bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific α‐Galactosidase Inhibitors, N‐Methylcalystegines Structure/Activity Relationships of Calystegines from Lycium Chinense
Authors: Asano Naoki , Kato Atsushi , Miyauchi Miwa , Kizu Haruhisa , Tomimori Tsuyoshi , Matsui Katsuhiko , Nash Robert J. , Molyneux Russell J.
Published: July 2004

An examination of the roots of Lycium chinense (Solanaceae) has resulted in the discovery of 14 calystegines, a cycloheptane bearing an amino group and three hydroxyl groups, and two polyhydroxylated piperidine alkaloids. Calystegines A7 and B5 in addition to the previously known calystegines A3, A5, A6, B1, B2, B3, B4, C1, C2, and N1, were isolated and determined as 1α,2β,4α‐trihydroxy‐nortropane and 1α,2β,4α,7α‐tetrahydroxy‐nortropane, respectively. L. chinense also had two polyhydroxytropanes bearing a methyl group on the nitrogen atom, unlike the previously reported nortropane alkaloids. They were established as N‐methylcalystegines B2 and C1, and their N‐methyl groups were found to be axially oriented from NOE experiments. 1α‐Amino‐3β,4β,5α‐trihydroxycycloheptane was also present in L. chinense and may be a biosynthetic precursor of the calystegines that occur in this plant. Two polyhydroxypiperidine alkaloids, fagomine and 6‐deoxyfagomine, were isolated. Calystegine B2 is a potent competitive inhibitor of almond β‐glucosidase (Ki= 1.9 μM) and coffee bean α‐galactosidase (Ki= 0.86 μM), while N‐methylcalystegine B2 was a more potent competitive inhibitor of the latter enzyme (Ki= 0.47 μM) than the parent compound but showed a marked lack of inhibitory activities towards most other glycosidases. Since this compound is a very specific inhibitor of α‐galactosidase and inhibits rat liver lysosomal α‐galactosidase with a Ki of 1.8 μM, it may provide a useful experimental model for the lysosomal storage disorder, Fabry's disease. The addition of a hydroxyl group at C6exo, as in calystegines B1 and C1, enhances the inhibitory potential towards β‐glucosidase and β‐galactosidase but markedly lowers or abolishes inhibition towards α‐galactosidase. Hence, the N‐methylation of calystegine C1 did not enhance its inhibition of α‐galactosidase. The chemical N‐methylation of calystegines A3 and B4 markedly enhanced inhibition of coffee bean α‐galactosidase, with Ki values of 5.2 μM and 36 μM, respectively, but almost eliminated their inhibitory potential towards β‐glucosidase and trehalase, respectively. Thus, methylation of the nitrogen atom significantly altered the specificity of the inhibitors.
Kinase‐Deficient Forms of Jak1 and Tyk2 Inhibit Interferon α Signaling in a Dominant Manner
Authors: Krishnan Kartik , Pine Richard , Krolewski John J.
Published: July 2004

Signaling by interferon α (IFNα), an extracellular factor that mediates a number of anti‐viral and growth‐suppressive effects, requires two members of the Janus family of tyrosine kinases (JAK family): Jak1 and Tyk2. IFNα treatment of cells induces the rapid tyrosine phosphorylation of these two kinases, two subunits of the IFNα receptor, and two members of the signal transducer and activator of transcription (STAT) family of latent transcription factors. These proteins are believed to be direct substrates of one or both JAKs. Though the requirement for both Jak1 and Tyk2 in the IFNα‐signaling cascade is well established, the order of activation and the relative contribution of the two kinases has not been elucidated completely. To address these questions, we have employed kinase‐deficient mutants of both enzymes. Both mutant kinases suppress transcriptional activation as measured by an IFNα‐dependent reporter‐gene assay. Furthermore, in transient‐transfection assays, the kinase‐deficient versions of Tyk2 and Jak1 can act independently to inhibit STAT phosphorylation. Thus, kinase‐deficient versions of JAK can act in a dominant‐negative fashion to suppress IFNα signaling. The effects of the overexpressed mutant kinases on the phosphorylation of the kinases themselves, however, are unequal, suggesting that Jak1 functions upstream of Tyk2.
Inositol Phospholipid 3‐Kinase is Activated by Cellular Stress but is not Required for the Stress‐Induced Activation of Glucose Transport in L6 Rat Skeletal Muscle Cells
Authors: Mcdowell Helen E. , Walker Trevor , Hajduch Eric , Christie Graham , Batty Ian H. , Downes C. Peter , Hundal Harinder S.
Published: July 2004

A characteristic response of cells subjected to a stress stimulus is a rapid activation of cellular glucose transport. The mechanisms governing this increase in glucose transport are poorly understood, but it has been suggested that the response may involve the intracellular‐signaling components that also participate in the hormonal activation of glucose transport. In skeletal muscle and fat tissue, inositol phospholipid 3‐kinase plays an integral role in the regulation of both basal and insulin‐stimulated glucose transport. In this study, we have investigated whether inositol phospholipid 3‐kinase is activated by chemical stress and, if so, whether it has a role to play in the stress‐induced increase in glucose transport in L6 muscle cells. Furthermore, we have attempted to assess the basis by which inositol phospholipid 3‐kinase may participate in the regulation of basal glucose transport. Acute exposure (30 min) of L6 muscle cells to 0.5 mM arsenite induced an 80% stimulation in glucose transport. This activation was due to a rise in the number of cell‐surface glucose transporters, based on an increase in the Vmax of glucose transport and the observation that arsenite increases the plasma membrane content of GLUT1 and GLUT4 glucose transporters by 95% and 60%, respectively, from an intracellular compartment. Arsenite induced rapid activation (<2 min) of inositol phospholipid 3‐kinase with an approximately fourfold increase in phosphatidylinositol 3,4,5‐trisphosphate (PtdIns3,4,5P3). In contrast, phosphatidylinositol 3‐phosphate (PtdIns3P) levels were unaffected. Prior treatment of L6 cells with 100 nM wortmannin suppressed the arsenite‐induced increase in Ptdms3,4,5P3 and reduced the cellular content of PtdIns3P by 50%. Under these conditions however, wortmannin failed to prevent the stress‐induced activation of glucose transport, but suppressed basal glucose transport by 60% with an IC50 of about 10 nM. In the absence of arsenite, wortmannin caused a dose‐dependent inhibition in the cellular levels of PtdIns3P and PtdIns3,4,5P3 with IC50 values of about 10 nM and 100 nM, respectively. In summary, the present results demonstrate that chemical stress activates inositol phospholipid 3‐kinase and glucose transport in L6 muscle cells, but unlike the hormonal responses of these cells the activation of inositol phospholipid 3‐kinase is not responsible for the stress‐induced increase in glucose transport. This implies that stress‐induced and hormonal stimulated increases in PtdIns3,4,5P3 levels are functionally distinct. By contrast, the maintenance of PtdIns3P levels, presumably involving a PtdIns‐specific, wortmannin‐sensitive inositol phospholipid 3‐kinase may be required to support basal glucose transport.
A Root‐Specific Iron‐Regulated Gene of Tomato Encodes a Lysyl‐tRNA‐Synthetase‐Like Protein
Authors: Giritch Anatoli , Herbik Alexandra , Balzer Hans‐Jörg , Ganal Martin , Stephan Udo W. , Bäumlein Helmut
Published: July 2004

The tomato mutant chloronerva exhibits a defect in iron‐uptake regulation. Despite high apoplastic and symplastic iron concentrations, the mutant shows characteristic symptoms of iron deficiency. Using a subtractive‐hybridisation approach, we have screened for cDNA clones specific for genes with altered expression in wild‐type versus mutant root tissue. Based on this clone collection, we have isolated and characterised a 2075‐bp full‐length cDNA encoding a lysyl‐tRNA‐synthetase‐like protein. The corresponding gene is localised as a single copy on chromosome 10. Its expression is strongly induced by changes in the iron status of the plant. This iron‐dependent regulation is superimposed upon a strict root specificity of gene expression. Possible functions of the gene product other than in protein biosynthesis will be discussed.
Structure of HOE/BAY 793 Complexed to Human Immunodeficiency Virus (HIV‐1) Protease in Two Different Crystal Forms Structure/Function Relationship and Influence of Crystal Packing
Authors: Lange‐Savage Gudrun , Berchtold Harald , Liesum Alexander , Budt Karl‐Heinz , Peyman Anusch , Knolle Jochen , Sedlacek Juraj , Fabry Milan , Hilgenfeld Rolf
Published: July 2004

Human immunodeficiency virus 1 (HIV‐1) protease is a prime target in the search for drugs to combat the AIDS virus. The enzyme functions as a C2‐symmetric dimer, cleaving the gag and gag‐pol viral polyproteins at distinct sites. The possession of a twofold axis passing through the active site, has led to the design of C2‐symmetrical inhibitors in the form of substrate‐based transition‐state analogs. One of the most active compounds of this class of inhibitors is HOE/BAY 793, which contains a vicinal diol central unit [Budt, K.‐H., Hansen, J., Knolle, J., Meichsner, C., Paessens, A., Ruppert, D. & Stowasser, B. & Winkler, I. (1990) European Patent application EP0428,849; Budt, K.‐H., Hansen, J., Knolle, J., Meichsner, C., Ruppert, D., Paessens, A. & Stowasser B. (1993) IXth International Conference on AIDS; Budt, K.‐H., Peyman, A., Hansen, J., Knolle, J., Meichsner, C., Paessens, A., Ruppert, D. & Stowasser, B. (1995) Bioorg. Med. Chem. 3, 559–571.] The structure of this inhibitor bound to HIV‐1 protease, in two different crystal forms, has been solved at 0.24‐nm resolution using X‐ray crystallography. In both forms, the details of the inhibitor‐protease interactions revealed an overall asymmetric binding mode, especially between the central diol unit and the active‐site aspartates. The main binding interactions comprise several specific H‐bonds and hydrophobic contacts, which rationalize many of the characteristics of the structure/activity relationship in the class of vicinal diol inhibitors. In a general analysis of the mobility of the flap regions, which cover the active site and participate directly in binding, using our structures and the HIV protease models present in the Brookhaven databank, we found that in most structures the flexibility of the flaps is limited by local crystal contacts. However, in one of the structures presented here, no significant crystal contacts to the flap regions were present, and as a result the flexibility of the inhibitor bound flaps increased significantly. This suggests that the mobility and conformational flexibility of the flap residues are important in the functioning of HIV‐1 protease, and must be considered in the future design of drugs against HIV protease and in structure‐based drug design in general.
The Ribosomal‐RNA‐Processing Pathway in Schizosaccharomyces Pombe
Authors: Good Liam , Intine Robert V. A. , Nazar Ross N.
Published: July 2004

In all cells, a long precursor RNA is processed into mature rRNAs for ribosome biogenesis. In eukaryotes, the complexity and speed of the overall process often has made it difficult to establish finer details of the maturation pathway. Since phylogenetic comparisons can provide evidence for critical events, the major rRNA processing pathway for the yeast Schizosaccharomyces pombe was determined using primer extension, nuclease protection and Northern‐hybridisational analyses. Transcript mapping of the 5′ external transcribed spacer revealed six cleavage sites which occur upstream of the mature 18S termini. Two of these sites as well as a site adjacent to the 18S termini are complementary to conserved Box sequences in the S. pombe U3 small nucleolar RNA. Transcript mapping of the internal transcribed spacers (1 and 2) suggest similar maturation schemes for the two spacers, in which an initial endonuclease cleavage is followed by processing to the mature termini. The mature 5′ termini of 25S rRNA appear to be heterogeneous in S. pombe, as has been demonstrated for 5.8S rRNA, suggesting an essential limiting structure in the ribosome‐integrated mature RNA. Together with our previous analysis of the 3′ external spacer region, the results reveal the major processing pathway for S. pombe and further support a maturation process which acts as a quality assurance mechanism.
Kinetics of Reciprocal Pro‐Urokinase/Plasminogen Activation
Authors: Petersen Lars C.
Published: July 2004

The two zymogens, plasminogen and pro‐urokinase plasminogen activator (pro‐uPA), constitute a system of reciprocal activation, since plasmin, generated by uPA‐catalysed plasminogen activation, can activate pro‐uPA to uPA. Two such zymogens, when mixed, will undergo autocatalytic, reciprocal activation resulting in generation of proteolytic activity. As an example of reciprocal zymaogen activation, the plasminogen/pro‐uPA system was analysed in terms of a kinetic model which describes the progression in activated enzymes. This model gave a detailed description of the progress curves in plasmin and uPA. It accounted for the effects of varying the concentration of the zymogens, and also for the effects of plasmin substrates and inhibitors in the reaction mixture. The model assumes non‐significant zymogen activity. It did not, however, exclude that a very low initial proteolytic activity, accounting for maximally 0.01% of that obtained when pro‐uPA is fully activated, could be attributed to a genuine pro‐uPA activity. Binding of the uPA receptor (uPAR) to pro‐uPA/uPA might affect separate steps of the reciprocal activation reaction, or it might induce a significant pro‐uPA activity. To distinguish between these possibilities the effect of a recombinant soluble (residues 1–277) form of uPAR, uPAR‐(1–277)‐peptide, on reciprocal pro‐uPA/plasminogen activation was studied. uPAR‐(1–277)‐peptide attenuated reciprocal zymogen activation, and the results suggested that this was due to a decreased accessibility of the pro‐uPA/uPAR‐(1–277)‐peptide complex to activation by plasmin. The uPAR‐(1–277)‐peptide in the presence of poly(D‐lysine) caused a 20‐fold enhancement of reciprocal zymogen activation. Kinetic analysis of separate activation steps revealed that this was due to a threefold stimulation of plasminogen activation by uPA/uPAR‐(1–277)‐peptide combined with a sixfold stimulation of plasmin's activation of pro‐uPA/uPAR‐(1–277)‐peptide. The results suggested that poly(D‐lysine) provided a template for a catalytically favourable interaction between plasminogen/plasmin and the uPAR‐(1–277)‐peptide complex with pro‐uPA/uPA. There was no indication of a significant uPAR‐(1–277)‐peptide‐induced enhancement of pro‐uPA activity.
Electron‐Transfer Properties of Pseudomonas Aeruginosa [Lys44, Glu64]azurin
Authors: Van Pouderoyen Gertie , Cigna Giulio , Rolli Gabriella , Cutruzzolà Francesca , Malatesta Francesco , Silvestrini Maria Chiara , Brunori Maurizio , Canters Gerard W.
Published: July 2004

In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron‐transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet‐visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron‐self‐exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild‐type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551, and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.
Kinetic Characterization of Individual Hexose Transporters of Saccharomyces Cerevisiae and their Relation to the Triggering Mechanisms of Glucose Repression
Authors: Reifenberger Elke , Boles Eckhard , Ciriacy Michael
Published: July 2004

In Saccharomyces cerevisiae, there are a large number of genes (HXT1‐HXT17/SNF3/RGT2) encoding putative hexose transporters which, together with a galactose permease gene (GAL2), belong to a superfamily of monosaccharide facilitator genes. We have performed a systematic analysis of the HXT1–7 and GAL2 genes and their function in hexose transport. Glucose uptake was below the detection level in the hxt1–7 null strain growing on maltose. Determination of the kinetic parameters of individual hexose transporter‐related proteins (Hxtp) expressed in the hxt null background revealed Hxt1p and Hxt3p as low‐affinity transporters (Km(glucose)= 50–100mM), Hxt2p and Hxt4p as moderately low in affinity (Km(glucose) about 10 mM), and Hxt6p, Hxt7p as well as Gal2p as high‐affinity transporters (Km(glucose)= 1–2 mM). However, Hxt2p kinetics in cells grown on low glucose concentrations showed a high‐affinity (Km =1.5 mM) and a low‐affinity component (Km= 60 mM). Furthermore, we investigated the involvement of glucose transport in glucose signalling. Glucose repression of MAL2, SUC2 and GAL1 was not dependent on a specific transporter but, instead, the strength of the repression signal was dependent on the level of expression, the properties of the individual transporters and the kind of sugar transported. The strength of the glucose repression signal correlated with the glucose consumption rates in the different strains, indicating that glucose transport limits the provision of a triggering signal rather then being directly involved in the triggering mechanism.
Identification of an Fe(III)‐dihydroxyphenylalanine Site in Recombinant Phosphomannose Isomerase from Candida albicans
Authors: Smith Jason J. , Thomson Andrew J. , Proudfoot Amanda E. I. , Wells Timothy N. C.
Published: July 2004

Candida albicans phosphomannose isomerase (PMI) is a zinc metalloprotein of known crystal structure. When heterologously overexpressed in Escherichia coli, a blue protein that contains up to 0.5 iron atoms/PMI molecule could be isolated, with absorption maxima at 420 nm and 680 nm. These bands are reminiscent of ferric catecholate complexes, an assignment that has been confirmed by resonance Raman spectroscopy, and by reaction with Arnow's reagent, which is specific for the presence of 3,4‐dihydroxy‐phenylalanine (Dopa). After enzymatic digestion of blue PMI, a peptide with the sequence DPHAXISG was isolated corresponding to residues Asp283 ‐Gly290 in the amino acid sequence of C. albicans PMI, where the unidentified residue X287 is encoded by a tyrosine codon. It is proposed that iron and oxygen bring about hydroxylation of Tyr287 in PMI and that Fe(III) subsequently chelates the Dopa residue to give the characteristic absorption spectrum. The EPR spectrum of the blue protein suggests three iron environments in the protein, two in axial environments with E/D values approximately equal to 0.06 and 0.12 and one rhombic species. The nature of the iron co‐ordination sites is discussed with the help of model systems and by comparison with other blue non‐heme iron proteins.
A partly Folded State of Acidic Fibroblast Growth Factor at Low Ph
Authors: Sanz Jesús M. , Giménez‐Gallego Guillermo
Published: July 2004

Acid denaturation of acidic fibroblast growth factor (aFGF) at low ionic strength was monitored by far‐ultraviolet circular dichroism and intrinsic fluorescence. The two spectroscopic probes displayed non‐coincident transitions, which suggested the accumulation of partly folded species around pH 4.0. Although under these conditions the fluorescence of aFGF resembled that of the unfolded form of the protein, far‐ultraviolet circular dichroism and proton nuclear magnetic resonance spectra indicated the presence of persistent secondary and tertiary structure. Moreover, at pH 4.0, aFGF showed cooperative thermal denaturation and interacted weakly with the hydrophobic probe N‐phenyl‐1‐naphthylamine, showing a relatively high level of structure that did not fit into the classical molten globule category. This intermediate is also capable of interacting with liposomes and might represent a membrane translocation‐competent form.
Molecular Cloning and Characterization of the Chromosomal Gene for Human Lactoperoxidase
Authors: Ueda Tadashi , Sakamaki Kazuhiro , Kuroki Tetsuo , Yano Ikuya , Nagata Shigekazu
Published: July 2004

Lactoperoxidase (LPO) is an oxidoreductase secreted into milk, and plays an important role in protecting the lactating mammary gland and the intestinal tract of the newborn infants against pathogenic microorganisms. In this study, the human LPO chromosomal gene was molecularly cloned, and its gene organization was determined. The human LPO gene was found to be arranged with the myeloperoxidase (MPO) gene in a tail‐to‐tail manner. Similar to the human MPO and eosinophil peroxidase (EPO) genes, the human LPO gene is split by 11 introns and spans 28 kb. Unlike most introns in mammalian gene, the 5′ splice donor sequence of intron 11 starts with GC instead of GT. When the minigene comprised of exon 11, intron 11 and exon 12 of the human LPO gene was introduced into COS cells, the correct splicing of the intron was found, suggesting the intron 11 of the human LPO gene is functional. The coding sequence of human LPO consists of 2136bp, and codes for a protein of 712 amino acids. The amino acid sequence of human LPO has 51% similarity with those of both human MPO and EPO, suggesting that these peroxidase genes have evolved from a common ancestral gene. On the other hand, the nucleotide sequences of the 5′ promoter regions of these peroxidase genes exhibit no similarity among them, which agrees with their tissue‐specific expression.
Glycosylation Analysis and Protein Structure Determination of Murine Fetal Antigen 1 (mFA1)
Authors: Krogh Thomas N. , Bachmann Elin , Teisner Børge , Skjødt Karsten , Højrup Peter
Published: July 2004

By means of sequence analysis, murine fetal antigen 1 (mFAl) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the δ‐like protein, stromal‐cell‐derived protein 1 (SCP‐1) and preadipocyte factor 1 (Pref‐1) gene products. The protein contains 36 cysteine residues arranged in six epidermal‐growth‐factor‐like domains. The purification of several C‐terminal peptides of varying lengths showed mFA1 to be C‐terminal heterogeneous. O‐linked glycosylations of the NeuNAcα2–3Galβ1‐ 3(NeuNAcα2–6)GalNAc type were present on all C‐terminal peptides at residues Thr235, Thr244 and Thr248, although glycosylation on Thr244 was only partial. Three N‐linked glycosylations were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and Asn151) were in the unusual Asn‐Xaa‐Cys motif. Fucosylated biantennary complex‐type and small amounts (less than 5%) of triantennary complex‐type structures were identified on the glycosylated asparagine residues using sequential exoglycosidase and endoglycosidase digestions combined with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). The presence of O‐linked monosaccharides (glucose attached to Ser71, Serl93 and fucose at Thr201) was tentatively ascertained by combining Edman degradation and MALDI‐MS. The results presented shows mFA1 to be the circulating heterogeneous cleavage products of the membrane‐bound protein encoded by the murine cDNAs dlk, pref‐1 and SCP‐1.
Secondary Structure and Shape of Plasma Sex Steroid‐Binding Protein — Comparison with Domain G of Laminin Results in a Structural Model of Plasma Sex Steroid‐Binding Protein
Authors: Beck Konrad , Gruber Tanja M. , Ridgway Catherine C. , Hughes William , Sui Li‐ming , Pétra Philip H.
Published: July 2004

We have analyzed the secondary structure, shape and dimensions of plasma sex steroid‐binding protein (SBP) by CD, size‐exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for β‐sheet structures. Analysis with different algorithms indicates 15%α‐helix, 43%β‐sheet and 10–16%β‐turn structures. An irreversible structural change is observed upon heating above 60 °C, which correlates with the loss of steroid‐binding activity. As the SBP sequence shows similarity with domains of several multidomain proteins, including laminins, we evaluated the structure of domain G of laminin‐1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolu‐tion results in 13%α‐helix, 32%β‐sheet and 15%β‐turn structures. Steroid‐binding assays indicate that laminin and fragments thereof have no activity. Size‐exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the steroid‐binding site located in the center of the rod‐like particle.
Unusual Characteristics of Amino‐Terminal and Hydrophobic Domains in Nuclear‐Encoded Thylakoid Signal Peptides
Authors: Brink Susanne , Bogsch Erik G. , Mant Alexandra , Robinson Colin
Published: July 2004

Thylakoid transfer signals carry information specifying translocation by either a Sec‐ or pH‐dependent protein translocator in the chloroplast thylakoid membrane, yet all resemble classical signal peptides in overall structural terms. Comparison of known transfer signals reveals two differences: (a) signals for the pH‐driven system invariably contain a critical twin‐arginine (Arg‐Arg) motif prior to the hydrophobic (H) domain, whereas known Sec‐dependent signals contain lysine, and (b) the H‐domains of Sec‐dependent signals are generally longer. Previous work has shown that a twin‐Arg motif before the H‐domain is critical for targeting by the pH‐dependent pathway; in this report we show that the charge characteristics of this region are not important for sorting by the Sec pathway. Twin‐Lys, twin‐Arg or single Arg are all acceptable to the Sec system, although single Lys/Arg is preferred. The single Lys in pre‐plastocyanin can even be replaced by an uncharged residue without apparent effect. We have also generated a pre‐plastocyanin mutant containing an H‐domain which, in terms of hydropathy profile, is identical to that of a pH‐dependent protein. This mutant is also transported efficiently by the Sec system, demonstrating that hydrophobicity per se is not a key sorting determinant. However, the characteristics of the H‐domain may be important in avoiding a different form of mis‐targeting: to the endoplasmic reticulum. Thylakoid signal peptides have undergone substantial structural changes during the evolution of the chloroplast from endosymbiotic cyanobacterium: plastid‐encoded and cyanobacterial signals contain H‐domains that are highly hydrophobic and enriched in Leu and aromatic residues, whereas nuclear‐encoded counterparts are Ala‐rich and far less hydrophobic. We speculate that this trend may reflect a need to avoid mistargeting through recognition by cytosolic signal recognition particle, which preferentially interacts with more hydrophobic signal peptides.
Conformational Changes of Arginine Kinase Induced by Photochemical Release of Nucleotides from Caged Nucleotides
Authors: Raimbault Cyrille , Besson Françoise , Buchet René
Published: July 2004

The conformations of arginine kinase (AK) in AK · Mg · ADP, AK · Mg · ATP, AK · Mg · ADP · NO3−, AK · Mg · ADP · Arg and AK · Mg · ADP · NO3−. Arg complexes were investigated by measuring their reaction‐induced infrared difference spectra (RIDS). The photochemical release of ATP from ATP[Et(PhNO2)] and of ADP from ADP[Et(PhNO2)] produced distinct RIDS of AK complexes, suggesting that binding of ADP and ATP promoted different structural alterations of the enzyme active‐site. Small infrared changes in the amide‐I region were observed, indicating that about 5 – 10 amino acid residues were involved in the nucleotide‐binding site. These infrared changes were due to the structural alteration of the peptide backbone caused by the nucleotide‐binding and to the coupling effects between the nucleotide‐binding site and the other substrate (Arg or NO3−)‐binding site. ATP binding to AK (as well as ADP‐binding to AK in the presence of NO3−) induced protonation of a carboxylate group of Asp or Glu, as evidenced by the appearance of the 1733‐cm−1 band, which was not observed with the AK · Mg · ADP, AK · Mg · ADP · Arg and AK · Mg · ADP · NO3−· Arg complexes. The RIDS of the AK · Mg · ADP · NO3· Arg complex showed new infrared bands at 1622cm−1 (negative) and at 1613 cm−1 (positive), which were not seen in the RIDS of other complexes (without NO3− or/and −Arg). In the transition‐state‐analog complex of AK, no protonation of the carboxylate residue (Asp or Glu) was observed, and the binding site of NO3− or the γ‐phosphate group of nucleotide was altered.
Conformation and Microenvironment of the Active Site of Xylose Reductase Inferred by Fluorescent Chemoaffinity Labeling
Authors: Rawat Urmila B. , Rao Mala B.
Published: July 2004

Conformation and microenvironment at the active site of xylose reductase (XR) from Neurospora crassa was probed with fluorescent chemoaffinity labeling (FCAL) using o‐phthalaldehyde as a chemical initiator. Formation of a single isoindole derivative resulted in complete inactivation of the enzyme as judged by spectroscopic and fluorescence studies. Kinetic analysis of the 2,4,6‐trinitrobenzenesulfonic‐acid‐modified XR implicated the presence of an essential lysine residue at the active site of XR. Modification of lysine in XR abolished the ability of the enzyme to form isoindole derivative, indicating that the lysine residue involved in the reaction with 2,4,6‐trinitrobenzenesulfonic acid and o‐phthalaldehyde is the same and that the probe o‐phthalaldehyde is directed to the active site.

Fluorescence studies revealed that inactivation of XR by Gdn/HCl precedes gross conformational change and the possibility of secondary‐conformational change was eliminated by acrylamide quenching studies. The enzyme inactivated by low concentrations of Gdn/HCl retained its ability to form the fluorescent XR‐isoindole derivative indicating that inactivation is not due to conformational changes at or near the active site of XR. Gdn/HCl also had no effect on the high‐affinity and low‐affinity NADPH‐binding sites of XR. Energy‐transfer experiments further revealed structural integrity at the active site of the Gdn/HCl‐inactivated XR. Changes in the fluorescence emission maximum of 1‐(β‐hydroxyethylthio)‐2‐β hydroxyethyl isoindole (EA adduct) in solvents of varying polarity was studied, the data obtained were utilized to interpret the fluorescence behaviour of XR‐isoindole derivative and assess the polarity at the active site. Experimental evidence presented here serves to suggest that the inactivation of XR by Gdn/HCl precedes conformational changes at the active site located in a microenvironment of low polarity.
Chemical, Spectroscopic and Structural Investigation of the Substrate‐Binding Site in Ascorbate Peroxidase
Authors: Hill Adrian P. , Modi Sandeep , Sutcliffe Michael J. , Turner Daniel D. , Gilfoyle David J. , Smith Andrew T. , Tam Beatrice M. , Lloyd Emma
Published: July 2004

The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine‐modification experiments. The binding interaction for the cyanide‐bound derivative (APX‐CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant, Kd, of 11.6 ± 0.4 μM (pH 7.002, μ= 0.10 M, 25.0°C). Individual distances between the non‐exchangeable substrate protons of APX‐CN and the haem iron were determined by paramagnetic‐relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90–1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular‐modelling studies, using the NNM‐derived distance restraints in conjunction with the crystal structure of the enzyme [Patterson, W. R. & Poulos, T. L. (1995)Biochemistry 34, 4331–4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class‐III peroxidases) is implicated.
Sequencing, Expression and Properties of Triosephosphate Isomerase from Entamoeba Histolytica
Authors: Landa Abraham , Rojo‐Domínguez Arturo , Jiménez Lucia , Fernández‐velasco D. Alejandro
Published: July 2004

We have isolated a cDNA clone of the glycolytic enzyme, triosephosphate isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleotides obtained by reverse translation of conserved poly‐peptide sequences, derived from TPIs of other organisms, were used to amplify a 450‐bp fragment using E. histolytica cDNA as a template. The fragment was used to screen a cDNA library. The isolated cDNA, encoding a protein of 261 amino acids, shares 43–52.6% positional identity with other known protozoan TPIs. The catalytic residues were conserved; nevertheless, several indels occurred at other regions in the protein sequence. The complete coding sequence of the E. histolytica TPI gene was cloned into the expression vector pRSET and expressed as a wild‐type TPI enzyme (E. histolytica TPI) and as a fusion protein with an N‐terminal tail of six histidine residues E. histolytica TPI‐His6); both recombinant proteins were purified.

Molecular modeling of E. histolytica TPI showed an identical topology to the known structures of other TPI molecules, but with a remarkable feature; more than 10 inserted residues are located in the same region of the molecular surface. Studies were performed to detect possible changes that might be caused by the inserted amino acids. The catalytic activity and oligomeric state of the purified protein were similar to that reported for TPI from other sources. In contrast, stability towards dilution, as well as thermal inactivation and unfolding assays, showed that E. histolytica TPI is significantly more stable towards denaturation than Trypanosoma brucei TPI.
Expression of Properly Folded Human Glutamate Decarboxylase 65 as a Fusion Protein in Escherichia Coli
Authors: Papouchado Mariana L. , Valdez Silvina N. , Ghiringhelli Daniel , Poskus Edgardo , Ermácora Mario R.
Published: July 2004

Autoantibodies to the islet‐cell 65‐kDa variant of glutamate decarboxylase (GAD65) are found in most insulin‐dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM‐preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti‐GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti‐GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large‐scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N‐terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.
Identification of A Putative Histidine Base and of a Non‐Protein Nitrogen Ligand in the Active Site of Fe‐Hydrogenases by One‐Dimensional and Two‐Dimensional Electron Spin‐Echo Envelope‐Modulation Spectroscopy
Authors: Van Dam Pieter J. , Reijerse Eduard J. , Hagen Wilfred R.
Published: July 2004

The active H‐cluster of the Fe‐hydrogenases from Megasphaera elsdenii and Desulfovibrio vulgaris (strain Hildenborough) has been investigated with one‐ and two‐dimensional pulsed EPR spectroscopy. In both complexes the coordination of a nitrogen‐containing ligand was found. The unusual quadrupole interaction parameters (D. vulgaris: quadrupole coupling constant, K= 1.20 MHz, asymmetry parameter η= 0.32, M. elsdenii: K= 1.23 MHz, η= 0.25) indicate a non‐protein type of nitrogen and are consistent with cyanide as ligand to the H‐cluster.

The additional interactions measured on the EPR signal of the inactivated H‐cluster in D. vulgaris hydrogenase are consistent with an imidazole interaction similar to that found in Rieske‐type iron‐sulfur clusters. Since a His residue near the putative H‐cluster binding motif of Cys residues, His371, is the only conserved His in Fe‐hydrogenases, it is a likely candidate for the base that accepts the proton in the heterolytic cleavage of molecular hydrogen. The inactivation of the enzyme is accompanied by direct binding of the imidazole ring to the H‐cluster.
1.8‐Å Crystal Structure of the Catalytic Domain of Human Neutrophil Collagenase (Matrix Metalloproteinase‐8) Complexed with a Peptidomimetic Hydroxamate Primed‐Side Inhibitor with a Distinct Selectivity Profile
Authors: Betz Michael , Huxley Philip , Davies Stephen J. , Mushtaq Yasmeen , Pieper Michael , Tschesche Harald , Bode Wolfram , Gomis‐Rüth F. Xavier
Published: July 2004

Matrix metalloproteinases (MMP) are zinc endopeptidases involved in tissue remodelling. They have been implicated in a series of pathologies, including cancer, arthritis, joint destruction and Alzheimer's disease. Human neutrophil collagenase represents one of the three interstitial collagenases that cleave triple‐helical collagen of type I, II and III. Its catalytic domain (residues Phe79–Gly242) has been heterol‐ogously expressed in Escherichia coli and crystallized as a non‐covalent complex with the hydroxamate inhibitor BB‐1909, which has distinct selectivity against different MMP, in a crystal form. The crystal structure, refined to 0.18‐nm resolution, shows that BB‐1909 is a right‐hand‐side inhibitor that binds to the S1′–S3′ subsites and coordinates to the catalytic Zn2+ in a bidentate manner via the hydroxyl and carbonyl oxygen atoms of the hydroxamate group in a similar manner to batimastat. The collagenase/BB‐1909 complex is described in detail and compared with the collagenase/batimastat complex. These studies provide information on MMP specificity and thus may assist the development of more‐selective MMP inhibitors.
Structural Elucidation of two Polysaccharides Present in the Lipopolysaccharide of a Clinical Isolate of Burkholderia Cepacia
Authors: Cérantola Stéphane , Montrozier Henri
Published: July 2004

Based on the sugar composition, methylation analyses and Smith degradation, supported by NMR spectroscopic analyses and fast‐atom‐bombardment MS experiments, the lipopolysacharide produced by a clinical isolate of Burkholderia cepacia was shown to contain two distinct polymers, both with linear trisaccharide repeating units; a major, containing D‐rhamnose and D‐galactose residues (2:1) with the structure

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and a minor repeating unit, constituted by D‐rhamnosyl residues, with the structure

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The Enzymology of Lysine Catabolism in Rice Seeds — Isolation, Characterization, and Regulatory Properties of a Lysine 2‐Oxoglutarate Reductase/Saccharopine Dehydrogenase Bifunctional Polypeptide
Authors: Gaziola Salete A. , Teixeira Cristiana M. O. , Luoli Juverlande , Sodek Ladaslav , Azevedo Ricardo A.
Published: July 2004

In plant, the catabolism of lysine has only been studied in some detail in maize. The enzymes lysine α‐oxoglutarate reductase (also known as lysine α‐ketoglutarate reductase; LOR) and saccharopine dehydrogenase (SDH), which convert lysine into saccharopine, and saccharopine into glutamic acid and 2–aminoadipate 6‐semialdehyde, respectively, were isolated from immature rice seeds and partially purified through a three‐step purification procedure involving ammonium sulphate precipitation, and anion‐exchange and gel‐filtration chromatographies, leading to a final yield of 30% for LOR and 24% for SDH. The molecular masses estimated by gel‐filtration chromatography on a Sephacryl S200 column and by native non‐denaturing PAGE using Ferguson plots were 203 kDa for both enzymes by gel‐filtration and 202 kDa for both enzymes by native non‐denaturing PAGE. A second band of LOR and SDH activities on native gels was observed for both enzymes with an estimated molecular mass of 396 kDa, which indicated a multimeric structure. Kinetic studies were consistent with an ordered sequence mechanism for LOR, where 2‐oxoglutarate is the first substrate and saccharopine is the last product.

The results observed for the LOR/SDH activity ratios during purification, the copurification in all three steps, the molecular masses, the relative mobilities on native non‐denaturing gels and the pl estimated for LOR and SDH suggest the existence of a bifunctional polypeptide containing LOR and SDH activities.
Activation of Progelatinase B (proMMP‐9) by Active Collagenase‐3 (MMP‐13)
Authors: Knäuper Vera , Smith Bryan , López‐Otin Carlos , Murphy Gillian
Published: July 2004

Human progelatinase B was activated by collagenase‐3 in a time‐dependent fashion. Activation proceeded through an intermediate form of Mr 86000 to the final active form of Mr 82000. N‐terminal amino acid sequence determination demonstrated that the Glu40–Met41 peptide bond was initially hydrolysed followed by cleavage of the Arg87–Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active collagenase‐3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by collagenase‐3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with collagenase‐3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C‐terminal domain interactions between progelatinase B and collagenase‐3, as assessed using wild‐type and C‐terminal deletion mutants of both enzymes.

Since elevated levels of both gelatinase B and collagenase‐3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix.
The Colchicine‐Binding and Pyrene‐Excimer‐Formation Activities of Tubulin Involve a Common Cysteine Residue in the β Subunit
Authors: Basusarkar Pampi , Chandra Suparna , Bhattacharyya Bhabatarak
Published: July 2004

Colchicine binding and pyrene excimer fluorescence of tubulin have been used to identify cysteine residue(s) essential for the colchicine binding activity of the protein. We report here that both the colchicine binding activity and the ability to form pyrene excimers of tubulin decay at an identical rate when the protein ages at 37°C. Glycerol, which stabilizes the colchicine binding site also stabilizes the excimer formation equally. Thus, these two properties of tubulin are correlated and are likely to belong to the same structural domain. In an attempt to identify the excimer‐forming Cys residues, we found that incubation of tubulin with N,N′ ethylenebis (iodoacetamide) causes a significant inhibition of excimer fluorescence. Incubation of tubulin with colchicine prior to this treatment fully retains excimer‐forming ability. It is known that Cys239 and Cys354 of β‐tubulin, which are about 0.9 nm apart in the native structure, are protected from ethylenebis(iodoacetamide) cross‐linking by incubation of tubulin with colchicine [Luduena, R. F. & Roach, M. C. (1981)Pharmacol. Ther. 49, 133–152], These residues must therefore be responsible for the excimer formation of tubulin with pyrene maleimide. Incubation of tubulin with ethylenebis(iodoacetamide) decreases the colchicine binding activity and the excimer formation at an identical rate. Since the alkylation of Cys239 of β‐tubulin (responsible for tubulin self‐assembly) has no effect on colchicine binding [Bai, R., Lin, C. M., Nguyen, N. Y., Liu, T. & Hamel, E. (1989)Biochemistry 28, 5606–5612], our results suggest that excimer formation and the colchicine binding site of tubulin share Cys354 of the β‐subunit. Determination of the number of essential Cys residue(s) for colchicine binding activity, using the statistical method of Tsou [Tsou, C. L. (1962)Sci. Sin. 11, 1535–1558], also shows only one essential Cys residue.
Identification and Activation of Profollipsin, a Latent Precursor form of Porcine Follipsin
Authors: Kihara Takahiro , Ohnishi Junji , Kohyama Kuniko , Moriyama Akihiko , Takahashi Takayuki
Published: July 2004

A latent protease has been identified in column fractions obtained during the purification of the porcine ovarian serine protease follipsin. The latent enzyme was readily activated by trypsin treatment. The trypsin‐activated enzyme was purified using a benzamidine‐Sepharose 6B column and was shown to be composed of two distinct, covalently associated polypeptides with Mr of 45000 and 32000. This polypeptide chain composition, together with its substrate specificity, inhibition profile using various protease inhibitors, cross‐reactivity with anti‐follipsin antibody, and ability to activate single‐chain precursor tissue plasminogen activator, indicated its identity as porcine follipsin. The activation of the enzyme with trypsin was found to occur by the hydrolysis of an internal peptide bond resulting in a two‐chain structure. Thus, we conclude that the latent enzyme is the inactive precursor form (profollipsin) of follipsin. The present study also shows that the follicular fluid of porcine ovary contains a profollipsin‐activat‐ing enzyme activity.