bluuebunny/crossref_metadata_2025 huggingface dataset:
An Ultracentrifugal Approach to Quantitative Characterization of the Molecular Assembly of a Physiological Electron‐Transfer Complex. The Interaction of Electron‐Transferring Flavoprotein with Trimethylamine Dehydrogenase
Authors: Wilson Emma K. , Scrutton Nigel S. , Cölfen Helmut , Harding Stephen E. , Jacobsen Michael P. , Winzor Donald J.
Published: July 2004

The interaction between two physiological redox partners, trimethylamine dehydrogenase and electron‐transferring flavoprotein, has been characterized quantitatively by analytical ultracentrifugation at 4°C. Analysis of sedimentation‐equilibrium distributions obtained at 15000 rpm for mixtures in 10 mM potassium phosphate, pH 7.5, by means of the psi function [Wills, P. R., Jacobsen, M. P. & Winzor, D. J. (1996) Biopolymers 38, 119–1301 has yielded an intrinsic dissociation constant of 3–7 μM for the interaction of electron‐transferring flavoprotein with two equivalent and independent sites on the homodimeric enzyme. This investigation indicates the potential of sedimentation equilibrium for the quantitative characterization of interactions between dissimilar macromolecules.
Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases
Authors: Wielgus‐Kutrowska Beata , Kulikowska Ewa , Wierzchowski Jacek , Bzowska Agnieszka , Shugar David
Published: July 2004

Nicotinamide 1‐β‐d‐riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine‐nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km, values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1–2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first‐order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP.

Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with K1 values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7‐methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Km values similar to those for inhibition of other natural substrates.

The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates.

The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme‐inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.
Phosphorylation of Vitronectin on Ser362 by Protein Kinase C Attenuates Its Cleavage by Plasmin
Authors: Gechtman Zeev , Shaltiel Shmuel
Published: July 2004

Vitronectin, found in the extracellular matrix and in circulating blood, has an important role in the control of plasminogen activation. It was shown to be the major protein substrate in human blood fluid for a protein kinase A (PKA) released from platelets upon their physiological stimulation with thrombin. Since vitronectin was shown to have only one PKA phosphorylation site, but to contain 2–3 mol cova‐lently bound phosphate, it was reasonable to assume that other protein kinases might phosphorylate vitronectin at other sites in the protein. We have reported earlier that human serum contains at least three protein kinases, one of which was found to be CAMP independent and to phosphorylate a repertoire of plasma proteins that was very similar to that obtained upon phosphorylation of human plasma with protein kinase C (PKC). Since there are now several examples of proteins with extracellular functions that are phosphorylated by PKC, we undertook to study the phosphorylation of vitronectin by PKC. Here, we show that vitronectin is a substrate for PKC, and characterize the kinetic parameters of this phosphorylation (Km≈ tenfold lower than the concentration of vitronectin in blood), indicating that, from the biochemical point of view, this phosphorylation can occur at the locus of a hemostatic event. We also identify Ser362 as the major PKC phosphorylation site in vitronectin, and confirm this localization by means of synthetic peptides derived from the cluster of basic amino acids in vitronectin surrounding Ser362. We show that the PKC phosphorylation at Ser362 alters the functional properties of vitronectin, attenuating its cleavage by plasmin at Arg361‐Ser362. This phosphorylation has the potential to regulate plasmin production from plasminogen by a feedback mechanism involving the above‐mentioned plasmin cleavage, a loosening of the vitronectin grip on inhibitor 1 of plasminogen activators, and a subsequent latency of this regulatory inhibitor.
The Neuronal Cell‐Adhesion Molecule Axonin‐1 is Specifically Released by an Endogenous Glycosylphosphatidylinositol‐Specific Phospholipase
Authors: Lierheimer Ruth , Kunz Beat , Vogt Lorenz , Savoca Reto , Brodbeck Urs , Sonderegger Peter
Published: July 2004

Axonin‐1, a member of the immunoglobulin/fibronectin type‐III family of cell‐adhesion molecules, occurs both as a glycosylphosphatidylinositol‐(glycosylPtdIns)‐anchored membrane‐bound and a soluble form. In vivo observations show that the major part of axonin‐1 is found in the soluble fraction and that soluble axonin‐1 perturbs neurite fasciculation and pathfinding in the developing chicken embryo. This has prompted further investigations into the mechanism of the axonin‐1 release. We demonstrate here that axonin‐1 released from dorsal root ganglion neurons contains ethanolamine and inositol, components of the glycosylPtdIns anchor. Secreted axonin‐1 does not exhibit the cross‐reacting determinant epitope, an indication that the cleavage of the anchor is not mediated by a phosphatidylinositol‐specific phospholipase C. Treatment of dorsal root ganglion neurons with 1,10‐phenanthroline, an inhibitor of glycosylPtd‐Ins‐specific phospholipase D, reduces the release of axonin‐1 by 56%. Moreover, glycosylPtdIns‐specific phospholipase D activity was detected in dorsal root ganglion neurons and brain. These results suggest that axonin‐1 is released from the membrane by an endogenously expressed glycosylPtdIns‐specific phospholipase Din vivoWith domain‐swaping experiments between axonin‐1 and its non‐released relative F11, deletion mutants and monoclonal antibodies, we demonstrate that the fourth fibronectin type‐III‐like domain of axonin‐1 is required for the generation of the soluble form of axonin‐1.
Inhibition of Cyclin‐Dependent Kinases by Purine Analogues
Authors: De Azevedo Walter Filgueira , Leclerc Sophie , Meijer Laurent , Havlicek Libor , Strnad Miroslav , Kim Sung‐Hou
Published: July 2004

Cyclin‐dependent kinases (cdk) control the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. A potent inhibitor of cdks, roscovitine [2‐(1‐ethyl‐2‐hydroxyethylamino)‐6‐benzylamino‐9‐isopropylpurine], was identified by screening a series ofC2,N6,N9‐sub‐stituted adenines on purified cdc2/cyclin B. Roscovitine displays high efficiency and high selectivity (Meijer, L., Borgne, A., Mulner, O., Chong, J. P. J., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, J.‐G. & Moulinoux, J.‐P. (1997)Eur. J. Biochem. 243, 527–536). It behaves as a competitive inhibitor for ATP binding to cdc2. We determined the crystal structure of a complex between cdk2 and roscovitine at 0.24‐nm (2.4 Å) resolution and refined to anRfactorof 0.18. The purine portion of the inhibitor binds to the adenine binding pocket of cdk2. The position of the benzyl ring group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP‐complex structure. Analysis of the position of this benzyl ring explains the specificity of roscovitine in inhibiting cdk2. The structure also reveals that the (R)‐stereoisomer of roscovitine is bound to cdk2. The (R)isomer is about twice as potent in inhibiting cdc2/cyclin B than the (S)‐isomer. Results from structure/activity studies and from analysis of the cdk2/roscovitine complex crystal structure should allow the design of even more potent cdk inhibitors.
Charge Reversal of a Critical Active‐Site Residue of Cytochrome‐c Peroxidase
Authors: Bujons Jordi , Dikiy Alexander , Ferrer Juan C. , Banci Lucia , Mauk A. Grant
Published: July 2004

A new variant of cytochrome‐c peroxidase in which the positively charged Arg48 present in the distal heme‐binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin‐centered π‐cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH‐linked spectroscopic species. The electronic absorption and 1H‐NMR spectra of the predominant form at low pH (HS1) are indicative of a high‐spin, pentacoordinate heme iron system. Near neutral pH, a second high‐spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low‐spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild‐type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild‐type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild‐type peroxidase is more related to the stability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Soc. 114, 6592–6593; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1993) Biochemistry 32, 9807–9818]. Stopped‐flow studies failed to detect even transient formation of a porphyrin‐centered radical following addition of hydrogen peroxide to the Fe(III)‐enzyme. The consequences of this drastic electrostatic modification of the active site on the steady‐state kinetics of the variant are relatively minor.
A Decade of Protein Engineering on Ribonuclease T1— Atomic Dissection of the Enzyme‐Substrate Interactions
Authors: Steyaert Jan
Published: July 2004

During the last decade, protein engineering has been used to identify the residues that contribute to the ribonulease‐T1‐catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2′‐oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the 2′‐oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and Phe100 further stabilize the transition state of the reaction. A functionally independent subsite, including Asn36 and Asn98, contributes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42–46, renders ribonuclease T1 guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face‐to‐face stacking interaction. Tyr45, often referred to as the lid of the guanine‐binding site, does not contribute to the binding of the base.
Interaction of N‐Tosyl‐l‐Phenylalanylchloromethane with Thermus Thermophilus Elongation Factor Tu
Authors: Nawrot Barbara , Hillenbrand Rainer , Limmer Stefan , Grillenbeck Norbert , Sprinzl Mathias
Published: July 2004

The interaction of N‐tosyl‐l‐phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF‐Tu) was studied by affinity labelling and NMR spectroscopy. TosPheCH2Cl binds to GDP and GTP conformers of EF‐Tu. The interaction of TosPheCH2Cl with EF‐Tu. GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF‐Tu GTP does not lead to covalent labelling. [A82]EF‐Tu, in which the Cys82 is replaced by Ala, has similar properties to wild‐type EF‐Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF‐Ts) and interaction with ribosomes. This structural change did not lead to changes, compared with wild‐type EF‐Tu · in the functionality of [A82]EF‐Tu, either in the GTP or in the GDP conformation. TosPheCH2Cl binds to EF‐Tu · GTP with a dissociation constant of 10μM. The interaction of TosPheCH2Cl with EF‐Tu promotes the hydration of the carbonyl group of TosPheCH2Cl. TosPheCH2Cl competes with aminoacyl‐tRNA for its binding site on EF‐Tu · GTP. Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl‐tRNA. TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl‐tRNA and binds to its binding site on EF‐Tu · GTP. This rather specific interaction with EF‐Tu · GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF‐Tu · GDP leads to alkylation of this residue.
Design and Solution Structure of a Partially Rigid Opioid Antagonist Lacking the Basic Center — Models of Antagonism
Authors: Crescenzi Orlando , Fraternali Franca , Picone Delia , Tancredi Teodorico , Balboni Gianfranco , Guerrini Remo , Lazarus Lawrence H. , Salvadori Severe , Temussi Piero A.
Published: July 2004

To discriminate between two general models of antagonism (participation and allosteric), an opioid antagonist lacking the basic nitrogen of tyramine was designed and characterized. Cyclo‐[Tyr(Me)2‐Tic‐], the diketopiperazine of 2,6‐dimethyltyrosyl‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid, is a partially rigid opioid antagonist; its pA2 (5.8) is one smaller than that of N,N‐bisallyl‐enkephalin but it has a very high binding affinity (10 nM) and has a δ selectivity (66 with respect to the binding to μ receptors) higher than that of naltrindole. The conformational state of this diketopiperazine, studied under a variety of solvent and temperature conditions by NMR and molecular dynamics, can be described in terms of only three conformers whose relative populations vary widely with solvent. Only one of the three conformers, characterized by a 90° arrangement of the aromatic rings of Tyr(Me)2 and Tic similar to those of rigid agonists and of the bioactive conformation of the corresponding linear antagonist, is consistent with the antagonist activity. This finding favors the participation model among the general mechanisms proposed to explain antagonism. Due to the simple composition of the conformational mixture and to the rigidity of the molecule, it is possible to propose a quantitative explanation for the discrepancy between the very high binding affinity (10 nM) and the fairly small in mouse vas deferens value (1.5 μM).
The Replacement of Lys620 by Serine Desensitizes Escherichia Coli Phosphoenolpyruvate Carboxylase to the Effects of the Feedback Inhibitors l‐aspartate and l‐malate
Authors: Yano Masato , Izui Katsura
Published: July 2004

Chemical modification of Escherichia coli phosphoenolpyruvate carboxylase (P‐pyruvate carboxylase) by 2,4,6‐trinitrobenzene sulfonate, a specific reagent for amino groups, causes desensitization to allosteric inhibitors, l‐aspartate and l‐malate, as well as inactivation. When l‐malate is included in the modification mixture, P‐pyruvate carboxylase was markedly protected from both desensitization and inactivation [Naide, A., Izui, K., Yoshinaga, T. & Katsuki, H. (1979) J. Biochem. (Tokyo) 85, 423–432]. To determine the lysine residue(s) involved in allosteric inhibition, the lysine residues that were protected from modification by l‐malate were investigated by analyzing trinitrophenylated peptides liberated by digestion with glutamyl endopeptidase (V8‐protease). The identified residues were Lys491, Lys620, Lys650, and Lys773. Each of these residues was individually replaced with an alanine or serine residue by site‐directed mutagenesis to produce mutant enzymes. The mutant enzyme whose lysine residue was replaced with serine ([Ser620]P‐pyruvate carboxylase) showed a marked desensitization to l‐aspartate and l‐malate, while retaining almost the same maximal catalytic activity as the wild‐type P‐pyruvate carboxylase. Essentially no changes in enzymatic properties were observed for the [Ala491]‐ and [Ala650]P‐pyruvate carboxylases, while for the [Ala620]‐ and [Ala773]P‐pyruvate carboxylases the polypeptides of the expected size were not significantly accumulated in the transformed E. coli cells, presumably due to intracellular degradation.
In Vitro Binding and Phosphorylation of Insulin Receptor Substrate 1 by the Insulin Receptor
Authors: Backer Jonathan M. , Wjasow Christina , Zhang Yitao
Published: July 2004

Insulin receptor substrate 1 (IRS‐1) is a major substrate of the insulin receptor in most cells. The N terminus of IRS‐1 contains a phosphotyrosine binding (PTB) domain and a pleckstrin homology (PH) domain, both of which have been identified as important for insulin‐stimulated phosphorylation in intact cells. The PTB domain binds to a phosphorylated motif, NPEY(P)960, that is present in the juxtamembrane region of the insulin receptor. A direct interaction between the PH domain of IRS‐1 and the receptor has not been demonstrated. In this study, we examine the role of the IRS‐1 PTB and PH domains during IRS‐1‐receptor binding and IRS‐1 phosphorylation in intact cells and in vitro. Abrogation of binding of the PTB domain to NPXY(P) by mutation of Tyr960 of the insulin receptor did not reduce the binding of phosphorylated IRS‐1 to insulin receptors in intact cells, and had no effect on binding of insulin receptors to IRS‐1 or on IRS‐1 phosphorylation in vitro. We examined the phosphorylation and receptor binding of a mutant recombinant IRS‐1 that lacks the N‐terminal PH domain (δPH‐IRS‐1). Although phosphorylation of fPH‐IRS‐1 by wild‐type or [Ala960]insulin receptors was similar to that of IRS‐1, binding of insulin receptor to PH‐IRS‐1 was markedly reduced relative to that to IRS‐1. We conclude that stable association of IRS‐1 with the insulin receptor is unaffected by disruption of PTB‐domain–Tyr960 interactions but requires the IRS‐1 PH domain, and that efficient phosphorylation of IRS‐1 in intact cells correlates with the formation of stable receptor IRS‐1 complexes.
Quantification of MyoD, Myogenin, MRF4 and Id‐1 by Reverse‐Transcriptase Polymerase Chain Reaction in Rat Muscles — Effects of Hypothyroidism and Chronic Low‐Frequency Stimulation
Authors: Kraus Bernhard , Pette Dirk
Published: July 2004

A highly sensitive method of reverse‐transcriptase polymerase chain reaction (RT‐PCR) was established to quantify transcript levels of the myogenic regulatory factors MyoD, myogenin and MRF4 (muscle regulatory factor 4) and for Id‐1 (inhibitor of differentiation), a putative negative regulator of myogenesis. The method was sensitive enough to detect mRNA amounts as low as 20 molecules. Measurements in 10 different skeletal muscles of the rat revealed that the amounts of the four factors differ by almost three orders of magnitude. Id‐1 is expressed at lowest levels (≈4×105 molecules/μg RNA) and MRF4 at highest levels (≈9×107 molecules/μg RNA). In general, myogenin and MyoD mRNAs were inversely distributed in slow and fast muscles. A correlation seemed to exist between the levels of MyoD and myosin heavy chain (MHC) IIb, the fastest MHC isoform. However, as revealed by changes in the expression levels of these two regulatory factors under conditions of hypothyroidism and chronic low‐frequency stimulation (CLFS), MyoD and myogenin did not seem to be strictly correlated with fast and slow myosins, respectively. Hypothyroidism led to pronounced depressions of MyoD, but only to small increases in myogenin mRNA in fast muscles. These changes were only slightly increased by CLFS. However, as previously shown, CLFS in combination with hypothyroidism induces in rat muscle pronounced fast to slow transitions in myosin expression [Kirschbaum, B. J., Kucher, H.‐B., Termin, A., Kelly, A. M. & Pette, D. (1990) J. Biol. Chem. 265, 13974–13980]. These findings suggest that MyoD and myogenin may not be causally related to the development and maintenance of fiber‐type diversities.
The Subunit Structure of Cytochrome‐C Oxidase from Tuna Heart and Liver
Authors: Arnold Susanne , Lee Icksoo , Kim Minjung , Song Eunsook , Linder Dietmar , Lottspeich Friedrich , Kadenbach Bernhard
Published: July 2004

Cytochrome‐c oxidase was isolated from tuna liver and heart, and the subunit composition was analysed by SDS/PAGE by two separation systems. Two additional subunits of the enzyme complex were immunoprecipitated from solubilized mitochondria with an antibody against bovine subunit IV. The N‐terminal and internal amino acid sequences of all nuclear‐coded subunits were determined after blotting onto poly(vinylidene difluoride) membranes or by tryptic hydrolysis of gel bands and HPLC separation of peptides, respectively. 13 subunits were identified with isoforms for subunits Va, VIc, VIIb and VIII. The isoforms for subunits Va and VIIb are found in liver and heart, isoforms for subunit VIc only in heart, and isoforms for subunit VIII only in liver. Isoforms for subunits Va, VIc and VIIb have not been described in other species. The postulated mechanism of thermogenesis in mammals, based on decreased H+/e− stoichiometry at high ATP/ADP ratios due to binding of ATP to the heart‐type subunit VIa [Frank, V. & Kadenbach, B. (1996) FEBS Lett. 382, 121–124], appears not to occur in tuna, because no isoforms of subunit VIa were found.
Rapid Induction of the Grp78 Gene by Cooperative Actions of Okadaic Acid and Heat‐Shock in 9L Rat Brain Tumor Cells
Authors: Chen Kuang‐Den , Hung Jan‐Jong , Huang Huei‐Luen , Chang Margaret Dah‐Tysr , Lai Yiu‐Kay
Published: July 2004

We have demonstrated that treatment with 200 nM okadaic acid (OA) for 1 h followed by a 15‐min heat shock (HS) at 45°C (termed OA→HS treatment) leads to a rapid transactivation of grp78, the gene for the 78‐kDa glucose‐regulated protein, in 9L rat brain tumor cells. The level of Grp78 mRNA rose 15‐fold in 60 min after the combined treatment. Nuclear extracts from cells subjected to OA→HS treatment, compared to those of treatment with OA or HS alone, exhibited an increased binding activity toward an oligonucleotide probe containing the cAMP‐responsive element‐like (CRE‐like, TGACGTGA) regulatory element in electrophoretic mobility shift assays (EMSA). The binding resulted in the formation of two protein‐EMSA probe complexes exhibiting different association and dissociation rates in kinetic studies. The protein factors in the upper band (complex I) and lower band (complex II) were identified as the activating transcription factor‐2 (ATF‐2) and the CRE binding factor 1 (CREB‐1), respectively, by antibody interference assays. In addition, the identity of CREB‐1 was confirmed by supershift analysis. The binding activity, as well as the transactivation of the grp78 gene, can be abolished by a 1‐h treatment with the cAMP‐dependent protein kinase (PKA) inhibitor but not with protein kinase C or Ca2+/calmodulin‐dependent protein kinase II inhibitors. Accumulation of steady‐state level of ATF‐2 was observed and was also modulated by treatment with H‐89, a PKA inhibitor. From these results, we conclude that the CRE‐like element plays an important role in the rapid transactivation of the grp78 gene and that the PKA signaling pathway is involved. In addition, PKA‐mediated transcriptional regulation of grp78 in OA→HS treatment is through regulation of protein phosphorylation as well as de novo synthesis of ATF‐2.
Activation of Partially Folded Mitochondrial Malate Dehydrogenase by Thioredoxin
Authors: Li Weikai , Churchich Jorge E.
Published: July 2004

CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure. Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme. A partially folded intermediate, possessing 25% of the α‐helix content of the native enzyme, is formed upon dilution. The partially folded intermediate binds the extrinsic probe 1‐anilinonaphtalene‐8‐sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum. Partially folded malate dehydrogenase is devoid of catalytic activity. In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin. In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed. Emission‐anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 μM. Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE. A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin.
Glycogen Biogenesis in Rat 1 Fibroblasts Expressing Rabbit Muscle Glycogenin
Authors: Skurat Alexander V. , Lim Soo‐Siang , Roach Peter J.
Published: July 2004

Glycogenin, a self‐glucosylating protein involved in the initiation of glycogen biosynthesis, varies in intracellular concentration from barely detectable in liver to a high level in muscle. The effect of increasing the glycogenin level on glycogen synthesis was studied in rat 1 fibroblasts stably overexpressing rabbit muscle glycogenin. In the presence of glucose, all of the expressed glycogenin was attached to polysaccharide and the free protein could only be detected by western blot analysis after incubation of cells in a glucose‐depleted medium or treatment of the cell extract with α‐amylase. In control cells, increased extracellular glucose concentrations promoted translocation of glycogen synthase from the soluble to the pellet fraction with an increase in the associated glycogen. Overexpression of glycogenin did not affect total intracellular glycogen and glycogen synthase levels at any concentration of glucose but significantly reduced glucose‐induced accumulation of insoluble glycogen and translocation of glycogen synthase. Immunofluorescence analysis revealed a diffuse cytoplasmic distribution of glycogenin expressed in rat 1 cells. In rat 1 cells incubated with glucose, discrete deposits of glycogen were detected by staining with HIO4/Schiff but this was eliminated by overexpressing glycogenin. Analysis of [14C]glucose‐or [35S]methionine‐labeled extracts from glycogenin‐expressing cells by continuous polyacrylamide gel electrophoresis and by two‐dimensional gel electrophoresis revealed a continuum of glycogenin‐containing species from low molecular mass to sizes significantly greater than 400 kDa. We conclude that (a) Overexpression of glycogenin does not enhance glycogen synthesis but causes production of more, smaller, glycogen molecules with a concomitant change in their intracellular localization; (b) glycogenin and elevated glucose have opposing effects on the distribution of glycogen and glycogen synthase in rat 1 cells; and (c) the biogenesis of glycogen in rat 1 cells occurs without the accumulation of any major intermediate form.
Calcium‐Binding Properties of the Third and Fourth Epidermal‐Growth‐Factor‐Like Modules in Vitamin‐K‐Dependent Protein S
Authors: Stenberg Yvonne , Julenius Karin , Dahlqvist Ingrid , Drakenberg Torbjörn , Stenflo Johan
Published: July 2004

Protein S is a plasma glycoprotein requiring vitamin K for normal biosynthesis and functioning as a cofactor of activated protein C, a regulator of blood coagulation. Protein S contains four modules that are similar to the epidermal growth factor (EGF) precursor. Qualitative Ca2+‐binding experiments have indicated that the EGF‐module region of bovine protein S harbors high‐affinity Ca2+‐binding sites. We have chemically synthesized the third and fourth EGF modules from human protein S, which both have the sequence motif associated with Ca2+‐binding and Asp/Asn β‐hydroxylation. Both modules were folded to a native conformation, as judged by immunochemical experiments and NMR spectroscopy. Ca2+ binding to the modules was monitored with 1H‐NMR spectroscopy. At physiological pH and 0.15 M NaCl, each module was found to have a single Ca2+‐binding site with low affinity, i.e. Kd values of 6.1 mM for the third and 8.6 mM for the fourth EGF module. At low salt conditions the Ca2+ affinities are 5.2 mM and 0.6 mM, respectively. This Ca2+ affinity is similar to that of the isolated N‐terminal EGF module from coagulation factors IX and X. The very high affinity Ca2+ binding to the EGF‐module region of protein S thus appears to be due to the influence of neighboring modules.
The Lantibiotic Mersacidin Inhibits Peptidoglycan Biosynthesis at the Level of Transglycosylation
Authors: Brötz Heike , Bierbaum Gabriele , Reynolds Peter E. , Sahl Hans‐Georg
Published: July 2004

The lantibiotic mersacidin has been previously reported to interfere with bacterial peptidoglycan biosynthesis, [Brötz, H., Bierbaum, G., Markus, A., Molitor, E. & Sahl, H.‐G. (1995)Antimicrob. Agents Chemother. 39, 714–719]. Here, we focus on the target reaction and describe a mersacidin‐induced accumulation of UDP‐N‐acetylmuramoyl‐pentapeptide, indicating that inhibition of peptidoglycan synthesis occurs after the formation of cytoplasmic precursors. In vitro studies involving a wall‐membrane particulate fraction of Bacillus megaterium KM demonstrated that mersacidin did not prevent the synthesis of lipid II [undecaprenyl‐diphosphoryl‐N‐acetylmuramoyl‐(pentapeptide)‐N‐acetylglucosamine] but specifically the subsequent conversion of this intermediate into polymeric nascent glycan strands by transglycosylation. Comparison with other inhibitors of transglycosylation shows that the effective concentration of mersacidin in vitro is in the range of that of the glycopeptide antibiotic vancomycin but 2–3 orders of magnitude higher than that of the competitive enzyme inhibitor moenomycin. The analogy to the glycopeptides may hint at an interaction of mersacidin with the peptidoglycan precursor rather than with the enzyme. Unlike vancomycin however, mersacidin inhibits peptidoglycan formation from UDP N‐acetylmuramoyl‐tripeptide and is active against Enterococcus faecium expressing the vanA resistance gene cluster. This indicates that the molecular target site of mersacidin differs from that of vancomycin and that no cross‐resistance exists between the two antibiotics.
Modulation of the Enzymatic Properties of Protein Phosphatase 2A Catalytic Subunit by the Recombinant 65‐kDa Regulatory Subunit PR65α
Authors: Turowski Patric , Favre Bertrand , Campbell Kathryn S. , Lamb Ned J. C. , Hemmings Brian A.
Published: July 2004

All protein phosphatase 2A (PP2A) holoenzymes contain a 36‐kDa catalytic subunit (PP2Ac) and a regulatory subunit of 6.5 kDa (PR6.5). We have studied the interaction between PP2Ac and PR65 in an in vitro system, using PP2Ac isolated from rabbit skeletal muscle and recombinant PR6Sa expressed in bacteria or insect cells. Bacterially expressed PR6Sa exhibited identical biochemical properties to the protein expressed and isolated from the baculoviral expression system. The association of recombinant PR6.5 with PP2Ac was very tight (KappD= 85 pM) and led to a suppression of PP2A activity, which was maximal (70–80 %) when phosphoproteins were used as substrates. When less‐structured or smaller substrates (such as phosphopeptides) were used, this inhibition was only 30 %. PR6.5 stimulated PP2Ac activity when the assays were performed in the presence of polycations. This indicates that the PR6.5 not only serves the previously predicted structural role as a molecular scaffold, but also allosterically modulates the enzymatic properties of PP2Ac. Furthermore, we identified a site of interaction between PP2Ac and PR6Sα by disruption of a stretch of basic amino acids by introduction of a glutamate at position 416. This produced an almost 100‐fold reduced affinity for PP2Ac and indicated that this basic motif is an important determinant for the interaction of PR6.5 and PP2Ac.
Lipopolysaccharide‐Binding Proteins and their Involvement in the Bacterial Clearance from the Hemolymph of the Silkworm Bombyx Mori
Authors: Koizumi Nobuo , Morozumi Asuka , Imamura Morikazu , Tanaka Emi , Iwahana Hidenori , Sato Ryoichi
Published: July 2004

Proteins having the ability to bind to Escherichia coli K12W3110 (rough (R) mutant) were isolated and purified by affinity precipitation from the larval hemolymph of the silkworm Bombyx mori. These proteins were found to consist of two components with molecular masses of 43 kDa and 40 kDa by SDS/ PAGE. They bound to all E. coli R mutants (Ra, Rb1, Rc, Rd1 and Re) and Salmonella minnesota R mutants. However, they did not bind to smooth types of the above bacteria. They bound to both lipopolysaccharide(LPS)‐coated and lipid‐A‐coated microtiter plates and have similar dissociation constants for LPS and lipid A. This indicates that the binding proteins recognize the lipid A portion of LPS and thus, we have named these proteins BmLBP (B. mori LPS‐binding proteins). We also found that BmLBP participated in the clearance of E. coli cells injected into the body cavity of the silkworm.
Molecular Characterisation of Integrin–Procollagen C‐Propeptide Interactions
Authors: Daviesz Dairyl , Calderwood David A. , Weston Susan A. , Takigawa Masarahu , Humphries Martin J.
Published: July 2004

The carboxyl‐terminal propeptide of type I procollagen (CPP‐I) plays a key role in regulation of collagen fibrillogenesis, and may exert feedback control of collagen biosynthesis. We have previously shown that CPP‐I is a ligand for the integrin α2β1 [Weston, S. A., Hulmes, D. J. S., Mould, A. P., Watson, R. B. & Humphries, M. J. (1994) Identification of the integrin α2β1 as a cell surface receptor for the C‐propeptide of type I procollagen, J. Biol. Chem. 269, 20982–20986] suggesting that some of the phenotypic effects of C‐propeptides may be mediated by adhesion receptors. Here we have extended this work to study the molecular basis of this interaction. We have broadened the ligand range by demonstrating that the C‐terminal propeptide of type II procollagen supports α2β1‐mediated binding of NHS human fibroblasts in cell attachment assays. Also, we have used function‐blocking antibodies in cell attachment and solid‐phase binding assays with purified integrin to expand the CPP‐I receptor family, showing that integrin α2β1 is also a receptor for CPP‐I. Integrin a‐subunit A‐domains are known to be major ligand‐binding sites and recombinant al and α2 subunit A‐domains were able to bind CPP‐I. Finally we have shown that peptides corresponding to potential integrin‐binding sequences in CPP‐I do not mediate integrin–CPP‐I adhesion. Taken together, these studies indicate that the interactions between C‐propeptides and integrins are more numerous than previously reported, that C‐propeptides are a new class of molecule which bind to A‐domains, and that the integrin–C‐propeptide interaction does not utilise established peptide motifs.
Enzymes of Octadecanoid Biosynthesis in Plants
Authors: Schaller Florian , Weiler Elmar W.
Published: July 2004

Octadecanoids, potent cyclic plant signaling molecules derived from α‐linolenic acid, are involved in the regulation of a multitude of physiological processes such as senescence, herbivore and pathogen defense, mechanoperception and morphogenesis. The first cyclic intermediate in the Vick‐Zimmerman pathway of octadecanoid biosynthesis is 12‐oxo‐phytodienoic acid. Its conversion to the end product of the pathway, jasmonic acid, a C12 compound, first proceeds through reduction to 3‐oxo‐2‐(pent‐2′‐enyl)‐cyclopentane‐1‐octanoic acid, which is then converted to jasmonic acid by three cycles of β‐oxidation. The first of these conversions is a decisive point in the biosynthetic sequence, in that it channels the octadecanoid into the pathway of β‐oxidation. 12‐Oxo‐phytodienoate reductase was purified to apparent homogeneity from a cell suspension culture of Corydalis sempervirens. The enzyme is soluble and a monomer of apparent molecular mass 41 kDa which prefers NADPH over NADH to reduce the 10,11‐double bond of 12‐oxo‐phytodienoic acid. The structure of the reaction product was proved by derivatization, GC/MS and NMR analysis. The enzyme accepts both the cis and the trans isomer of 12‐oxo‐phydodienoic acid, with a preference for the cis‐isomer (6:1). 12‐Oxo‐phytodienoate reductase will also convert the synthetic substrate 2‐cyclohexenone to cyclohexanone, but the enzyme did not reduce some other cyclic α,β‐unsaturated ketones tested (the plant hormone abscisic acid or the steroids testosterone and progesterone). Characteristic parameters of 12‐oxo‐phytodienoate reductase were determined.
1H, 15N and 13C NMR Assignments, Secondary Structure and Overall Topology of the Escherichia Coll Glgs Protein
Authors: Beglova Natalia , Fischer Daniela , Hengge‐Aronis Regine , Gehring Kalle
Published: July 2004

GlgS is a 7892‐Da protein which is involved in glycogen biosynthesis in bacteria. We report the 1H, 15N and 13C NMR assignments of the backbone and side‐chain resonances at 25°C and pH 6.7 from two‐dimensional homonuclear and three‐dimensional heteronuclear NMR experiments. The secondary structure of the protein was determined using sequential and medium‐range NOE correlations, vicinal 3JNH‐Hα coupling values and amide proton exchange rates. The secondary structure obtained is consistent with the secondary chemical shifts of 1Hα, 13Cα and 13C = O. It was found that the secondary structure of GlgS comprises two amphipathic helices (AsnlO–Met21 and Glu39–Arg60), one short highly hydrophobic helix (Ile30–Val33), a short extended β‐strand‐like fragment (Arg26–Asp29) and two type I β‐turns (His22–Gly25 and Thr34–Met37). An overall topology of GlgS is suggested based on long‐range NOEs. The elements of secondary structure form a sandwich in which the β‐strand and the short hydrophobic helix are positioned between the two amphipathic helices.
Overexpression of the Coenzyme‐F420‐Dependent N5,N10‐Methylenetetrahydromethanopterin Dehydrogenase Gene from the Hyperthermophilic Methanopyrus Kandleri
Authors: Klein Andreas R. , Thauer Rudolf K.
Published: July 2004

The mtd gene encoding coenzyme‐F420‐dependent N5,N10‐methylenetetrahydromethanopterin dehydrogenase (Mtd) in the hyperthermophilic Methanopyrus kandleri has been cloned, sequenced and functionally overexpressed in Escherichia coll. The overproduced enzyme was purified in a 90% yield to apparent homogeneity by means of only one chromatographic step. Its thermostability properties and most of its catalytic properties were the same as those of the native enzyme purified directly from M. kandleri. Only the dependence of the activity on the concentration of lyotropic salts differed slightly. Northern blot analysis revealed that in M. kandleri the mtd gene is monocistronically transcribed.
Reversible Folding of UDP‐galactose 4‐epimerase from Escherichia coli
Authors: Dutta Samrajnee , Maiti Nilesh Ranjan , Bhattacharyya Debasish
Published: July 2004

UDP‐galactose 4‐epimerase from Escherichia coli is a homodimer of 39‐kDa subunits having 1 or 2 molecules of NAD bound non‐covalently/dimer. The enzyme can be dissociated and denatured by 8 M urea at pH 7.0 to a state having only 15% of residual secondary structure. Dilution of the denaturant by 20 mM potassium phosphate, pH 8.5, leads to functional reconstitution of the enzyme. No addition of extraneous NAD is required for reactivation, indicating a strong affinity of the cofactor for refolded molecule. The reactivation follows a second‐order kinetics (k= 1.2±0.07×103 M−1 S−1 at 25°C) with an energy of activation of 23.79 ± 0.33 kJ/mol. The native, denatured and renatured states of the enzyme were characterized by far‐ultraviolet CD spectra for secondary structure; protein fluorescence, interaction with extrinsic fluorescence probe ANS (1‐anilino 8‐naphthalene sulfonic acid) and ultraviolet absorption spectra for tertiary structure and size‐exclusion HPLC, gel‐filtration chromatography and light‐scattering for quaternary structure. The folding process could be broadly divided into two distinct steps: (a) regain of secondary structure and dimerization were fast and were complete within 2 min and 9 min, respectively, and (b) regain of catalytic activity was slow and was complete by 45 min. No active holoenzyme could be identified. It appears that generation of the NAD‐binding site and subsequent assembly of NAD is the rate‐limiting step expressing catalytic activity.
Sequence Specificity of C‐Terminal Src Kinase (Csk)
Authors: Ruzzene Maria , Songyang Zhou , Marin Oriano , Donella‐Deana Arianna , Brunati Anna Maria , Guerra Barbara , Agostinis Patrizia , Cantley Lewis C. , Pinna Lorenzo A.
Published: July 2004

An eicosapeptide encompassing the C‐terminal tail of c‐Src (Tyr527) which is conserved in most Src‐ related protein kinases, is phosphorylated by C‐terminal Src kinase (CSK) and by the two Src‐related protein kinases c‐Fgr and Lyn, with similar kinetic constants. Two related peptides reproducing the C‐terminal segments of c‐Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8, 117–124] AFLEDSCTGTEPLYQRGENL (mutant number 28) and AFLEDNFTGTKPQYHPGENL (mutant number 29), proved a better and a much worse substrates, respectively than the wild‐type peptide, with either CSK or the two Src kinases. By changing individual residues in the best peptide substrate, it was shown that the main element responsible for its improved phosphorylation is leucine at position –1 (instead of glutamine), while lysine at position –3 (instead of glutamate) has a detrimental effect, possibly accounting for the negligible phosphorylation of peptide derived from mutant number 29. By contrast to most peptide substrates, including the Src C‐terminal peptides, which exhibit relatively high Km values, a polyoma‐virus‐middle‐T‐antigen‐(mT)‐derived peptide with tyrosine embedded in a highly hydrophobic sequence (EEEPQFEEIPIYLELLP) exhibits with CSK a quite low Km value (63 μM). Consistent with this, the optimal sequence selected by CSK in an oriented peptide library is XXXIYMFFF. This is different from sequences selected by Lyn (DEEIYEELX) and c‐Fgr (XEEIYGIFF), although they all share a high selection for a hydrophobic residue at n–1. In sharp contrast, TPKIIB/p38Syk, related to the catalytic domain of p72syk, selects acidic residues at nearly all positions, n–1 included.

These data support the notion that the features determining the specific phosphorylation of the C‐terminal tyrosine residue of Src do not reside in the primary structure surrounding the target tyrosine. They also show that this site does not entirely fulfil the optimal consensus sequence recognized by CSK, disclosing the possibility that as yet unrecognized CSK targets structurally unrelated to the C‐terminal tyrosine residue of Src kinases may exist.
Structural Analysis of the O‐Antigenic Polysaccharide from the Enteropathogenic Escherichia coli 0142
Authors: Landersjö Clas , Weintraub Andrej , Widmalm Göran
Published: July 2004

The polysaccharide part of the lipopolysaccharide obtained from the enteropathogenic Escherichia coli O142 has been isolated, and its structure determined. Together with 1H‐NMR and 13C‐NMR spectros‐copy, sugar and methylation analyses show that the polysaccharide is composed of repeating pentasaccha‐ride units. Sequential information on the O‐polysaccharide was obtained by two‐dimensional NMR techniques, namely heteronuclear‐multiple‐bond‐connectivity and NOESY experiments. The repeating unit of the O‐polysaccharide of E. coli strain O142 has the following structure:
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Evolution of the Enzymatic Characteristics of C4Phosphoenol Pyruvate Carboxylase
Authors: Svensson Per , Biasing Oliver Ernst , Westhoff Peter
Published: July 2004

C4phosphoenolpyruvate (P‐pyruvate) carboxylases have evolved from ancestral C3P‐pyruvate carboxylases during the evolution of C4 photosynthesis (Lepiniec et al., 1994). To meet the requirements of a new metabolic pathway, the C4 enzymes have gained distinct kinetic and regulatory properties compared to C3 enzymes. Our interest is to deduce the structure responsible for these C4‐specific properties. As a model system, the orthologous ppcA P‐pyruvate carboxylases of Flaveria trinervia (C4) and Flaveria pringlei (C3) were investigated by expressing them in Escherichia coli using their cDNAs. The Km (P‐pyruvate) was about ten times higher for the C4 enzyme (650 μM) than for the C3 enzyme (60 μM). The activation by glucose 6‐phosphate, which was shown by a decrease in the Km (P‐pyruvate), was about twice for the C4 enzyme and three times for the C3 enzyme. The C3 enzyme showed a very high sensitivity to L‐malate with an I0.5 (50% inhibition) value of 80 μM malate, whereas the C4 enzyme was much less sensitive with a I0.5 value of 1.2mM malate. To locate the structural positions responsible for these differences in kinetic and regulatory properties, chimeras of these 95% identical enzymes were made. In this study, the first 437 residues of the 966‐amino‐acid protein were interchanged. The results showed that the N‐terminal part of the enzyme was responsible for a small but significant part of the kinetic difference observed between these two isoenzymes. Additionally, the results suggest that the N‐terminus was the site for glucose 6‐phosphate activation and was also responsible for the observed difference in activation by this sugar phosphate. The difference in inhibition by L‐malate, however, is suggested to originate mainly from the C‐terminal part of the enzyme.
Human Pancreatic Ribonuclease
Authors: Bal Harshawardhan P. , Batra Janendra K.
Published: July 2004

Mammalian ribonucleases constitute one of the fastest evolving protein families in nature. The addition of a four‐residue carboxyl‐terminal tail: Glu‐Asp‐Ser‐Thr (EDST) in human pancreatic ribonuclease (HPR) in comparison with bovine pancreatic RNase (RNase A) could have adaptive significance in humans. We have cloned and expressed human pancreatic ribonuclease in Escherichia coli to probe the influence of the four‐residue extension and neighboring C‐terminal residues on the biochemical properties of the enzyme. Removal of the C‐terminal extension from HPR yielded an enzyme, HPR‐(1–124)‐peptide, with enhanced ability to cleave poly(C). HPR‐(1–124)‐peptide also exhibited a steep increase in thermal stability mimicking that known for RNase A. Wild‐type HPR had significantly low thermal stability compared to RNase A. The study identifies the C‐terminal boundary in the human pancreatic ribonuclease required for efficient catalysis.