bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional molecular mass of binding sites for [3H]dihydrotetrabenazine and [3H]reserpine and of dopamine β‐hydroxylase and cytochrome b561 from chromaffin granule membrane as determined by radiation inactivation
Authors: GASNIER Bruno , ELLORY J. Clive , HENRY Jean‐Pierre 
Published: March 2005

The monoamine transporter of chromaffin granule membrane has two distinct high‐affinity binding sites for tetrabenazine and reserpine, which can be assayed by [3H]dihydrotetrabenazine and [3H]reserpine binding, respectively. The functional molecular mass of the components bearing these sites has been investigated by the radiation inactivation technique. The decline of [3H]dihydrotetrabenazine binding activity with increasing radiation doses followed a single exponential, from which a functional molecular mass of 68 kDa was derived for tetrabenazine binding sites. [3H]Reserpine binding activity declined in a more complex way; however, under conditions where high‐affinity reserpine binding sites were specifically assayed, the decline was also exponential, corresponding to a functional molecular mass of 37 kDa for these sites. The figures obtained for high‐affinity tetrabenazine and reserpine binding sites are consistent with previous values obtained by photoaffinity of tetrabenazine and serotonin binding sites, respectively. It is thus concluded that the monoamine transporter has an oligomeric structure.By the radiation inactivation technique, cytochrome b561 and dopamine β‐hydroxylase have functional molecular masses of 25 and 123 kDa, respectively. The latter value might be attributed to the dimeric form of the enzyme.
Nucleotide sequence of barley chymotrypsin inhibitor‐2 (CI‐2) and its expression in normal and high‐lysine barley
Authors: WILLIAMSON Martin S. , FORDE Janice , BUXTON Bernadette , KREIS Martin 
Published: March 2005

cDNA clones for chymotrypsin inhibitor‐2 (CI‐2) have been isolated from an endosperm‐specific library of barley using a synthetic oligonucleotide probe. The nucleotide sequences of several of the cDNAs predict an open reading frame (beginning with an ATG codon) which encodes a protein of 84 residues (Mr 9380). In the longest clone another ATG codon is present, a further 69 nucleotides upstream. The nucleotide sequence between these two ATG codons predicts an amino acid sequence with the characteristics of a signal peptide, as found in other cloned plant protease inhibitors. However, it contains an in‐frame TAA stop codon, which is also present in all of the shorter cDNAs which extend into this region. From in vitro translation experiments, using mRNAs synthesized from cDNAs, we conclude that, in vitro, translation of all or the vast majority of CI‐2 mRNAs begins at the second ATG codon, 31 nucleotides downstream from the ochre stop codon.Southern blotting of genomic DNA shows that CI‐2 is encoded by a small multigene family, while sequence analysis of the cDNAs shows that at least two sub‐families of mRNAs, which are more than 90% homologous, are present in the endosperm. Northern blotting analysis shows that related but different sequences are present in leaf and shoot RNA populations.Further Northern blot hybridizations using RNA from the normal line, Sundance, and the high‐lysine barley mutant, Hiproly, show that endosperms of the latter contain greatly increased levels of CI‐2 mRNA. This correlates with the increased amount of CI‐2 protein deposited in Hiproly, and demonstrates that the differential expression of CI‐2 in the two genotypes is controlled at the level of transcription and/or stability of the mRNA. In contrast, the abundance of CI‐2 mRNAs in leaves and shoots is not affected.
The effect of monovalent and divalent cations on the ATP‐dependent Ca2+‐binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+‐transport ATPase
Authors: MEDDA Pankaj , FASSOLD Elisabeth , HASSELBACH Wilhelm 
Published: March 2005

The coupling of Ca2+ movements and phosphate fluxes as well as the time‐dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg‐dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 μM does not allow more than one reaction cycle to occur. The respective fractions of ADP‐sensitive and ADP‐insensitive phosphoenzyme have been determined. The chosen experimental conditions (0–1 C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively.It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P ⇀ E2P transition, E2P hydrolysis, and E2⇀ E1 transformation). Mg2+ represents the sole ionic co‐factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P ⇀ E2P transition rate (including Ca2+ translocation and Ca2+ release from low‐affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded.We find Ca2+ release to occur time‐coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low‐affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19–49, Academic Press, Orlando].
Regulation of the cardiac calcium channel by protein phosphatases
Authors: HESCHELER Jürgen , KAMEYAMA Masaki , TRAUTWEIN Wolfgang , MIESKES Gottfried , SÖLING Hans‐Dieter 
Published: March 2005

The calcium current (ICa) through the L‐type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285–293]. We investigated the possible contribution of the ‘catalytic subunits’ of protein phosphatase 1 and 2A in the down‐regulation of the cardiac calcium channel. Single guinea‐pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 μM) as well as 2A (2.3 μM) completely abolished the increase of ICa induced by isoprenaline (0.05 μM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor‐2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash‐out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor‐2. (4) Perfusion of the myocyte under basal conditions with adenosine 5′‐[γ‐thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 μM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min.From these results we make the following conclusions. (1) The calcium channel from guinea‐pig myocytes is regulated by phosphorylation‐dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down‐regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.
The peptidyltransferase centre of the Escherichia coli ribosome
Authors: TATE Warren P. , SUMPTER Vicki G. , TROTMAN Clive N. A. , HEROLD Marzel , NIERHAUS Knud H. 
Published: March 2005

Modification of the Escherichia coli 50S ribosomal subunit with histidine‐specific diethyl pyrocarbonate affects peptide bond formation and release‐factor‐dependent peptidyl‐tRNA hydrolysis.
Unmodified L16 can restore activity to a split protein fraction from the altered subunit but other proteins of the core also contain histidine residues important for the activity of the peptidyltransferase centre.
When isolated and purified by centrifugation, particles reconstituted with unmodified proteins and modified L16 do not retain the altered L16. The modified protein does mediate the partial restoration of peptide bond formation and release‐factor‐2 activities to these particles. It must be exerting its effect during the assembly of the peptidyltransferase centre in the reconstituted particle.
A particle could be reconstituted which lacks L16 and has significant activity in peptide bond formation and peptidyl‐tRNA hydrolysis. L16 stimulates these activities.
A tighter ribosomal binding of the release factor 2, dependent upon the absence of protein L11, can in part compensate for the loss of activity of the peptidyltransferase centre when it is assembled with either modified L16 or in the absence of L16. The protein and its histidine residue seem important, therefore, for the peptidyltransferase centre to be formed in the correct conformation but not essential for activity once the centre is assembled.
Characterisation of a type‐I collagen trimeric cross‐linked peptide from calf aorta and its cross‐linked structure
Authors: HENKEL Werner , GLANVILLE Robert W. , GREIFENDORF Dieter 
Published: March 2005

A collageneous trimeric cross‐linked peptide has been isolated from the insoluble matrix of calf aorta, using trypsin solubilisation, and purified by gel filtration, cation‐exchange chromatography and reversed‐phase HPLC. Molecular mass and amino acid composition indicated that the C‐terminal, non‐helical region of type I collagen in its dimer form, designated as [Colc(I)]2, is cross‐linked to a tryptic peptide TN(I) from the N‐terminal helical cross‐link region of an adjacent type I molecule, forming the cross‐linked peptide [Colc(I)]2× TN(I).Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross‐linking domains ColC(I) and TN(I) and furnished the first direct chemical evidence for the 4D staggered arrangement of type I molecules within native fibers.The trifunctional cross‐linking amino acid pyridinoline was shown to occur in the peptide, confirming the peptides three‐chain structure. Pyridinoline was isolated from the cross‐linked peptide by preparative amino acid analysis and reversed‐phase HPLC and identified by (a) its ultraviolet absorption spectra, (b) its fluorescence excitation and emission spectra and, for the first time, (c) its time‐of‐flight secondary ion‐mass spectrum. The high sensitivity of the latter method, exceeding that of fast‐atom‐bombardement mass spectroscopy by three orders of magnitude, allowed detection of pyridinoline in the picomole range.The occurrence of pyridinoline in non‐stoichiometric amounts, the presence of hydroxylysine in hydrolysates of all cross‐linked peptides and the finding that hydrolysates also contained an unidentified component indicated that there is at least one cross‐link form that is different from pyridinoline and is hydrolysable.
Immunological evidence for the existence of H1‐like histone in yeast
Authors: SREBREVA Ljuba , ZLATANOVA Jordanka , MILOSHEV George , TSANEV Roumen 
Published: March 2005

In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt.Perchloric‐acid‐extracted proteins from whole cells contain only two fractions which comigrate with ‘authentic’ yeast high‐mobility‐group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross‐reaction with anti‐(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1.The isolation of ‘authentic’ yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti‐HMG antiserum whereas the richer preparation also gives considerable cross‐reaction with the anti‐H1 antiserum. Immunoblotting analysis performed on the salt‐extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti‐H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.
Inhibition of leukotriene D4 catabolism by D‐penicillamine
Authors: HUBER Michael , KEPPLER Dietrich 
Published: March 2005

Inhibition of the catabolism of the most biologically potent cysteinyl leukotriene, LTD4, was studied in rat hepatoma cells in vitro and in the rat in vivo. LTD4 dipeptidase, an ectoenzyme on the surface of AS‐30D hepatoma cells, exhibited an apparent Km value of 6.6μM for LTD4. D‐Penicillamine and L‐penicillamine inhibited this enzyme activity with apparent Ki values of 0.46 mM and 0.21 mM respectively. Bestatin, an inhibitor of the aminopeptidase activity of hepatoma cells, did not affect LTD4 hydrolysis at concentrations as high as 5 mM, indicating that the aminopeptidase did not contribute to LTD4 catabolism.In the rat in vivo, D‐penicillamine also inhibited LTD4 catabolism. After intravenous injection of [3H]LTC4 an accumulation of [3H]LTD4 and a retarded formation of [3H]LTE4 were observed in the circulating blood after D‐penicillamine pretreatment. Within 1 h after intravenous [3H]LTC4 injection, about 80% of the administered radioactivity was recovered in bile. After D‐penicillamine pretreatment [3H]LTD4 was the major biliary leukotriene metabolite, whereas in untreated controls leukotriene metabolites more polar than LTC4 predominated in bile.After stimulation of endogenous leukotriene production in vivo by platelet‐activating factor, N‐acetyl‐LTE4 was the major cysteinyl leukotriene detected in bile. D‐Penicillamine treatment prior to platelet‐activating factor resulted in the accumulation of LTD4, which under these circumstances was the major endogenous leukotriene metabolite detected in bile.
DNA methylation in wheat
Authors: THEISS Gudrun , SCHLEICHER Roland , SCHIMPFF‐WEILAND Gabriele , FOLLMANN Hartmut 
Published: March 2005

The origin and function of the large amount of 5‐methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300‐fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE‐cellulose, Sephadex G‐75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in double‐stranded DNAs isolated from wheat, maize, calf thymus or bacteria using S‐adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de novo and in maintenance methylation. A relative molecular mass of 50000–55000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of Mr= 50000 and one other component (Mr= 35000).The preference for endogenous, double‐stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA.
Mitochondrial phosphate carrier
Authors: LIGETI Erzsébet , FONYÓ Attila 
Published: March 2005

1. The phosphate carrier of isolated rat liver mitochondria was studied.2. To investigate the effect of the substrate on the external side of the transport protein, a technique was required by which the extramitochondrial phosphate concentration could be changed without any effect on either the phosphate content or the pH value in the intramitochondrial compartment. This was found when non‐respiring mitochondria were incubated with the ionophore nigericin.3. Using this method variation of the phosphate concentration outside the mitochondria did not protect the essential SH groups located at the external surface of the phosphate carrier.4. Interrelations within the phosphate carrier unit were investigated in mitochondria in which only one of the two essential thiol groups of the carrier unit was free. These mitochondria were however able to carry out phosphate transport.5. Alterations of the intramitochondrial pH‐induced modification of the position of the single free sulfhydryl group at the external surface of the membrane similarly to the changes which occur in intact mitochondria where both SH groups are free [E. Ligeti and A. Fonyó (1984) Eur. J. Biochem. 139, 279–285].6. It is suggested that the sulfhydryl groups essential for the transport function do not participate in the formation of the substrate binding site of the carrier protein. They probably have a role in the conformational change necessary for the translocation step. The possibility of two equivalent but independently functioning parts within the carrier unit is raised.
The effect of thrombin on the complex between factor VIII and von Willebrand factor
Authors: HAMER Rob J. , KOEDAM Johannes A. , BEESER‐VISSER Nel H. , SIXMA Jan J. 
Published: March 2005

Purified human factor FVIII (FVIII; 6000–8000 U/mg) was radiolabeled and bound to immobilized von Willebrand factor (vWF). The complex was incubated with human thrombin. Thrombin induced a release of 65% of the radioactivity initially bound. Released FVIII fragments and fragments remaining bound during incubation with thrombin were analyzed using gel electrophoresis. This led to the following observations. Released fragments largely consisted of Mr‐70000 and Mr‐50000 fragments; Mr‐90000 and Mr‐80000 fragments were only found in the fractions remaining bound to vWF and decreased with time. In contrast to these digestion products of FVIII, the Mr‐42000 heavy‐chain fragment remained bound to vWF, comprising the larger part of the radioactivity after a 2‐h incubation. No thrombin‐induced cleavages were observed in vWF. Furthermore, vWF‐coated wells preincubated with thrombin were still able to bind 125I‐FVIII.These results implicate a new concept for the activation of vWF‐bound FVIII. Activation is a multistep process in which several cleavages are necessary to produce and release a coagulant‐active FVIII molecule (FVIIIa), which is probably an Mr‐50000/70000 heterodimer. Inactivation of FVIIIa is likely to be the result of a nonproteolytic dissociation due to loss of the joining divalent cation(s).
Cycloheximide induces accumulation of vasoactive intestinal peptide (VIP) binding sites at the cell surface of a human colonic adenocarcinoma cell line (HT29‐D4)
Authors: LUIS José , MARTIN Jean‐Michel , EL BATTARI Assou , FANTINI Jacques , GIANNELLINI Fernand , MARVALDI Jacques , PICHON Jacques 
Published: March 2005

Incubation of monolayers of HT29‐D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 μM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand.The increase in 125I‐VIP‐binding capacity was dose‐dependent between 0.35 μM and 17.5 μM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5–100 μM) a plateau corresponding to a twofold increase in VIP‐binding capacity was reached independently of the extent of protein synthesis inhibition.We found that VIP receptors of HT29‐D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide).After inactivation of 90% of cell‐surface VIP receptors by α‐chymotrypsin, we observed a biphasic kinetic of reappearance of VIP‐binding sites. 40% of VIP‐binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide‐treated cells. However, the slow recovery was inhibited in cycloheximide‐treated cells probably because a pool of immature receptors was depleted by the drug before the α‐chymotrypsin treatment.Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.
Vasodilator‐stimulated protein phosphorylation in platelets is mediated by cAMP‐ and cGMP‐dependent protein kinases
Authors: WALDMANN Rainer , NIEBERDING Maria , WALTER Ulrich 
Published: March 2005

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin‐regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 μM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masscs, Mr, of 240000, 68 000, 50 000, and 22 000 in intact platelets.These proteins were also phosphorylated in response to low concentrations (1–2 μM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 μM) and the 8‐bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50000 which was also phosphorylated in response to low concentrations (1–2 μM) of cGMP in platelet membranes. An additional protein (Mr 24000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50000 was stimulated both in intact platelets by cyclic‐nucleotide‐elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50‐kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50‐kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50‐kDa protein phosphorylated in platelet membranes by cGMP‐ and cAMP‐dependent protein kinases, respectively.Regulation of protein phosphorylation mediated by cAMP‐ and cGMP‐dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.