bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of four tyrosine protein kinases from the particulate fraction of rat spleen
Authors: BRUNATI Anna Maria , PINNA Lorenzo A. 
Published: March 2005

Four distinct tyrosine protein kinases active on poly(Glu4,Tyr1) and angiotensin II, and operationally termed TPK‐I, TPK‐IIA, TPK‐IIB and TPK‐III have been resolved and partially purified from rat spleen particulate fraction by combining DEAE‐Sepharose, heparin‐Sepharose, phosphocellulose and polylysine‐agarose chromatographies. Once partially purified all of them are free of Ser/Thr‐specific protein kinase activity as judged using casein, histones, protamine and the peptide Arg‐Arg‐Ala‐Ser‐Val‐Ala as substrates.TPK‐I (apparent molecular mass 64 kDa, by gel filtration) and TPK‐IIA (54 kDa) share several properties, including substrate specificity and stimulation by heparin; the latter however is much more responsive to polylysine then the former (10‐ and 3‐fold maximum stimulation, respectively). Conversely TPK‐IIB (51 kDa) is markedly inhibited by heparin and it is also characterized by its unique substrate specificity: unlike the other three tyrosine protein kinases it by far prefers the tetrapeptide Glu‐Tyr‐Ala‐Ala over the decapeptide Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly and readily phosphorylates band‐3 protein of red cell membrane. The unusual preference for Mg2+ over Mn2+ as activator and the capability to phosphorylate calmodulin distinguish TPK‐III (61 kDa) from the other isoenzymes. Moreover TPK‐III is insensitive to heparin and polylysine and is inhibited by quercetin much more efficiently than the other enzymes (I50=10 μM).Upon incubation with [γ‐32P]ATP, TPK‐I, TPK‐IIA and TPK‐III give rise to alkali‐stable radiolabeled components of 61, 55 and 52 kDa respectively, as evaluated by PAGE/SDS. In every case such a radiolabeling takes place also in the presence of a large excess of phosphorylatable substrate (angiotensin II) while it is readily reversed by isotopic dilution with 10‐fold excess unlabeled ATP, supporting the view that it represents an autophosphorylation process. No (auto)phosphorylation product(s) could be detected in TPK‐IIB even if its amount, in terms of catalytic activity, was 10‐fold higher than that of the others.
Dual mechanism of phosphatidylinositol hydrolysis by substance P in brain
Authors: CATALÁN R. Edgardo , MARTÍNEZ Ana M. , ARAGONÉS M. Dolores , MIGUEL Begoña G. , ROBLES Antonio , HERNANDEZ Félix 
Published: March 2005

The treatment of cerebral cortex slices with substance P caused alterations in the phospholipid levels. A significant loss of phosphatidylinositol in a dose‐dependent manner was observed. In contrast, the levels of the major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were enhanced by the peptide.The effect of substance P on the fatty acid composition of phospholipids was also studied. The most relevant event was the decrease in the content of both stearic and arachidonic acids of phosphatidylinositol. This decrease was more evident at the lowest substance P concentration tested (65 pM).These results are consistent with the phosphatidylinositol breakdown caused by substance P in some tissues. Furthermore, our data indicate that this breakdown is selective depending on the peptide dose. Thus, in the presence of very low doses of substance P (65 pM) a preferential degradation of 1‐acyl(predominantly stearoyl)‐2‐arachidonoylglycerophosphoinositol molecular species occurs, whereas high doses of the peptide (0.65 μM) induce a generalized hydrolysis of phosphatidylinositol without showing any preference towards molecular species rich in arachidonic acid.Hence we describe for the first time a dual, dose‐dependent mechanism for phosphatidylinositol hydrolysis by substance P, suggesting the possibility that either phospholipase A2 or phospholipase C activation is involved.
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.
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.
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.
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 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.
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 .
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.
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

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.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.
Influence de la polymérisation de la d‐aminoacide oxidase sur le comportement de l'enzyme immobilisée sur chitosane par fixation covalente
Authors: LEMAINQUE Arnaud , BRAUN Jacques , LE GOFFIC François 
Published: March 2005

Influence of polymerization of the d‐amino‐acid oxidase on the behaviour of the enzyme covalently immobilized on chitosan. d‐Amino‐acid oxidase is a flavoprotein using FAD as cofactor. The enzyme has been immobilized in the presence of FAD on a non‐porous matrix: chitosan. This support is covalently bound to the enzyme with glutaraldehyde as cross‐linking reagent. It is characterized by a good mechanical resistance to mechanical stirring. The enzymatic assays have been performed in batch reactor with d‐phenylglycine as substrate by a spectrophotometric method which is based on the variation of the absorbance at 252 or 280 nm.The behaviour of the biocatalysts has been studied during repeated assays of 1 h at 25 °C in the absence of exogeneous FAD. The experimental results have been compared with those obtained with the soluble enzyme tested in the presence or in the absence of FAD. The dependence of d‐amino‐acid oxidase on FAD concentration has been studied. Immobilized enzyme on chitosan appears to be less sensitive to the association‐dissociation equilibrium of FAD. This property and the capacity of the enzyme to polymerize spontaneously in solution according to the experimental conditions have been established. The fact that the enzyme can exist in various oligomeric forms is of major importance because its catalytic expression is dependent of this phenomenon. The polymerization is known to be responsible for a decrease of the maximal rate V of the enzyme. It has also been shown that in the same way this decrease was accompanied by an improvement of the affinity of enzyme for substrates. Furthermore, the value of the dissociation constant of the apoenzyme‐FAD complex is significantly smaller as the degree of polymerization is high. The conclusion is that the dissociation of the cofactor can be avoided if the immobilization step is carried out at high concentration of enzyme which is favourable to its polymerization.