bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Human Small‐Ribosomal‐Subunit Proteins by N‐Terminal and Internal Sequencing, and Mass Spectrometry
Authors: Vladimirov Sergey N. , Ivanov Anton V. , Karpova Galina G. , Musolyamov Alekxander K. , Egorov Tsezi A. , Thiede Bernd , Wittmann‐Liebold Brigitte , Otto Albrecht
Published: July 2004

Reverse‐phase HPLC was used to fractionate 40S ribosomal proteins from human placenta. Application of a C4, reverse‐phase column allowed us to obtain 27 well‐resolved peaks. The protein composition of each chromatographic fraction was established by two‐dimensional polyacrylamide gel electrophoresis and N‐terminal sequencing. N‐terminally blocked proteins were cleaved with endoproteinase Lys‐C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40S human ribosomal subunit in the HPLC elution profile. By matrix‐assisted laser‐desorption ionization mass spectrometry the masses of the 40S proteins were determined and checked for the presence of post‐translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post‐translational modifications.
Purification and Characterisation of an α‐Glucan Phosphorylase from the Thermophilic Bacterium Thermus thermophilus
Authors: Boeck Birgit , Schinzel Reinhard
Published: July 2004

An α‐glucan phosphorylase has been purified 4500‐fold from the thermophilic bacteria Thermus thermophilus. In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose. T. thermophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5′‐phosphate content (1 M pyridoxal‐P/M sub‐unit), subunit molecular mass (about 90 kDa) and inhibitor constants. The optimum temperature of T. thermophilus phosphorylase was observed at 70°C in the pH range 5.5–6.5. While at 25°C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70°C seems to be a dimer. Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other α‐glucan phosphorylases. Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (Vmax= 18 U/mg). Correspondingly, maltotriose functions as primer in the synthesis direction. Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenate acting as a substrate indicate that the active site structure of T. thermophilus phosphorylase differs from that of known α‐glucan phosphorylases.
A H2O‐Producing NADH Oxidase from the Protozoan Parasite Giardia Duodenalis
Authors: Brown David M. , Upcroft Jacqueline A. , Upcroft Peter
Published: July 2004

We describe the purification of a H2O‐producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N‐terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a Km, value of 4.2 μM for NADH and 16 μM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O˙−2 nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (CU2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin: NAD(P)+ oxido‐reductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.
Analysis of Nucleotide Binding by a Vacuolar Proton‐Translocating Adenosine Triphosphatase
Authors: Webster Lorna C. , Apps David K.
Published: July 2004

The vacuolar‐type proton‐translocatine adenosine triphosphatase from bovine adrenal secretory granules (chromaffin granules) was purified and reconstituted into proteoliposomes. The binding of nucleotides to the enzyme was studied by quantifying their effects on the rate of inactivation by N‐ethylmaleimide (MalNEt) of ATP‐dependent proton translocation, and by direct measurement of the binding of [3H]MgADP. The results of these experiments are consistent with a model of the enzyme that had been developed as a result of kinetic experiments, the features of which are that the enzyme exists in two states, each containing three nucleotide‐binding sites on catalytic subunits, and that nucleoside diphosphates regulate the enzyme by binding with high affinity to a single site in the inactive T state of the enzyme.

Under the conditions of the experiments, MalNEt inactivated the ATPase in a pseudo‐first order reaction. Rate constants of inactivation were reduced in the presence of MgADP, MgIDP and free ADP; the kinetics of protection suggested that the two conformational states of the enzyme were inactivated at different rates and also confirmed the existence of two different types of binding site for MgADP. Low nucleotide concentrations afforded partial protection from MalNEt; this was ascribed to binding of nucleotide to the regulatory site causing a shift in the conformational equilibrium towards the T state, which was more slowly inactivated than the unliganded R state of the enzyme. At higher nucleotide concentrations, binding at the catalytic site afforded complete protection from MalNEt. Protection by MgADP[S] and magnesium 2′‐ and 3′‐O ‐[4‐benzoylbenzoyl]adenosine 5′‐triphosphate showed simpler kinetics but was also consistent with previously reported kinetic results.

Analysis of subunit labelling with [3H]MalNEt showed that the three 72‐kDa (catalytic) subunits were alkylated by MalNEt with similar rate constants, consistent with a symmetrical arrangement of the catalytic subunits, in contrast to the situation in F‐type ATPases.

Analysis of the binding of [3H]MgADP also confirmed the results of kinetic experiments. MgADP was shown to bind to the enzyme with an apparent dissociation constant of about 66 nM; assuming that the nucleotide binds only to the T‐state, the true dissociation constant is < 1 nM. Using Blue Native polyacrylamide gel electrophoresis to separate the holo‐ATPase from the membrane sector, the stoichiometry of binding was calculated to be 0.6 mol/mol enzyme, confirming the existence of a single regulatory site for MgADP. However, binding of MgADP to the enzyme was much slower than could be accounted for by the measured dissociation constants, suggesting that it is rate limited by a step such as a protein conformational change. Treatment designed to remove endogenous nucleotide had no effect on the rate or extent of binding of MgADP.
Properties of the Purified, Recombinant, NADP(H)‐Binding Domain III of the Proton‐Translocating Nicotinamide Nucleotide Transhydrogenase from Rhodospirillum Rubrum
Authors: Diggle Christine , Bizouarn Tania , Cotton Nick P. J. , Jackson J. Baz
Published: July 2004

Transhydrogenase comprises three domains. Domains I and III are peripheral to the membrane and possess the NAD(H)‐ and NADP(H)‐binding sites, respectively, and domain II spans the membrane. Domain III of transhydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli, and purified. The purified protein was associated with substoichiometric quantities of tightly bound NADP+ and NADPH. Fluorescence spectra of the domain III protein revealed emissions due to Tyr residues. Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nucleotide are spatially close. The rate constants for NADP+ release and NADPH release from domain III were 0.03 S‐1 and 5.6×10‐4S‐1, respectively. In the absence of domain II a mixture of the recombinant domain III protein, plus the previously described recombinant domain 1 protein, catalysed reduction of acetylpyridine–adenine dinucleotide (AcPdAD+) by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP+ from domain III. Similarly, the mixture catalysed reduction of thio‐NADP1 by NADH (forward transhydrogenation) at a rate limited by release of thio‐NADPH from domain III. The mixture also catalysed very rapid reduction of AcPdAD1 by NADH, probably by way of a cyclic reaction mediated by the tightly bound NADP(H). Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain III and vice versa indicated (a) that during reduction of AcPdAD1 by NADPH, a single domain I protein can visit and transfer H‐ equivalents to about 60 domain III proteins during the time taken for a single domain III to release its NADP+, whereas (b) the cyclic reaction is rapid on the timescale of formation and breakdown of the domain I. III complex. The rate of the hydride transfer reaction was similar in the domain I. III complex to that in the complete membrane‐bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I · III complex due to the greatly decreased rates of release of NADP+ and NADPH. It is concluded that, in the complete enzyme, conformational changes in the membrane‐spanning domain II, which result from proton translocation, lead to changes in the binding affinity of domain III for NADP+ and for NADPH.
Substrate Specificity of Chlorophyll(ide) b Reductase in Etioplasts of Barley (Hordeurn Vulgare L.)
Authors: Scheumann Verena , Ito Hisashi , Tanaka Ayumi , Schoch Siegrid , Rüdiger Wolfhart
Published: July 2004

Enzyme activity of chlorophyll(ide) b reductase is present in etioplasts. Recently the conversion of chlorophyllide b to chlorophyll a via 71‐hydroxychlorophyll a was demonstrated in barley etioplasts. We used zinc pheophorbide b for a detailed investigation of the reduction of the 7‐formyl group to the 7l‐hydroxy compound in intact barley etioplasts. The reaction proceeded likewise before esterification and after esterification with phytyl diphosphate. The metal‐free pheophorbide b, that is not accepted by chlorophyll synthase for esterification, is reduced to 71‐hydroxypheophorbide a to a small extent. The zinc (132S)‐pheophorbide b is at least equally well accepted for reduction as the epimer with the 132R configuration of natural chlorophyll b. The reaction requires NADPH or NADH, although the latter is less effective. ATP is not required for the first step to the 71‐hydroxy compound. The significance of chlorophyll b reduction for acclimation from shade to sun leaves and for chlorophyll degradation is discussed.
The Bound Adenine Nucleotides of Purified Bovine Mitochondrial ATP Synthase
Authors: Beharry Seelochan , Bragg Philip D.
Published: July 2004

The experiments in this study were directed towards defining the nucleotide content of purified beef‐heart mitochondrial F1F0 ATP synthase during binding and hydrolysis of ATP. The purified, soluble synthase as prepared contained 2 mol ATP and 2 mol ADP/mol enzyme. Three of these four nucleotides were exchangeable on incubation with radiolabelled MgATP. Passage of the ATP synthase through a column of Sephadex G‐50 readily removed 1 mol ADP/mol. The remaining bound nucleotides were not displaced by incubation with 1 mM GTP or 5 mM sodium sulfite, the latter an activator of the ATPase activity of the synthase. Incubation of the synthase with 250 μM MgATP in the presence of 3 mM sodium azide, an inhibitor of the ATPase, resulted in the transitory formation of a form of the enzyme in which 5–6 nucleotide‐binding sites were loaded with ATP and/or ADP, thus showing that the ATP synthase, like the soluble F1 ATPase, contained a minimum of six nucleotide‐binding sites. The presence of an ATP‐regenerating system during incubation with MgATP resulted in the loading of 5–6 sites to yield a form of the enzyme containing 3–4 mol ATP and 2 mol ADP/mol synthase even after passage through a centrifuged column. Following hydrolysis of the medium MgATP, the enzyme reached a stable form containing 2 mol ATP and 2 mol ADP/mol synthase. Like the form of the enzyme originally prepared, 1 mol ADP/mol synthase was readily released. However, this ADP remained bound to the synthase in the presence of GTP if azide was present. These results are discussed in the context of current ideas about nucleotide‐binding sites on the F1 ATPase portion of the F1F0 ATP synthase. It is concluded that the properties of the sites on the F1F0 synthase show some differences from those on the F1 ATPase.
The Osmotic Properties and Free Energy of Formation of the Actomyosin Rigor Complexes from Rabbit Muscle
Authors: Magri Ermes , Cuneo Paola , Trombetta Giorgio , Grazi Enrico
Published: July 2004

We have studied the osmotic properties of the calcium‐regulated actomyosin complexes from skeletal muscle at the protein osmotic pressure of 18 kPa and at different actin‐to‐myosin molar ratios. Essentially, protein solutions were equilibrated against a solution of poly(ethylene glycol) 40000 of known macromolecular osmotic pressure. At the end of the equilibration the water and the protein masses of the protein solutions were determined gravimetrically and the protein molar concentration was calculated.

In this reconstructed system we have found following, at the actin‐to‐molar ratio of 2.6 (the most likely stoichiometry of these two proteins in the dense region of the A band) the average distance between the myosin filaments is 34.2 nm, this equals the interfilament distance in the intact fibre of muscle in rigor, at the sarcomere length of 3.38 μm. The formation of the F‐actin‐myosin and of the tropomyosin–F–actin‐myosin rigor complexes involves the largest free energy changes, ‐5.38 kJ/mol myosin and ‐5.67 kJ/mol myosin, respectively.

The formation of the troponin–tropomyosin–F‐actin–myosin(Ca) rigor complex from myosin and troponin–tropomyosin–F‐actin(Ca) occurs with the free energy change of ‐3.43 kJ/mol myosin. Of these ‐3.43 kJ, ‐1.81 kJ are provided by the endergonic conversion of troponin–tropomyosin–F‐actin(EGTA) into troponin–tropomyosin–F‐actin(Ca). The transition of myosin and of troponin–tropomyosin–F‐actin(EGT.4) into the ‐F‐actin‐myosin(Ca) rigor complex is accompanied by a 5.8% increase of volume. The increase of volume is due to a large influx of water, which is essentially protein‐hydration water.
The Mechanism of Proline/Glutamate Antiport in Rat Kidney Mitochondria
Authors: Atlante Anna , Passarella Salvatore , Pierro Pasqualina , Di Martino Catello , Quagliariello Ernesto
Published: July 2004

Proline/glutamate antiport in rat kidney mitochondria has been studied in terms of two different features: energy dependence and glutamate‐carrier contribution to accomplish proline movement across the mitochondrial membrane.

Energy dependence of the proline/glutamate antiporter in rat kidney mitochondria has been investigated by means of both spectroscopic measurements and isotopic techniques, using either normal or [14C]glutamate‐loaded mitochondria. The sensitivity of the proline/glutamate antiport to the ionophores valinomycin and nigericin, under conditions in which Dψ and DpH are selectively affected, shows that the exchange is energy dependent. Measurements of both membrane potential and proton movement across the mitochondrial membrane suggest that proline/glutamate antiport is driven by the electrochemical proton gradient via the Dψ dependent proline/glutamate translocator and DpH‐dependent glutamate/OH‐ carrier. Such a carrier provides for re‐uptake of glutamate that has already passed out of the mitochondria in exchange with incoming proline, made possible by the existence of a separate pool of glutamate in the intermembrane space, directly shown by means of HPLC measurements.
Cloning, Sequencing and Sites of Expression of Genes for the Hydroxyarginine‐Containing Adhesive‐Plaque Protein of the Mussel Mytilus galloprovincialis
Authors: Inoue Koji , Takeuchi Yasuhiro , Miki Daisuke , Odo Satoshi , Harayama Shigeaki , Waite J. Herbert
Published: July 2004

A segment of Mytilus galloprovincialis foot protein 3 (Mgfp‐3) cDNA was amplified by means of reverse‐transcription (RT)/PCR with degenerate primers. The 5′ and 3′ regions of the cloned segment were amplified by means of rapid amplification of cDNA ends. The 5′‐region clones had almost identical nucleotide sequences, but two sequences were found among 3′‐region clones. The Mgfp‐3 coding region was amplified between a 5′ untranslated sequence and one of two 3′ untranslated sequences. Two cDNA clones which encoded variants Mgfp‐3A and Mgfp‐3B, were isolated. These two clones encoded proteins with 70 and 77 amino acid residues, of which the first 24 residues are predicted to be signal peptides. The existence of additional variants was suggested by the sequences of other clones. Thus, it was suggested that Mgfp‐3 genes constitute a gene family. RTPCR of RNA from developing larvae indicates that Mgfp‐3 genes are transcribed after settlement. RT/PCR of RNA from major organs and in situ hybridization of the foot indicate that Mgfp‐3 genes are transcribed in a limited part of the foot, i.e., the phenol gland and a distal part of the accessory gland.
Stimulation of ADP‐Ribosyl Cyclase Activity of the Cell Surface Antigen CD38 by Zinc Ions Resulting from Inhibition of Its NAD+ Glycohydrolase Activity
Authors: Kukimoto Iwao , Hoshino Shin‐ichi , Kontani Kenji , Inageda Kiyoshi , Nishina Hiroshi , Takahashi Katsunobu , Katada Toshiaki
Published: July 2004

The lymphocyte cell surface antigen, CD38, which has an amino acid sequence similar to Aplysia ADP‐ribosyl cyclase, catalyzes not only the hydrolysis of NAD+ and 1‐(5‐phospho‐β‐d‐ribosyl)adenosine 5′‐phosphate cyclic anhydride (cyclic ADP‐ribose) but also the formation of cyclic ADP‐ribose from NAD+. To characterize the bifunctional enzyme properties, we produced the recombinant CD38 fused with a maltose‐binding protein (MBP‐CD38). Zinc ions stimulated the ADP‐ribosyl cyclase activity of MBP‐CD38, but inversely inhibited its NAD+ glycohydrolase activity which was approximately 100‐fold dominant to the cyclase activity in the absence of Zn2+. Such dual effects of Zn2+ were also observed in the native membrane‐bound CD38 of HL‐60 cells which had been caused to differentiate by retinoic acid. Zinc ions inhibited the NAD+ glycohydrolase reaction catalyzed by MBP‐CD38 in an uncompetitive manner, whereas they enhanced the ADP‐ribosyl cyclase reaction without affecting the Km value for NAD+. There was an increase in the fluorescence intensity of a hydrophobic fluorescent probe, 8‐anilino‐1‐naphthalenesulfonate, in the presence of MBP‐CD38. The fluorescence increase was further enhanced by the addition of Zn2+ with a shift in the maximum emission wavelength from 484 nm to 470 nm, suggesting that Zn2+ caused conformational changes of MBP‐CD38. These results indicate that Zn2+ directly interacts with CD38 to stimulate its ADP‐ribosyl cyclase with inhibition of its NAD+ glycohydrolase, probably due to prevention of the access of water molecule to an intermediate of the enzyme‐substrate complex.
Differential Expression of Superoxide Dismutases Containing Ni and Fe/Zn in Streptomyces Coelicolor
Authors: Kim Eun‐Ja , Kim Hyoung‐Pyo , Hah Yung Chil , Roe Jung‐Hye
Published: July 2004

Streptomyces coelicolor contains two distinct superoxide dismutase (SOD) activities detected on native PAGE. The level of each changed differently depending on growth media and scarcely responded to paraquat, a superoxide‐generating agent. The total SOD activity doubled in late exponential phase compared with that in mid‐exponential phase and less than double upon treatment with plumbagin, another superoxide‐generating agent. The two SODs from S. coelicolor ATCC 10147 (Müller) strain were purified to near homogeneity. SOD1, a tetramcr of 13.4‐kDa subunits, was found to be a novel type of SOD containing 0.74 mol nickel/mol subunit as determined by atomic absorption spectroscopy. SOD2, a tetramer of 22.2‐kDa subunits, was found to contain 0.36 mol iron and 0.26 mol zinc/mol subunit. The N‐terminal amino acid sequences of both SODs were determined. SOD2 is similar to manganese‐containing superoxide dismutases (MnSODs) and iron‐containing superoxide dismutases (FeSODs) from other organisms, whereas SOD1 is less similar to known SODs but still contains a few conserved amino acids. The effects of metals and chelating agents on the expression of these two SODS were examined. The presence of nickel at micromolar concentrations in growth media induced the expression of SOD1 (nickel‐containing superoxidc dismutase; NiSOD), whereas the expression of SOD2 (iron/zinc‐containing superoxide dismutase; FeZnSOD) was repressed. The changes in SOD activities were positively correlated with the amount of each enzyme as determined by immunoblotting, suggesting that metals do not modulate the activity per se but the amount of each protein.
Interfacial Air/Water Proton Conduction from Long Distances by Sulfolobus solfataricus Archaeal Bolaform Lipids
Authors: Vilalta Isabelle , Gliozzi Alessandra , Prats Michel
Published: July 2004

The stability, structural organization, and the ability to transfer protons long distances have been investigated in monolayers formed from archaeal bolaform lipids at the air/water interface. The lipids employed were the fractions GroR2Gro (R represents an acyl group with variable chain length typically consisting of 0–4 cyclopentane rings and 40 isoprenoid residues) and GroR2GroNon‐ol (Non‐ol represents nonitol) extracted from Sulfolobus solfataricus by hydrolysis of the cytoplasmic membrane. GroR2‐GroNon‐ol films exhibit a very peculiar behaviour: the monolayer surface pressure increases with time, regardless of its low or high initial value. This finding is related to the possibility of GroR2GroNon‐ol molecules to assume an upright (a metastable) or a U‐shaped (stable) configuration. In the gaseous state and in the collapsed state of the film, no lateral proton conduction was observed. However, in the pressure range 0 < π < 25 mN/m for GroR2Gro and 0 < π < 30 mN/m for GroR2GroNon‐ol monolayers, a lateral proton conduction at the air/water interface was observed.

The structural organization of these bipolar lipids at the air/water interface can be related to the lateral proton conduction; it is possible to conclude that whatever configuration these lipids may adopt, they are able to structure the air/water interface in a hydrogen bond network that supports lateral proton conduction. This process may be ascribed to a percolation phenomenon occurring when the polar lipid head groups form a structured lattice of hydrogen bonds.
Characterization of a Ribosomal Inhibitory Polypeptide of Protein Phosphatase‐1 from Rat Liver
Authors: Beullens Monique , Stalmans Willy , Bollen Mathieu
Published: July 2004

About 4% of the spontaneous phosphorylase phosphatase activity in a rat liver extract was associated with the ribosomal fraction and stemmed from both protein phosphatase‐1 (PP‐1) and protein phosphatase‐2A (PP‐2A). However, after repeated washing, only PP‐1 remained bound to the ribosomes. The activity of ribosome‐associated PP‐I (PP‐1R) was partially latent and could be increased 2–3‐fold by incubation with trypsin and an additional 50% by incubation with low concentrations of exogenous type‐1 catalytic subunit. In contrast, incubation of the ribosomal fraction with MgATP resulted in a 50% drop in the activity of PP‐1R.

We have purified from a ribosomal extract a basic polypeptide (pI ≥ 10.5) of 23 kDa that potently inhibited PP‐1. This ribosomal inhibitor of PP‐1, termed RIPP‐1, was at least 30‐times less efficient in inhibiting other major Ser/Thr protein phosphatases (PP‐2A, PP‐2B and PP‐2C). RIPP‐1 was identified as a non‐competitive inhibitor of PP‐1 with a substrate‐dependent potency. The lowest Ki (approximately 20 nM) was obtained with phosphorylase and myelin basic protein as substrates. Besides instantaneously inhibiting the type‐1 catalytic subunit, RIPP‐1 also converted the catalytic subunit in a time‐dependent manner (t1/2= 45 min at 25°C) into a less active conformation. Unlike the inhibition, this slow inactivation was not reversed by the removal of RIPP‐1. We propose that RIPP‐1 accounts, at least in part, for the latency of PP‐1R.
Dissociation of Protamine‐DNA Complexes by Xenopus Nucleoplasmin and Minichromosome Assembly in vitro
Authors: Ruiz‐Lara Simón A. , Cornudella Luís , RodrÍguez‐Campos Antonio
Published: July 2004

Nucleoplasmin, an acidic thermostable protein abundant in the nucleus of Xenopus laevis oocytes, has been found to dissociate complexes of pUC19 DNA and protein φ1, an intermediate protamine present in ripe sperm from the mollusc Mytilus edulis. Cruder preparations of nucleoplasmin, such as the amphibian oocyte S150 extract and its thermostable fraction, also dissociate the heterologous DNA‐φ1 complexes and, in addition, promote the assembly of plasmid DNA into a minichromosome displaying regular nucleosomal periodicity, as revealed by micrococcal nuclease digestion. In contrast, purified nucleoplasmin complemented with rat hepatocyte core histone octamers in the presence of DNA topoisomerase I, although capable of inducing nucleoprotein formation onto the complexed DNA, fails to position nucleosomes at the native spacings seen in chromatin in vivo. These data favour the existence of a general mechanism to bring about, in a concerted manner, removal of sperm specific nuclear proteins and reconstitution of somatic chromatin following fertilization.
Biochemical Studies of Two Rat Acyl‐CoA Synthetases, ACS1 and ACS2
Authors: Iijima Hiroaki , Fujino Takahiro , Minekura Hiroyuki , Suzuki Hiroyuki , Kang Man‐Jong , Yamamoto Tokuo
Published: July 2004

Two types of acyl‐CoA synthetase (ACS), designated ACS1 and ACS2, are structurally similar isozymes with different tissue distributions. The two enzymes are organized into the following five regions: an NH2 terminus; two luciferase‐like regions; a linker connecting the luciferase‐like regions; a COOH terminus. Under the control of a lac promoter, rat ACS1 and ACS2 were overproduced in Escherichia coli and purified to homogeneity. The specific activities of the purified ACS1 and ACS2 were 26.2 μmol · min−1· mg−1 and 7.4 μmol · min−1· mg−1, respectively, and the most efficiently utilized saturated fatty acids were those with 10–18 carbon atoms. Among unsaturated fatty acids with 16–22 carbon atoms, the most preferred substrates were palmitoleate, oleate and linoleate for ACS1, and, for ACS2, oleate, arachidonate, eicosapentaenoate and docosahexaenoate. To determine the functionally important regions in the ACS isozymes, we constructed five ACS1 mutants lacking each of the five regions. Introduction of these mutants into E. coli revealed that all five regions in ACS1 are required for functional expression of the enzyme in E. coli; deletion of any one of the five regions almost completely abolished the enzyme activity.
Crystal Structure of Flavodoxin from Desulfovibrio desulfuricans ATCC 27774 in Two Oxidation States
Authors: Romero Antonio , Caldeira Jorge , Legall Jean , Moura Isabel , Moura José J. G. , Romao Maria J.
Published: July 2004

The crystal structures of the flavodoxin from Desulfovibrio desulfuricans ATCC 27774 have been determined and refined for both oxidized and semi‐reduced forms to final crystallographic R ‐factors of 17.9% (0.8–0.205‐nm resolution) and 19.4% (0.8–0.215‐nm resolution) respectively. Native flavodoxin crystals were grown from ammonium sulfate with cell constants a=b= 9.59 nm, c= 3.37 nm (oxidized crystals) and they belong to space group P3221. Semireduced crystals showed some changes in cell dimensions: a=b= 9.51 nm, c= 3.35 nm. The three‐dimensional structures are similar to other known flavodoxins and deviations are found essentially in the isoalloxazine ring environment. Conformational changes are observed between both redox states and a flip of the Gly61‐Met62 peptide bond occurs upon one‐electron reduction of the FMN group. These changes influence the redox potential of the oxidized/semiquinone couple.

Modulation of the redox potentials is known to be related to the association constant of the FMN group to the protein. The flavodoxin from D. desulfuricans now studied has a large span between E2 (oxidized → semiquinone) and E1 (semiquinone → hydroquinone) redox potentials, both these values being substantially more positive within known flavodoxins. A comparison of their FMN environment was made in both oxidation states in order to correlate functional and structural differences.
Protein Phosphatase Activity of Abscisic Acid Insensitive 1 (ABII) Protein from Arabidopsis Thaliana
Authors: Bertauche Nathalie , Leung Jeffrey , Giraudat Jérôme
Published: July 2004

Mutations at the ABII (abscisic acid insensitive 1) locus of the plant Arabidopsis thaliana cause a reduction in sensitivity to the plant hormone abscisic acid. The sequence of ABII predicts a protein composed of an N‐terminal domain that contains motifs for an EF‐hand Ca2+‐binding site, and a C‐terminal domain with similarities to protein serine/threonine phosphatases 2C. We report here two sets of experimental evidence that indicate that ABI1 has typical protein phosphatase 2C activity. First, expression of the ABI1 C‐terminal domain partially complemented the temperature‐sensitive growth defect of a Saccharomyces cerevisiae protein phosphatase 2C mutant. Second, recombinant proteins that contained the ABI1 C‐terminal domain displayed in vitro phosphatase activity towards 32P‐labelled casein, and this activity displayed Mg2+ or Mn+ dependence and okadaic acid insensitivity typical of protein phosphatases 2C. Characterisation of recombinant proteins that contained various portions of ABI1 indicated that the putative EF‐hand motif is unlikely to mediate Ca2+ regulation of the ABI1 phosphatase activity at physiological Ca2+ concentrations, and may represent an EF‐hand analogue rather than an EF‐hand homologue. The abil‐1 mutation appeared to cause significant reduction in the phosphatase activity of ABI1. These results are discussed in relation to the dominant phenotype of abil‐1 over the wild‐type allele in plants, and to the possible role of ABI1 in abscisic acid signalling.
Conservation of Aconitase Residues Revealed by Multiple Sequence Analysis
Authors: Frishman Dmitrij , Hentze Matthias W.
Published: July 2004

Aconitases have recently regained much attention, because one member of this family, iron regulatory protein‐1 (IRP‐1), has been found to play a dual role as a cytoplasmic aconitase and a regulatory RNA‐binding protein. This finding has highlighted a novel role for Fe‐S clusters as post‐translational regulatory switches. We have aligned 28 members of the Fe‐S isomerase family, identified highly conserved amino acid residues, and integrated this information with data on the crystallographic structure of mammalian mitochondrial aconitase. We propose structural and/or functional roles for the previously unrecognized conserved residues. Our findings illustrate the value of detailed protein sequence analysis when high‐resolution crystallographic data are already available.
Identification of the Biosynthetic Leukotriene C4 Export Pump in Murine Mastocytoma Cells as a Homolog of the Multidrug‐Resistance Protein
Authors: Leier Inka , Jedlitschky Gabriele , Büchler Markus , Buchholz Ulrike , Brom Manuela , Keppler Dietrich
Published: July 2004

A membrane glycoprotein of 190 kDa has been identified previously by photoaffinity labeling as a candidate for the ATP‐dependent export pump for leukotriene C4 in mastocytoma cells [Leier, I., Jedlitschky, G., Buchholz, U. & Keppler, D. (1994) Eur. J. Biochem. 220, 599–606]. The present study indicates that this protein represents the murine homolog of the human multidrug resistance protein (MRP). In immunoblot analyses several polyclonal anti‐MRP antibodies and one monoclonal antibody recognized the protein of 190‐kDa in plasma membranes of mastocytoma cells. Immunoprecipitation after photoaffinity labeling with [3H]leukotriene C4 precipitated the labeled 190‐kDa glycoprotein. Deglycosylation by glycopeptide N‐glycosidase F of mastocytoma membrane proteins was performed in comparison with membranes from MRP‐overexpressing cells and resulted in a reduction of the molecular mass of 190 kDa by about 20 kDa in all membrane preparations. The expression of the murine mrp gene in the mastocytoma cells was analyzed by amplification and sequencing of two mrp cDNA fragments in the first nucleotide binding domain (182 bp) and in a domain proximal to the 3′‐end (291 bp). The deduced amino acid sequences of these fragments were identical with murine Mrp and 86.7% and 89.7% identical with the corresponding sequences of human MRP. These results indicate that the ATP‐dependent release of leukotriene C4 by murine mastocytoma cells is mediated by murine Mrp.
Catalytic Activity of Complex I in Cell Lines that Possess Replacement Mutations in the ND Genes in Leber's Hereditary Optic Neuropathy
Authors: Majander Anna , Finel Moshe , Savontaus Marja‐Liisa , Nikoskelainen Eeva , WikströM Måten
Published: July 2004

Short‐chain ubiquinone analogues act as electron acceptors and as inhibitors in the lymphoblast mitochondria of ND1/3460 mutants, which indicates structural changes in the ubiquinone‐binding domain of Complex I in this mutant. The ND4/1778 mutant and two secondary ND5 mutants studied are associated with reductions of at least 50, 35 and 30% in the catalytic rate constant, respectively. However, the efficiency of oxidative phosphorylation is unaffected in all these ND mutants. The rate of respiration is only slightly limited by Complex I in lymphoblast mitochondria. Consequently, there is a 30–35% reduction in the electron flow through Complex I compared with that through Complex II, and an increased lactate/pyruvate ratio, in the ND1 and ND4 mutants, but these factors were unaffected in the secondary ND5 mutants. Energy metabolism is thus less severely affected in the secondary mutants than in the primary mutants, which supports the division into these two categories. An increased ubiquinone‐10 content in the mitochondrial membrane of all the mutants, and enhanced succinate dehydrogenase and citrate synthase activities in the ND4 mutant, are proposed to be compensatory changes. The efficiency of these changes and the level of kinetic limitation of respiration by Complex I in each tissue are proposed to determine the clinical development of the disease.
Kinetic Analysis of Synthetic Analogues of Linear‐Epitope Peptides of Glycoprotein D of Herpes Simplex Virus Type 1 by Surface Plasmon Resonance
Authors: Lasonder Edwin , Schellekens Gerard A. , Koedijk Danny G. A. M. , Damhof Ria A. , Welling‐Wester Sytske , Feijlbrief Matty , Scheffer Albert J. , Welling Gjalt W.
Published: July 2004

The interaction between mAb A16 and glycoprotein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface‐plasmon‐resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11–19 of gD. In a previous study, three critical residues, Asp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15‐amino‐acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino‐acid‐replacement study of the epitope gD‐(9–19)‐peptide and of a gD‐(9–19)‐peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D‐amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon‐resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD‐(9–19)‐peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD‐(11–17)‐peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11–17, identified Argl6 as an essential residue and suggested that Asp 13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide.
A Deletion in the First Cysteine‐Rich Repeat of the Low‐Density‐Lipoprotein Receptor Leads to the Formation of Multiple Misfolded Isomers
Authors: Djordjevic Julianne T. , Bieri Stephan , Smith Ross , Kroon Paulus A.
Published: July 2004

The ligand‐binding domain of the low‐density‐lipoprotein (LDL) receptor comprises seven cysteine‐rich repeats, each approximately 40 amino acids long. The deletion of two amino acids (Asp26 and Gly27) from the first of these repeats (LB1), leads to a defective LDL receptor, and the clinical syndrome of familial hypercholesterolemia [Leitersdorf, E., Hobbs, H. H., Fourie, A. M., Jacobs, M., van der Westhuyzen, D. R. & Coetzee, G. A. (1988) Proc. Natl Acad. Sci. USA 85, 7912–7916]. Receptors which reach the cell surface fail to bind IgG‐C7, a conformation‐specific monoclonal antibody directed to LB1. To determine the effects of the two‐amino‐acid deletion on the folding of the LB1 of the LDL receptor, we have expressed LBI and the mutant repeat, des‐Asp26, Gly27‐LB1, as recombinant (rLB1 and des‐Asp26, Gly27‐rLB1) peptides, and have determined their ability to fold in vitro. Unlike rLB1, which folded into a single isomer that was recognized by IgG‐C7 and had three disulfide bonds, des‐Asp26, Gly27‐rLB1 folded into an equilibrium mixture of four isomers. Each of these isomers contained three disulfide bonds, but none were recognized by IgG‐C7. We suggest that mutant LDL receptors in the endoplasmic reticulum (ER) of the cell also fold into an equilibrium mixture of distinct receptor molecules, each with an abnormally folded isomer of des‐Asp26, Gly27‐LB1, and that the retarded transport of receptors to the cell surface arises because only a subset of the isomers reaches the cell surface.
The Influence of the Peptide Chain on the Kinetics and Stability of Microperoxidases
Authors: Spee Johan H. , Boersma Marelle G. , Veeger Cees , Samyn Bart , Van Beeumen Jozef , Warmerdam Gertrüd , Canters Gerard W. , Van Dongen Walter M. A. M. , Rietjens Ivonne M. C. M.
Published: July 2004

Microperoxidases with increasing lengths of the peptide attached to the heme moiety have been isolated after proteolytic digestion of horse‐heart cytochrome c (microperoxidases 6, 8, and 11) and of cytochrome c550 from Thiobacillus versutus (microperoxidase 17). The different microperoxidases catalyze the H2O2‐dependent para ‐hydroxylation of aniline relatively efficiently but are rapidly inactivated under turnover conditions. The horse‐heart cytochrome‐c ‐derived microperoxidases have identical values for Vmax but show a decrease of the Km, for aniline and a higher stability when the attached peptide is longer. The kinetic constants obtained for microperoxidase 17, differ markedly from the microperoxidases derived from horse‐heart cytochrome c. Possible factors underlying the observed differences are discussed.
Mathematical Model for Evaluating the Krebs Cycle Flux with Non‐Constant Glutamate‐Pool Size by 13C‐NMR Spectroscopy
Authors: Tran‐Dinh Son , Beganton Frankie , Nguyen Trong‐Thuc , Bouet Francoise , Herve Martine
Published: July 2004

A practical method using matrix operations is proposed for studying the isotopic transformation of glutamate, or any other metabolite isotopomers, in the Krebs cycle. Two mathematical models were constructed for evaluating the Krebs cycle flux where the enrichment of [2‐13C]acetyl‐CoA is not 100% and the total glutamate concentration remains constant or varies during incubation.

A comparative study of [1‐13C]glucose metabolism was subsequently carried out using Saccharumyces cerevisiae cells from two different strains (ATCC‐9763 and NCYC‐239) by 13C‐NMR spectroscopy and biochemical techniques. The results show that there are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2‐oxoglutarate dehydrogenase and hence the flux in the Krebs cycle is negligible. With [1‐13C]glucose as a carbon source, the 13C‐NMR spectra of glutamate exhibit the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1‐13C]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The second cell type is represented by NCYC‐239. The C2 and C3 areas are equivalent and smaller than the C4 resonance. The results suggest that labeled metabolites enter the Krebs cycle only at the citrate level via acetyl‐CoA, 2‐oxoglutarate dehydrogenase is present but pyruvate carboxylase is virtually absent or inactivated. When both are incubated with glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchange with the Krebs cycle in NCYC‐239 cells is about 2.6% and the reduction in glutamate concentration is about 0.5%/min. Using our model, with a variable glutamate pool size, good agreement between the theoretical and experimental data is obtained.
X‐Ray Structure of Yeast Inorganic Pyrophosphatase Complexed with Manganese and Phosphate
Authors: Harutyunyan Emil H. , Kuranova Inna P. , Vainshtein Boris K. , Höhne Wolfgang E. , Lamzin Victor S. , Dauter Zbigniew , Teplyakov Alexei V. , Wilson Keith S.
Published: July 2004

The three‐dimensional structure of the manganese‐phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4‐Å resolution. X‐ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation. There is one dimeric omolecule in the asymmetric unit. The upper estimate of the root‐mean‐square coordinate error is 0.4 Å using either the σA plot or the superposition of the two crystallographically independent subunits. The good agreement between the coordinates of the two subunits, which were not subjected to non‐crystallographic symmetry restraints, provides independent validation of the structure analysis. The active site in each subunit contains four manganese ions and two phosphates. The manganese ions are coordinated by the side chains of aspartate and glutamate residues. The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains. The phosphates are bridged by two of the manganese ions. The outer phosphate is exposed to solvent. The inner phosphate is surrounded by all four manganese ions. The ion‐binding sites are related to the order of binding previously established from kinetic studies. A hypothesis for the transition state of the catalytic reaction is put forward.
Limited Proteolysis of β‐Conglycinin and Glycinin, the 7S and 11S Storage Globulins from Soybean [Glycine Max (L.) Merr.]
Authors: Shutov Andrei D. , Kakhovskaya Irina A. , Bastrygina Angela S. , Bulmaga Valentina P. , Horstmann Christian , Müntz Klaus
Published: July 2004

The G2 (A2B1a) glycinin subunit from soybean (Glycine max L. Merr.) was purified and renatured to the homohexameric holoprotein. This protein along with purified β‐conglycinin were subjected to limited proteolysis by trypsin. The generated polypeptide fragments were separated via SDS/PAGE and the amino acid sequence of the N‐terminals was determined. Four cleavage points were detected in the α‐chain A2 of glycinin as well as in the α′‐chain of β‐conglycinin. From the known three‐dimensional structure of 7S globulin and the hypothetical model of 7S globulin‐like 11S globulin structure, it was possible to draw the conclusion that two distinct types of susceptible sites for proteolytic cleavage are characteristic of the subunits of both globulins. The first includes the sequences linking N‐ and C‐terminal domains of both globulins and the sequence of N‐terminal extensions of 70‐kDa subunits from the vicilin‐like 7S globulins. The second type includes the loop between β‐strands E and F of the N‐terminal domain of 11S globulins and of the C‐terminal domain of 7S globulins. A statistically significant similarity was found between the N‐terminal extension of the α′‐chain of β‐conglycinin and the interdomain linker regions of soybean glycinin and pea legumin. It is proposed that the three sequence regions which form the first type of susceptible sites are of similar structural function and might have evolved from the N‐terminal segment of a putative single‐domain ancestor.
Kinetics of Phosphorolysis of 3‐(β‐d‐Ribofuranosyl)Adenine and 3‐(β‐d‐Ribofuranosyl)Hypoxanthine, Non‐Conventional Substrates of Purine‐Nucleoside Phosphorylase
Authors: Bzowska Agnieszka , Kulikowska Ewa , Poopeiko Nicolai E. , Shugar David
Published: July 2004

The properties of two non‐conventional substrates of the calf‐spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3‐(β‐d‐ribofuranosyl)adenine (RibfAde) and 3‐(β‐d‐ribofuranosyl)hypoxanthine (RibfHyp), are described. In contrast to Ado, RibfAde is a substrate for the mammalian enzyme. With the calf enzyme, the pseudo‐first‐order rate constants (Vmax/Km) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E. coli PNP the Corresponding values are 22% and 30%, respectively. The Michaelis constants (Km) for RibfAde were 800 μM (calf PNP) and 150 μM (E. coli PNP). For RibfHyp, the corresponding Km values were 220 μM and 260 μM.

Two well‐characterized inhibitors of calf spleen PNP [9‐(2‐fluoro‐3,4‐dihydroxybutyl)guanine] and E. coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibfHyp with the same inhibition constants as for Ino. Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3‐β‐nucleosides occurs at the same active site as for the natural substrate Ino.

In particular, the substrate properties of both 3‐β‐nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9‐β‐nucleosides in the conformation anti to the pyrimidine ring.

The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes.