bluuebunny/crossref_metadata_2025 huggingface dataset:
Deoxycytidylate hydroxymethylase gene of bacteriophage T4
Authors: LAMM Norbert , WANG Yeong , MATHEWS Christopher K. , RÜGER Wolfgang
Published: March 2005

We describe two approaches to cloning and over‐expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild‐type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42. Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8‐kb fragment of cytosine‐substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild‐type allele by site‐directed mutagenesis. Both gene‐42‐containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed.

dCMP hydroxymethylase purified from one of the over‐expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N‐terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42.
A maximum of two tryptophan residues in gene‐32 protein from phage T4 undergo stacking interactions with single‐stranded polynucleotides
Authors: CASAS‐FINET José R. , TOULMÉ Jean‐Jacques , SANTUS René , MAKI August H.
Published: March 2005

The effect of specific photochemical and radiochemical modification of tryptophyl and cysteinyl residues of the gene 32 protein (gp 32) of bacteriophage T4 on its affinity towards single‐stranded polynucleotides has been investigated. Oxidation of Cys residues of gp 32 by the free‐radical anion I−2 induces a partial loss of the protein affinity, probably by affecting the metal‐binding domain which includes three of the four cysteine residues of gp 32. Ultraviolet irradiation of gp 32 in the presence of trichloroethanol results in the modification of three of its five Trp residues and total loss of the protein binding. Analysis of the relative affinity of ultraviolet‐irradiated gp 32 for single‐stranded polynucleotides suggest that modification of a Trp of enhanced reactivity occurs first and has no effect on the protein binding. Radiochemical modification of three Trp residues of gp 32 by (SCN)−2 results in total loss of activity. Complexation of gp 32 with denatured DNA prior to γ‐irradiation protects two Trp residues and prevents the protein inactivation. These results suggest that at most two Trp residues are involved in stacking interactions with nucleic acid bases. However, time‐resolved spectroscopic methods which allow us to monitor selectively the stacked tryptophan residues have not yielded evidence of more than a single residue undergoing such interactions.
Polypeptide elongation factor Tu from Halobacterium marismortui
Authors: GUINET Françoise , FRANK Rainer , LEBERMAN Reuben
Published: March 2005

A GDP‐binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high‐salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF‐Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF‐Tus, and by immunological studies. Like some other EF‐Tus the native protein can be cleaved with trypsin without concomitant loss of GDP‐binding ability. The molecular mass of this hEF‐Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein posesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS‐PAGE, are described.
Purification of the β subunit of the fibronectin receptor
Authors: TARONE Guido , MASCARELLO Patrizia , ZIBETTI Maurizio , GIANCOTTI Filippo G.
Published: March 2005

In this work we describe a method for purification of the β subunit of the mouse fibronectin receptor (CP135). Cellular glycoproteins were isolated from a detergent extract of SR‐Balb tumor cell membranes by two steps of affinity chromatography on lentil lectin‐Sepharose and wheat‐germ‐agglutinin–agarose. This material was subsequently bound to an Affi gel 102 column and eluted by increasing salt concentration. Most of the GP135 was eluted at 80 mM sodium chloride together with a few other components. A final step of hydroxyapatite chromatography in sodium dodecyl sulphate allowed elution of GP135 as a single chromatographic peak. Fractions containing GP135 were identified at each chromatographic step by immunoblotting with a specific antiserum. By this procedure GP135 was purified to homogeneity as judged by SDS‐PAGE analysis of 125I‐labelled material.
Rapid activation of human platelets by low concentrations of low‐density lipoprotein via phosphatidylinositol cycle
Authors: KNORR Marcus , LOCHER Rudolf , VOGT Esther , VETTER Wilhelm , BLOCK Lutz H. , FERRACIN Fabrizia , LEFKOVITS Hana , PLETSCHER Alfred
Published: March 2005

The interaction of low‐density lipoprotein (LDL) with the human platelet was investigated with regard to saturable high‐affinity binding, shape change, cytosolic free Ca2+ concentration, phosphatidylinositol (PtdIns) turnover, and thromboxane B2 biosynthesis. The experiments show that LDL, at a concentration approximately 100 times lower than in plasma, causes platelet activation concomitantly with stimulation of the PtdIns cycle and thromboxane B2 formation, similarly to other activators of platelets. The effects of LDL were inhibited by high‐density lipoprotein. The results suggest that activation of platelets by low concentrations of LDL may play a role in pathophysiological conditions and that platelet can serve as a model for studying the influence of LDL on various target cells.
Structural and transcriptional characterization of the external spacer of a ribosomal RNA nuclear gene from a higher plant
Authors: DELCASSO‐TREMOUSAYGUE Dominique , GRELLET Françoise , PANABIERES Franck , ANANIEV Evgueni D. , DELSENY Michel
Published: March 2005

A lambda recombinant phage, carrying a radish rDNA fragment spanning the complete external spacer and its borders, has been isolated and characterized by sequencing. The fragment is 2911 bp long and includes 486 bp of the 3′ end of the 25s rRNA sequence, 2349 bp of spacer and the first 76 bp of the 5′ end of the 18s rRNA sequence. The spacer can be divided into three regions: two unique domains flanking a 830‐bp region of repeated sequences. Seven repeats ranging from 80 to 103 bp can be recognized. They are separated by short arrays of 12–21 adenylic residues. Each repeat slightly differs from the others by single‐nucleotide changes or short deletions. Examination of single‐nucleotide changes common to two units suggests that a duplication arose during the evolution of this sequence. The repeated region was subcloned and used as a probe to demonstrate that it is highly species‐specific: in stringent conditions it does not cross‐hybridize with the spacer of ribosomal genes from closely related species such as Brassica. Transcription products, starting or finishing within the spacer sequence, were mapped by northern blotting, primer extension and S1 mapping. Two major precursors were identified starting respectively at positions 2095 and 2280. The region surrounding the start at 2095 presents extensive homology with an analogous region in maize, rye, mung bean, Xenopus and tse‐tse fly. However, longer transcripts can be detected. Several 3′ ends downstream of the 25s terminus were also observed. Taken together these results indicate that rDNA transcription and pre‐rRNA processing in plants are more complex than anticipated from previous studies.
Modulation of the substrate specificity of the polycation‐stimulated protein phosphatase from Xenopus laevis oocytes
Authors: HERMANN Jacques , CAYLA Xavier , DUMORTIER Kathelijn , GORIS Jozef , OZON René , MERLEVEDE Wilfried
Published: March 2005

A polycation‐stimulated (PCS) protein phosphatase was isolated in high yield (280 μg/100 g ovaries) from Xenopus laevis oocytes through a procedure involving a tyrosine‐agarose hydrophobic chromatography. The 220‐kDa enzyme contains a 35‐kDa and a 62‐kDa subunit. It was identified as the low‐Mr polycation‐stimulated (PCSL) protein phosphatase. The labile p‐nitrophenyl phosphatase activity, copurifying with the phosphorylase phosphatase activity, can be increased severalfold by preincubating the purified enzyme with ATP, its analogues or PPi. This activation is time‐dependent and accompanied by a parallel decrease of the phosphorylase phosphatase activity. Although the stimulation was antagonized by metal ions during the preincubation, the basal and ATP‐stimulated p‐nitrophenyl phosphatase requires Mg2+ or Mn2+ in the assay, with pH optima of 8.5–9 and 7.5 respectively.
Isolation and characterization of a cDNA clone corresponding to bovine cellular retinoic‐acid‐binding protein and chromosomal localization of the corresponding human gene
Authors: NILSSON Magnus H. L. , SPURR Nigel K. , SAKSENA Pushpa , BUSCH Christer , NORDLINDER Hans , PETERSON Per A. , RASK Lars , SUNDELIN Johan
Published: March 2005

A bovine adrenal cDNA library was constructed and a clone corresponding to cellular retinoic‐acid‐binding protein (CRABP) mRNA was isolated and sequenced. The insert of the clone corresponds to 75 bp of the 5′ untranslated portion, the whole translated and the complete 3′ untranslated portion of the bovine CRABP mRNA. A genomic Southern blot, probed with CRABP cDNA, indicated that only one copy of the gene is present in the human genome. Hybridizing bands in restricted chicken and fish DNA were also observed. Using the CRABP cDNA as probe we have located the human CRABP gene to chromosome 3 in hybridizations to mouse‐human, hamster‐human and rat‐human cell hybrids. In situ hybridizations on rat testis cells probed with CRABP and cellular retinol‐binding protein antisense mRNA indicate that both proteins are expressed in tubuli cells.
Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L27
Authors: TANAKA Tatsuo , KUWANO Yuh , ISHIKAWA Kiichi , OGATA Kikuo
Published: March 2005

We constructed cDNA libraries from 8‐9‐S poly(A)‐rich RNA from regenerating rat liver, isolated clones specific for ribosomal protein L27 and determined the nucleotide sequences of the cDNA clones. The longest cDNA consists of 15 base pairs from the 5′ leading sequence, the entire coding sequence of 411 base pairs, and the 3′ trailing sequence of 59 base pairs including the poly(A) tail. The primary structure of protin L27 was deduced from the sequence of nucleotides. Protein L27 contains 135 amino acids and has a molecular mass of 15666 Da. The amino‐terminal sequence agreed well with the published partial amino acid sequence and the calculated amino acid composition is also consistent with the reported composition determined for the hydrolyzate of L27.
Yeast ubiquinol: cytochrome c oxidoreductase is still active after inactivation of the gene encoding the 17‐kDa subunit VI
Authors: SCHOPPINK Peter J. , HEMRIKA Wieger , REYNEN J. Marleen , GRIVELL Leslie A. , BERDEN Jan A.
Published: March 2005

The single nuclear gene encoding the 17‐kDa subunit VI of yeast ubiquinol: cytochrome c oxidoreductase has been inactivated by one‐step gene disruption. Disruption was verified by Southern blot analysis of nuclear DNA and immunoblotting. Cells lacking the 17‐kDa protein are still capable of growth on glycerol and they contain all other subunits of complex III at wild‐type levels, implying that the 17‐kDa subunit is not essential for either assembly of complex III, or its function. In vitro, electron transport activity of complex III of mutant cells is about 40% of the wild‐type complex, but for the total respiratory chain no significant difference in activity was measured between mutant and wild type. The energy‐transducing capacity of the complex is not reduced in the absence of the 17‐kDa protein. In a relatively high proportion of the transformants, disruption of the 17‐kDa gene was accompanied by the appearance of a second mutation causing a petite phenotype. In these cells which lack cytochrome b, the presence of the 17‐kDa protein (after complementation) results in stabilization of cytochrome c1.
Inactivation of flavocytochrome b2 with fluoropyruvate
Authors: URBAN Philippe , LEDERER Florence
Published: March 2005

Fluoropyruvate inactivated oxidized flavocytochrome b2 (baker's yeast l‐lactate dehydrogenase) in a biphasic process yielding convex semilog plots of residual activity versus time. At each reagent concentration, rate constants k1 and k2 for the two phases could be calculated by simulation studies using one of the schemes proposed by Ray and Koshland [J. Biol. Chem. (1961) 236, 1973–1979]: E → E1 (fully active) → E2 (inactive). When plotted as a function of reagent concentration, the values of k2, but not those of k1, showed a saturation effect. Inactivation was slowed down by d‐lactate, a competitive inhibitor, and completely prevented by enzyme reduction. While no enzyme chemical modification could be demonstrated for the first step, the inactivation event of the second step could be ascribed to alkylation of a histidine belonging to proteolytic fragment β of the enzyme. The only histidine present in the fragment sequence is His‐373. In the enzyme three‐dimensional structure [Xia et al. (1987) Proc. Natl Acad. Sci. USA 84, 2629–2633] His‐373 is well located, close to the cofactor, to play the role of the active‐site base required by the chemical mechanism. Alternative chemical interpretations of the kinetic scheme are discussed, so is the difference between flavocytochrome b2 inactivation by fluoropyruvate and bromopyruvate.
Purification and characterization of a novel protein from bovine aorta that inhibits coagulation
Authors: REUTELINGSPERGER Chris P. M. , KOP Jos M. M. , HORNSTRA Gerard , HEMKER H. Coenraad
Published: March 2005

A novel inhibitor of blood coagulation has been isolated from the intima of bovine aorta. The inhibitor, vascular anticoagulant (VAC), has been purified to an active fraction that contains two Coomassie‐blue‐staining bands (Mr= 34000 and Mr= 32000, as judged by sodium dodecyl sulfate/polyacrylamide electrophoresis). Both bands are single‐chain proteins, having no glycoprotein features. Furthermore, they do not contain any detectable 4‐carboxyglutamic acid residues. Both proteins have an identical isoelectric pH of approximately 4.5. VAC binds in the presence of calcium ions to a bilayer consisting of 20% dioleoylglycerophosphoserine and 80% dioleoylglycerophosphocholine with a Kd= 6 nM. The binding is dependent on the calcium concentration: half‐saturation of binding occurs at a calcium concentration of 0.8 mM. The binding is completely reversible with EDTA. Furthermore the phospholipid/VAC ratio at saturation was n= 112 and n= 32 mol/mol for 0.5 mM Ca2+ and 2 mM Ca2+, respectively. Binding does not occur between VAC and pure dioleoylglycerophosphocholine. In a system with purified coagulation factors VAC inhibits the activation of prothrombin by factor Xa and calcium only in the presence of negatively charged phospholipids. VAC decreases the Vmax and increases the Km, of the factor‐Xa‐catalyzed prothrombin activation. Based on these results, we conclude that we have purified from bovine aortic intima an anticoagulant protein, which exerts its activity through a calcium‐dependent binding to negatively charged phospholipids, and thus interferes with the assembly of prothrombinase on the phospholipid surface.
The kinetics of glucose–fructose oxidoreductase from Zymomonas mobilis
Authors: HARDMAN Michael J. , SCOPES Robert K.
Published: March 2005

Glucose–fructose oxidoreductase operates by a classic ping‐pong mechanism with a single site for all substrates: glucose, fructose, gluconolactone and sorbitol. The Km values for these substrates were determined. The values of kcat are 200 s−1 and 0.8 s−1 for the forward and reverse directions respectively.

The overall catalytic process consists of two half‐reactions with alternate reduction of NADP+ and oxidation of NADPH tightly bound to the enzyme. Reduction of enzyme‐NADP+ by glucose and oxidation of enzyme‐NADPH by gluconolactone involve single first‐order processes. The values of the rate constants at saturating substrate are 2100 s−1 and 8 s−1 respectively; deuterium isotope effects indicate that these are for the hydrogen transfer step. Oxidation of enzyme‐NADPH by fructose is first order with a limiting rate constant of at least 430 s−1. The reaction of enzyme‐NADP+ with sorbitol is biphasic, with rate constants for both phases less than 1s−1. This behaviour is explained by a mechanism in which the slow cyclisation of the acyclic form of fructose follows its dissociation from the enzyme.

The rate‐determining steps for the overall reaction are probably dissociation of gluconolactone in the forward direction and hydrogen transfer from sorbitol to enzyme‐bound NADP+ in the reverse direction.
Electrostatic potential of macromolecules measured by pKa shift of a fluorophore
Authors: FRIEDRICH Klaus , WOOLLEY Paul
Published: March 2005

We have investigated the use of the pH‐sensitive fluorescein label as a probe for electrostatic potential in macromolecules. The practicality of this technique is demonstrated by its application to the 16S RNA molecule. The dependence of the electrostatic potential upon ionic conditions and upon the presence of ribosomal proteins and the state of the RNA was studied. The combination of electrostatic and anisotropy data emphasizes the r61e of the 30S ribosomal proteins, rather than of the renaturation of the 16S RNA or the presence of the 50S subunit, in shaping the environment of the 3′ terminus of the 16S RNA in the active ribosome.
Catabolism of Ap4A and Ap3A in whole blood
Authors: LÜTHJE Jürgen , OGILVIE Adaling
Published: March 2005

Adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and adenosine(5′)triphospho(5′)adenosine (Ap3A) are stored in large amounts in human platelets. After activation of the platelets both dinucleotides are released into the extracellular milieu where they play a role in the modulation of platelet aggregation and also in the regulation of the vasotone. It has recently been shown that the dinucleotides are degraded by enzymes present in the plasma [Luthje, J. & Ogilvie, A. (1987) Eur. J. Biochem. 169, 385–388]. The further metabolism as well as the role of blood cells has not been established. The dinucleotides were first degraded by plasma phosphodiesterases yielding ATP (ADP) plus AMP as products which were then metabolized to adenosine and inosine. The nucleosides did not accumulate but were very rapidly salvaged by erythrocytes yielding intracellular ATP as the main product.

Although lysates of platelets, leucocytes and red blood cells contained large amounts of Ap3 A‐degrading and Ap4A‐degrading activities, these activities were not detectable in suspensions of intact cells suggesting the lack of dinucleotide‐hydrolyzing ectoenzymes. Compared to ATP, which is rapidly degraded by ectoenzymes present on blood cells, the half‐life of Ap4A was two to three times longer.

Since the dinucleotides are secreted together with ADP and ATP from the platelets, we tested the influence of ATP on the rate of degradation of Ap4A. ATP at concentrations present during platelet aggregation strongly inhibited the degradation of Ap4A in whole blood. It is suggested that in vivo the dinucleotides are protected from degradation immediately after their release. They may thus survive for rather long times and may act as signals even at sites far away from the platelet aggregate.
A monoclonal antibody recognising the site of limited proteolysis of protein kinase C
Authors: YOUNG Susan , ROTHBARD Johnathan , PARKER Peter J.
Published: March 2005

A monoclonal antibody to protein kinase C is described that recognises the site of limited proteolysis on the native enzyme. Binding of the antibody to the purified kinase in vitro blocks partial proteolysis by trypsin, and introduction of the Fab fragment into a rodent glioma cell line inhibits phorbol‐ester‐induced down‐regulation of the kinase. These observations are discussed in the context of the domain structure of protein kinase C and the agonist‐induced proteolysis of the kinase in vivo.
Regulation of synthesis and secretion of major rat acute‐phase proteins by recombinant human interleukin‐6 (BSF‐2/1L‐6) in hepatocyte primary cultures
Authors: ANDUS Tilo , GEIGER Thomas , HIRANO Toshio , KISHIMOTO Tadamitsu , TRAN‐THI Thuy‐Anh , DECKER Karl , HEINRICH Peter C.
Published: March 2005

The regulation of the three major acute‐phase proteins α2‐macroglobulin, cysteine proteinase inhibitor and α1‐antitrypsin by recombinant human interleukin‐1β, recombinant human interleukin‐6 and recombinant human tumor necrosis factor α was studied in rat hepatocyte primary cultures. Synthesis and secretion of the acute‐phase proteins was measured after labeling with [35S]methionine and immunoprecipitation.

Incubation of hepatocytes with interleukin‐6 led to dose‐dependent and time‐dependent changes in the synthesis of the three major acute‐phase proteins and albumin, similar to those occurring in vivoduring experimental inflammation. α2‐Macroglobulin and cysteine proteinase inhibitor synthesis was induced 54‐fold and 8‐fold, respectively, 24 h after the addition of 100 units/ml interleukin‐6. At the same time synthesis of the negative acute‐phase protein albumin was reduced to 30% of controls. Half‐maximal effects were achieved with 4 units interleukin‐6/ml. Interleukin‐1β had only a partial effect on the regulation of the four proteins studied: only a twofold stimulation of α2‐macroglobulin and a 60% reduction of albumin synthesis were observed. Tumor necrosis factor α did not alter the synthesis of acute‐phase proteins.

The stimulation of α2‐macroglobulin and cysteine proteinase inhibitor synthesis by interleukin‐6 was inhibited by interleukin‐1β in a dose‐dependent manner.

In pulse‐chase experiments the effect of interleukin‐1β, interleukin‐6 and tumor necrosis factor α on the secretion of acute‐phase proteins was examined. Interleukin‐6 markedly accelerated the secretion of total proteins and α2‐macroglobulin, whereas the secretion of cysteine proteinase inhibitor, α1 antitrypsin and albumin was not affected. The inhibition of N‐glycosylation by tunicamycin abolished the effect of interleukin‐6 on the secretion of α2‐macroglobulin, indicating a possible role of interleukin‐6 on N‐glycosylation.
The auto‐oxidation of tetrahydrobiopterin
Authors: DAVIS Michael D. , KAUFMAN Seymour , MILSTIEN Sheldon
Published: March 2005

The product of the aerobic oxidation of tetrahydrobiopterin, quinonoid dihydrobiopterin, is unstable and rapidly rearranges to form a 7,8‐dihydropteridine. Kaufman [Kaufman, S. (1967) J. Biol. Chem. 242, 3934–3943] identified the stable product produced in 0.1 M phosphate pH 6.8, as 7,8‐dihydrobiopterin. However, Armarego et al. [Armarego, W. L. F., Randles, D. and Taguchi, H.(1983) Eur. J. Biochem. 135 393–403] questioned this assignment because they found that the dihydroxypropyl group on C‐6 was eliminated and 7,8‐dihydropterin was the predominant product when the aerobic oxidation was performed in 0.1 M Tris pH 7.6. In the present study we demonstrate that the rearrangement of the unstable quinonoid dihydrobiopterin results in a mixture of these two 7,8‐dihydropteridines at neutral pH, 25°C. Furthermore, we find that the loss or retention of the alkyl side‐chain is not solely dependent on the pH of the reaction mixture, as was previously assumed by Armarego et al., but rather is strongly influenced by the temperature and the type of buffer. In addition, we describe a new method for quantifying the relative amounts of these two 7,8‐dihydropteridines in mixtures of unknown concentrations. This method relies on multicomponent analysis of second derivative spectra and results in values which agree with the concentrations determined directly by HPLC.
Structure and activity of trypsin in reverse micelles
Authors: WALDE Peter , PENG Qiaoqian , FADNAVIS Nitin W. , BATTISTEL Ezio , LUISI Pier Luigi
Published: March 2005

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely Nα‐benzoyl‐l‐Arg ethyl ester, Nα‐benzoyl‐l‐Phe‐l‐Val‐l‐Arg p‐nitroanilide (BzPheValArg‐NH‐Np) in AOT and Nα‐benzyloxycarbonyl‐l‐Lys p‐nitrophenyl ester (ZLysO‐Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of α‐chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo, (wo= [H2O]/[AOT]). Whereas in the case of BzPheValArg‐NH‐Np kcat is much smaller than in water, in the case of ZLysO‐Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped‐flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate‐limiting step (acylation rather than hydrolysis). As in the case of α‐chymotrypsin, the maximal found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo= 13 approx., whereas λmax shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied. The stoichiometry of inhibition and the general inhibition behaviour is as in water, suggesting that exchange and recognition of macromolecules in reverse micelles are as fast as in the case of low‐molecular‐mass inserted molecules.
The function of galactosyl phosphorylpolyprenol in biosynthesis of lipoteichoic acid in Bacillus coagulans
Authors: YOKOYAMA Kohei , ARAKI Yoshio , ITO Eiji
Published: March 2005

Incubation of UDP‐[14C]galactose with membranes of Bacillus coagulans led to the formation of a radioactive glycolipid, which was tentatively characterized as β‐galactosyl phosphorylpolyprenol (Gal‐P‐prenol) on the basis of its chromatographic behavior and data from structural analysis of its sugar 1‐phosphate moiety. The sugar moiety of [14C]Gal‐P‐prenol was shown to be incorporated into a membrane‐bound polymer, which coincided with the diacyl form of lipoteichoic acid in its chromatographic behavior on columns of Sephacryl S‐300, DEAE‐Sephacel and octyl‐Sepharose. Hydrogen fluoride hydrolysis of the polymer afforded an α‐galactoside identical with Gal(α1 → 2)Gro obtained from lipoteichoic acids. The incorporation of galactose residues from [14C]Gal‐P‐prenol into the polymer was greatly enhanced by exogenous lipoteichoic acids, especially of the diacyl and monoacyl forms. The optimal pH and metal concentration for the Gal‐P‐prenol formation, respectively, were found to be 8.4 and 10 mM (MgCl2), whereas those for the transfer of galactose from this lipid intermediate to polymer were 4.5 and 16 mM (CaCl2).

The above results lead to the conclusion that Gal‐P‐prenol serves as the direct galactosyl donor in the synthesis of lipoteichoic acids in B. coagulans.
Transcriptional regulation of genes for plant‐type ribulose‐1,5‐bisphosphate carboxylase/oxygenase in the photosynthetic bacterium, Chromatium vinosum
Authors: VALLE Estela , KOBAYASHI Hirokazu , AKAZAWA Takashi
Published: March 2005

The content of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in the photosynthetic purple sulfur bacterium, Chromatium vinosum, grown either heterotrophically or autotrophically, was highly correlated with the level of 2.0‐kb mRNA encoding genes for both large (rbcL) and small (rbcS) subunits. This result indicates the transcriptional regulation of Rubisco biosynthesis in Chromatium cells. In the analysis of transcripts for rbcL and rbcS in Escherichia coli transformed by a plasmid bearing both genes downstream of E. coli tac promoter (pCKS1), the mRNAs were found to be the same sizes as those from Chromatium. However, we were unable to detect mRNA for Rubisco in E. coli harboring a plasmid containing the genes for Rubisco and its own promoter without any E. coli promoters (pCUB1). In the in vitro transcription experiment of pCKS1 and pCUB1 by E. coli RNA polymerase, it was observed that the enzyme could not recognize the Rubisco promoter. Therefore, we have purified RNA polymerase from Chromatium cells and developed a homologous in vitro transcription system. We have detected factor(s) for transcriptional regulation from either heterotrophically or autotrophically grown cells of Chromatium using the homologous in vitro transcription system.
14,15‐Dihydroxy‐5,8,10,12‐eicosatetraenoic
Authors: WETTERHOLM Anders , HAEGGSTRÖM Jesper , HAMBERG Mats , MEIJER Johan , RÅDMARK Olof
Published: March 2005

When 14C‐labeled (14S, 15S)‐14,15‐trans‐oxido‐5,8‐cis‐10,12‐trans‐eicosatetraenoic acid (14,15–leukotriene A4) was incubated with cytosolic epoxide hydrolase purified from mouse liver, one major radiolabeled product appeared. The structure was assigned as (14R, 15S)‐14,15‐dihydroxy‐5,8‐cis‐10,12‐trans‐eicosatetraenoic acid (14,15‐DHETE), based on analytical data as well as enzyme mechanistic considerations. The formation of this compound was dependent on time and enzyme concentration and was abolished after heat treatment of the enzyme. The apparent Km and Vmax values at 37°C were 11 μM and 900 nmol × mg−1× min−1 respectively. This enzymatic hydrolysis of 14,15‐leukotriene A4 respresents an additional mode of formation for 14,15‐DHETE, a compound previously found to modulate functions of human leukocytes.
Fur (ferric uptake regulation) protein and CAP (catabolite‐activator protein) modulate transcription of fur gene in Escherichia coli
Authors: DE LORENZO Victor , HERRERO Marta , GIOVANNINI Fabio , NEILANDS J. B.
Published: March 2005

A fusion between the fur (ferric uptake regulation) gene, known to mediate negative regulation of iron absorption in Escherichia coli, and lacZ was constructed in vitro.β‐Galactosidase levels of cells harboring this fusion were under the control of sequences contained in a 185‐bp DNA fragment located upstream of the fur structural gene. The fusion was prepared in multicopy (pVLN102 plasmid) and low‐copy‐number states, the latter constructed as a λ phage lysogen carrying a fur′‐′lacZ insert. DNase I footprinting experiments with purified Fur protein, performed on a 250‐bp restriction fragment carrying the promoter region of the fusion, showed the presence of a single Fur‐protected site overlapping the – 10 region of a potential promoter sequence. Examination of the DNA sequences located upstream of the fur gene revealed a possible binding site for the catabolite‐activator protein (CAP). β‐Galactosidase synthesis of E. coli cells harboring the fusion were measured in fur, crp and cya genetic backgrounds and compared with the corresponding levels in wild‐type strains. The data obtained indicate a moderate autoregulation of fur expression by its gene product and also a significant stimulation by the cAMP‐CAP system. Transcription start sites were mapped by primer‐extension experiments with total RNA obtained in vivo from cells harboring pVLN102. The results show that transcription of the fur gene is initiated from at least two different sites separated by 6 bp, which appear to originate from two overlapping promoters sensitive to catabolic activation.
Interaction of influenza virus with gangliosides and liposomes containing gangliosides
Authors: SLEPUSHKIN Vladimir A. , STAROV Alexander I. , BUKRINSKAYA Alice G. , IMBS Andrei B. , MARTYNOVA Maria A. , KOGTEV Leonid S. , VODOVOZOVA Elena L. , TIMOFEEVA Natalya G. , MOLOTKOVSKY Julian G. , BERGELSON Lev D.
Published: March 2005

It has already been shown that influenza virus binds unspecifically to liposomes containing ganglioside GM1 whereas with gangliosides GD1b and GT1b binding occurs in a specific and saturable manner [Slepushkin et al. (1986) Biol. Membr. 3, 229–235]. In the present study the mode of interaction between influenza virus and various gangliosides or phospholipid liposomes containing cholesterol and gangliosides has been investigated.

The influence of exogenous gangliosides on the structure of the viral envelope was studied using fluorescent and photoactivatable phospholipids incorporated into the viral membrane. With both types of probes maximal effects of gangliosides were caused by GT1b. Addition of that ganglioside resulted in a marked decrease in the fluorescence polarization (P) of fluorescent labeled virus as well as in substantial changes in the binding of photoactivatable analogues of sphingomyelin and phosphatidylcholine to virus proteins, mainly hemagglutinin. The effects of GT1b and GD1b on P value were comparable, whereas gangliosides with other oligosaccharide chains caused much smaller changes in P. Furthermore GT1b but not GM1b influenced phospholipid‐hemagglutinin cross‐linking.

Interaction of the virus with large unilamellar liposomes was monitored by two fluorescence assays based on resonance‐energy transfer from the tryptophans and tyrosines of viral proteins to vesicles labeled with a triacylglycerol (anthrylvinyldioleoylglycerol) or from these labeled vesicles to virions labeled with a perylenoyl derivative of galactosylcerebroside (PGalSph). A third fluorescence assay was based on relief of self‐quenching in PGalSph‐labeled virions, upon low‐pH‐induced virus‐liposome fusion. With all three fusion assays the changes of fluorescence caused by GT1b were more pronounced than those induced by GM1. On the other hand, virus‐induced release of [14C]glucose from multilamellar liposomes was enhanced by GM1 but not by GT1b or GD1b.

It is concluded that the interaction of GT1b or GD1b with virus hemagglutinin induces a rearrangement of the viral lipids rendering lipid bilayer areas of the viral envelope significantly fluid, which in turn promotes fusion of the virus with target membranes. Probably virus‐liposome fusion and virus‐induced liposome leakage are brought about by different mechanisms depending on specific or unspecific binding of the virions to the target.
A light‐dependent dicyclohexylcarbodiimide‐sensitive Ca‐ATPase activity in chloroplasts which is not coupled to proton translocation
Authors: PICK Uri , WEISS Meira
Published: March 2005

The early observation of light‐dependent Ca‐ATPase activity in chloroplast thylakoids [Avron, M. (1962) J. Biol. Chem. 237, 2011–2017] has been reinvestigated. It is demonstrated that in contrast to light‐triggered Mg‐ATP activity, Ca‐ATPase activity is strictly dependent on , the transthylakoid membrane electrochemical potential gradient, since (a) there is an absolute requirement for continuous illumination; (b) electron‐transport mediators that catalyze proton uptake, like phenazinemethosulphate, methylviologen of ferricyanide, are essential and (c) uncouplers inhibit the activity. The Ca‐ATPase activity is essentially unaffected by dithiols, but is inhibited by CF0‐CF1 inhibitors including tentoxin, dicyclohexylcarbodiimide and antisera to CF1.

Addition of Ca‐ATP to thylakoids does not induce ΔpH or ΔΨ (the electrical potential gradient) formation either in the light or following preillumination with dithiols, demonstrating that it is not coupled to proton translocation. It is also demonstrated that Ca‐ATP or Ca‐ADP does not induce a proton leak through CF0‐CF1.

It is concluded that the Ca‐ATPase activity in chloroplast thylakoid reflects a partial reaction of ATP synthesis catalyzed by CF0‐CF1, which is internally uncoupled from proton translocation but is dependent on energization by a transmembrane .
The N terminus of laminin A chain is homologous to the B chains
Authors: HARTL Lydia , OBERBÄUMER Ilse , DEUTZMANN Rainer
Published: March 2005

A major proteolytic fragment (E1/E1‐4) of the basement membrane protein laminin, comprising the three short arms with some terminal globules missing, was isolated by elastase digestion, and partial protein sequence data were determined for several tryptic peptides. Sequences which corresponded to A‐chain structures were used to synthesize oligonucleotides for the construction and screening of a primer‐extended cDNA library from mouse PYS‐2 cells. A clone of 1.1 kb was obtained and shown by sequencing to correspond to the 5′ end of the 10‐kb mRNA of the A chain of laminin. The clone contains 77 nucleotides of 5′ untranslated sequence and a region coding for 334 amino acids, including a presumptive signal peptide of 24 amino acids. The sequence is 30% homologous to the corresponding N‐terminal part of the B1 chain of laminin, suggesting the same structure for both domains. The data present further evidence for a recent structural model which postulates that each of the three laminin polypeptide chains forms a distinct short arm.
Structure of the horseradish peroxidase isozyme C genes
Authors: FUJIYAMA Kazuhito , TAKEMURA Hiroshi , SHIBAYAMA Shiro , KOBAYASHI Kazuo , CHOI Jong‐Kyu , SHINMYO Atsuhiko , TAKANO Mitsuo , YAMADA Yasuyuki , OKADA Hirosuke
Published: March 2005

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19–23 (1976)] with additional sequences at the N and C terminal. All three inserts included in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91–94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S–S‐bond‐forming Cys, were conserved in the three isozymes, but a few N‐glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5′ upstream and a poly(A) signal 3′ downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.
Cloning and nucleotide sequence of a heat‐stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum
Authors: FUKUSUMI Shoji , KAMIZONO Akito , HORINOUCHI Sueharu , BEPPU Teruhiko
Published: March 2005

A highly heat‐stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686‐amino‐acid protein of relative molecular mass 81200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2‐terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2‐terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH‐stability and heat‐stability, were examined. The amylase was found to be a highly liquefying‐type.
Palmitate binding to serum albumin, measured by rate of dialysis
Authors: BRODERSEN Rolf , HONORÉ Bent , ANDERSEN Signe
Published: March 2005

Dialysis experiments were performed with an acetylcellulose membrane between two identical sample solutions; a trace amount of radiolabelled palmitate was added on one side and the rate of dialytic equilibration of the label was measured. By comparison with rates measured in standard experiments, using pure albumin solutions, we obtained the reserve albumin concentration for binding of palmitate, previously defined as the concentration of a pure standard albumin which binds the labelled ligand as tightly as it is bound in the sample. Two techniques were developed, one for 1‐ml sample volumes, another for 25 μl.

Reserve albumin for binding of palmitate, measured in pure albumin solutions, decreased equally on addition of palmitate and stearate, slightly less with oleate and still less with linoleate, indicating that palmitate and stearate are bound competitively while interaction with binding of the unsaturated acids is less pronounced. Chloride ions compete with binding of palmitate.

Reserve albumin concentration in serum samples from 33 male adults was 420 ± 59 μM (mean ± SD), and in 33 females, 351 ± 50 μM. Umbilical cord sera from ten newborn infants averaged 172 ± 56 μM. The reserve albumin concentrations in the sera of newborn infants are low compared with the normal albumin concentration, which is about 600 μM. This appears to indicate the presence of an albumin species with lower affinity for palmitate.
Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets
Authors: TYSNES Ole‐Bjørn , VERHOEVEN Adrie J. M. , HOLMSEN Holm
Published: March 2005

Human platelets were labelled with [32P]Pi and [3H]glycerol before gel filtration. In unstimulated cells, the specific 32P radioactivity in phosphatidic acid (PtdOH) was similar to that of phosphatidylinositol (PtdIns) but only 4% of that of the γ‐phosphate of ATP. Upon 3 min of stimulation with 0.5 U/ml of thrombin, there was a 20‐fold increase in specific 32P radioactivity of PtdOH which approached that of the ATP γ‐phosphate. Based on constant rates of synthesis and removal, this thrombin‐induced increase in specific 32P radioactivity in PtdOH allowed us to calculate the flux of phosphate through PtdOH upon stimulation. Synthesis and removal occurred at rates of 107 and 52 nmol min−1/1011 cells, respectively. The specific [3H]glycerol radioactivity was similar in PtdIns, phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate in unstimulated platelets. In PtdOH, it was 50% of that of the inositol phospholipids. Thrombin stimulation induced no changes in the specific 3H radioactivity of the inositol phospholipids whereas specific [3H]PtdOH increased to the level of these lipids. It is concluded that PtdIns, PtdInsP and PtdInsP2 exist in a metabolic homogenous pool in human platelets.
Identification of a myometrial oxytocin‐receptor protein
Authors: FAHRENHOLZ Falk , HACKENBERG Mario , MÜLLER Michael
Published: March 2005

The specific binding of [3H]oxytoxin to uterine membrane peparations derived from different species at late pregnancy was examined. The highest receptor density (bmax value) was found in membranes derived from the myometria of guinea pigs between day 60 post‐conception (bmax= 3.6 ± 0.1 pmol/mg) and day 65 (bmax= 4.4 ± 0.1 pmol/mg). The similarity of Kd values for oxytocin binding (Kd= 2.6 ± 0.2 nM) and for vasopressin binding (Kd= 2.1 ± 0.4 nM) to the same membranes derived from a guinea pig myometrium indicate a homogenous population of high‐affinity binding sites which do not discriminate between these two hormones. Competitive binding experiments with specific oxytocin agonists containing either sarcosine or N‐methylalanine in the place of Pro7 demonstrated that these myometrial receptors have the pharmacological properties of oxytocin receptors.

The analogue of 1‐deamino‐[8‐lysine]vasopressin containing a photoreactive azidophenylamidino group at the sidechain of Lys8 retained roughly the same receptor affinity as oxytocin. In photoaffinity labelling experiments with the tritium‐labelled analogue a membrane protein from guinea pig myometrium with an apparent relative molecular mass Mr of 78000 ± 5000 (n= 13) was preferentially labelled. The labelling of this protein was completely suppressed by a 100‐fold molar excess of either oxytocin, or [Sar7]oxytocin or [Thr4, Sar7]oxytocin, but not by other peptide hormones. These results provide evidence that the labelled 78000‐Mr protein is a myometrial oxytocin‐receptor protein.
A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor
Authors: CONLON J. Michael , THIM Lars
Published: March 2005

image
.

There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin‐releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1‐P1′ reactive site lysine‐isoleucine (or arginine‐isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine‐asparagine bond. This region does, however, show limited homology to the reactive centre of human α1‐protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.