bluuebunny/crossref_metadata_2025 huggingface dataset:
Calorimetric and infrared spectroscopic studies of the interaction of α‐tocopherol and α‐tocopheryl acetate with phospholipid vesicles
Authors: VILLALAIN José , ARANDA Francisco J. , GÓMEZ‐FERNÁNDEZ Juan C.
Published: March 2005

The location of α‐tocopherol and α‐tocopheryl acetate in the membrane has been a subject of considerable interest, not only because of their relevant physiological function, but also for understanding their interaction with the phospholipids. We have examined this question by using reconstituted systems including dipalmitoyl‐glycerophosphocholine multibilayer vesicles to which α‐tocopherol and α‐tocopheryl acetate were incorporated. Differential scanning microcalorimetry measurements showed that both compounds were capable of modifying the thermotropic properties of the pure phospholipid, so that the pretransition disappears at low concentrations of these terpenoid molecules. The enthalpy corresponding to the main transition decreases as the concentration of α‐tocopherol increases and the transition peak is progressively shifted to lower temperatures. α‐Tocopheryl acetate gave the same type of effect but less marked than in the case of α‐tocopherol. Fourier‐transform infrared spectroscopic measurements were also made on these systems and the temperature dependence of the infrared spectra was studied. A comparison of the spectroscopic data showed that, in agreement with the calorimetric results, α‐tocopherol remarkably perturbs the thermotropic phase transition of dipalmitoylglycerophos‐phocholine. It was concluded from a study of the CH2 stretching bands that α‐tocopherol induced changes in frequency and in bandwidth parameters. However, the changes in frequency and bandwidth with respect to temperature are not concerted; this is a consequence of the existence of more than one phase in the presence of α‐tocopherol. From the study of the CH2 scissoring band, it was concluded that α‐tocopherol disrupts the acyl chain packing present in pure dipalmitoylglycerophosphocholine below the onset temperature of the gel‐to‐liquid‐crystal transition. The effect of α‐tocopheryl acetate is of a similar type of that of α‐tocopherol but weaker with respect to these stretching and scissoring CH2 bands. Spectral changes were also found in the C=O stretching mode of the phospholipid in the presence of α‐tocopherol and these changes were attributed to perturbations in the C1‐C2 bonds of sn‐2‐acyl chains of the phospholipid. These effects were much weaker in the case of α‐tocopheryl acetate; it is suggested that this may be due to a specific interaction between α‐tocopherol and the polar region of the phospholipid. This interaction seems to be absent in the systems containing α‐tocopheryl acetate. The different locations in the membrane of α‐tocopherol and α‐tocopheryl acetate that can be inferred from these results are discussed.
Orthophosphate – pyrophosphate exchange catalyzed by soluble and membrane‐bound inorganic pyrophosphatases
Authors: DE MEIS Leopoldo , BEHRENS Maria Isabel , CELIS Heliodoro , ROMERO Irma , GÓMEZ PUYOU Marietta Tuena , PUYOU Armando GÓMEZ
Published: March 2005

A comparative study of the orthophosphate—pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane‐bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed.

In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO42− or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7–8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were inclded in the assay medium. In the presence of 6.7–8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO42− and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24–0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total, Pi, HPO42− and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5–7.5 mM free Mg2+ was very high and could not be measured.

In the presence of 0.24–0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate mesured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5–8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.
Polysaccharide from coccoliths (CaCO3 biomineral)
Authors: KOK Dik J. , BLOMEN Leo J. M. J. , WESTBROEK Peter , BIJVOET Olav L. M.
Published: March 2005

A polysaccharide associated with coccoliths of the marine alga Emiliania huxleyi (coccoliths are elaborately shaped calcite biominerals) was isolated and its influence on the crystallization of calcium oxalate monohydrate crystals was studied. Crystallization was monitored in a carefully controlled system by measuring the incorporation of 45Ca tracer from a supersaturated solution into seed crystals of calcium oxalate monohydrate in the absence and in the presence of polysaccharide. The method allowed differentiation between effects on solubility, growth and agglomeration of crystals. At the very low concentrations used in this study, the polysaccharide had no significant effect on the solubility product; it strongly inhibited the growth and strongly stimulated the agglomeration of the crystals. Thus, the two processes of growth and agglomeration, being both crystal‐surface‐related processes, may react in opposite directions upon surface adhesion of the additive. This finding opens new insights on how a mineralization process may be controlled. The inhibitory effect on growth is shown to proceed through a monolayer type of adsorption of the polysaccharide onto the crystal surface. The portion of the polysaccharide used for the stimulatory effect on agglomeration shows a different type of adsorption, whereby less crystal surface is covered per molecule of polysaccharide. This strongly suggests, that the mechanism whereby agglomeration is stimulated operates through ‘viscous binding’, with the polysaccharide bridging the gap between two crystal surfaces. In the discussion these findings are related to some possible biological functions of the polysaccharide.
The effect of the inverted repeat structure on the production of the cloned Bacillus amyloliquefaciensα‐amylase
Authors: KALLIO Pauli
Published: March 2005

An inverted repeat structure from Bacillus natto preceding the Bacillus subtilisα‐amylase gene has been suggested to be responsible for the enhancement of α‐amylase production [Yamazaki et al. (1983) J. Bacteriol. 156, 327–337]. A similar inverted repeat region has also been found upstream from the Bacillus amyloliquefaciensα‐amylase gene and shown to function as a transcription termination signal of an upstream operon of 2.2 × 103 bases (2.2 kb) (Kallio et al., following paper in this journal). The removal of this DNA region reduced the level of α‐amylase production by 70% and led to concomitant formation of a readthrough transcript arising from the promoter of the 2.2‐kb operon. To test whether the readthrough transcript affected the α‐amylase production, the promoter of the 2.2‐kb operon was removed from plasmid constructions carrying either intact or deleted inverted repeat sequences. When cultures carrying the above plasmids were assayed for α‐amylase activity, both constructions produced equal amounts of α‐amylase. Thus, the inverted repeat structure preceding the α‐amylase promoter does not, as such, enhance the α‐amylase production. Instead, its presence prevents the inhibition of α‐amylase expression caused by the readthrough transcripts from the upstream promoter.
Isolation and characterization of a 2.2‐kb operon preceding the α‐amylase gene of Bacillus amyloliquefaciens
Authors: KALLIO Pauli , ULMANEN Ismo , PALVA Ilkka
Published: March 2005

A DNA region of 2.8 × 103 base pairs (2.8 kb) upstream of the Bacillus amyloliquefaciensα‐amylase gene has been isolated. This DNA gave rise to a 2.2‐kb transcript. The 3′ end of the transcript was mapped with S1 nuclease and shown to terminate 49 base pairs upstream of the‐35 region of the α‐amylase promoter. In B. subtilis minicells this 2.2‐kb transcript coded for three different polypeptides, thus indicating a polycistronic operon‐type structure. The location and the order of the polypeptides were established using DNA deletions. The joining of the 2.2‐kb operon to the downstream α‐amylase gene in the plasmid pUB110 did not have any significant effect on the level of expression of the α‐amylase.
A transcription terminator in the 5′ non‐coding region of the tyrosyl tRNA synthetase gene from Bacillus stearothermophilus
Authors: WAYE Mary M. Y. , WINTER Greg
Published: March 2005

The 5′ non‐coding region of the tyrosyl‐tRNA synthetase gene (tyrS) of Bacillus stearothermophilus is 324 nucleotides long. It contains a premature terminator and a strong promoter: these were identified in vitro by RNA run‐off experiments and in Escherichia coli by construction of specific mutants. The terminator consists of a stem and loop structure followed by the string of T residues characteristic of rho‐independent termination. This is preceded by another stem and loop structure which may permit the formation of an anti‐terminator. Neither the promoter nor the premature terminator appears to be regulated by the tyrosyl‐tRNA synthetase in vitro or in E. coli.
1H‐NMR assignment and secondary structure of a Herpes simplex virus glycoprotein D‐1 antigenic domain
Authors: WILLIAMSON Michael P. , HALL Michael J. , HANDA Balraj K.
Published: March 2005

The peptide αAhx‐Met‐Ala‐Asp‐Pro‐Asn‐Arg‐Phe‐Arg‐Gly‐Lys‐Asp‐Leu‐Pro‐Val‐Leu‐Asp‐Gln‐ Leu‐Thr‐Asp‐Pro‐Pro‐αAhx (ɛAhx = 6‐aminohexanoyl), the antigenic sequence 11–32 from Herpes simplex virus glycoprotein D‐1, has been synthesised. Its 1H‐NMR spectrum has been assigned by a combination of two‐dimensional techniques in H2O and 2H2O. Its secondary structure has been defined by nuclear Overhauser effects and amide proton exchange rates, and also to some extent chemical shifts, coupling constants and amide proton temperature coefficients. These latter parameters are shown to be less reliable as guides to secondary structure. The peptide has a helical (type I/III) turn at residues Pro‐14—Asn‐15 and helical structure at residues Lys‐20–Val‐24, in rapid equilibrium with random‐coil structure. A β‐turn at residues Arg‐18—Gly‐19 may be present as a minor component. These locations of secondary structure correspond with previously determined regions of antigenic activity.
Monoclonal antibodies inhibitory to human plasmin
Authors: SIM Pheng‐Siew , FAYLE David R. H. , DOE William F. , STEPHENS Ross W.
Published: March 2005

Four monoclonal antibodies raised against purified human plasminogen were characterized for their effects on the activation of plasminogen and on three enzymic properties of plasmin: (a) thioesterolysis, (b) fibrinolysis, (c) conversion of high‐Mr urokinase to its low‐Mr form.

None of the monoclonal antibodies inhibited plasminogen (plg) activation by urokinase. The monoclonal antibodies characterized in this study fell into three groups. Anti‐plg 1 inhibited (a), (b) and (c), while anti‐plg 2 inhibited activities (a), (b) and (c) to varying degrees but also formed complexes with plasmin that were stable to sodium dodecyl sulphate. Anti‐plg 3 and anti‐plg 4 inhibited only activity (c).

Selective use of these monoclonal antibodies demonstrated unequivocally that plasmin mediates the activation of the proenzyme form of urokinase‐type plasminogen activator. Besides their use in affinity chromatography, therefore, these antibodies are valuable for defining the role of plasmin in the mechanisms of extracellular matrix degradation.
Estimation of inter‐binding‐site distances in sarcoplasmic reticulum (Ca2++Mg2+)‐ATPase using Eu(III) luminescence energy transfer
Authors: HERRMANN Tom R. , GANGOLA Preeti , SHAMOO Adil E.
Published: March 2005

We have used several trivalent lanthanides as probes for the high‐affinity Ca(II)‐binding site of the Ca(II)+Mg(II)‐ATPase of skeletal muscle sarcoplasmic reticulum. The luminescent probes Eu(III) and Tb(III) were excited directly with pulsed laser light and the energy transfer efficiencies to several lanthanide acceptors were measured, under conditions in which most donor‐acceptor pair occupied high‐affinity Ca(II) sites. We obtain an inter‐ionic site distance of about 0.8–0.9 nm. Energy transfer measurements were also done with Eu(III) in at least one Ca(II) site and bidentate Cr‐ATP comlex at the ATP hydrolytic site. Quenching of Eu(III) luminescence by Cr‐ATP was total under these conditions. We calculate an upper limit of 1.0 nm for the distance from the Ca(II) site(s) to the complexed Cr(III) ion at the hydrolytic site.
Peptide uptake by Salmonella typhimurium The periplasmic oligopeptide‐binding protein
Authors: HILES Ian D. , HIGGINS Christopher F.
Published: March 2005

The uptake of most peptides, including many peptide antibiotics, by the oligopeptide permeases of Escherichia coli and Salmonella typhimurium requires the function of a specific peptide‐binding protein (the OppA protein) located within the periplasm. The OppA protein is the largest and most abundant periplasmic substrate‐binding protein known and has an unusually broad substrate‐binding specificity. The OppA protein has been purified to homogeneity and anti‐OppA antibodies have been raised. The sequence of the OppA protein has been deduced from the nucleotide sequence of the oppA gene. This protein is unrelated to any other known periplasmic substrate‐binding protein, either immunologically or in its amino acid sequence. The role of this protein in peptide transport is discussed.
Brain pyridoxal kinase
Authors: KERRY Julie Ann , ROHDE Manfred , KWOK Francis
Published: March 2005

Pyridoxal kinase has been purified 9000‐fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE‐cellulose chromatography, affinity chromatography and Sephadex G‐100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10–25% gradient polyacrylamide gel electrophoresis and Sephadex G‐200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.
The role of compound III in reversible and irreversible inactivation of lactoperoxidase
Authors: HUWILER Martin , JENZER Herbert , KOHLER Heinz
Published: March 2005

In the presence of iodide (I−, 10 mM) and hydrogen peroxide in a large excess (H2O2, 0.1–10 mM) catalytic amounts of lactoperoxidase (2 nM) are very rapidly irreversibly inactivated without forming compound III (cpd III). In contrast, in the absence of I− cpd III is formed and inactivation proceeds very slowly. Increasing the enzyme concentration up to the micromolar range significantly accelerates the rate of inactivation. The present data reveal that irreversible inactivation of the enzyme involves cleavage of the prosthetic group and liberation of heme iron. The rate of enzyme destruction is well correlated with the production of molecular oxygen (O2), which originates from the oxidation of excess H2O2. Since H2O2 and O2per se do not affect the heme moiety of the peroxidase, we suggest that the damaging species may be a primary intermediate of the H2O2 oxidation, such as oxygen in its excited singlet state (1ΔgO2), superoxide radicals (O−2), or consequently formed hydroxyl radicals (OH⋅).
Purification, subunit composition and regulatory properties of the ATP · Mg2+‐dependent form of type I phosphoprotein phosphatase from bovine heart
Authors: PRICE Daniel J. , TABARINI Diane , LI Heng‐Chun
Published: March 2005

The ATP · Mg2+‐dependent phosphoprotein phosphatase has been purified from bovine heart to nearhomogeneity. It is a heterodimer (75 kDa) consisting of a catalytic (C) subunit (40 kDa) and a regulatory (R) subunit (35 kDa). The R subunit, which is identical to inhibitor‐2, is transiently phosphorylated during activation of the enzyme catalyzed by phosphatase‐1 kinase (FA). Maximal activation requires preincubation of the phosphatase with FA and ATP · Mg2+. However, relatively low yet definitively demonstrable basal activity can be expressed by Mg2+ alone (ranging from 3% to 10% of the FA· ATP · Mg activity, depending on the degree of endogeneous proteolytic damage of the phosphatase during purification), but not by either FA or ATP alone. Limited trypsinization results in a rapid and total degradation of the R subunit and partial degradation of the 40‐kDa C subunit to active proteins of 35–38 kDa. The resulting ‘nicked’ C subunit of 35–38 kDa is no longer dependent on FA for activation and can be fully activated by Mg2+ (or Mn2+) alone. Endogenous proteolytic damage of the R subunit also results in an increase of activity that can be expressed by M2+ alone with a concomitant decrease of the FA‐dependent activation. Although Mn2+ is slightly more effective than Mg2+ in expressing the holoenzyme basal activity, the activation by Mn2+ is only about 60% of that of Mg2+ when FA and ATP are also present. In the activation by adenosine 5′‐[γ‐thio]triphosphate (ATP[γS]), Co2+ is the most effective cofactor. The activation by ATP[γS] · Co2+ is more than 50% of that by ATP · Mg2+. The present studies indicate that Mg2+ is the natural divalent cation for the FA‐catalyzed activation in which Mg2+ plays two distinctly different roles: (a) it forms Mg2+· ATP which serves as a substrate for the kinase; (b) it acts as an essential cofactor for the catalytic function of the phosphatase. The discrepancies between the results obtained by this and other laboratories with respect to the effectiveness of Mg2+ and ATP[γS] in the activation of the phosphatase are discussed.
Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator
Authors: NORRMAN Bo , POHL Gunnar , JÖRNVALL Hans , WALLÉN Per
Published: March 2005

Tissue plasminogen activator was treated with Sepharose‐bound trypsin or chymotrypsin. Trypsin rapidly converted the one‐chain activator to the two‐chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active‐site labeling revealed that after the conversion to the two‐chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded.

Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50000/52000 and 17000/20000 respectively. N‐terminal sequence analysis identified cleavage sites at peptide bonds 420–421 and 423–424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations.

However, treatment of two‐chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin‐digested one‐chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420–421 and 423–424, convert the active one‐chain activator into an ‘inactive’ zymogen, which is again ‘activated’ by plasmin cleavage.
Structure and properties of casein kinase‐2 from Saccharomyces cerevisiae
Authors: MEGGIO Flavio , GRANKOWSKI Nikodem , KUDLICKI Wieslaw , SZYSZKA Ryszard , GASIOR Eugeniusz , PINNA Lorenzo A.
Published: March 2005

A type‐2 casein kinase (YCK‐2), lacking the 25‐kDa autophosphorylatable β subunit characteristic of animal casein kinases‐2, has been obtained in a nearly pure form from Saccharomyces cerevisiae and was compared with liver casein kinase‐2 (LCK‐2). A 22‐kDa phosphorylatable protein, copurifying with YCK‐2, can be removed by ultracentrifugation at low ionic strength and is shown by several criteria to be unrelated to the β subunit of LCK‐2. The native Mr of YCK‐2, deprived of the 22‐kDa phosphoprotein, is about 150000. Limited proteolysis experiments show that YCK‐2 included 37‐kDa catalytic subunits, which can be converted into still active 35‐kDa proteolytic derivatives. These data are consistent with a homotetrameric quaternary structure as opposed to the heterotetrameric subunit composition α2β2 of LCK‐2 and other animal casein kinases‐2. Although many properties of YCK‐2 and LCK‐2, including substrate specificity, inhibition by heparin, polyglutamic acid and quercetin and stimulation by polyamines, are similar; their stability under denaturating and dissociating conditions and their response to polybasic peptides are quite different. In particular YCK‐2 is more readily denaturated than LCK‐2 by heating and exposure to urea, sodium dodecylsulphate and deoxycholate while its activity is inhibited by 100–150 mM NaCl, which conversely stimulates LCK‐2 activity 2–3‐fold. The Km value of the synthetic peptide substrate Ser‐(Glu)5 for YCK‐2 is not significantly changed by the addition of polylysine. On the contrary the Km value of the same peptide substrate for LCK‐2 decreases approximately tenfold upon addition of polylysine, which also prevents the fast autophosphorylation of the kinase at its β subunit. These data suggest that the β subunit of animal CK‐2 may play a role in determining both the stability of the enzyme and its regulation and that, consequently, the different properties of YCK‐2 may be at least in part accounted for by its lack of β subunits.
Proteins from rat liver cytosol which stimulate mRNA transport
Authors: SCHRÖDER Heinz C. , ROTTMANN Michael , BACHMANN Michael , MÜLLER Werner E. G. , McDONALD Alexander R. , AGUTTER Paul S.
Published: March 2005

Two polysome‐associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA‐transport‐stimulatory proteins. Their interaction with the mRNA‐translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down‐regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35000‐Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA‐binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.
Expression of intracellular fibrinogen on the surface of stimulated platelets
Authors: COURTOIS Gilles , RYCKEWAERT Jean‐Jacques , WOODS Virgil L. , GINSBERG Mark H. , PLOW Edward F. , MARGUERIE Gérard A.
Published: March 2005

Participation of fibrinogen in platelet aggregation is contingent upon the capacity of various stimuli to induce specific receptors for the molecule on the surface of the cell. The interaction of fibrinogen with this receptor results directly in platelet aggregation, and dissociation of fibrinogen is associated with disaggregation. While the role of exogenous fibrinogen in this process has been fully documented, the mechanisms which control the surface exposure of platelet fibrinogen are less understood. In the present study Fab fragments of antibodies monospecific for fibrinogen have been used to examine the surface expression of intracellular fibrinogen and its involvement in platelet aggregation. Radiolabelled Fab fragments did not interact with non‐stimulated platelets but significant binding was observed when the cells were stimulated by ADP, thrombin, collagen and Ca ionophore A23187. Binding was specific for fibrinogen, was not observed with thrombasthenic platelets and was dependent upon the presence of extracellular calcium. With all stimuli tested, the binding of the Fab probe to platelets correlated with platelet secretion. At the following concentrations of stimuli: 30 μM ADP, 4 μg/ml collagen, 3 μM A23187 and 0.05 U/ml thrombin, the immune Fab fragments inhibited platelet aggregation. A monoclonal antibody to glycoprotein IIb/IIIa complex and a synthetic peptide γ400–411, that inhibited the interaction of plasma fibrinogen with platelets, did not inhibit the binding of 125I‐FAB fragments. Taken together these results support the hypothesis that endogenous fibrinogen becomes surface‐expressed during stimulation of the cell and can support platelet aggregation, particularly that induced by low concentrations of stimuli. The mechanism for the surface expression of platelet fibrinogen may be distinct from that for the binding of plasma fibrinogen.
Conformational analysis of the octamer d(G‐G‐C‐C‐G‐G‐C‐C) in aqueous solution
Authors: RINKEL Lambertus J. , SANDERSON Mark R. , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

Proton NMR studies in H2O and 2H2O were carried out on the self‐complementary DNA octamer d(G‐G‐C‐C‐G‐G‐C‐C) and compared with similar studies on the dimethylated analogue d(G‐G‐m5C‐m5C‐G‐G‐C‐C) [Sanderson, M. R., Mellema, J.‐R., van der Marel, G. A., Wille, G., van Boom, J. H. & Altona, C. (1983) Nucleic Acids Res. 11, 3333–3346]. Base, H1′, H2′ and H2” resonances were assigned by means of two‐dimensional nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments. Chemical shift vs temperature profiles were used to analyze the temperature‐dependent conformational behaviour and to extract thermodynamic parameters for the duplex‐to‐coil transition. Analysis of proton‐proton couplings were used to discriminate between J1′2′ and J1′2″ and between the H2′ and H2″; resonances as well as to obtain conformational parameters of the sugar rings. From the NOESY and COSY experiments, the temperature profiles and the analysis of the coupling data it is concluded that: (a) the compound adopts a B‐DNA‐type helix in solution; (b) the sugar rings in the intact duplex display limited conformational freedom; (c) methylation of the cytosine bases at the C5 position has no measurable effect on the conformational behaviour of the octamer, nor on the conformation of the sugar rings; however, methylation does increase the stability of the duplex in aqueous solution under conditions of low salt concentration.
Structural characteristics and classfication of some tRNA‐binding sites of elongating Escherichia coli ribosome
Authors: ABDURASHIDOVA Gulnara G. , BASKAYEVA Irena O. , CHERNYI Alexey A. , KAMINIR Lev B. , BUDOWSKY Edward I.
Published: March 2005

Ultraviolet(254 nm)‐irradiation‐induced cross‐linkages in ribosomal complexes allowed identification of proteins in contact with tRNA at different elongation steps. Both the set and the ratio of cross‐linked proteins, i.e. the structural characteristics of the tRNA‐binding sites of the ribosome, were shown to depend strongly not only on the position of the mRNA codon with which tRNA interacts as a component of a ribosomal complex, but also on its functional state, i.e. on the elongation step. A new classification of tRNA‐binding sites of ribosome is suggested.
Elicitation of thiomolybdates from the iron‐molybdenum cofactor of nitrogenase
Authors: NEWTON William E. , GHELLER Stephen F. , HEDMAN Britt , HODGSON Keith O. , LOUGH Samuel M. , McDONALD John W.
Published: March 2005

Aerial oxidation of the iron‐molybdenum cofactor (FeMoco) of Azotobacter vinelandii nitrogenase has been shown to yield either the tetrathiomolybdate ion ([MoS4]2−) or the oxotrithiomolybdate ion ([MoOS3]2−), depending on the reaction conditions. Thus, when N‐methylformamide (NMF) solutions of FeMoco either were titrated with measured aliquots of air or were diluted with air‐saturated NMF, [MoOS3]2− was found to be the predominant product while dilution of NMF solutions of FeMoco with air‐saturated methanol produced [MoS4]2− almost exclusively. Similar aerial oxidation of solutions of chemically synthesized Fe‐Mo‐S clusters showed that significant information about the molybdenum environment in these species could be deduced from the nature of the elicited thiomolybdates. The differences in decomposition products as a function of solvent are postulated to be due to the loss through precipitation of the reducing agent sodium dithionite on addition of methanol but not NMF. These overall decomposition results are discussed in the context of recent X‐ray absorption spectroscopic data which suggest the presence of an ‘MoS3’ core in FeMoco. A possible mechanism wherby [MoS4]2− might be rapidly formed from this core is presented.
The hydrophobic moment of the amphipathic helix of salmon calcitonin and biological potency
Authors: EPAND Richard M. , SEYLER Jay K. , ORLOWSKI Ronald C.
Published: March 2005

The formation of an amphipathic helix in the central portion of calcitonin contributes to the potency of this hormone. We have synthesized a number of analogs of salmon calcitonin, containing deletions in the region of the peptide which is thought to form an amphipathic helix. There is no direct relationship between the hydrophobic moment of the helix and the biological activity of the peptide. For example, salmon des‐Leu19‐calcitonin and des‐Ser13‐calcitonin both have lower helical hydrophobic moments but have greater or equal biological potency compared with the native hormone. We suggest that other conformational features, such as flexibility and helixforming potential, are also important in determining biological potency.
The phosphoenolpyruvate‐dependent fructose‐specific phosphotransferase system in Rhodopseudomonas sphaeroides
Authors: LOLKEMA Juke S. , ROBILLARD George T.
Published: March 2005

Redox titrations of the fructose‐specific carrier protein, EFruII, in Rhodopseudomonas sphaeroides show that only the reduced form of the enzyme is active. The oxidized form of the enzyme can still be phosphorylated but is unable to transfer the phosphoryl group to fructose. The redox properties of the enzyme change upon phosphorylation. The reduction rate of EFruII is slower than that of EFruII‐P, whereas the opposite is true for the oxidation rate. Consequently the midpoint potential of the redox centre is more negative on EFruII than EFruII‐P, most likely due to an upwards pK shift of the thiols upon phosphorylation. The measurements indicate that the phosphotransferase system is regulated by the redox potential in a way that is dependent on the substrate concentrations. We propose that the change in the midpoint potential during turnover could be a mechanism for an electron transport function of the carrier.

The binding of Zn2+ protects the carrier dithiol against oxidation but the presence of Zn2+ does not stimulate the reduction of the oxidized carrier.
Photosystem I reaction centers from maize bundle‐sheath and mesophyll chloroplasts lack subunit III
Authors: NECHUSHTAI Rachel , SCHUSTER Gadi , NELSON Nathan , OHAD Itzhak
Published: March 2005

Phtosystem I reaction centers were isolated from mesophyll and bundle‐sheath chloroplasts of the C4 maize plant. Both preparations were found to be free of chlorophyll b and to have the same spectral properties and chlorophyll/P700 ratio as photosystem I reaction centers isolated from C3 plants. Photosystem I reaction centers from both mesophyll and bundle sheath were found to consist of six subunits with apparent molecular masses of about 70 kDa, 20 kDa, 17 kDa, 16 kDa, 10 kDa and 8 kDa, corresponding to photosystem I reaction center subunits I, II, IV, V, VI and VII of spinach, as tested by their immunological cross‐reactivity with antibody raised against the respective spinach subunits. No cross‐reactivity was found with antibodies raised against subunit III of spinach, either in whole thylakoids or purified reaction centers of both bundle‐sheath and mesophyll chloroplasts. It is concluded that photosystem I reaction centers of bundle‐sheath and mesophyll thylakoids of maize are identical and lack the polypeptide corresponding to subunit III present in all C3 plants so far tested.
Membrane‐bound hydroxylases in elicitor‐treated bean cells
Authors: BOLWELL G. Paul , DIXON Richard A.
Published: March 2005

1. Treatment of cell‐suspension cultures of bean (Phaseolus vulgaris cv. Canadian Wonder) with an elicitor preparation heat‐released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the activities of two microsomal oxygenases, cinnamic acid 4‐hydroxylase, involved in accumulation of wall‐bound phenolics and phytoalexins, and proline 2‐oxoglutarate dioxygenase (prolylhydroxylase) involved in the post‐translational modification of hydroxyproline‐rich glycoproteins.

2. An anti‐(cytochrome P‐450) monoclonal antibody, originally raised against rat cytochrome P‐450 isoform c, has been shown to bind to bean microsomes and recognise in Western blots an Mr‐48 000 polypeptide, which comigrates with a haeme‐containing protein on SDS/polyacrylamide gel analysis and which has been tentatively identified as a cytochrome P‐450 capable of the hydroxylation of cinnamic acid.

3. A preparation of proline 2‐oxoglutarate dioxygenase purified to homogeneity was used to immunise rabbits for the production of antiserum. An elicitor‐induced polypeptide of Mr 65000 was identified as prolyl hydroxylase while an antigenically related polypeptide of Mr 60000 was also immunoprecipitated but not induced by elicitor treatment.

4. Use of the two antibodies has demonstrated rapid transient increases in the synthesis of the Mr 48000 and Mr 65000 oxygenases in vivo and for mRNAs as measured in in vitro translations, particularly for the putative cytochrome P‐450. These increases slightly precede corresponding changes in the synthesis of the soluble enzyme phenylalanine ammonia‐lyase, in common with which these oxygenases probably share a mechanism of gene activation underlying the increased activities seen in response to elicitor treatment.
A camel milk whey protein rich in half‐cystine
Authors: BEG Obaid Ullah , von BAHR‐LINDSTRÖM Hedvig , ZAIDI Zafar H. , JÖRNVALL Hans
Published: March 2005

The amino acid sequence of a recently isolated camel milk protein rich in half‐cystine has been determined by peptide analyses. The 117‐residue protein has 16 half‐cystine residues, concluded to correspond to disulfide bridges and suggesting a tight conformation of the molecule.

Comparisons of the structure with those of other proteins reveal several interesting relationships. The camel protein is clearly homologous with a previously reported rat whey phosphoprotein of possible importance for mammary gland growth regulation, and with a mouse protein of probable relationship to neurophysins. The camel, rat and mouse proteins may represent species variants from a rapidly evolving gene. Residue identities in pairwise comparisons are 40% for the camel/rat proteins and 33% for the camel/mouse proteins, with 38 positions conserved in all three forms. The camel protein also reveals an internal repeat pattern similar to that for the other two proteins.

The homology between the three milk whey proteins has wide implications for further relationship. Thus, previously noticed similarities, involving either of the milk proteins, include limited similarities to casein phosphorylation sites for the camel protein, to neurophysins in repeat and half‐cystine patterns for the mouse and rat proteins, and to an antiprotease for the rat protein. These similarities are reinforced by the camel protein structure and the recognition of the three whey proteins as related. Finally a few superficial similarities with the insulin family of peptides and with some other peptides of biological importance are noticed. Combined, the results relate the camel protein in a family of whey proteins, and extend suggestions of relationships with some binding proteins.
Identification of two distinct regions within the binding sites for fibrinogen and fibronectin on the IIb–IIIa human platelet membrane glycoprotein complex by monoclonal antibodies P2 and P4
Authors: McGREGOR John L. , McGREGOR Lilian , BAUER Anne‐Sylvie , CATIMEL Bruno , BROCHIER Jean , DECHAVANNE Marc , CLEMETSON Kenneth J.
Published: March 2005

The effect of two monoclonal antibodies P2 (LyP 2) or P4 (LyP 4), specific for the platelet membrane glycoprotein IIb/IIIa complex, on binding of 125I‐labelled fibrinogen or 125I‐labelled fibronectin to thrombin‐stimulated platelets was studied. These monoclonal antibodies are directed against different determinants on the IIb‐IIIa complex and react only with the complex and not with the individual glycoproteins. Fibrinogen binding to thrombin‐stimulated platelets was significantly inhibited by P2 but not by P4. Fibronectin binding to thrombin‐stimulated platelets was significantly inhibited by P4 but only poorly by P2. These results indicate the presence of specific regions on the glycoprotein IIb‐IIIa complex which act as binding sites for fibrinogen or fibronectin. Other authors [Haverstick et al. (1985) Blood 66, 946–952; Ginsberg et al. (1985) J. Biol. Chem. 260, 4133–4138] have shown that a tetrapeptide, Arg‐Gly‐Asp‐Ser, inhibited the binding of fibrinogen, fibronectin, and von Willebrand factor (vWf) to stimulated platelets and that fibrinogen competes with vWf and fibronectin for binding. These findings, together with previous studies, therefore indicate the presence of specific regions as well as a common region in the binding sites for fibrinogen and fibronectin on the IIb‐IIIa complex.
Differential inhibition of prostaglandin and superoxide production by dexamethasone in primary cultures of rat Kupffer cells
Authors: DIETER Peter , SCHULZE‐SPECKING Agnes , DECKER Karl
Published: March 2005

Dexamethasone inhibited the stimulus‐induced prostaglandin E2 formation by rat Kupffer cells in primary culture, e.g. after treatment with zymosan, phorbol ester, calcium ionophore A23187, platelet‐activating factor or lipopolysaccharide. Prostaglandin E2 production from added free arachidonic acid was not influenced by the hormone. The time course, as well as the partial inhibition of the hormone effect by actinomycin D and cycloheximide, point to the hormone‐induced formation of a protein which regulates phospholipase A2.

The hormone did not affect the phagocytotic activity of the Kupffer cells. The quantity of [3H]arachidonic acid incorporated into phospholipids was also not altered by dexamethasone. After stimulation with zymosan, [3H]arachidonic acid was liberated from phosphatidylcholine only.

Superoxide generation by rat Kupffer cells was induced by zymosan, phorbol ester and, to a much smaller extent, by platelet‐activating factor. A23187 and lipopolysaccharide were without effect. In contrast to prostaglandin formation, the generation of superoxide was not influenced by dexamethasone.

These results indicate that in cultured rat Kupffer cells prostaglandin formation and superoxide generation are independently triggered processes.
Regulated expression of cloned human fetal Aγ‐globin genes introduced into murine erythroleukemia cells
Authors: CHARNAY Patrick , HENRY Lynda
Published: March 2005

We have analysed the expression of cloned human fetal γ‐globin genes introduced into murine erythroleukemia cells by a protoplast fusion procedure. Both the wild‐type aγ‐globin gene and a mutant derivative related to a phenotype of hereditary persistence of fetal hemoglobin were studied. In both cases the level of γ‐globin mRNA increased by a factor of approximately sevenfold when erythroid cell differentiation was induced by treatment with hexamethylenebisacetamide. Thus, the regulation of the expression of the cloned fetal Aγ‐globin gene in murine erythroleukemia cells resembled that of cloned adult β‐globin genes.
Modification of the actin interface of skeletal myosin subfragment‐1 by treatment with dibromobimane
Authors: MORNET Dominique , UE Kathleen , CHAUSSEPIED Patrick , MORALES Manuel F.
Published: March 2005

Recently, by treating the head portion of skeletal myosin subfragment‐1 (S1) with the bifunctional agent dibromobimane, we introduced an intramolecular covalent cross‐link which resulted in the stabilisation of an internal loop in the heavy chain structure of the head [Mornet et al. (1984) Proc. Natl Acad. Sci. USA 82, 1658–1662]. In order to define the functional properties of this new S1 conformational state, we have first determined the experimental conditions for the optimum modification of S1 by dibromobimane. We finally settled on a 60% yield of cross‐linked S1.

Because the modification occurs between the 50‐kDa and the 20‐kDa tryptic heavy chain fragments which have been postulated to be involved in the interaction of native S1 with actin, we have investigated the association of dibromobimane‐treated S1 with actin, using chemical cross‐linking of their rigor complex with 1‐ethyl‐3‐[3‐(dimethylamino)propyl] carbodiimide. The cross‐linked species obtained were analyzed by polyacrylamide gel electrophoresis and compared with those known for unmodified S1. The carbodiimide‐catalyzed linkage between actin and dibromobimane‐modified S1 led to a singlet protein band migrating with an apparent molecular mass of 155 kDa, in contrast to the usual doublet bands of 175 kDa and 185 kDa produced with native S1. This result suggests that a change has occurred at the actin interface on the dibromobimane‐treated S1 heavy chain.

The covalent complex generated by carbodiimide cross‐linking between actin and dibromobimane‐modified S1 (27‐kDa + 50‐kDa + 20‐kDa fragments) was submitted to chemical hydrolysis with hydroxylamine. The nature of the products identified is consistent with the conclusion that the internal freezing of the heavy chain structure by dibromobimane induces the loss of the ability to cross‐linkage of the actin site on the 20‐kDa domain but does not affect the conformation of the second site on the 50‐kDa segment, which becomes the unique actin region cross‐linkable by actin.
The aromatic 1H‐NMR spectrum of plasminogen kringle 4
Authors: RAMESH Vasudevan , GYENES Marianne , PATTHY László , LLINÁS Miguel
Published: March 2005

The isolated kringle 4 domain of human plasminogen has been compared with homologous structures from bovine and porcine sources, both free and in the presence of the ligand 6‐aminohexanoic acid, by two‐dimensional 1H‐NMR spectroscopies at 300 MHz and 600 MHz. The chemical‐shift‐correlated, spin‐echo‐correlated, and double‐quantum‐correlated aromatic spectra of the three proteins reveal that the globular conformation of the fourth kringle is closely maintained throughout the set of homologs. Direct comparison shows that the three conserved Trp residues (at sites 25, 62 and 72) which exhibit highly non‐degenerate subspectra, find themselves in similar intramolecular environments. In particular, proton Overhauser experiments reveal that the close steric interaction between the Trp‐II (Trp62 or Trp25) indole group and the aromatic ring at site 74 (Tyr74 or Phe74) is strictly preserved. This feature forces the kringle inner loop, closed by the Cys51‐Cys75 link, to fold back onto itself so as to place the site 74 residue proximal to the Cys22‐Cys63 bridge.

Single‐residue substitutions enable unambiguous assignments of His‐I to His3, Tyr‐III to Tyr41 and Tyr‐IV to Tyr74. From this direct evidence, comparison with the kringle 1 spectrum, and the previously reported chemical modification of Tyr‐II (Tyr50) [Trexler M., Bänyai L., Patthy L., Pluck N. D. & Williams R. J. P. (1985) Eur. J. Biochem. 152, 439–446], Tyr‐I and Tyr‐V (the latter, an immobile ring on the 600‐MHz time scale) could be assigned to Tyr2 and Tyr9, respectively. Since Trp‐III has previously been assigned to Trp72 at the lysine‐binding site, the present study completes the assignment of 10 out of 12 aromatic spin systems in the kringle 4 1H‐NMR spectrum; the only ambiguity which remains concerns the Trp‐I and Trp‐II indole spin systems, which are totally identified but as yet only tentatively assigned to Trp25 and Trp62, respectively.
Characterization of a β‐galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase
Authors: CRENON Isabella , LADANT Daniel , GUISO Nicole , GILLES Anne‐Marie , BÂRZU Octavian
Published: March 2005

A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and β‐galactosidase activities, was purified and characterized. This protein, obteined by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth‐residue of β‐galactosidase through a pentapeptide Val‐Gly‐Asp‐Pro‐Val. The fusion protein was less stable than the native β‐galactosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and β‐galactosidase. ‘Truncated’ adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild‐type adenylate cyclase. Photoirradiation of the hybrid protein with 8‐azidoadenosine 5′‐triphosphate inactivated the adenylate cyclase activity, leaving intact the β‐galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.
Molecular cloning and sequencing of salmon gonadotropin β subunit
Authors: TRINH Khiet‐Yen , WANG Nam C. , HEW Choy L. , CRIM Laurence W.
Published: March 2005

Gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin‐A affinity chromatography. The α and β subunits were dissociated and fractionated by C‐18 reverse‐phase high‐performance liquid chromatography with a 0.1% trifluoroacetic acid/acetonitrile gradient. An enriched cDNA library was screened for the β‐GTH gene(s) using two synthetic oligonucleotides based on partial protein data. A positive, full‐size clone (E3) was identified and sequenced. It contains 657 base pairs and codes for a 142‐amino‐acid precursor protein. The mature salmon β‐GTH (119 amino acids) is structurally homologous to human luteinizing hormone and chorionic gonadotropin. The effect of testosterone implantation on pituitary GTH and β‐GTH mRNA was examined with radioimmunoassay and Northern blot analysis. There was a corresponding increase in both the pituitary GTH and mRNA levels.