despite some properties being similar to those of villin, crayfish actin modulator did not cross‐link actin filaments. It is regarded in principle as a gelsolin‐type protein, but with characteristic functional deviations from vertebrate gelsolin.
Evidence for a pyrimidine‐nucleotide‐specific initiation site (the i site) on Escherichia coli RNA polymerase
Authors: Reddy Padmalatha S. , Chatterji Dipankar
Published: March 2005

Escherichia coli RNA polymerase has two sites, the i and i+1, for the binding of the first two substrates. The i site is template‐ and Mg2+‐independent and purine‐nucleotide‐specific, whereas the i+1 site is template‐ and Mg2+‐dependent and shows no nucleotide preference. The specificity of the i site for purine nucleotides is well in accord with the fact that most promoters initiate with a purine nucleotide. But there are a few promoters that initiate with a pyrimidine nucleotide. Dinucleotide synthesis at these promoters is completely inhibited by rifampicin. Earlier studies have failed to identify an i site for pyrimidine nucleotides. In this paper, using a fluorescent analog of UTP, namely uridine 5′‐[γ‐(5‐sulfonic acid) naphthylamidate]‐triphosphate, abbreviated as UTP[AmNS], we are able to show its binding to RNA polymerase, with a Kd of 0.8 μM, in the absence of Mg2+ and template. This suggests the presence of an i pyrimidine nucleotide site. The fact that UTP‐[AmNS] is capable of initiating RNA synthesis from the i site is further evidenced by the abortive transcription analyses at the lac promoter. Fluorescence titration studies performed in the presence and absence of purine initiator molecules indicate that this site is different from the i purine site. Scatchard analysis of the above data indicates the presence of a single binding site for UTP[AmNS] in the absence of Mg2+. Moreover UTP[AmNS] binds to the core enzyme with a Kd of 3.0 μM implying that, unlike the i purine nucleotide site, the σ protein confers a tighter binding of UTP‐[AmNS] to the low‐Kd site. Forster's energy transfer measurements using UTP[AmNS] as the donor and rifampicin as the acceptor have been used for estimation of the distance of the i pyrimidine nucleotide site from the rifampicin site. From these measurements, we infer that there is no direct interference of rifampicin with the first phosphodiester bond between two pyrimidine nucleotides.
Gene‐Targeting Approaches in the Study of Cellular Processes Involved in Growth or Differentiation
Authors: Yamada Gen , Sugimura Kazuhisa , Stuart Edward T.
Published: March 2005

Contents Development of ES cell technology–Cytokines (neutrophins, neurokines, and interleukins) and their receptors–Proto‐oncogenes and tumour‐suppressor genes–Developmental control genes–Concluding remarks.
The Energetics and Sodium‐Ion Dependence of N5‐Methyltetrahydromethanopterin:Coenzyme M Methyltransferase Studied with Cob(I)Alamin as Methyl Acceptor and Methylcob(III)Alamin as Methyl Donor
Authors: Weiss David S. , Gärtner Peter , Thauer Rudolf K.
Published: March 2005

N
5‐Methyltetrahydromethanopterin: coenzyme M methyltransferase from methanogenic Archaea is a membrane‐associated enzyme complex that uses a methyl‐transfer reaction to drive an energy‐conserving sodium‐ion pump. Methyl transfer occurs in two steps, first from N5‐methyl‐tetrahydromethanopterin (CH3‐H4MPT) to an enzyme‐bound cob(I)amide prosthetic group, and secondly from the methylated cobamide to coenzyme M (H‐S‐CoM). In this study, we report that methyltransferase can also use exogenous cob(I)alamin and methylcob(III)alamin as methyl acceptor and methyl donor, respectively. The enzyme catalyzes methylcob(III)alamin formation from CH3‐H4MPT and cob(I)alamin (reaction a), and methyl‐coenzyme M formation from methyl‐cob(III)alamin and H‐S‐CoM (reaction b). Both reactions were shown to be reversible. Reaction a was catalyzed at approximately the same rate (3 U/mg) and reaction b at approximately 10% the rate (0.3 U/mg) of the physiological reaction, namely methyl transfer from CH3‐H4MPT to H‐S‐CoM. The free energy changes (ΔG°′)associated with reactions a and b were both between –10 kJ/mol and ‐20 kJ/mol, consistent with a free energy change of approximately–30 kJ/mol determined for the physiological reaction. Reaction b but not reaction a was sodium‐ion dependent. Assuming that methylation of exogenous cob(I)alamin and demethylation of exogenous methylcob(III)alamin mimic methylation and demethylation of the enzyme‐bound corrinoid prosthetic group, it can be inferred that methyl transfer from the methylated cobamide prosthetic group to H‐S‐CoM is a site of coupling with sodium‐ion translocation.
Catalytic Properties of the Plant Cytochrome P450 CYP73 Expressed in Yeast
Authors: Pierrel Marie Agnès , Batard Yannick , Kazmaier Michael , Mignotte‐Vieux Claudia , Durst Francis , Werck‐Reichhart Danièle
Published: March 2005

The catalytic properties of CYP73, a cinnamate 4‐hydroxylase isolated from Helianthus tuberosus tuber [Teutsch, H. G., Hasenfratz, M. P., Lesot, A., Stoltz, C., Garnier, J. M., Jeltsch, J. M., Durst, F. & Werck‐Reichhart, D. (1993) Proc. Natl Acad. Sci. USA 90, 4102–4106] and expressed in an optimised yeast system [Urban, P., Werck‐Reichhart, D., Teutsch, G. H., Durst, F., Regnier, S., Kazmaier, M. & Pompon, D. (1994) Eur. J. Biochem. 222, 843–850] have been investigated. Microsomes from transformed yeast catalysed trans‐cinnamate hydroxylation with high efficiency. CYP73 was highly specific for its natural substrate, and did not catalyse oxygenation of p‐coumarate, benzoate, ferulate, naringenin or furanocoumarins. No metabolism of terpenoids or fatty acids, known substrates of plant P450s, was observed. CYP73 however demethylated the natural coumarin herniarin into umbelliferone. In addition, it was shown to oxygenate five xenobiotics and mechanism‐based inactivators, including the herbicide chlorotoluron. All substrates of CYP73 were small planar aromatic molecules. Comparison of the kinetic parameters of CYP73 for its various substrates showed that, as expected, cinnamate was by far the best substrate of this P450. The physiological and toxicological significance of these observations are discussed.
Analysis of the Thermodynamic Linkage of DNA Binding and Ion Binding for Dimeric and Tetrameric forms of the Lac Repressor
Authors: Stickle Douglas F. , Liu Gang , Fried Michael G.
Published: March 2005

The salt concentration dependences of the observed association constants (Kobe) for the binding of wild‐type lac repressor tetramer and the dimeric lacl ‐18 mutant repressor to lactose operator DNA were compared. For both proteins, the data are consistent with a class of linkage models in which ion binding by the protein is driven by differences in the ionic concentrations in bulk solution and in the volume near the DNA surface. The models that best agree with the data are those in which ion‐binding reactions are cooperative. In spite of close agreement between these models and the data, the determination of ion stoichiometries and apparent ion‐binding affinities requires additional mechanistic or structural information. The simplest ion‐binding mechanism consistent with the data is compatible with a current structural model of the repressor‐operator complex. At salt concentrations in excess of 50 mM, at which cation displacement from the DNA and anion displacement from the protein are expected to dominate, similar ion stoichiometries are found for the DNA binding of dimeric and tetrameric repressors. This supports the notion that the DNA contacts of these proteins are homologous. At lower salt concentrations, in which cation binding by the proteins is expected to be significant, the net ion stoichiometry of wild‐type repressor binding is slightly greater than that of the lacl ‐18 mutant. This difference may reflect the availability of ion‐binding sites in the distal subunits of tetramer that are not present in the dimer, or may be a consequence of the involvement of ion binding in the dimer/monomer equilibrium.
A Model for the Denaturation and Aggregation of β‐Lactoglobulin
Authors: Roefs Sebastianus P. F. M. , De Kruif Kees G.
Published: March 2005

A quantitatively correct kinetic model for the temperature‐induced denaturation and aggregation of β‐lactoglobulin is presented. The model recognizes an initiation, a propagation and a termination step by analogy with polymer radical chemistry. The decrease in native β‐lactoglobulin is predicted to follow order 3/2, in agreement with experimental results. The size of the protein polymer particles is predicted to be proportional to the square root of the initial β‐lactoglobulin concentration. The scattered light intensity is proportional to the product of concentration and size of the protein polymer particles. The initial increase in scattering intensity of the particles therefore scales with the initial squared β‐lactoglobulin concentration. The influence of other reaction conditions, e.g. ionic strength and pH, can be incorporated via the reaction constants of the reaction kinetic pathway.
Naturally Occurring Human Glutathione S‐transferase GSTP1‐1 Isoforms with Isoleucine and Valine in Position 104 Differ in Enzymic Properties
Authors: Zimniak Piotr , Nanduri Bindu , Pikuła Sławomir , Bandorowicz‐Pikuła Joanna , Singhal Sharad S. , Srivastava Sanjay K. , Awasthi Sanjay , Awasthi Yogesh C.
Published: March 2005

Glutathione S‐transferase P1‐1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. & Kurosky, A. (1990) Arch. Biochem. Biophys. 278, 398–408]. In the present report, the enzymic properties of these two proteins are compared. [I104]glutathione S‐transferase P1‐1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated to replace Ile104 by Val104, and [V104]glutathione S‐transferase P1‐1 was expressed and isolated as described for [I104]glutathione S‐transferase P1‐1. The two enzymes differed in their specific activity and affinity for electrophilic substrates (KM values for 1‐chloro‐2,4‐dinitrobenzene were 0.8 mM and 3.0 mM for [I‐104]glutathione S‐transferase P1‐1 and [V‐104]glutathione S‐transferase P1‐1, respectively), but were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half‐lives at 45°C of 19 min and 51 min, respectively. The resistance to heat denaturation was differentially modulated by the presence of substrates. These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate‐binding site, and may also influence activity by interacting with residues directly involved in substrate binding.
Solvent Hydrogen Isotope Effects and Anion Inhibition of CO2 Hydration Catalysed by Carbonic Anhydrase from Pisum sativum
Authors: Johansson Inga‐Maj , Forsman Cecilia
Published: March 2005

Chloroplast carbonic anhydrase from Pisum sativum has been studied to elucidate the catalytic mechanism and to test if the mechanism proposed for human carbonic anhydrase II is also valid for pea carbonic anhydrase. The catalytic activity was found to depend on the chemical nature of the buffer. Barbital buffer gives the highest turnover number at infinite buffer concentration and the lowest Km value with respect to the buffer, while the kinetic parameters obtained in the imidazole‐type buffer, 1‐methylimidazole, do not differ from those obtained using the biological‐type buffer Mops. The anion inhibition of CO2 hydration was investigated using SCN− at pH 6–9. The binding of the anion was found to be pH dependent with the strongest interaction at low pH. We obtained an uncompetitive inhibition pattern at high pH and noncompetitive inhibition patterns at pH 7 and low pH. The catalytic mechanism was further tested by measurements of the solvent hydrogen isotope effects on the kinetic parameters for CO2 hydration. The observed effects were comparatively small with a kcat value of approximately 2 irrespective of the pH. The effect on kcat/Km and on Km changes when going from high pH to pH 7 and low pH. At high pH, the solvent isotope effect in Km is at least 3, giving a value below 1 for kcat/Km, while at pH 7 and low pH the major effect is found in kcat/Km with values of 2.6 and 2.9.

The dependence of the CO2‐hydration activity on the buffer concentration is in agreement with a ping‐pong mechanism with buffer acting as a second substrate. This is analogous to the behaviour of human carbonic anhydrase II. The inhibition patterns and the observed isotope effects at high pH can also be explained within the framework of the catalytic mechanism for human carbonic anhydrase II, with a rate‐determining and buffer‐dependent part. The results are consistent with a mechanism involving a proton transfer that contributes to rate limitation. However, the isotope effects found at pH 7 and low pH indicate that some part of the mechanism has changed. Moreover, we cannot decide whether the mechanism for pea carbonic anhydrase involves an internal proton‐shuttle group, or if the buffer molecule acts in a direct proton transfer from the zinc‐coordinated water.
Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase
Authors: Benguría Alberto , Hernández‐Perera Octavio , Martínez‐pastor María Teresa , Sacks David B. , Villalobo Antonio
Published: March 2005

An epidermal‐growth‐factor(EGF)‐receptor preparation isolated by calmodulin‐affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3–8‐fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium‐binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 32P/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used.
Cloning and Sequencing of a Rhodothermus marinus Gene, bglA, Coding for a Thermostable β‐Glucanase and its Expression in Escherichia coli
Authors: Spilliaert Rémi , Hreggvidsson Gudmundur Oli , Kristjansson Jakob Kristjan , Eggertsson Gudmundur , Palsdottir Astridur
Published: March 2005

A gene library of the thermophilic eubacterium, Rhodothermus marinus, strain 21, was prepared in pUC18 and used to transform Escherichia coli. Of 5400 transformants, two produced halos on lichenan plates after Congo‐red staining. Restriction mapping showed that the two clones shared an overlapping 1200‐bp DNA fragment, which was used for DNA sequencing. Five potential methionine (Met) translational‐initiation codons were identified. A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids. The molecular mass of the mature enzyme was estimated to be 29.7 kDa. A comparison of the primary protein sequence of β‐glucanase of Rhodothermus marinus with other glycosyl hydrolases showed 38.5% identity to the C‐terminal part of the β‐1,3‐glucanase of Bacillus circulars and limited identity to bacterial endo‐β‐1,3–1,4‐glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial β‐glucanases. A gene fragment of 889 bp containing the catalytic domain was overexpressed in E. coli using the pET23, T7‐phage RNA polymerase system. The enzyme showed activity on lichenan, β‐glucan and laminarin but not on CMC cellulose or xylan. The expressed enzyme was purified by heat treatment of the host. The enzyme had a temperature and pH optima of 85°C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80°C and have a half life of 3 h at 85°C.
Comparison of Plasminogen Binding and Activation on Extracellular Matrices Produced by Vascular Smooth Muscle and Endothelial Cells
Authors: Stack M. Sharon , Rinehart Alex R. , Pizzo Salvatore V.
Published: March 2005

Plasminogen is the zymogen form of the serine proteinase plasmin. Although plasmin functions primarily as a fibrinolytic enzyme, recent evidence from numerous laboratories indicates that plasmin is also active in extracellular‐matrix (ECM) proteolysis. The role of plasmin in ECM degradation suggests that activation of plasminogen may be regulated by interaction with components of the ECM. In the current study, we have investigated binding and kinetic interactions between plasminogen, plasminogen activators and ECM synthesized by either vascular smooth muscle cells (SMCECM) or endothelial cells (ECECM). We report binding of plasminogen, tissue‐type plasminogen activator (t‐PA) and urinary‐type plasminogen activator (u‐PA) to intact SMCECM with concentrations of ligand yielding half‐maximal binding (B50) of 34, 5 and 15 nM, respectively. ECECM bound only plasminogen and t‐PA, with B50 values of 32 nM and 10 nM, respectively. The initial rate of t‐PA‐catalyzed plasminogen activation was enhanced 41‐fold in the presence of SMCECM and 27‐fold on ECECM. In contrast, u‐PA‐catalyzed activation on SMCECM and ECECM was increased only 1.5‐fold or 3‐fold, respectively. These data suggest that the ECM may provide an alternative surface for assembly and regulation of plasminogen activation.
Structural Characteristics of Cross‐Linking Sites in type V Collagen of Bone
Authors: Niyibizi Christopher , Eyre David R.
Published: March 2005

To understand the role of type V collagen and its spatial interrelationship with type I collagen in bone matrix, the molecule's covalent intermolecular cross‐links were structurally characterized. Type V collagen containing α1(V), α2(V) and α1(XI) chains was isolated from bovine bone and reacted with NaB3H4 to label the cross‐linking residues. Radiolabeled native molecules and isolated a chains were treated with sodium metaperiodate to cleave the divalent cross‐linking bonds. Sequence analysis of the periodate‐released peptides matched two of them to α1(V) and α1(XI) aminopropeptide domains. A third peptide was derived from the α1(I) carboxytelopeptide domain of type I collagen. This latter peptide, therefore, came from a site of heterotypic cross‐linking between types I and V collagens and accounted for about 15% of the total cross‐linked peptides. Sequence analysis of isolated cross‐linked tryptic peptides defined the helical sites of attachment of the periodate‐released telopeptides and revealed that the putative aminoproteinase‐cleavage sites in the α1(V) and α1(XI) chains are located in the molecule interior to the cross‐linking residue. These data imply that type V collagen molecules in the extracellular matrix are primarily cross‐linked to each other in a head‐to‐tail linear polymer that is linked laterally to type I collagen molecules in copolymeric fibrils.
Identification of Functional Domains in the Cytoskeletal Protein Talin
Authors: Niggli Verena , Kaufmann Stephan , Goldmann Wolfgang H. , Weber Thomas , Isenberg Gerhard
Published: March 2005

The cytoskeletal protein talin potentially plays a key role in actin–membrane linkage. It is able to nucleate actin filament growth in vitro while binding simultaneously to lipid bilayers. Thrombin digestion of human platelet talin yields two polypeptide domains of 200 kDa and 47 kDa. We have purified these fragments and analyzed their functional properties: the 200‐kDa fragment was active in nucleating actin filament formation and reduced the viscosity of filamentous actin, comparable to the effects of the intact protein. The 47‐kDa fragment was inactive in this respect.

However, the 47‐kDa polypeptide, but not the 200‐kDa fragment, interacted specifically with large liposomes containing acidic phospholipids. This is demonstrated by selective, hydrophobic photolabeling of the 47‐kDa fragment using phosphatidylserine liposomes containing trace amounts of a photoactivatable phospholipid analogue and by selective co‐sedimentation of this domain with the liposomes. The 200‐kDa fragment, whether alone or in conjunction with the small fragment, neither incorporated significant amounts of label nor co‐sedimented with the liposomes.

We thus are able to attribute specialized functions to distinct domains on the talin molecule. These enable the protein to interact simultaneously with actin filaments and lipid membranes.
Mouse Very‐Low‐Density‐Lipoprotein Receptor (VLDLR) cDNA Cloning, Tissue‐specific Expression and Evolutionary Relationship with the Low‐density‐lipoprotein Receptor
Authors: Oka Kazuhiro , Ishimura‐Oka Kazumi , Chu Mei‐jin , Sullivan Merry , Krushkal Julia , Li Wen‐Hsiung , Chan Lawrence
Published: March 2005

The very‐low‐density‐lipoprotein receptor (VLDLR) is a recently described lipoprotein receptor that shows considerable similarity to the low‐density‐lipoprotein receptor (LDLR). This receptor has been suggested to be important for the metabolism of apoprotein‐E‐containing triacylglycerol‐rich lipoproteins, such as very‐low‐density‐lipoprotein (VLDL), β‐migrating VLDL and intermediate‐density lipoprotein. cDNA clones that code for the VLDLR were isolated from a mouse heart cDNA library. The deduced amino acid sequence predicts a mature protein of 846 amino acids preceded by a 27‐residue signal peptide. Three mRNA species for the VLDLR with sizes of 3.9, 4.5 and 7.9 kilobases were present in high concentration in heart and muscle, which utilize triacylglycerols as an energy source. VLDLR mRNA is also detected in decreasing amounts in kidney, brain, ovary, testis, lung and adipose tissue. It is essentially absent in liver and small intestine. The amino acid sequence of the VLDLR is highly conserved among rabbit, human and mouse. VLDLR contains five structural domains very similar to those in LDLR, except that the ligand‐binding domain in VLDLR has an eightfold repeat instead of a sevenfold repeat in LDLR. Sequence conservation among animal species is much higher for the VLDLR than the LDLR. Sequences of the VLDLR from three vertebrate species and the LDLR from five vertebrate species were aligned and a phylogenetic tree was reconstructed. Although both receptors contain five domains and share amino acid sequence similarity, our computations showed that they diverged before the divergence between mammals and amphibians. In addition, sequence comparison of both receptor sequences suggests that the rabbit is evolutionarily closer to man than to the mouse. These results are consistent with the hypothesis that the VLDLR and the LDLR have evolved from a common ancestral gene to play distinct roles in lipoprotein metabolism and that the metabolic handling of triacylglycerol by the body via the VLDLR is a highly conserved mechanism.
Involvement of a Cystatin‐α‐Sensitive Cysteine Proteinase in the Degradation of Native l‐Lactate Dehydrogenase and Serum Albumin by Rat Liver or Kidney Lysosomes
Authors: Ohshita Takeyuki , Kido Hiroshi
Published: March 2005

A lysosomal cysteine proteinase plays a critical role in the lysosomal degradation of native llactate dehydrogenase. The effects of a recombinant cystatin α and other cysteine proteinase inhibitors on the degradation of unlabeled native llactate dehydrogenase by total lysosomal enzymes were examined in vitro.lLactate dehydrogenase was inactivated, its 35‐kDa subunit disappeared and the final amino acid degradation products were produced during an incubation with disrupted lysosomes in vitro. These processes were all markedly suppressed by a small amount of cystatin α without inhibiting the activities of the known lysosomal cysteine proteinases, cathepsins B, H, I., and J. These results suggest that a cysteine proteinase, which is highly sensitive to cystatin α and distinct from other known lysosomal cysteine proteinases, is involved in the lysosomal degradation of native llactate dehydrogenase. An llactate‐dehydrogenase‐inactivating enzyme in the extracts of lysosomes was partially purified. It was separated from cathepsin J using a Sephacryl S‐200 gel‐filtration column and was further separated from cathepsin H by DEAE‐Sephadex A‐50 anion‐exchange column chromatography. The inactivation and degradation of llactate dehydrogenase by this partially purified enzyme were all markedly suppressed by a low level of cystatin α without inhibiting the activities of both cathepsins B and L. The degradation of rat serum albumin by the partially purified enzyme was also inhibited by the same concentration of cystatin α. It is concluded that a cystatin‐α‐sensitive cysteine proteinase, other than cathepsins B, H, L and J, is present in lysosomes and functions in the lysosomal degradation of at least native llactate dehydrogenase and serum albumin.
Isolation and Characterisation of Arcelin‐5 Proteins and cDNAs
Authors: Goossens Alain , Geremia Roberto , Bauw Guy , Van Montagu Marc , Angenon Geert
Published: March 2005

Arcelins are seed storage proteins present in some wild bean accessions (Phaseolus vulgaris). They are implicated in the resistance phenotype of these wild beans towards the Mexican bean weevil. Arcelin 5, one of six arcelin electrophoretic variants, has been characterised in detail. The purified arcelin‐5 protein fraction contained two major polypeptides of 32.2 and 31.5 kDa, designated arcelin 5a and arcelin 5b, respectively, and one minor polypeptide of 30.8 kDa, designated arcelin 5c. The three polypeptides have an identical isoelectric point and are identical for their first nine N‐terminal amino acids. Arcelin 5a and arcelin 5b are glycoproteins whereas arcelin 5c is not glycosylated. Native arcelin 5 has a molecular mass corresponding to a dimer form. Using amino acid sequence analysis and PCR techniques, two different arcelin‐5 cDNA sequences were obtained, designated arc5‐I and arc5‐II. Both encode proteins of 261 amino acids with a signal peptide of 21 amino acids. The identity between the two is 99% at the DNA level and 97% at the level of the deduced amino acid sequences. The arc5‐I and arc5‐II cDNAs encode arcelin 5a and arcelin 5b, respectively. Sequence comparisons and protein characteristics show clearly that arcelin 5 is related to, but distinct from, other arcelin variants and lectins of P. vulgaris.
Mapping of Human Granulocyte‐Macrophage‐Colony‐Stimulating‐Factor Domains Interacting with the Human Granulocyte‐Macrophage‐Colony‐Stimulating‐Factor‐Receptor α‐Subunit
Authors: Brown Christopher B. , Pihl Carin E. , Kaushansky Kenneth
Published: March 2005

The high‐affinity granulocyte‐macrophage‐colony‐stimulating‐factor (GM‐CSF) receptor (R) is composed of at least two subunits, termed α and β. The α subunit is crucial for initiating ligand/receptor interaction and for ligand specificity. The experiments reported in this study sought to identify the domains of human (h)GM‐CSF which are responsible for interaction with hGM‐CSFRα. Anti‐(human‐GM‐CSF) mAb were used as competitors in a 125I‐GM‐CSF receptor‐binding assay on cells expressing the recombinant hGM‐CSFRα chain. Inhibition of 125I‐GM‐CSF binding to the GM‐CSFRα chain was demonstrated by mAb which mapped to the middle third of the third α helix (amino acids 78–83), the distal two‐thirds of the third α helix and the initial 7 residues of the loop between the third and fourth helix (amino acids 78–94), and the extreme carboxy terminus. No inhibition of binding occured with an antibody whose domain begins in the first β‐pleated sheet (amino acids, 39–43), continues through the second helix (amino acids 55–64) and into the proximal third of the third helix (to amino acid 77). mAb which mapped to the distal half of the fourth helix and the carboxy‐terminal tail (amino acids 110–127) increased the binding of 125I‐GM‐CSF to GM‐CSFRα. Due to the known discontinous epitopes the possibility that the distal portion of the fourth helix contributes to binding cannot be eliminated. However, the domains of hGM‐CSF most clearly involved in binding to the hGM‐CSFRα are the distal two‐thirds of the third a helix, the immediate downstream residues and the extreme carboxy terminus of hGM‐CSF.
Pre‐Steady‐State MgATP‐Dependent Proton Production and Electron Transfer by Nitrogenase from Azotobacter vinelandii
Authors: Duyvis Martina G. , Wassink Hans , Haaker Huub
Published: March 2005

MgATP‐dependent pre‐steady‐state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o ‐cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP‐dependent proton production by reduced nitrogenase proteins at 20.0°C is 14±24 s−1. No proton production with a rate constant comparable to the observed rate constant of electron transfer (kobs≈ 100 s−1) was detected. The extent of the observed MgATP‐dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7±0.3 mol H+produced/mol MoFe protein with oxidized Fe protein, and 1.1±0.1 mol H+produced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electrons transferred/H+uptake was calculated to be 1.2±0.2.

After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton‐production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP‐dependent proton production (kobs≈ 35 s−1). The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein; with dithionite‐reduced Fe protein a value of 3.1±0.4 mol H+produced/mol MoFe protein was found (or 0.5±0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite‐reduced Fe protein with MgADP resulted in the production of 0.17±0.05 mol H+/mol Fe protein.

All reported absorbance changes were absent when the experiments were performed in a buffered solution.

The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s−1 which is followed by H+ production (kobs≈ 14 s−1). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP‐induced proton production decreases. When electrons are transferred, stoichiometrically less protons are produced.
Electron Paramagnetic Resonance Studies of the Methylmalonyl‐CoA Mutase Reaction
Authors: Zhao Ymin , Abend Andreas , Kunz Meinrad , Such Peter , Rétey János
Published: March 2005

The substrate‐dependent homolysis of the cobalt‐carbon bond and generation of organic radicals in the coenzyme‐B12–methylmalonyl‐CoA‐mutase complex have been demonstrated by EPR measurements. Both the natural substrate methylmalonyl‐CoA, its 13C‐substituted analogue and the non‐hydrolysable synthetic substrates succinyl‐dethia(carba)‐CoA, succinyl‐dethia(dicarba)‐CoA and 4‐carboxy‐2‐oxo‐butyl‐CoA induced similar but not identical EPR signals. 3‐Carboxypropyl‐CoA, a novel competitive inhibitor, has been synthesised. Its Ki, value of 89 ± 6 μM was in the same range as the Km, of succinyl‐CoA. Using [5′‐3H]adenosylcobalamin, an enzyme‐dependent tritium transfer to the inhibitor has been shown. The enzyme‐coenzyme‐inhibitor complex also exhibited EPR signals that were less structured and less intensive than the corresponding signals with active substrates.

These results prove that the inhibitor also induces cobalt‐carbon bond homolysis and undergoes reversible hydrogen transfer but not rearrangement.
Effects of Cadmium on the Control and Internal Regulation of Oxidative Phosphorylation in Potato Tuber Mitochondria
Authors: Kesseler Adolf , Brand Martin D.
Published: March 2005

The effect of cadmium on the distribution of control over oxidative phosphorylation in potato tuber mitochondria was quantified by measuring control coefficients using top‐down metabolic control analysis. Oxidative phosphorylation was divided into three subsystems, namely substrate oxidation, the phosphorylation reactions and the proton leak. The control exerted by each of these subsystems over the system fluxes, the value of the protonmotive force and the effective P/O ratio was quantified in the presence of different concentrations of free cadmium (up to 21 μM). Cadmium is known to stimulate the proton leak and inhibit the substrate oxidation reactions, but it had little effect on the distribution of control over the system variables except to shift the pattern to lower rates. Control exerted by particular subsystems appeared to change or to stay the same as cadmium was varied, depending on whether the control coefficients were presented as a function of respiration rate or protonmotive force. The regulatory strength of protonmotive force on the system variables was also calculated, as partial internal response coefficients. These coefficients changed with ATP turnover rate and with cadmium concentration, showing how the internal regulation of oxidative phosphorylation shifts under different conditions. The values of control coefficients and partial internal response coefficients show where control lies and how intermediates regulate the system variables under different conditions of ATP demand and external effector (i.e. cadmium) concentration. However, they are not useful for identifying the sites of action of external effectors, for which elasticity and regulation analysis must be used.