bluuebunny/crossref_metadata_2025 huggingface dataset:
A Potassium‐Channel Toxin From the Sea Anemone Bunodosoma Granulifera, An Inhibitor for Kv1 Channels — Revision of the Amino Acid Sequence, Disulfide‐Bridge Assignment, Chemical Synthesis, and Biological Activity
Authors: Cotton Jöel , Crest Marcel , Bouet Françoise , Alessandri Nicole , Gola Maurice , Forest Eric , Karlsson Evert , Castaneda Olga , Harvey Alan L. , Vita Claudio , Ménez Andre
Published: July 2004

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37‐amino‐acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C‐terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C.

The toxin BgK was synthesized according according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2‐Cys37, Cys11‐Cys30 and Cys20‐Cys34. The toxin contains a C‐terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C‐terminus in either the carboxylate or carboxamido form.

Synthetic BgK inhibits binding of 125I‐α‐dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi‐K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage‐dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker‐related Kv1 channels are equally affected by BgK, while the Shaw‐related channel Kv3.1 is insensitive up to 0.125 pM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd= 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker‐related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ.

The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demostrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.
Flexibility and Fine Structure of Smooth‐Muscle α‐Actinin
Authors: Winkler Jörg , Lünsdorf Heinrich , Jockusch Brigitte M.
Published: July 2004

The microfilament protein α‐actinin exists as a dimer. The N‐terminal regions of both polypeptides, arranged in antiparallel orientation, comprise the actin‐binding regions, while the C‐terminal, larger parts consist of four spectrin‐like repeats that interact to form a rod‐like structure. To elucidate the fine structure of smooth‐muscle α‐actinin, we used energy‐filtered transmission electron microscopy in conjunction with negative staining. Survey pictures of the protein purified from chicken gizzard revealed discrete, elongated particles whose length and width varied with the ionic strength of the buffer. It was determined to to 29.3 nm × 4.8 nm in 0.05 M KC1 and 32.6 nm × 4.4 nm in 0.15 M KC1. Both ends of the molecule displayed hook‐like structures consisting of globular domains, which were highly variable in their orientation with respect to the long axis of the molecule. Their location at the ends of the molecule, and the finding that these hooks were missing from particles obtained by thermolysin digestion indicated that they probably correspond to the N‐terminal actin‐binding regions. The rod‐like center of the molecule revealed discrete globular masses which probably comprise the spectrin‐like repeats. Their arrangement was compatible with the interpretation that three spectrin repeats of each polypeptide chain can form pairs with the respective sequences of the other chain. The rod‐like 53‐kDa fragment obtained after thermolysin digestion largely retained this structural organization but appeared wider (22.5 nm × 5.9 nm). Our results help to clarify previous discrepancies on the quatenary organization of α‐actinin and suggest that effective actin‐binding and cross‐linking of α‐actinin is based on the high flexibility of the terminal hooks.
TheDrosophila Lsp‐1βGene
Authors: Massey Holman C. , Kejzlarová‐Lepesant Jana , Willis Rebecca L. , Castleberry Alecia B. , Beneš Helen
Published: July 2004

InDrosophila melanogaster, metamorphosis and reproduction are thought to be supported in large by two immunologically distinct hexameric storage proteins (hexamerins), larval serum protein 1 (LSP‐1), a mixed hexamer of three closely related subunits, Lsp‐1(α, β and γ) and larval serum protein 2 (LSP‐2), a homohexamer of Lsp‐2 subunits. To understand the structural and functional differences between these two storage hexamers, the nucleotide sequence of the coding region of theLsp‐1βgene was determined for comparison with LSP‐2 and a number of other arthropod hexamerins. The G+C content of the coding sequence is 55%, with 92.8% of the codons containing G or C in the third position. Conceptual translation of theLsp‐1βopen reading frame revealed a 789‐amino‐acid polypeptide of 94465 Da. The amino acid sequence of Lsp‐1β is 65.8% identical to that of calliphorin, the major hexamerin of the blowfly,Calliphora vicina, and only 35.2% identical toDrosophilaLsp‐2. This greater similarity to calliphorin is also reflected in high aromatic amino acid and methionine contents, in contrast to LSP‐2 which is enriched to a lesser extent only in aromatic amino acids. Lsp‐1β is also more closely related to calliphorin with respect to the protein domain structure, the presence of a single intron in its gene, and the absence of glycosylation sites. However, phylogenetic analysis based on multiple alignments revealed that LSP‐1/calliphorin and LSP‐2 form a distinct dipteran clade whose members are more similar to each other than to any previously sequenced lepidopteran hexamerin or arthropod hemocyanin.
Tissue‐Specific Expression of Promoter Regions of the αr1(VI) Collagen Gene in Cell Cultures and Transgenic Mice
Authors: Braghetta Paola , Vitale Paola , Piccolo Stefano , Bonaldo Paolo , Fabbro Carla , Girotto Davide , Volpin Dino , Bressan Giorgio Maria
Published: July 2004

Cis‐acting regions regulating transcription of the α1(VI) collagen chain have been investigated in vitro by transfection of promoter‐CAT (where CAT is chloramphenicol acetyltransferase) constructs in different types of cultured cells and in vivo in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomic and cDNA sequences in which small deletions of the collagenous domain had been engineered. 215 bp of 5′‐flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transgenic lines, indicating that this fragment contains the basal promoter, but not activator sequences. Constructs with 0.6 and 1.4kb of the 5′‐flanking region produced significantly higher CAT activity in transfected cells and were expressed in tissues of about 30% of transgenic lines. Although CAT activity was totally unrelated to the pattern of expression of the α1(VI) mRNA, these results suggest the presence of an activator(s) between −0.2 and −0.6 kb from the transcription start site. When the promoter size was increased to 5.4 or 6.5 kb, CAT activity was stimulated several fold relative to the construct p1.4CAT and p4.0CAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimulation was, however, not observed in C2C12 myoblasts. Transgenic mice generated with 6.SCAT construct or minigenes, containing 6.2 kb of promoter, exhibited very high levels of expression, which was similar to the relative amount α 1 (VI) mRNA in the majority of tissues, with the exception of lung, adrenal gland and uterus. CAT activity in tissues was 100–1000‐fold higher than that measured in transgenic mice with shorter promoter (0.6 or 1.4kb). Since expression of minigenes was determined by RNase protection assay, the levels of mRNA per transgene copy were compared to those of the chromosomal gene and found to be always less than one quarter. These data suggest that the region ‐4.0/‐5.4 contains an important activator(s) sequence which induces transcription in several, but not all, type VI collagen‐producing tissues. Finally, analysis with the longest promoter fragment (7.5 kb) revealed a complex effect of the region ‐6.5/‐7.5 on α1(VI) chain transcription. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblasts and activating in chondroblasts in vitro, whereas transgenic animals generated with 7.5CAT construct produced a pattern of expression comparable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreased. However, the ratio of transgene/chromosomal gene expression was not constant, but varied in a way dependent on the tissue. This observation suggests that the fragment studied contains key sequences for the age‐dependent regulation of the α1(VI) gene. No phenotypic alterations were induced by the presence of mutations in the minigenes.
Surface Plasmon Resonance Analysis of gp17, a Natural CD4 Ligand from Human Seminal Plasma Inhibiting Human Immunodeficiency Virus Type‐1 gp120‐Mediated Syncytium Formation
Authors: Autiero Monica , Gaubin Muriel , Mani Jean‐Claude , Castejon Christophe , Martin Michelle , El Marhomy Sandrine , Guardiola John , Piatier‐Tonneau Dominique
Published: July 2004

We have previously isolated from human seminal plasma a CD4 ligand, the gp17 glycoprotein, which shares sequence identity with three previously identified proteins: secretory actin‐binding protein (SABP) from seminal plasma, gross‐cystic‐disease fluid protein‐15 (GCDFP‐15) and prolactin‐inducible protein (PIP) from breast tumor cells. Functions of these glycoproteins are unknown. To further characterize the physical interaction between gp17 and CD4 we used surface plasmon resonance and demonstrated that gp17‐CD4 binding affinity is high. Competition experiments indicated that gp17 interferes with human immunodeficiency virus (HIV) envelope protein/CD4 binding, although it binds to a site distinct from but close to the gp120‐binding site. We observed moreover that gp17 inhibits syncytium formation between transfected cells expressing the wild‐type HIV‐1 envelope glycoprotein and CD4, respectively. Our results suggest that gp17, which may function as an immunomodulatory CD4‐binding factor playing a role at insemination, may also play a role in controlling HIV spread in the sexual tract.
Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum
Authors: Rees‐Milton Karen , Thorne Mark , Greasley Peter , Churchich Jorge , Gore Michael G.
Published: July 2004

Mg2+ ions, essential for the catalytic activity of mammalian inositol monophosphatase, increase the ellipticity in the near‐ultraviolet region of the CD spectrum of the enzyme. These spectral changes are not affected by the additional presence of substrate and are reversed if EDTA is added to the solution of enzyme and metal ions. Titration of the spectral perturbation at 275 nm shows that this binding occurs with a dissociation constant (Kd) around 275 μM, 292 μM and 302 μM for the wild‐type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes respectively. The source of the spectroscopic change at 275 nm is not Trp219.

The addition of Mg2+ also causes a decrease in ellipticity over most of the far‐ultraviolet region of the spectrum (between 205–240 nm). The Kd values describing the binding of Mg2+ ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild‐type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes, respectively, each showing an approximate 12% change in ellipticity. In the additional presence of 10 mM Pi there is a fourfold increase in the affinity of wild‐type enzyme for Mg2+.

It is concluded that CD spectral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high‐affinity metal‐binding site (site 1). The spectral changes around 225 nm are associated with interactions at a lower‐affinity site normally occupied by the Mg2+ ion which is reflected by the Km value for this metal ion.

Other metal ions such as Ca2+ and Tb3+ (but not Mn2+ or Zn2+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal changes are greater for Mg2+ or Tb3+ (25%) ions than for Ca2+ (8.5%), although two Ca2+‐binding sites with Kd values of 20 μM and 100 μM have been identified.

The uncompetitive inhibitor Li+ causes little change in the near‐ultraviolet spectrum in the absence or presence of either substrate or Pi. However, in contrast to other metal ions, Li+ ions elicit a 10% increase in ellipticity at 220 nm with a Kd of 0.8 mM.
D‐2‐Hydroxy‐4‐Methylvalerate Dehydrogenase from Lactobacillus Delbrueckii Subsp. Bulgaricus— II. Mutagenic analysis of catalytically important residues
Authors: Bernard Nathalie , Johnsen Keyji , Gelpi Josep Lluis , Alvarez Josep A. , Ferain Thierry , Garmyn Dominique , Hols Pascal , Cortes Antonio , Clarke Anthony R. , Holbrook J. John , Delcour Jean
Published: July 2004

Five residues involved in catalysis and coenzyme binding have been identified in D‐2‐hydroxy‐4‐methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD‐dependent D‐2‐hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 105‐fold compared with the wild‐type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their kcatKm ratios (200–800‐fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the α‐carboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104‐fold reduced kcatKm due to both an increased Km and a reduced kcat for 2‐oxo‐4‐methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L‐lactate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7‐fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild‐type but showed a sevenfold increased Km for NADH and a 100‐fold increase in Kd for NADH together with 10–30‐fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D‐2‐hydroxy‐4‐methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate‐limiting processes in catalysis at pH 6.5.
Leukocyte Adhesion
Authors: Gahmberg Carl G. , Tolvanen Martti , Kotovuori Pekka
Published: July 2004

Leukocyte adhesion is of pivotal functional importance and this has resulted in extensive research and rapid develoment in the field. Leukocyte adhesion involves members of three molecular families: integrins, members of the immunoglobulin superfamily and carbohydrate binding selectins and sialoadhe‐sins. Recently, considerable structural information on leukocyte integrins and members of the immunoglobulin superfamily of adhesion molecules has been obtained. This fact, combined with the identification of several novel adhesion molecules, has increased our understanding of how they function at the molecular level. Furthermore, the important issue of how integrins are activated to become adhesive is rapidly advancing. It is clearly evident that the knowledge accumulated from basic research will increasingly be applied in clinical medicine. In this review we focus on two important families of adhesion molecules, the leukocyte‐specific β2‐integrins and their ligands, the intercellular adhesion molecules. Emphasis is put on their structural/functional relationships, their mode of regulation and on novel adhesion molecules recently discovered.
Investigation of Different Recombinant Isoforms of Grass Group‐V Allergens (Timothy Grass Pollen) Isolated by Low‐Stringency cDNA Hybridization — Antibody Binding Capacity and Allergenic Activity
Authors: Gehlhar Kirsten , Petersen Arnd , Schramm Gabriele , Becker Wolf‐Meinhard , Schlaak Max , Bufe Albrecht
Published: July 2004

A cDNA library of timothy grass pollen was screened for homologous isoforms of major group‐V allergens by low stringency hybridization with a Phi p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group‐V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group‐V allergens was shown to be in the same range for both subgroups.

Analysis of allergenic B‐cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine‐release and skin‐prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group‐V isoallergens in grass pollen, although they can be structurally different, induce a similar B‐cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type‐I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.
Solution Structure of Lqh‐8/6, a Toxin‐Like Peptide from a Scorpion Venom
Authors: Adjadj Elisabeth , Naudat Vincent , Quiniou Eric , Wouters Danièle , Sautière Pierre , Craescu Constantin T.
Published: July 2004

Lqh‐8/6 is a minor fraction isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus. Here we describe the purification, amino acid sequencing and solution structure determination by NMR and molecular modeling of this peptide. Lqh‐8/6 is a small polypeptide (38 residues) which contains 8 half‐cystines and is highly similar to another venom component, chlorotoxin. Standard homonuclear methods were used to sequentially assign the proton NMR spectra and to collect spatial restraints for structure determination. Two populations, identified early in the assignment step, are in slow interconversion on the NMR timescale. The two conformers were shown to originate from a cis/trans peptidyl‐prolyl isomerization. Using a distance geometry program and simulated annealing protocol under the NMR restraints we obtained 10 final structures for the major conformation (trans isomer). None of the structures showed NOE violations larger than 0.05 nm, and the rmsd value relative to the mean structure (considering the main chain atoms in well‐defined secondary structure) is 0.07 nm. The three‐dimensional structure contains a short α‐helix strapped on a small antiparallel β‐strand and an N‐terminal extended fragment. The sequence/structure and structure/function relationships of the new scorpion toxin‐like peptide are discussed in the context of the present structure determination. This toxin shows a stable, highly populated cis conformer of a peptidyl‐prolyl peptide bond.
Cell‐Surface Acceleration of Urokinase‐Catalyzed Receptor Cleavage
Authors: Høyer‐Hansen Gunilla , Ploug Michael , Behrendt Niels , Rønne Ebbe , Danø Keld
Published: July 2004

The urokinase‐type plasminogen activator (uPA) binds to a specific cell‐surface receptor, uPAR. On several cell types uPAR is present both in the full‐length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either directly or indirectly mediated by uPA itself. In a soluble system, uPA can cleave purified uPAR, but the low efficiency of this reaction has raised doubts as to whether uPA is directly responsible for uPAR cleavage on the cells. We now report that uPA‐catalyzed cleavage of uPAR on the cell surface is strongly favored relative to the reaction in solution. The time course of uPA‐catalyzed cleavage of cell‐bound uPAR was studied using U937 cells stimulated with phorbol 12‐myristate 13‐acetate. Only 30 min was required for 10 nM uPA to cleave 50% of the cell‐bound uPAR. This uPA‐catalyzed cleavage reaction was inhibited by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high‐efficiency cleavage of cell‐bound uPAR. Furthermore, amino‐terminal sequence analysis revealed that uPAR(2+3), purified from U937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occusred at two positions in the hinge region connecting domain 1 and 2, between Arg83‐Ala84 and Arg89‐Ser90, respectively. The uPA‐catalyzed cleavage of uPAR is a new negative‐feedback regulation mechanism for cell‐surface plasminogen activation. We propose that this mechanism plays a physiological role at specific sites with high local concentrations of uPA, thus adding another step to the complex regulation of this cascade reaction.
Heterodisulfide Reductase from Methanol‐Grown Cells of Methanosarcina Barkeri is not a Flavoenzyme
Authors: Künkel Andreas , Vaupel Martin , Heim Steffen , Thauer Rudolf K. , Hedderich Reiner
Published: July 2004

Heterodisulfide reductase from methanol‐grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane‐bound enzyme composed of a 46‐kDa subunit MbHdrD and a 23‐kDa subunit MbHdrE. The enzyme has been shown to contain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. In addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED revealed that both subunits also lack an FAD‐binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum (MtHdr), which is a flavo iron‐sulfur protein composed of the subunits MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin‐binding site. Sequence comparisons revealed that the N‐terminal third of MbHdrD, which contained two sequence motifs for [4Fe‐4S] clusters, is similar to MtHdrC and that the C‐terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrBC are structurally equivalent subunits. MbHdrE shows sequence similarity to b‐type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that MbHdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since MbHdrD contains only iron‐sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron‐transfer reactions has to be considered such as operative in ferredoxin:thioredoxin reductases from chloroplasts and cyanobacteria.
Complex ATP‐Activation Kinetics of Plant H+‐Transporting ATPase may or may not Require Two Substrate Sites
Authors: Roberts Gretel , Beaugé Luis
Published: July 2004

The complex ATP‐activation kinetics of plant H+‐ATPase requires two ATP effects on the enzyme. They may result from the simultaneous existence of two ATP sites or a single site that consecutively changes its properties. We describe here three main models for ATP binding to the plant H+‐ATPase. Considering the experimental data there are some restrictions in their application. A system with two simultaneous catalytic sites with cooperative binding is possible provided the substrate exerts regulatory properties, and when ES (or SE) leads to a slower velocity path than SES (v1 < v2). In other words, simple cooperativity does not work. A system with two substrate sites, one of which is catalytic and the other is always and only regulatory (i.e. it affects the overall reaction rate), offers two alternatives: one with potential cooperative binding (the system does not discriminate between binding to the catalytic and regulatory sites; and the other with intrinsically different affinities of catalytic and regulatory sites (i.e. the system discriminates between binding to the two binding sites). Here it is also obligatory that ES (or SE) leads to a slower‐velocity path than SES (v1 < v2). Thirdly, a system is possible with single ATP domain that is consecutively catalytic and regulatory as the cycle proceeds. These three mechanisms give rise to equivalent rate equations. Therefore, there is no way to distinguish between them on the basis of kinetic studies. Another conclusion drawn from modeling these schemes is that the form of the plots might resemble but not correspond to certain cooperativity type. For instance, for two substrate sites, a true negative cooperativity for substrate binding can mimic positive cooperativity if the v1 velocity pathway is much slower than the v2 one.
Enzymatic Semisynthesis of Dicarba Analogs of Calcitonin
Authors: čeřovský Václav , Wünsch Erich , Brass Johann
Published: July 2004

The semisynthesis of eel[L‐α‐aminosuberic acid1.7]calcitonin (elcatonin) was accomplished by α‐chymotrypsin‐catalyzed coupling of two peptide segments in a single reaction without the protection of any functional group. The eel calcitonin‐(10–32)‐peptide was prepared by a gene manipulation. The esters of cyclic desamino nonapeptide (segment 1–9) were synthesized by the conventional solution method including a thermolysin‐mediated resolution of DL‐α‐aminosuberic acid via one‐step tripeptide synthesis leading to the 7–9 sequence. The main aim of this work was to determine the conditions for protease‐catalyzed segment condensation while avoiding a concurrent cleavage of other proteolytically labile peptide bonds in the hormone. The α‐chymotrypsin condensation strategy under usual conditions led to a complicated mixture of split products with an insignificant amount of the required peptide. When the coupling reaction was carried out at 0°C, the reaction resulted in a satisfactory yield of elcatonin with the complete conversion of the acyl donor (1–9 segment) accompanied by negligible concurrent peptide bond digestion. The same strategy was employed for the preparation of analogous dicarba salmon calcitonin using a synthetic elcatonin‐(10–32)‐peptide. Both calcitonin analogs exhibited hypocalcemic activity corresponding to the international standard of elcatonin. We demonstrate in this work a peptide synthesis based on the combination of genetic engineering, chemical synthesis and proteinase‐catalyzed segment condensation. This approach enables effective incorporation of an unnatural amino acid into calcitonins without the side‐chain protection.
Activity of Tethered Human Immunodeficiency Virus 1 Protease Containing Mutations in the Flap Region of One Subunit
Authors: Tözsér József , Yin Fay H. , Cheng Yin‐Shyun E. , Bagossi Péter , Weber Irene T. , Harrison Robert W. , Oroszlan Stephen
Published: July 2004

The tethered‐dimer protease of human immunodeficiency virus 1 (HIV‐1) [Cheng Y.‐S. E., Yin, F. H., Foundling, S., Blomstrom, D. & Kettner, C. A. (1990) Proc. Natl Acad. Sci. USA 87, 9660–9664] and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coll. These mutant enzymes showed various degrees of self‐processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in Km and decrease in kcat values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2′ substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen‐bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.
The Fidelity of 3′ Misinsertion and Mispair Extension During DNA Synthesis Exhibited by two Drug‐Resistant Mutants of the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 with Leu74→Val and Glu89→Gly
Authors: Rubinek Tami , Bakhanashvili Mary , Taube Ran , Avidan Orna , Hizi Amnon
Published: July 2004

The relatively low fidelity of DNA synthesis characteristic to the reverse transcriptases (RTs) of the AIDS‐causing viruses, human immunodeficiency viruses types 1 and 2 (HIV‐1 and HIV‐2, respectively) was implicated as a dominant factor that contributes to the genetic hypervariability of these viruses. The formation of 3′‐mispaired DNA and the subsequent extension of this DNA were shown to be key determinants that lead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV‐1 RT, we have conducted mutational studies aimed at identifying amino‐acid residues involved in affecting the fidelity of DNA synthesis by the enzyme. We have recently found that two mutants of HIV‐1 RT, which show resistance to nucleoside analog inhibitors ([Leu184)RT and [Phe183]RT), exhibit in vitro error proneness of DNA synthesis lower than that of wild‐type enzyme [Bakhanshvili, M., Avidan, O. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis, FEBS Lett. 391, 257–262]. Using both criteria, the current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild‐type RT counterpart. In the current study we have analyzed two additional drug‐resistant mutants of HIV‐1 RT, [Val74]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis: 3′ mispair formation and elongation of 3‐mismatched DNA. The current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild‐type RT counterpart, using both criteria. Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA synthesis in HIV‐1 RT is wild‐type > [Val74]RT > [Gly89]RT mutant. The results further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and extend a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting and maintaining HIV virions with high fidelity and drug‐resistant RTs to suppress the subsequent appearance of virions resistant to other drugs.
Characterization of the Basal and Pheromone‐Stimulated Phosphorylation States of Ste12p
Authors: Hung Wesley , Olson K. Amy , Breitkreutz Ashton , Sadowski Ivan
Published: July 2004

The Saccharomyces cerevisiae transcription factor Ste12p is required for basal and activated expression of pheromone‐responsive genes, and for invasive growth in haploid cells. In diploid yeast, Ste12p is implicated in pseudohyphal development. The ability of Ste12p to effect these various responses in three different cell types must require stringent regulation of its transcriptional activation function and interaction with additional transcription factors. We have examined the phosphorylation state of Ste12p in untreated and pheromone‐treated haploid cells, and found eight constitutively phosphorylated peptides. Phosphorylation at the constitutive sites does not require the protein kinases of the pheromone‐response pathway. Treatment of haploid yeast with mating pheromone causes the appearance of novel relatively minor phosphorylations on Stel2p. Brief [35S]methionine labeling reveals novel pheromone‐dependent, electrophoretically slower migrating Ste12p species. Similarly, the sole difference we observe in tryptic phosphopeptides generated from Ste12p from pheromone‐treated and untreated cells is the transient appearance of two novel minor hydrophobic phosphopeptides. The pheromone‐dependent phosphorylation of Ste12p requires an intact pheromone‐response pathway and localization of Ste12p to the nucleus, but does not require the Ste12p DNA‐binding domain. We conclude from these experiments that the pheromone‐response pathway induces the formation of specific hyperphosphorylation on Stel2p, which can only be detected as apparently minor modifications in vivo. We argue that, if Ste12p is regulated by direct pheromone‐responsive phosphorylation, then that phosphorylation must be represented by the two novel phosphopeptides. However, we cannot exclude the possibility that pheromone‐responsive transcription is controlled by direct phosphorylation of a target other than Stel2p.
Transferred Nuclear Overhauser Enhancement (NOE) and Rotating‐Frame NOE Experiments Reflect the Size of the Bound Segment of the Forssman Pentasaccharide in the Binding Site of Dolichos Biflorus Lectin
Authors: Casset Florence , Imberty Anne , Pérez Serge , Etzler Marilynn E. , Paulsen Hans , Peters Thomas
Published: July 2004

A complex between the Forssman pentasaccharide α‐D‐GalNAc‐(1→3)‐β‐D‐GalNAc‐(1→3)‐α‐D‐Gal‐(1→4)‐β‐D‐Gal‐(1→4)‐D‐Glc and the seed lectin from Dolichos biflorus was studied using transfer‐NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non‐reducing disaccharide moiety α‐D‐GalNAc‐(1→3)‐β‐D‐GalNAc compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the non‐reducing disaccharide moiety α‐D‐GalNAc‐(1→3)‐β‐D‐GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non‐reducing disaccharide fragment is buried in the lectin‐binding pocket, whereas the reducing trisaccharide portion α‐D‐Gal‐(1→4)‐β‐D‐Gal‐(1→4)‐D‐Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton‐proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment α‐D‐GalNAc‐(1→3)‐β‐D‐GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.
Domain‐Specific N‐Glycosylation of the Membrane Glycoprotein Dipeptidylpeptidase IV (CD26) Influences its Subcellular Trafficking, Biological Stability, Enzyme Activity and Protein Folding
Authors: Fan Hua , Meng Wenmao , Kilian Christiane , Grams Sabine , Reutter Werner
Published: July 2004

Dipeptidyl peptidase IV (DPPIV, CD26) is an N‐glycosylated type II plasma membrane protein. The primary structure of rat wild‐type DPPIV contains eight potential N‐glycosylation sites. To investigate the role of N‐glycosylation in the function of DPPIV, three of its asparagine residues were separately converted to glutamine by site‐directed mutagenesis. The resulting N‐glycosylation mutants of rat DPPIV were studied in stable transfected Chinese hamster ovary cells. All three N‐glycosylation mutants of DPPIV showed a reduced half‐life, as well as differing degrees of inhibition of the processing of their N‐glycans. Mutation of the first (Asn83→Gln) or eighth (Asn686→Gln) N‐glycosylation site had only a small effect on its enzymatic activity, cell‐surface expression and dimer formation, whereas the mutation of the sixth N‐glycosylation site (Asn319→Gln) abolished the enzymatic activity, eliminated cell‐surface expression and prevented the dimerization of the DPPIV protein. The mutant [Gln319]DPPIV is retained in the cytoplasm and its degration was drastically increased. Our data suggest that the N‐glycosylation at Asn319 is involved in protein trafficking and correct protein folding.
Methyl Phenylalkanoates as Substrates to Probe the Active Sites of Esterases
Authors: Kroon Paul A. , Faulds Craig B. , Brézillon Christophe , Williamson Gary
Published: July 2004

We have used methyl esters of phenylalkanoic acids to probe the active site of two esterases (FAE‐III and CinnAE) from Aspergillus niger. Only methyl 4‐hydroxy‐3‐methoxycinnamate and 4‐hydroxy‐3‐methoxyphenylpropionate out of 19 substrates tested were significant substrates for both enzymes (kcat values about 102 s−1 and 103 s−1, respectively). Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Differences in Km values for FAE‐III were small (0.45–2.08 mM) but there were two orders of magnitude difference in Kcat values (12.1–1063 s−1), whereas for CinnAE, there were large differences in values for both Km (0.014–1.32 mM) and kcat (41.3‐1410 s−1)‐ Lability of the ester bonds, as estimated from second‐order rate constants (A:2) for chemical reaction with sodium hydroxide, did not correlate to kcat for CinnAE (r= 0.33) or for FAE‐III (r= 0.43). Maintaining the phenylpropenoate structure but altering the substitutions on the aromatic ring demonstrated the following: a 3‐methoxy group is essential for FAE‐III activity, whereas a 3‐methoxy group precluded activity of CinnAE, with the exception of methyl 4‐hydroxy‐3‐methoxycinnamate which was a relatively poor substrate for CinnAE; (b) increasing the number of methoxy substitutions increased the activity of FAE‐III, and decreased the activity of CinnAE; (c) 4‐hydroxy substituents, and additional hydroxy substituents, increased the activity of CinnAE, but decreased that of FAE‐III; (d) the rate of hydrolysis with sodium hydroxide of the methyl esters in general is decreased by hydroxy substitutions on the aromatic ring but increased by methoxy substitutions. Analysis of kinetic data obtained in the presence of inhibitors indicated that substrate analogs were able to bind to both free CinnAE and to a CinnAE‐substrate complex, but conversely, were only able to bind to free FAE‐III. The results show that the specificities of the two A. niger esterases are complementary. The rate of hydrolysis by this class of carboxylic ester hydrolase does not depend on the intrinsic lability of the ester bond, but depends on both the distance between the aromatic ring and the ester bond, and the substitutions on the aromatic ring.
Kinetics of Folding of the Myosin Rod
Authors: Bechet Jean‐Jacques , Nozais Muriel
Published: July 2004

The kinetics of the unfolding and refolding of the myosin rod have been studied by fluorescence and circular dichroism techniques, at different concentrations of protein and guanidine hydrochloride. The unfolding of the myosin rod was fast and at least biphasic in 2–3 M denaturant, with an initial immediate phase followed by a slower low‐amplitude first‐order phase. The refolding of the rod in 0.4–2 M guanidine hydrochloride was also at least biphasic; an initial immediate phase preceded a slow second‐order phase. At the final denaturant concentration of 0.8 M, the amplitude of the burst phase was weakly dependent on the protein concentration and the rate constant of the refolding slow phase was optimal. These data are incorporated into a folding mechanism with at least three states. The high rates of the first steps of unfolding and refolding may be relevant for the functioning of the native myosin molecule by allowing a transient separation of the two strands of the myosin tail.
Mechanism‐Based Inactivation of Bovine Cytochrome P‐450U11β by 18‐Unsaturated Progesterone Derivatives
Authors: Delorme Cécile , Piffeteau Annie , Sobrio Franck , Marquet Andrée
Published: July 2004

Two 18‐unsaturated progesterone derivatives, 18‐vinylprogesterone (18‐VP) and 18‐ethynylprogester‐ one (18‐EP) have proved to be potent inhibitors of the bovine cytochrome P‐45011β the enzyme involved in the last steps of aldosterone biosynthesis [Delorme, C., Piffeteau, A., Viger, A. & Marquet, A. (1995) Eur. J. Biochem. 232, 247–256]. In the present study, we demonstrate that these two compounds exhibit the characteristics of mechanism‐based inactivators of this enzyme. Inactivation followed pseudo‐first‐order and saturation kinetics. The kinetic parameters of inactivation were ki= 0.11 min−1 and Ki= 4 μM for 18‐VP, and ki, = 0.12 min−1 and 22 μM for 18‐EP. Inactivation of P‐45011β activity was strictly dependent on the presence of NADPH. Protection by the substrate deoxycorticosterone was observed, demonstrating a selective modification at the substrate‐binding site. With radiolabeled 18‐VP, inactivation was shown to be irreversible with a stoichiometry of 1.4mol bound [3H]18‐VP/mol inactivated cytochrome P‐45011β SDS/PAGE analysis of the [3H] 18‐VP–inactivated enzyme showed that, under conditions preventing heme dissociation, the P‐45011β band was labeled, while no labeling of the apoprotein was observed under denaturating conditions. Furthermore, the loss of catalytic activity could be correlated with the destruction of the P‐45011β chromophore evaluated by the FeII‐CO versus FeII difference spectra. These arguments led us to propose that 18‐vinylprogesterone inactivates cytochrome P‐45011β by heme destruction rather than by protein modification.
Plasminogen Activation by Pro‐Urokinase in Complex with its Receptor — Dependence on a Tripeptide (Spectrozyme Plasmin)
Authors: Wang Jieyi , Mazar Andrew , Quan Nancy , Schneider Andrew , Henkin Jack
Published: July 2004

The intrinsic activity of single‐chain pro‐urinary‐type plasminogen activator (pro‐uPA) and whether its receptor (uPAR) potentiates this activity remains controversial. In this report, the pro‐uPA/uPAR‐(1–281)‐peptide complex in solution is shown to have equivalent plasminogen‐activator activity to that of active two‐chain uPA (tc‐uPA). However, the activity of the complex was dependent on a synthetic tripeptide, Spectrozyme plasmin (Spl, H‐d‐2‐aminohexanoic acid(Ahx)‐hexatyrosyl‐lysine‐p‐nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins and detergents. The pro‐uPA/uPAR‐(1–281)‐peptide complex at 1 nM displayed similar activity to that of tc‐uPA for either [Glu1]plasminogen or [Lys77]plasminogen in chromogenic assays with Spl present as the plasmin substrate. When assayed with another plasmin substrate, S2251, the pro‐uPA/uPAR‐(1–281)‐peptide complex was unable to activate plasminogen. The pro‐uPA/uPAR‐(1–281)‐peptide complex and tc‐uPA also showed a similar extent of plasminogen activation as measured by SDS/PAGE, when incubated with plasminogen and Spl in the presence of 100 u. M aprotinin, and plasminogen activation by pro‐uPA alone was also stimulated in the presence of Spl in this assay. Activation of plasminogen by the pro‐uPA/uPAR‐(1–281)‐peptide strictly required the presence of Spl, and pro‐uPA remained in single‐chain form during these assays. This activity of the pro‐uPA/uPAR‐(1–281)‐peptide complex but not that of tc‐uPA was completely inhibited by human serum albumin, bovine serum albumin, Tween‐80, Triton X‐100, and Pluronic‐F68. Taken together, the data indicates that uPAR‐(1–281)‐peptide itself is not sufficient to augment pro‐uPA activity and the presence of an effector molecule (e.g. Spl) is required to elicit the full plasminogen‐activator activity of the pro‐uPA/uPAR‐(1–281)‐peptide complex. It remains to be seen whether there is a physiological counterpart to this phenomenon.
The AMP‐Activated Protein Kinase
Authors: Hardie D. Grahame , Carling David
Published: July 2004

A single entity, the AMP‐activated protein kinase (AMPK), phosphorylates and regulates in vivo hydroxymethylglutraryl‐CoA reductase and acetyl‐CoA carboxylase (key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively), and probably many additional targets. The kinase is activated by high AMP and low ATP via a complex mechanism, which involves allosteric regulation, promotion of phosphorylation by an upstream protein kinase (AMPK kinase), and inhibition of dephosphorylation. This protein‐kinase cascade represents a sensitive system, which is activated by cellular stresses that deplete ATP, and thus acts like a cellular fuel gauge. Our central hypothesis is that, when it detects a ‘low‐fuel’ situation, it protects the cell by switching off ATP‐consuming pathways (e.g. fatty acid synthesis and sterol synthesis) and switching on alternative pathways for ATP generation (e.g. fatty acid oxidation). Native AMP‐activated protein kinase is a heterotrimer consisting of a catalytic α subunit, and β and γ subunits, which are also essential for activity. All three subunits have homologues in budding yeast, which are components of the SNF1 protein‐kinase complex. SNF1 is activated by glucose starvation (which in yeast leads to ATP depletion) and genetic studies have shown that it is involved in derepression of glucose‐repressed genes. This raises the intriguing possibility that AMPK may regulate gene expression in mammals. AMPK/SNF1 homologues are found in higher plants, and this protein‐kinase cascade appears to be an ancient system which evolved to protect cells against the effects of nutritional or environmental stress.