bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin
Authors: De Beer Tonny , Van Zuylen Carol W. E. M. , Leeflang Bas R. , Hård Karl , Boelens Rolf , Kaptein Robert , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: July 2004

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Am52 of its α subunit (αhCG) is essential for signal transduction, whereas the N‐glycan at Am78 stabilizes the structure of the protein. In this study, an almost complete 1H‐NMR and a partial 13C‐NMR spectral assignment for the amino acids and the N‐glycans of αhCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of αhCG in solution, which was determined based on NOE data, is partially similar to that of the α subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 33–58. In the crystal structure of hCG, residues 33–37 and 54–58 of the a subunit are part of an intersubunit seven‐stranded β‐barrel and residues 41–47 constitute a 310‐helix. In contrast, in free αhCG in solution, amino acids 33–58 are part of a large disordered loop, indicating that in intact hCG interactions with the β subunit of hCG stabilize the conformation of the α subunit. The NMR data of αhCG and its deglycosylated counterpart are very similar, indicating that removal of carbohydrate residues other than GlcNAc‐1 does not notably affect the conformation of the protein part. However, numerous 1H‐NOES between the GlcNAc‐1 residue at Am78 and several amino acid residues show that this GlcNAc residue is tightly packed against the protein, being an integral part of the structure of the α subunit. 1H‐NOES across the glycosidic linkages of the glycan, resonance‐line widths, and 1H and 13C chemical shifts of the other monosaccharides suggest that the remainder of the glycans at Asn78, and the glycans at Asn52 are largely extended in solution.
An Insect‐Specific Toxin from Centruroides noxius Hoffmann
Authors: Selisko Barbara , Garcia Consuelo , Becerril Baltazar , Delepierre Muriel , Possani Lourival D.
Published: July 2004

Scorpion toxins acting on sodium channels differ in their specificity. Toxic peptides specific towards mammals and arthropods (insects and/or crustaceans) have been described. Because of the similar three‐dimensional fold of these peptides, the molecular base of their specificity is thought to reside in certain differences at the level of amino acid residues especially within or near the binding site of the toxin to the particular ion channel. The cDNA, amino acid sequence and biological activity of an insect‐specific toxin, Cn10, from the scorpion Centruroides noxius Hoffmann is reported. The electrostatic potential surface around a three‐dimensional model of Cn10 was calculated. It revealed that residues Tyr4, Lys13, Ile18, Leu19, Gly20, Lys43, Leu44, Thr57, Tyr58, Pro59, Thr64 and Cys65, situated at the side of the toxin proposed in the literature to bind to the sodium channel, constitute a positive surface region. Therefore, they may form the site that binds to the channel. Cn10 was included in a comparative analysis of two groups of natural variants, highly similar peptides of the genus Centruroides with specificities towards mammals or arthropods. A number of surface‐accessible residues, consistently different between the two groups and situated near the putative binding site, may be of importance for the specificity of the analyzed toxins.
Regulation of Membrane‐Type Matrix Metalloproteinase‐1 Expression by Growth Factors and Phorbol 12‐Myristate 13‐Acetate
Authors: Lohi Jouko , Lehti Kaisa , Westermarck Jukka , Kähäri Veli‐Matti , Keski‐Oja Jorma
Published: July 2004

Overexpression of membrane‐type matrix metalloproteinase (MT‐MMP‐1) results in the activation of both endogenous and exogenous 72‐kDa gelatinase. To understand the effects of MT‐MMP‐1 on 72‐kDa gelatinase activation, we analyzed its expression in human fibroblasts and HT‐1080 fibrosarcoma cells. Both cell types expressed the MT‐MMP‐1 mRNA constitutively at a considerable level and treatment of cells with PMA enhanced the expression about 2–3‐fold. Concanavalin A treatment increased MT‐MMP‐1 mRNA levels in fibroblasts about 4‐fold. Induction of MT‐MMP‐1 by phorbol 12‐myristate 13‐acetate (PMA) required protein synthesis as shown by cycloheximide inhibition. The induction was also inhibited by dexamethasone. Analysis of MT‐MMP‐1 mRNA stability using actinomycin D indicated that the half‐life was rather long and not affected by PMA, suggesting transcriptional regulation. Only HT‐1080 cells had significant 72‐kDa gelatinase processing activity after treatment with PMA or concanavalin A, while fibroblasts were virtually negative. Immunoblotting analysis of fibroblast lysates indicated that MT‐MMP‐1 was present mainly in a 60‐kDa form. PMA and concanavalin A caused 2–4‐fold increases in its protein levels, while in HT‐1080 cells PMA, concanavalin A, or overexpression of MT‐MMP‐1 did not significantly enhance the level of the 60‐kDa protein. Instead, an immunoreactive, proteolytically processed 43‐kDa form was observed, and its appearance correlated to 72‐kDa gelatinase processing activity. Thus 72‐kDa gelatinase activation, while enhanced by MT‐MMP‐1 expression, needs additional co‐operating factors.
Functional Characterization of an Extreme Thermophilic Class II Fructose‐1,6‐Bisphosphate Aldolase
Authors: De Montigny Chantal , Sygusch Jurgen
Published: July 2004

Fructose‐1,6‐bisphosphate aldolase activity has been isolated and purified to homogeneity from the extreme thermophile eubacteria Thermus aquaticus. The homogeneous enzyme is a class II aldolase as fructose‐1,6‐bisphosphate cleavage activity was strongly inhibited by EDTA, and activated by Co2+ metal ion. Taq aldolase is a stable tetramer with estimated molecular mass of 165 kDa. The enzyme is thermostable and is not inactived after heating at 90°C for 2 h but looses 80% of activity after 1 h at 97°C. The pH profile corresponding to maximal aldolase activity is displaced to more acidic values compared to other class II aldolases. Enzyme activation by both detergents and alcohols and chromatographic behaviour on hydrophobic stationary phases is consistent with presence of hydrophobic surface regions on the soluble enzyme. Kinetic behaviour of T. aquaticus aldolase at high fructose‐l, 6‐bisphosphate concentrations indicates significant negative cooperativity. The Taq aldolase NH2‐terminal sequence was determined and compared with available sequences from other class II aldolases. Significant sequence similarity was found between Tag aldolase and the thermostable aldolase from Bacillus stearothermophilus.
Dipeptidyl‐Peptidase IV‐β, a Novel form of Cell‐Surface‐Expressed Protein with Dipeptidyl‐Peptidase IV Activity
Authors: Jacotot Etienne , Callebaut Christian , Blanco Julià , Krust Bernard , Neubert Klaus , Barth Alfred , Hovanessian Ara G.
Published: July 2004

The T‐cell activation antigen CD26, is a type II membrane glycoprotein with intrinsic dipeptidyl‐peptidase IV (DPP IV) activity, characterized by its capacity to cleave off N‐terminal dipeptides containing proline as the penultimate residue. Independent of its catalytic activity, CD26 has also been characterized as adenosine deaminase binding protein. By using CD26 negative human C8166 cells, here we describe the existence of another cell‐surface protein which manifests CD26‐like DPP IV activity. For convenience, this protein will be referred to as DPP IV‐β. Consistent with the cell‐surface expression of DPP IV‐β, intact C8166 cells manifested a high level of DPP IV, whereas, they manifested poor activity against substrates of DPP II known to have an intracellular localization. A partially purified preparation of CD26 from human MOLT4 cells, and the DPP IV‐β expressed on intact cells were found to possess similar catalytic activity and pH optimum. In addition, cell‐surface CD26 and DPP IV‐β on intact MOLT4 and C8166 cells, respectively, resisted digestion by proteolytic enzymes such as trypsin and proteinase K. However, adenosine deaminase activity was not detectable on the surface of C8166 cells in contrast to CD26 positive MOLT4 cells. In accord with this, 125I‐labeled adenosine deaminase which binds CD26 was found not to bind DPP IV‐β. Gel‐filtration experiments using 0.5% Triton X‐100 extracts from C8266 and MOLT4 cells, revealed that the apparent molecular mass of DPP IV‐β is 82 kDa, whereas that of CD26 is 110 kDa as expected. Taken together, our results suggest that DPP IV‐β is a CD26‐like protein which could be characterized by distinct properties.
Site‐Specific Replacement of Amino Acid Residues in the CD Site of Rat Parvalbumin Changes the Metal Specificity of this Ca2+/Mg2+‐Mixed Site Toward a Ca2+‐Specific Site
Authors: Pauls Thomas L. , Durussel Isabelle , Clark Ian D. , Szabo Arthur G. , Berchtold Martin W. , Cox Jos A.
Published: July 2004

Rat parvalbumin (PV) and oncomodulin (OM) display considerable sequence similarity and structural similarity, but differ in the affinity and selectivity of metal binding to their CD site, a Ca2+/Mg2+‐mixed site in PV and a Ca2+‐specific site in OM. In an attempt to identify the structural basis for these differences, mutations were introduced in the previously generated [W102]PV mutant, which contains a unique tryptophan as a conformational‐sensitive fluorescent probe inside the hydrophobic core. In the present report, we substituted selected amino acid residues in the CD site of PV by those present at identical positions in OM. One mutant protein, named [F66, W102]PV, has one new substitution in which isoleucine at position 66 was exchanged by phenylalanine. The second mutant protein, [I46, I50, L58, F66, W102]PV, has four new substitutions, namely V46→I, L50→I, I58→L and I66→F. Tryptophan fluorescence and difference spectrophotometry indicated that the mutations do not alter significantly the hydrophobic core. Both mutant proteins display two metal‐binding sites of identical affinities with intrinsic affinity constants K′Ca2+ of 2.9×107 M−1 for [F66, W102]PV and 1.7×107 M−1 for [I46, I50, L58, F66, W102]PV and K′Mg2+ of 3.1×104 M−1 for [F66, W102]PV and 1.9×104 M−1 for [I46, I50, L58, F66, W102]PV. Thus, the five‐residue substitution, but not the two‐residue one, leads to a small decrease of affinity compared to [W102]PV (K′Ca2+= 2.7×107 M−1, K′Mg2+= 4.4 × 104 M−1). Despite these similarities, the Mg2+ effect on Ca2+ binding is different for the two mutant parvalbumins: the Ca2+‐binding isotherms of [F66, W102]PV undergo a parallel shift upon increasing Mg2+ concentrations, which indicates that the Mg2+ effect on the two Ca2+‐binding sites is the same and quantitatively very similar to that described for [W102]PV. In [I46, I50, L58, F66, W102] PV, Mg2+ antagonizes the binding of the second Ca2+ (likely at the EF site) much more than that of the first Ca2+ (likely the CD site). According to the competition equation, the two sites display KMg2+· compet values of 390 M−1 and 3.9×103 M−1 respectively. These data indicate that (a) the single 166→F mutation does not modify the cation binding parameters. (b) Multiple modifications in the hydrophobic core still do not change the affinity for Ca2+ and Mg2+, but strongly affect the Mg2+ antagonism and probably the selectivity of the CD site.
Overexpression of the Nocardia lactamduransα‐Aminoadipyl‐Cysteinyl‐Valine Synthetase in Streptomyces lividans
Authors: Coque Juan José R. , De La Fuente Juan Luis , Liras Paloma , Martín Juan F.
Published: July 2004

Formation of the tripeptide δ‐(l‐α‐aminoadipyl)‐l‐cysteinyl‐d‐valine (Aad‐Cys‐Val) is catalyzed by a multienzyme peptide synthetase encoded by the pcbAB gene in producers of β‐lactam antibiotics. The pcbAB gene of Nocardia Lactamdurans was overexpressed in Streptomyces Lividans giving a high Aad‐Cys‐Val synthetase activity. The synthetase was purified 2785‐fold to near homogeneity showing a molecular mass of 430 kDa by SDS/PAGE. The protein was identified in the gels with antibodies to Aad‐Cys‐Val synthetase and by the formation of aminoacyl‐synthetase thioester complex with [14C]valine. The purified synthetase used α‐aminoadipic acid or its lactam 6‐oxopiperidine 2‐carboxylic acid but was unable to use piperideine 6‐carboxylic acid or pipecolic acid as substrates to form Aad‐Cys‐Val. l‐Cysta‐thionine, (2‐amino‐2‐carboxyethyl)‐l‐homocysteine, was used as substrate and formed Aad‐Cys‐Val with the same efficiency as l‐cysteine. The product of the reaction eluted with authentic Aad‐Cys‐Val. The synthetase preparation was able to hydrolyze l‐cystathionine by a pyridoxal‐phosphate‐independent mechanism which is not inhibited by propargylglycine, to form Aad‐Cys‐Val.
Cloning, Expression and Properties of the α′ Subunit of Casein Kinase 2 from Zebrafish (Danio Rerio)
Authors: Antonelli Marcelo , Daniotti José L. , Rojo Daniel , Allende Catherine C. , Allende Jorge E.
Published: July 2004

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (α and/or α′) and regulatory subunits (β2). The α and α′ subunits are encoded by different genes but are very similar in amino acid sequence, except that α′ is normally considerably shorter. There have been extensive biochemical studies with recombinant α and β subunits of many species, but only one previous description of the activity of an isolated recombinant α′ subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (α, α′, and β), site‐directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263–273). In the present work, the isolation and bacterial expression of a cDNA coding for the α′ subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the α′ subunits of human and chicken, and 82% identical to the sequenced portion of the CK2α subunit of zebrafish. The recombinant α′ subunit has apparent Km, values for ATP (6 μM), GTP (20 μM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant α subunit of Xenopus laevis. The α′ subunit kcat, was 7.2 min‐1 which is again similar to that of Xenopus laevisα subunit (7.5 min‐1). The α′ subunit also behaved similarly to CK2α with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However α′ kinase activity was less sensitive to poly(U) inhibition than α, it was more heat stable than α, and α′ was slightly more sensitive to KCl inhibition than α. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory β subunit which shifted the optimal salt concentration of the phosphorylating activity. The α′2β2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the α2β2 enzyme was stimulted by KCl concentrations up to 150 mM and required 180 mM for inhibition.

Another important difference between α and α′ is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory β subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2β produced maximal stimulation of both α′ (D. rerio) and α (X. laevis), however the activity levels with α′ were stimulated 20‐fold by β while the addition of β stimulated α (X. luevis) only 7–8‐fold.
Mechanisms of Inhibition and Uncoupling of Respiration in Isolated Rat Liver Mitochondria by the General Anesthetic 2,6‐Diisopropylphenol
Authors: Kigoulet Michel , Devin Anne , Avéret Nicole , Vandais Bénédicte , Guérin Bernard
Published: July 2004

We investigated the effects of 2,6‐diisopropylphenol on oxidative phosphorylation of isolated rat liver mitochondria. Diisopropylphenol strongly inhibits state‐3 and uncoupled respiratory rates, when glutamate and malate are the substrates, as a direct consequence of the limitation of electron transfer at the level of complex I. In addition, diisopropylphenol acts as an uncoupler in non‐phosphorylating mitochondria, which leads to an increase in respiratory rate and a large decrease in proton‐motive force. However, such effects cannot be due to the classical protonophoric property of this drug, since addition of ADP plus oligomycin before diisopropylphenol avoids this increase in proton permeability, and in phosphorylating mitochondria, the ATP/O ratio is not significantly affected by diisopropylphenol addition. In the absence of added ADP, diisopropylphenol modifies some mitochondrial ATPases in such a way that they become insensitive to oligomycin and unable to couple proton movement to ATP synthesis or hydrolysis. However, these modified enzymes can catalyse passive proton permeability, which leads to uncoupling. Addition of ADP before diisopropylphenol prevents these changes. We propose that ADP induces a change in conformation of ATPase, which leads to insensitivity of this complex towards diisopropylphenol.

In conclusion, we show that diisopropylphenol has two main effects on rat liver mitochondria: inhibition of the respiratory chain at the level of complex I level and modification of ATPase such that, in the absence of phosphorylation, it catalyses a H+ leak, which becomes negligible when oxidative phosphorylation is functional.
The Carotenoid 7, 8‐Dihydro‐ψ end Group can be Cyclized by the Lycopene Cyclases from the Bacterium Erwinia Uredovora and the Higher Plant Capsicum Annuum
Authors: Takaichi Shinichi , Sandmann Gerhard , Schnurr Georg , Satomi Yoshiko , Suzuki Akashi , Misawa Norihiko
Published: July 2004

The genes for geranylgeranyl diphosphate synthase (crtE) and phytoene synthase (crtB) from the epiphytic bacterium Erwinia uredovora and the phytoene desaturase gene from the photosynthetic bacterium Rhodobacter capsulatus (Rc‐crtI) were introduced into Escherichia coli, which resulted in the accumulation of the acyclic carotenoid, neurosporene. Further introduction of the lycopene cyclase gene from E. uredovora (crtY) or the higher plant Capsicum annuum (Icy) resulted in the production of a bicyclic carotenoid, 7,8‐dihydro‐β‐carotene, via monocyclic β‐zeacarotene. ζ‐Carotene was also found to be cyclized to bicyclic 7,8,7′,8′‐tetrahydro‐β‐carotene by the Erwinia cyclase. These results indicate that both lycopene cyclases can cyclize a 7,8‐dihydro‐ψ end group to a 7,8‐dihydro‐β end group, in addition to the usual cyclization of the ψ end group to the β end group. Furthermore, β‐carotene hydroxylase from Enwnia (CrtZ) was able to add a hydroxyl group to the 7,8‐dihydro‐β end group and the β end group.
Associations Between Erythrocyte Band 3 Protein and Aldolase in Detergent Solution
Authors: Huber Elfriede , Bäumert Hans Georg , Spatz‐kümbel Gerhard , Schubert Dieter
Published: July 2004

The cytoplasmic domain of band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified band 3, aldolase is bound exclusively or at least predominantly to the band 3 tetramer (but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers.

The results described, together with those reported previously, point at a prominent role of the band 3 tetramer in ligand binding.
Temperature‐Dependent Conformational Change of Bacteriorhodopsin as Studied by Solid‐State 13C NMR
Authors: Tuzi Satoru , Naito Akira , Saitô Hazime
Published: July 2004

Cross‐polarization and dipolar‐decoupled magic‐angle spinning 13C‐NMR spectra of [3‐13C]Ala‐labelled bacteriorhodopsin were obtained for hydrated purple membrane in the temperatures range 23°C to –110°C. Well‐resolved 13C‐NMR signals were observed either at ambient temperature or at –20°C but were broadened considerably at lower temperature below –40°C. This situation was interpreted in terms of the presence of exchange processes with a rate constant of 102 S−1 at ambient temperature among several conformations slightly different from each other. We found that such an exchange process was strongly influenced by the manner of organization of the lipid bilayers depending upon the presence or absence of cations responsible for electric shielding of negative charge at the polar head groups. The manner of organization of the lipid bilayers was conveniently characterized by a characteristic temperature at which the methyl peaks of fatty acyl groups of lipids in the purple membrane were suppressed due to interference of motional frequency with the decoupling frequency (10–100 kHz) for preparations containing 10 mM NaCl or CaCl2. No such spectral change in the absence of these cations was noted even if a preparation was cooled to –110°C. The secondary structures of [3‐13C]Ala‐labelled bacteriorhodopsin was not always identical at temperatures between ambient and low temperatures, since the 13C chemical shifts and relative peak intensities for purple membrane preparations containing these salts changed with temperature in the range –110°C to 23°C. In particular, we found that some residues involving Ala residues at the αII‐helix and loop region were converted at temperatures below –60°C to a conformation involving αI‐helix. In other words, some portion of the α‐helical conformation of bacteriorhodopsin proposed from results obtained by cryoelectron microscopy, at very low temperatures, is not always retained at ambient temperature.
Membrane Modifications in Human Erythroleukemia K562 Cells During Induction of Programmed Cell Death by Transforming Growth Factor β1 or Cisplatin
Authors: Maccarrone Mauro , Nieuwenhuizen Willem F. , Dullens Hub F. J. , Catani M. Valeria , Melino Gerry , Veldink Gerrit A. , Vliegenthart Johannes F. G. , AgrÒ Alessandro Finazzi
Published: July 2004

Transforming growth factor β1 (TGFβ1) and cisplatin induce apoptosis (programmed cell death, PCD) in human erythroleukemia K562 cells in an additive manner. After PCD was induced in K562 cells, analysis of phospholipid composition, fatty acids and cholesterol content in their membranes showed a decrease in phosphatidylethanolamine and an increase in phosphatidylserine, cardiolipin and phosphatidic acid. Moreover, cisplatin but not TGFβ1 enhanced sphingomyeline levels in apoptotic cells, whereas TGFβ1 increased the amount of linoleic acid and, more remarkably, of cholesterol. The combination TGFβ+ cisplatin produced membrane changes similar to those provoked by each inducer individually. Furthermore, the specific activities of 5‐lipoxygenase and cytosolic phospholipase A2, both modulating the physical properties of membranes and membrane‐lipid‐mediated intracellular signalling, were enhanced by treatment with TGFβ1 or TGFβ1 + cisplatin. These findings highlight the profound changes in cell membranes during the biochemical events of the apoptotic pathway.
The Role of Controlled Proteolysis in Cell‐Cycle Regulation
Authors: Udvardy Andor
Published: July 2004

Cyclins and cyclin‐dependent kinases are key regulators of the cell cycle. The binding of different cyclins, required to activate the catalytically inactive cyclin‐dependent kinases, determines the substrate specificity of the enzymes. Cyclin‐dependent‐kinase inhibitors have an adverse effect, blocking the catalytic activity of cyclin‐activated cyclin‐dependent kinases. The cell cycle is a cyclic process of successive transient activation or inactivation of cyclin‐dependent kinases by association with different cyclin regulatory subunits or cyclin‐dependent kinase inhibitors. As the concentration of cyclin‐dependent kinases is fairly constant during the cell cycle and exceeds the total amount of cyclins present in the cell, the exchange of regulatory subunits is determined by the availability of the different cyclins. Transcriptional control of cyclin gene expression is the most decisive factor determining the total amount of different cyclins synthesized. The actual concentration of a cyclin, however, is always the result of an equilibrium between the rates of its synthesis and degradation. While cyclin gene expression has long been known to be cell‐cycle controlled, the idea of the rapid destruction of cyclins or cyclin‐dependent‐kinase inhibitors as an equally important factor contributing to the progress of the cell cycle is more recent. The role of controlled proteolysis in the regulation of cell cycle is discussed in this review. Two general features of this regulation are worth mentioning: cyclin‐dependent kinases activated by different cyclin regulatory subunits have a central role both in the transcriptional regulation of their own genes and in the regulated, selective destruction of cyclins or cyclin‐dependent kinase inhibitors; transcriptional regulation of cyclin gene expression ensures fine‐tuned, continuous changes, and controlled proteolysis generates abrupt, irreversible transitions. The progress of the cell cycle is based on a delicate balance of the these mutual, but opposite regulations.
Identification and Characterization of a Rice Cysteine Endopeptidase that Digests Glutelin
Authors: Kato Hideki , Mtnamikawa Takao
Published: July 2004

Little or no endopeptidase activity was detected in extracts from storage organs of dark‐grown rice seeds until day 6 or post‐imbibition, and the activity expressed per seed increased notably after day 9, reached a maximum on day 18, then decreased. Two major endopeptidases, REP‐1 and REP‐2, were present in the 40–75% saturated ammonium sulfate fraction from day‐9 germinated seeds, and could be separated by hydrophobic column chromatography. REP‐1 was further purified to a single polypeptide of 36 kDa. REP‐1 digested in vitro both the acidic and basic subunits of rice glutelin, the major seed storage protein of rice. Determination of the N‐terminal amino acid sequence and experiments with protease inhibitors indicated that REP‐1 is a cysteine endopeptidase. The nucleotide sequence of a full‐length REP‐1 cDNA was determined by a combination of screening of cDNA libraries from rice seeds and the 5′ rapid amplification of cDNA ends technique.
Molecular Characterisation of Formamidase from Methylophilus methylotrophus
Authors: Wyborn Neil R. , Mills James , Williams Steven G. , Jones Colin W.
Published: July 2004

A 3.2‐kbp PstI fragment of DNA encoding formamidase from the methylotrophic bacterium Methylophilus methylotrophus which had previously been cloned (pNW3) [Wyborn, N. R., Scherr, D. J. & Jones, C. W. (1994) Microbiology 140, 191–1951, was subcloned as a 2.3 kbp HindIII fragment (pNW323). Nucleotide sequencing showed that the subclone contained two genes which encoded formamidase (fmdA) and a possible regulatory protein (fmdB). Predicted molecular masses for FmdA and FmdB were 44438 Da (compared with approximately 44500 Da by electrospray mass spectrometry and 51000 Da by SDSPAGE of the purified enzyme) and 12306 Da, respectively. The derived amino acid sequence of formamidase was supported by N‐terminal amino acid sequencing of the enzyme and of proteolytic fragments prepared from it using V8 endoproteinase and was 57% similar to that of the acetamidase from Mycobacterium smegmatis. The structural similarities between these two enzymes, and their existence as a separate class of bacterial amidase, were confirmed by immunological investigations.
A Comparison of the Fine Saccharide‐Binding Specificity of Dioclea grandiflora Lectin and Concanavalin A
Authors: Gupta Dipti , Oscarson Stefan , Raju T. Shantha , Stanley Pamela , Toone Eric J. , Brewer C. Fred
Published: July 2004

The lectin from the seeds of Dioclea grandiflora (DGL) is a Man/Glc‐specific tetrameric protein with physical and saccharide‐binding properties reported to be similar to that of the jack bean lectin concanavalin A (ConA). Unlike other plant lectins, both DGL and ConA bind with high affinity to the core trimannoside moiety, 3,6‐di‐O‐(α‐d‐mannopyranosyl)‐α‐d‐mannopyranoside, which is present in all asparagine‐linked carbohydrates. In the present study, hemagglutination inhibition techniques have been used to investigate binding of DGL and ConA to a series of mono‐ and dideoxy analogs of methyl 3,6‐di‐O‐(α‐d‐mannopyranosyl)‐α‐d‐mannopyranoside and to a series of asparagine‐linked oligomannose and complex oligosaccharides and glycopeptides. The results indicate that both DGL and ConA recognize epitopes on all three residues of the trimannoside: the 3‐, 4‐, and 6‐hydroxyl groups of the α(1–6)Man residue, the 3‐hydroxyl group of the α(1–3)Man residue, and the 2‐ and 4‐hydroxyl groups of the central Man residue of the core trimannoside. However, unlike ConA, DGL does not bind to biantennary complex carbohydrates. This was confirmed by showing that biantennary complex glycopeptides do not bind to a DGL‐Sepharose affinity column. Unlike ConA, DGL does not show enhanced affinity for a large N‐linked oligomannose carbohydrate (Man9 glycopeptide) relative to the trimannoside. Thus, DGL and ConA share similar epitope recognition of the core trimannoside moiety. However, they exhibit differences in their fine specificities for larger N‐linked oligomannose and complex carbohydrates.
Characterization of the Chicken Fatty Acid Synthase Gene 5′ Part and Promoter Region
Authors: Le Fur Nathalie , El Khadir‐Mounier Catherine , Powell Rohan S. , Diot Christian , Mallard Jacques , Douaire Madeleine
Published: July 2004

Fatty acid synthase activity has been shown to be regulated mainly at the transcriptional level under both dietary and hormonal influences. As a first step towards elucidating the factors involved, we isolated and characterized chicken genomic clones encompassing the 5′ part of the chicken fatty acid synthase gene and its flanking region. The entire region of the cloned DNA spans 30 kb, and the first three exons of the gene were mapped to a 6.3‐kb genomic fragment. The transcription initiation site was determined after subcloning the cDNA which encodes the 5′ end of the mRNA. The first exon, which was 129 bp long, was located approximately 5.3 kb upstream of the second exon, which contained the start codon. In the 5′ flanking region, putative TATA and CAAT boxes were located 30 and 92 bp, respectively, upstream of the transcription initiation site. The 5′ flanking region contained numerous sequences corresponding to consensus binding sites for transcription factors. Various lengths of flanking sequences extending up to 1028 bp upstream of the transcription initiation site and containing 100 bp of the first exon were linked to the bacterial chloramphenicol acetyltransferase gene; in this study, these constructs were analyzed in transient transfection assays in human hepatoma cells. The proximal 125‐bp sequence upstream of the transcription start site was shown to be a basal promoter. The cloning and characterization of the chicken fatty‐acid synthase gene provides some further insight into the regulation of fatty acid synthesis in birds as compared to mammals.
Substitutions at C2′ of Daunosamine in the Anticancer Drug Daunorubicin Alter Its DNA‐Binding Sequence Specificity
Authors: Gao Yi‐gui , Priebe Waldemar , Wang Andrew H.‐J.
Published: July 2004

In the search for new generations of anthracycline drugs, lower cytotoxic side effect and higher activity toward resistant cancer cells are two major goals. A new anthracycline drug, WP401 (2′‐bromo‐4′‐epidaunorubicin, α‐munno configuration), exhibits promising activity toward multidrug‐resistant cells. In contrast, the related compound WP400 (2′‐bromo‐4′‐epidaunorubicin, α‐gluco configuration), is significantly less cytotoxic. To establish the structural and molecular bases of this observation, we performed X‐ray diffraction analyses of the complexes between WP401 and four DNA hexamers CGTACG, CGATCG, CGCGCG, and CGGCCG. Their crystal data (space group P41212, a=b≈ 2.8 nm, c≈ 5.3 nm) are similar to those of other daunorubicin/doxorubicin complexes. The refined crystal structures at 0.18‐nm resolution revealed that two WP401 drug molecules bind to the duplex, with the aglycons intercalated between the CpG steps and their modified daunosamines in the minor groove. The bulky bromine atom at the C2′ position caused the daunosamine of the bound WP401 to adopt a different conformation from that of the bound daunorubicin. In the presence of formaldehyde, WP401 formed a covalent adduct with CGGCCG more readily than with CGCGCG. This is the opposite of what is seen for daunorubicin and doxorubicin. Thus modifications at the C2′ position of daunosamine modulate the sequence specificity of the formalde‐hyde‐crosslinking reactions between anthracyclines and DNA.
A Lipid‐Anchored Binding Protein is a Component of an ATP‐Dependent Cellobiose/Cellotriose‐Transport System from the Cellulose Degrader Streptomyces reticuli
Authors: Schlösser Andreas , Schrempf Hildgund
Published: July 2004

During cultivation in the presence of cellobiose or crystalline cellulose, Streptomyces reticuli expresses an inducible uptake system that transports cellobiose (Km, 4 μM), cellotriose and, to a lesser degree, cellotetraose and cellopentaose. Cellobiose uptake is dependent on ATP and inhibited by N‐ethylmaleimide. A binding protein was identified in its palmitylated form in the cytoplasmic membrane of mycelia. It could be extracted with the detergent Triton X‐100 and purified by two subsequent anion‐exchange chromatographies. It showed highest affinity (Kd, 1.5 μM) for cellobiose and cellotriose. The data suggest that cellobiose/cellotriose uptake is mediated by a membrane‐anchored lipoprotein as a component of an ATP‐binding‐cassette‐transporter system.
Domain Structure, Stability and Interactions in Streptokinase
Authors: Medved Leonid V. , Solovjov Dmitry A. , Ingham Kenneth C.
Published: July 2004

The structural organization of streptokinase was established through detailed study of its denaturation by differential scanning calorimetry. Streptokinase exhibited a complex endotherm whose shape was sensitive to changing pH. In all cases the endotherms were easily described by four two‐state transitions indicating unambiguously the presence of four independently folded domains in the molecule. Two of them were slightly destabilized by lowering pH from 7.0 to 3.8 while the other two were stabilized in this pH range. Two proteolytic fragments of streptokinase were examined, a 37‐kDa fragment beginning at Ile1 with a cleavage following Phe62, and a 17‐kDa fragment beginning at Lys147. At pH 8.5, three two‐state transitions were observed in the former and two in the latter indicating this many domains in each and suggesting that the fragments are formed by a step‐wise removal of individual domains from the parent molecule. Comparison of the melting of these fragments with that of streptokinase allowed the first two transitions in the parent protein to be assigned to the melting of two NH2‐terminal domains and the two higher‐temperature transitions to the melting of the two COOH‐terminal domains. The latter two domains strongly interact with each other since the absence of the most stable extreme COOH‐terminal domain in both fragments resulted in a strong destabilization of its neighbor whose melting occurred with a midpoint near room temperature. The two NH2‐terminal domains seem to be more independent. One of them melts similarly in the parent protein and both fragments while the other, formed by the 1–146 region, is less stable in the 37‐kDa fragment. This destabilization is most probably due to the cleavage after Phe62 which, based on the sequence similarity of streptokinase with serine proteases, may be part of a surface‐oriented loop.
Up‐Regulation of an Adult Cuticular Gene by 20‐Hydroxyecdysone in Insect Metamorphosing Epidermis Cultured in vitro
Authors: Braquart Christine , Bouhin Hervé , Quennedey André , Delachambre Jean
Published: July 2004

Pupal forewing epidermis of the Coleoptera, Tenebrio molitor, was used to develop an in vitro system to study the hormonal control of metamorphosis at the cellular and molecular levels. Exposure to 1 μM 20‐hydroxyecdysone for 48 h caused the formation of a typical adult cuticle. Under these conditions the expression of ACP‐20, an adult‐specific cuticular gene, was fivefold higher than in absence of exogenous hormone. This stimulation was also observed when a higher level of 20‐hydroxyecdysone was maintained, and prevented by protein inhibitors, indicating that 20‐hydroxyecdysone does not act directly on this gene. Exposure to 20‐hydroxyecdysone followed by exposure in hormone‐free medium caused the cessation of this stimulation, showing the requirement of the 20‐hydroxyecdysone continuous presence for stimulating ACP‐20 gene expression. Thus, unlike the other cuticular protein genes so far studied, its expression is not repressed by 20‐hydroxyecdysone, and does not need the decline in ecdysteroids titer.
Recombinant Soluble β‐1,4‐Galactosyltransferases Expressed in Saccharomyces cerevisiae
Authors: Malissard Martine , Borsig Lubor , Di Marco Stefania , Grütter Markus G. , Kragl Udo , Wandrey Christian , Berger Eric G.
Published: July 2004

β‐1,4‐Galactosyltransferase (Gal‐T, EC 2.4.1.38) transfers galactose (Gal) from JDP‐Gal to N ‐acetyl‐d‐glucosamine or a derivative GlcNAc‐R. Soluble Gal‐T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three‐dimensional analysis, the human enzyme (hGal‐T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal‐T (rGal‐T) and purified. The purification protocol was based on chromatography on concanavalin‐A–Sepharose followed by affinity chromatographies on GlcNAc–Sepharose and α‐lactalbumin–Sepharose. Analysis by SDS/PAGE revealed hyper‐glycosylation at the single N‐glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal‐T. The N‐glycan could be removed by treatment with endoglycosidase H (endo H). The N‐deglycosylated form of rGal‐T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N‐glycosylation consensus sequence by site‐directed mutagenesis changing Asn69→Asp. The soluble mutated enzyme (N‐deglycosylated rGal‐T) was expressed in S. cerevisiae and its production scaled up to 60 U. N‐deglycosylated rGal‐T was purified to electrophoretic homogeneity. When analyzed by IEF, N‐deglycosylated rGal‐T was resolved in two bands. The O‐glycans could be removed by jack bean α‐mannosidase treatment and the completely deglycosylated Gal‐T appeared homogeneous by IEF. The kinetic parameters of N‐deglycosylated rGa1‐T were shown not to differ to any significant extent from those of the hGal‐T. No significant changes in CD spectra were observed between hGal‐T and N‐deglycosylated rGal‐T. Light‐scattering analysis revealed dimerization of both enzymes. These data indicate that N‐deglycosylated rGal‐T was correctly folded, homogeneous and thus suitable for crystallization experiments.
The Mutation Met121→His Creates a Type‐1.5 Copper Site in Alcaligenes denitrificans Azurin
Authors: Kroes Sandra J. , Hoitink Carla W. G. , Andrew Colin R. , Ai Jingyuan , Sanders‐Loehr Joann , Messerschmidt Albrecht , Hagen Wilfred R. , Canters Gerard W.
Published: July 2004

The Cu ligand Met121 in azurin of Alcaligenes denitrificans was mutated to His. The spectroscopic and mechanistic properties of [M121H]azurin appear to be pH dependent with a pKa of 3.8 due to the ionization of His121. The [M121H]azurin mutant exhibits two major distinct metal‐site‐coordination geometries which coexist in solution according to a pH‐dependent equilibrium. Both species have been spectroscopically characterized by ultraviolet–visible, EPR and resonance Raman spectroscopies. At neutral pH, His121 is deprotonated and acts as the fourth ligand of the Cu; the spectroscopic characteristics of the Cu site at this pH are halfway between those of a type‐1 and a type‐2 Cu site, and the site is referred to as a type‐1.5 or intermediate Cu site. The spectral data are compatible with a tetrahedral geometry of this site. At low pH, the spectroscopic data indicate that [M121H]azurin has a trigonal type‐1 rhombic Cu site.
Intramolecular Disulfide Bonds Enhance the Antimicrobial and Lytic Activities of Protegrins at Physiological Sodium Chloride Concentrations
Authors: Harwig Sylvia S. L. , Waring Alan , Yang Hye Jin , Cho Yoon , Tan Leonie , Lehrer Robert I.
Published: July 2004

Protegrins are 2‐kDa antimicrobial peptides that contain 16–18 amino acid residues and two intramolecular disulfide bonds. We studied the contribution of these disulfide bonds to the bactericidal activity of protegrins in physiological concentrations of NaCl by comparing protegrin PG‐1 with variants that lacked one or both cysteine disulfides. Whereas the bactericidal and liposome‐lytic properties of protegrin PG‐1 were enhanced by adding 100 mM NaCl to the phosphate‐buffered medium, NaCl addition strongly inhibited the effects of its linearized, disulfide‐free variant, [A6, A8, A13, A15]protegrin‐1. Whereas protegrin PG‐1 manifested β‐sheet structure by CD (circular dichroism) and ATR‐FTIR (attenuated‐total‐reflectance‐Fourier‐transform‐infrared) spectroscopy in buffer or membrane‐mimetic environments, [A6, A8, A13, A15]protegrin‐1 manifested disordered structure in phosphate buffer and α‐helical characteristics in membrane‐mimetic environments. Both single‐disulfide protegrin variants, [A8, A13]protegrin‐1 and [A6, A15]protegrin‐1, assumed β‐sheet conformations with liposomes that simulated bacterial membranes, and both retained substantial bactericidal activity when 100 mM NaCl was present. These findings demonstrate that the intramolecular disulfide bonds of protegrins are required for their antiparallel β‐sheet conformation in membrane‐mimetic environments and for their potent antimicrobial activity in media containing NaCl concentrations comparable to those found in serum and extracellular fluids.
Multiple Solution Conformations of the Integrin‐Binding Cyclic Pentapeptide Cyclo(‐Ser‐d‐Leu‐Asp‐Val‐Pro‐)
Authors: Viles John H. , Mitchell John B. O. , Gough Sharon L. , Doyle Paul M. , Harris C. John , Sadler Peter J. , Thornton Janet M.
Published: July 2004

The aqueous solution structure of the cyclic pentapeptide cyclo(‐Ser‐d‐Leu‐Asp‐Val‐Pro‐) has been determined by two‐dimensional 1H‐NMR spectroscopy, combined with a conformational search and distance‐geometry calculations. As many as five conformers in slow exchange were observed, and the rate of interconversion between components was measured from the build‐up rates of exchange peaks. NMR data allowed the structures of the two predominant conformers to be determined. The major component (66%) contained a cis‐proline as part of a type‐VIa2 β‐turn encompassing residues Asp‐Val‐cis‐Pro‐Ser. The second component (16%) contained only trans‐amide bonds, and a type‐VIII, β‐turn formed by residues Val‐Pro‐Ser‐d‐Leu. These structures are discussed in relation to the (φ,ψ) space available to the cyclic pentapeptide, determined by a conformational search, and in relation to previously published cyclic‐pentapeptide structures. The molecule exhibits activity in a scintillation‐proximity assay for the inhibition of the interaction between the integrin very‐late antigen‐4 (VLA‐4; α4β1) and vascular‐cell‐adhesion molecule‐1 (VCAM‐1). The structure/activity relationship of the LDV sequence is discussed and related to the recently published X‐ray structure of VCAM‐1. The relevance of the work to the design of anti‐inflammatory drugs is discussed.
The Hemolysin B Protein, Expressed in Saccharomyces cerevisiae, Accumulates in Binding‐Protein(BiP)‐Containing Structures
Authors: Kölling Ralf , Hollenberg Cornelis P.
Published: July 2004

The hemolysin B (HlyB) protein of Escherichia coli, a member of the ABC‐transporter family, was expressed in Saccharomyces cerevisiae and tested for its ability to complement a defect in the a‐factor transporter Ste6. We found that HlyB was not able to restore mating ability to a STE6 deletion strain. The HlyB protein did not co‐fractionate with Ste6 on sucrose gradients, indicating that improper localization of the HlyB protein could contribute to the lack of complementation. Immunofluorescence experiments suggest that HlyB is localized to structures derived from the endoplasmic reticulum (ER). The HlyB‐expressing cells revealed a perinuclear staining typical of ER‐localized proteins and intensely staining ring‐like structures (HlyB‐bodies). Double‐label immunofluorescence experiments show that the HlyB structures also contain the ER binding protein (BiP), the product of the kAR2 gene. The HlyB protein, however, did not co‐fractionate with another ER marker protein, the NADPH cytochroine c reductase. The HlyB bodies could be derivatives of a novel compartment of the early secretory pathway which contains BiP but not other resident ER proteins. In this case, HlyB could serve as a tool for the biochemical characterization of this compartment.
Does the N‐Terminal Pyroglutamate Residue have Any Physiological Significance for Crab Hyperglycemic Neuropeptides?
Authors: Chung J. Sook , Webster Simon G.
Published: July 2004

A characteristic feature of all crustacean hyperglycemic hormones (CHH) is that they are always present in the sinus gland as multiple forms or isoforms. The amino acid sequence of the minor form of CHH from the green shore crab, Carcinus maenas, was determined by automated microsequencing and MS, and was almost identical to that of the major form, except that the N‐terminal residue was glutamine rather than pyroglutamate. Limited analysis (electrospray MS and amino acid composition) of the two corresponding forms of CHH from the edible crab, Cancer pagurus, suggested a similar phenomenon in this species. For C. maenas, both forms were indistinguishable in terms of their ability to cause sustained hyperglycemia in vivo and repression of ecdysteroid synthesis in vitro. Similarly, the two forms were immunologically identical in RIA, and exhibited similar binding characteristics in competitive‐receptor‐binding assays. CD studies showed only minor differences in presumed secondary structure. In vitro release experiments with isolated sinus glands demonstrated that both forms are probably released in a stoichiometric manner and that both peptides are present in the haemolymph at the same ratio as that in the sinus gland. Preliminary results suggest that the in vivo clearance/degradation rates of both peptides are similar. The unblocked (Gln) terminus is of particular significance, since the presence of this amino acid indicates that this peptide is derived from a precursor that does not possess the same structure of those of established preproCHH, or that N‐terminal processing is slow, which results in the presence of unblocked CHH in sinus glands. The similar biological activity of the unblocked CHH to that of the blocked CHH suggests that the N‐terminal pyroglutamate residue has no obvious biological significance (with respect to the known functions of CHH), an observation which is in contrast to the widely accepted paradigms concerning the stability and biological activity of N‐terminally blocked and unblocked peptides.