bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross‐linking of α and γ‐thrombin to distinct binding sites on human platelets
Authors: JANDROT‐PERRUS Martine , DIDRY Dominique , GUILLIN Marie‐Claude , NURDEN Alan T. 
Published: March 2005

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross‐linking with the membrane‐impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross‐linking of membrane glycoproteins. The proteins covalently linked to 125I‐labelled α and γ‐thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I‐α‐thrombin was detected in high‐molecular‐mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr∼ 400000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78‐kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active α‐thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine‐protease complex. The 240‐kDa complex was formed with 0.8‐100 nM α‐thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl‐inactivated α‐thrombin. After analysis of Triton‐X‐100‐soluble extracts of cross‐linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I‐α‐thrombin. These were a precipitate containing GPIIb‐IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240‐kDa complex. Significantly, 125I‐γ‐thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240‐kDa and 78‐kDa molecular mass range were not observed.We conclude that several binding sites for α‐thrombin are present at the platelet surface, and that GPIb is one of them. The studies with γ‐thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.
The control of glycogen metabolism in yeast
Authors: FRANÇOIS Jean , VILLANUEVA Maria Elena , HERS Henri‐Géry 
Published: March 2005

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6‐monophosphate and of fructose 2,6‐bisphosphate. The activity of glycogen synthase increased about 4‐fold and that of glycogen phosphorylase decreased 3–5‐fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6‐phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26°C, but were much more pronounced at the nonpermissive temperature of 35°C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate‐cyclase‐deficient mutant (cyr1), able to incorporate exogenous cyclic AMP.When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic‐AMP‐dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6‐bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6‐bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35°C without affecting cyclic AMP concentration.
The control of glycogen metabolism in yeast
Authors: FRANÇOIS Jean , HERS Henri‐Géry 
Published: March 2005

Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate‐cyclase‐deficient mutant that had been preincubated at the restrictive temperature of 35°C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20‐fold less active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6‐phosphate, the two forms of enzyme had a similar affinity for UDP‐Glc but differed essentially by their Vmax. Glucose 6‐phosphate had no effect on synthase a, but increased both Vmax and Km, of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km, of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20‐fold more active than synthase b.When an extract of cells that had been preincubated in the absence of glucose was gel‐filtered and then incubated at 30°C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6‐phosphate. When a gel‐filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30°C in the presence of ATP‐Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6‐phosphate and only slightly and irregularly stimulated by cyclic AMP.
Potential role for prostaglandin F2α, D2, E2 and thromboxane A2 in mediating the metabolic and hemodynamic actions of sympathetic nerves in perfused rat liver
Authors: IWAI Masaru , GARDEMANN Andreas , PÜSCHEL Gerhard , JUNGERMANN Kurt 
Published: March 2005

In isolated rat liver perfused at constant pressure perivascular nerve stimulation caused an increase of glucose and lactate output and a reduction of perfusion flow. The metabolic and hemodynamic nerve effects could be inhibited by inhibitors of prostanoid synthesis, which led to the suggestion that the effects of nerve stimulation were, at least partially, mediated by prostanoids [Iwai, M. & Jungermann, K. (1987) FEBS Lett. 221, 155–1601. This suggestion is corroborated by the present study.

Prostaglandin D2, E2 and F2α as well as the thromboxane A2 analogue U46619 enhanced glucose and lactate release and lowered perfusion flow similar to nerve stimulation.
The extents, the kinetics and the concentration dependencies of the metabolic and hemodynamic actions of the various prostanoids were different. Prostaglandin F2α and D2 caused relatively stronger changes of metabolism, while prostaglandin E2 and U46619 had stronger effects on hemodynamics. Prostaglandin F2α, elicited greater maximal alterations than D2 with similar half‐maximally effective concentrations. Prostaglandin F2α mimicked the nerve actions on both metabolism and hemodynamics best with respect to the relative extents and the kinetics of the alterations.
The hemodynamic effects of prostaglandin F2α could be prevented completely by the calcium antagonist nifedipine without impairing the metabolic actions of the prostanoid. Apparently, prostaglandin F2α influenced metabolism directly rather than indirectly via hemodynamic changes.

The present results, together with the previously described effects of prostanoid synthesis inhibitors, suggest that prostanoids, probably prostaglandin F2α and/or D2, could be involved in the actions of sympathetic hepatic nerves on liver carbohydrate metabolism. Since prostanoids are synthesized only in non‐parenchymal cells, nervous control of metabolism appears to depend on complex intra‐organ cell‐cell interactions between the nerve, non‐parenchymal and parenchymal cells.
Physiological stresses inhibit guanine‐nucleotide‐exchange factor in Ehrlich cells
Authors: ROWLANDS Anne G. , MONTINE Kathleen S. , HENSHAW Edgar C. , PANNIERS Richard 
Published: March 2005

Previously, we have shown that phosphorylation of the eukaryotic initiation factor eIF‐2α increases under several physiological stresses in which protein synthesis is inhibited in Ehrlich ascites tumor cells. As phosphorylated eIF‐2 [eIF‐2(αP)] is a potent inhibitor of guanine nucleotide exchange factor (GEF), it seemed likely that it was responsible for the inhibition. We have assayed GEF activity levels in extracts prepared from Ehrlich cells exposed to three such stresses, namely heat shock, serum deprivation and glutamine deprivation. Activity was estimated by the ability of GEF to enhance the release of [α‐32P]GDP from purified eIF‐2 [a modification of the reticulocyte lysate assay of Matts, R. L. & London, I. M. (1984) J. Biol. Chem. 259, 6708]. GEF activity was reduced from control values in extracts of heat‐shocked cells and serum‐deprived cells, concomitant with increased eIF‐2α phosphorylation. Inhibition of GEF activity in heat‐shocked and serum‐deprived cells was reversed to control levels by increasing the concentration of purified eIF‐2. GDP added as substrate in the GEF assay. Since we have shown elsewhere that eIF‐2(αP). GDP inhibits GEF by competition with eIF‐2. GDP, the complete reversal of inhibition of GEF activity in heat‐shocked and serum‐deprived cells indicates that inhibition is due solely to phosphorylation of eIF‐2α. In glutamine‐deprived cells phosphorylation of eIF‐2α was increased modestly and GEF activity was reduced but GEF activity could not be fully reversed by addition of eIF‐2 GDP, suggesting that GEF may also be regulated in other ways.There are greater amounts of GEF relative to eIF‐2 in Ehrlich cells (approximately 50%) compared with rabbit reticulocytes (approximately 20%). This explains the efficient rates of protein synthesis in control Ehrlich cells even though they have 30% of their eIF‐2 phosphorylated which is enough to inhibit GEF and initiation in reticulocytes completely but only enough to trap approximately 60% of the GEF in Ehrlich cells.
Cell‐free‐synthesized interleukin‐6 (BSF‐2/IFN‐β2) exhibits hepatocyte‐stimulating activity
Authors: GEIGER Thomas , ANDUS Tilo , BAUER Joachim , NORTHOFF Hinnak , GANTER Ursula , HIRANO Toshio , KISHIMOTO Tadamitsu , HEINRICH Peter C. 
Published: March 2005

Secretory products of cultured human blood monocytes contain a hepatocyte‐stimulating factor which is able to induce the acute‐phase proteins α2‐macroglobulin and fibrinogen in rat liver cells. Total RNA was isolated from unstimulated and lipopolysaccharide‐stimulated human monocytes and translated in a reticulocyte lysate. The capability of the cell‐free synthesized proteins to induce the acute‐phase proteins α2‐macroglobulin and fibrinogen was assayed in rat hepatocyte primary cultures and in the rat hepatoma cell line Fao. The products translated from the mRNA of lipopolysaccharide‐stimulated human monocytes induced mRNAs for α2‐macro‐globulin and fibrinogen and therefore contain hepatocyte‐stimulating factor. The translation products of unstimulated monocytes had no effect.A cDNA containing the coding sequence for interleukin‐6 (B‐cell stimulatory factor 2, interferon‐β2/26‐kDa protein, interleukin HP1) derived from human T‐cells cloned into the transcription vector pGEM4 was transcribed in vitro. Translation of the isolated RNA in a reticulocyte lysate led to the synthesis of a protein of about 25 kDa. This cell‐free synthesized interleukin‐6 exhibited hepatocyte‐stimulating activity measured by the induction of β‐fibrinogen mRNA in Fao cells. Using an antibody against interleukin‐6, two proteins of 22 kDa and 23 kDa were immunoprecipitated from the culture medium of lipopolysaccharide‐stimulated human monocytes. These two proteins were not synthesized by unstimulated monocytes.When total RNA from unstimulated human monocytes and lipopolysaccharide‐stimulated human monocytes and lymphocytes was subjected to Northern analysis and hybridized with the interleukin‐6 cDNA, a strong hybridization signal corresponding to an RNA of about 1300 bases was detected only in the RNA from lipopolysaccharide‐stimulated human monocytes, indicating that human monocytes express the interleukin‐6 gene after stimulation. The data presented in this paper strongly suggest that hepatocyte‐stimulating factor from human monocytes and interleukin‐6 from T‐cells are identical.
Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl‐CoA carboxylase
Authors: MUNDAY Michael R. , CAMPBELL David G. , CARLING David , HARDIE D. Grahame 
Published: March 2005

We have examined the sites phosphorylated on acetyl‐CoA carboxylase by three protein kinases which have been shown to inactivate the enzyme, i.e. cyclic‐AMP‐dependent protein kinase, acetyl‐CoA carboxylase kinase‐2 (ACK2, purified from rat mammary gland) and the AMP‐activated protein kinase (formerly called acetyl‐CoA carboxylase kinase‐3, purified from rat liver). Each protein kinase phosphorylates two out of three sites (termed 1–3) which have been established by amino acid sequencing. The two sites phosphorylated by each kinase can be recovered on separate peptides, TC1 and TC2, derived by combined digestion of the native enzyme by trypsin and chymotrypsin: TC1 = Ser‐2Ser(P)‐Met‐3Ser(P)‐Gly‐Leu; TC2 = Arg‐Met‐1Ser(P)‐Phe‐Cyclic‐AMP‐dependent protein kinase phosphorylates sites 1 and 2 exclusively, whereas the AMP‐activated protein kinase phosphorylates sites 1 and 3, plus at least one other minor site. ACK2 phosphorylates site 1 and, more slowly, an unidentified site(s) within TC1.We have also established the structures of the single major phosphopeptides (T1 and C1 respectively) which are recovered by HPLC after acetyl‐CoA carboxylase phosphorylated by cyclic‐AMP‐dependent protein kinase is digested with trypsin or chymotrypsin alone. T1 is related to TC1, and has the structure: Ser‐Ser(P)‐Met‐Ser‐Gly‐Leu‐His‐Leu‐Val‐Lys. C1 is identical with TC2.We have carried out studies on the correlation of the activity of acetyl‐CoA carboxylase with the occupancy of sites 1, 2 and 3 during phosphorylation by each of the three protein kinases. The results suggest that phosphorylation of site 3 is primarily responsible for the large decrease in Vmax produced by the AMP‐activated protein kinase, while phosphorylation of site 1 may be primarily responsible for the increase in A0.5 for citrate and more modest depression of Vmax produced by cyclic‐AMP‐dependent protein kinase and ACK2.Our results emphasize that amino acid sequence information is essential in the unequivocal interpretation of data from phosphopeptide mapping experiments and allow a more complete interpretation of previous data on phosphorylation of acetyl‐CoA carboxylase in intact cells. They also open the way to experiments which could establish the physiological roles of these protein kinases in the control of fatty acid synthesis.
Analysis of sites phosphorylated on acetyl‐CoA carboxylase in response to insulin in isolated adipocytes
Authors: HAYSTEAD Timothy A. J. , CAMPBELL David G. , HARDIE D. Grahame 
Published: March 2005

We have examined the sites phosphorylated on acetyl‐CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P‐labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the pep tide containing the sites phosphorylated in vitro by casein kinase‐2. The other may correspond to the T site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme‐bound phosphate (equivalent to < 0.1 mol phosphate/mol 240‐kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic‐AMP‐dependent protein kinase and calmodulin‐dependent multiprotein kinase respectively.We have sequenced peptides T4a and T4b from acetyl‐CoA carboxylase derived from control and insulin‐treated adipocytes, and also after phosphorylation in vitro with casein kinase‐2 and the calmodulin‐dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe‐Ile‐Ile‐Gly‐Ser4‐Val‐Ser5‐Gln‐Asp‐Asn‐Ser6‐Glu‐Asp‐Glu‐Ile‐Ser‐Asn‐Leu‐. Site 5 was phosphorylated by the calmodulin‐dependent protein kinase and site 6 by casein kinase‐2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P‐labelling of T4a. We were unable to isolate sufficient quantities of the other insulin‐sensitive peptide to determine its sequence.Our results are consistent with the idea that insulin activates either casein kinase‐2, or a protein kinase which has the same specificity as casein kinase‐2. The function of this modification is not clear, since phosphorylation by casein kinase‐2 has no direct effect on acetyl‐CoA carboxylase activity.
Tertiary structure of Escherichia coli tRNAThr3 in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase
Authors: THEOBALD Anne , SPRINGER Mathias , GRUNBERG‐MANAGO Marianne , EBEL Jean‐Pierre , GIEGE Richard 
Published: March 2005

The solution structure of Escherichia coli tRNAThr3 (anticodon GGU) and the residues of this tRNA in contact with the α2 dimeric threonyl‐tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N‐7 positions in guanosines and N‐3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N‐7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNAThr3. The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNAAsp, tRNAPhe and tRNATrp, already mapped with these probes.The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNAThr3 shows some similarities to that of yeast tRNAPhe and mammalian tRNATrp, especially in the D‐arm (positions 19 and 24) and with tRNATrp, at position 50, the junction between the variable region and the T‐stem. In the T‐loop, tRNAThr3, similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNAThr3 is unique as far as very strong protections are also found for phosphates 55 to 58 in the T‐loop. Compared with yeast tRNAAsp, the main differences in reactivity concern phosphates 19, 24 and 50.Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNAPhe. A striking conformational feature of tRNAThr3 is found in the 3′‐side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30–40 base pairs.Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl‐tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extra‐loop, and in the amino acid accepting region. The involvement of the anticodon of tRNAThr3 in the recognition process with threonyl‐tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA‐like structure present in the operator region of the thrSmRNA.
Purification and characterization of natural and recombinant human plasminogen activator inhibitor‐1 (PAI‐1)
Authors: ALESSI Marie Christine , DECLERCK Paul J. , DE MOL Maria , NELLES Luc , COLLEN Désiré 
Published: March 2005

Human plasminogen activator inhibitor‐1 (PAI‐1) was purified from the conditioned medium of endotoxin‐stimulated umbilical vein endothelial cell cultures by combinations of zinc‐chelate‐Sepharose chromatography, gel filtration on Sephacryl S‐300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA‐7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 μ;g/ml PAI‐1. The yield of PAI‐1 was 15 – 100 μ;g/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr= 46000 both under reducing and non‐reducing conditions. On gel filtration on Sephacryl S‐300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI‐1, and one with Mr= 46000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high‐Mr fraction revealed several bands including a main 46000‐Mr component, which reacted with anti‐(PAI‐1) antibodies on immunoblotting and neutralized tissue‐type plasminogen activator (t‐PA). The active high‐Mr fraction and the reactivated low‐Mr fraction of PAI‐1 inhibited t‐PA very rapidly with an apparent second‐order rate constant of (1.5–4) × 107 M−1 S−1.The cDNA of endothelial cell PAI‐1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high‐Mr and a low‐Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high‐Mr fraction was separated into the Mr‐46000 low‐Mr PAI‐1 and two other components with Mr 65000 and one barely entering the gel.When reactivated low‐Mr PAI‐1 was added to plasma, PAI activity and PAI‐1 antigen eluted with an apparent Mr 300000 on gel filtration, indicating that active PAI‐1 complexes with one or more binding proteins in plasma.