bluuebunny/crossref_metadata_2025_split huggingface dataset:
Properties of monoclonal antibodies to nicotinic acetylcholine receptor from chick muscle
Authors: MEHRABAN Fuad , KEMSHEAD John T. , DOLLY J. Oliver 
Published: March 2005

1. Four stable, hybrid‐cell lines secreting monoclonal antibodies to distinct determinants on the nicotinic acetylcholine receptor from chick muscle have been established. These were characterised by the following criteria: immunoglobulin isotype, ability to produce experimental autoimmune myasthenia gravis in mice and reactivity towards homologous and hetereologous acetylcholine receptor proteins.2. Two monoclonal antibodies were found to inhibit the reaction of α‐bungarotoxin with homologous acetylcholine receptor; in addition one of these, on binding to receptor‐toxin, induced a rapid dissociation of the complex (t1/2= 0.5 h at 23 °C).3. Three of the antibody preparations recognised epitopes on this receptor from muscle of other species and two of these caused experimental autoimmune myasthenia gravis in BALB/c mice following passive transfer.4. The latter two recognised to significant extents the α‐bungarotoxin binding component purified from chick optic lobe and brain cortex.5. Sedimentation analysis demonstrated that two of the monoclonal antibodies form a distinct size (s20, w = 12S) of complex with the receptor of chick muscle which most probably corresponds to a 1:1 attachment of antibody and receptor; this may involve cross‐linking of two determinants within the same oligomer. A similar observation was made with the α‐bungarotoxin binding component from optic lobe using one of the cross‐reacting antibodies. Another monoclonal antibody was found to be capable of forming much heavier complexes with the receptor from chick muscle, these are thought to involve inter‐molecular cross‐linking of oligomers.6. The observed properties of these antibodies are discussed in relation to their myasthenogenicity and with reference to the extent of structural similarities between the peripheral nicotinic acetylcholine receptor and the α‐bungarotoxin binding protein from brain.
Role of the ATPase of sugar‐cane vacuoles in energization of the tonoplast
Authors: THOM Margaret , KOMOR Ewald 
Published: March 2005

Vacuoles of sugar‐cane suspension cells contained a tonoplast‐bound ATPase which was exclusively located on the cytoplasmic side of the vacuole. Vanadate and diethylstilbestrol had little effect on the vacuolar ATPase. ATP was the optimum substrate for the tonoplast ATPase, but there was also evidence for tonoplast‐bound GDP‐hydrolyzing and GTP‐hydrolyzing enzymes which can interfere with the ATPase assay. Other phosphate anhydrides and esters were not hydrolyzed.The addition of MgATP polarized the tonoplast from about 0 mV to an interior‐positive value of about +20 mV; MgADP and MgGTP had much less effect; MgGDP and ATP (in the absence of magnesium) had no effect on the membrane potential. The polarization of the tonoplast was insensitive to valinomycin, nigericin, and inhibitors of plasmalemma ATPase, but was strongly reduced by the uncoupler carbonylcyanide m‐chlorophenyl‐hydrazone. These data are interpreted as evidence for the action of tonoplast‐bound ATPase as a pump which translocates protons into the vacuoles.The activity of the ATPase was highly specific for MgATP2‐; the other important ionic states of ATP: ATP4−, HATP3−, MgHATP−, and Mg2ATP neither stimulated nor inhibited. The same was true for Mg2+. Since the protons were not brought to the catalytic site by protonation of the substrate, the tonoplast‐ATPase may pick up the proton for translocation from the cytoplasm. The saturation kinetics for MgATP2− hydrolysis were biphasic, the higher affinity ATPase with Km value of 0.7 mM seems to be the physiologically relevant activity.
Incorporation of 1,N6‐ethanoadenosine into the 3′ terminus of tRNA using T4 RNA ligase 1. Preparation of yeast tRNAPhe derivatives
Authors: PAULSEN Harald , WINTERMEYER Wolfgang 
Published: March 2005

The 3′‐terminal A‐C‐C‐A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6‐ethenoadenosine (ɛA) or 2‐aza‐1,N6‐ethenoadenosine (aza‐ɛA). T4 RNA ligase was used to join the nucleoside 3′,5′‐bisphosphates to the 3′ end of the tRNA which was shortened by one [tRNAPhe(‐A)] or four [tRNAPhe(‐ACCA)] nucleotides. It was found that the base‐paired 3′‐terminal cytidine 72 in tRNAPhe(‐ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A‐C‐C terminus of tRNAPhe(‐A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double‐stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single‐stranded acceptors.The replacement of the 3′‐terminal adenosine 76 with ɛA and aza‐ɛA leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu‐specific tRNAs both from yeast and Escherichia coli) are also inactivated by ɛA incorporation.Replacement of adenosine 73 followed by repair of the C‐C‐A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of ɛA and aza‐ɛA at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the ɛA‐labeled tRNAPhe is complexed with phenylalanyl‐tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3′ terminus is not changed appreciably in these complexes.
Localization of Lys‐51 of L7/L12 on 50S ribosomes from Escherichia coli
Authors: MAASSEN J. Antonie , SCHOP Enno N. , MÖLLER Wim 
Published: March 2005

Ribosomal protein L7/L12 from Escherichia coli was modified specifically at Lys‐51 with 4‐(6‐formyl‐3‐azido‐phenoxy)butyrimidate.Reconstitution of ribosomal cores, lacking L7/L12, with imidate‐modified L7/L12 resulted in back formation of 50S particles which were fully active in elongation‐factor‐dependent processes. By use of the formylazidophenoxy moiety as hapten, the position of Lys‐51 of L7/L12 on the 50S ribosome was determined by immune electron microscopy. The results show that an L7/L12 dimer is present in the L7/L32 stalk in such a way that Lys‐51 is located at the far cytoplasmic end of the stalk. The experimental data are discussed in relation to a proposed model for the L7/L12 dimer.
Protein synthesis during chloroplast development in Spirodela oligorhiza
Authors: De HEIJ Hans T. , JOCHEMSEN Aart‐Gerrit , WILLEMSEN Peter Th. J. , GROOT Gert S. P. 
Published: March 2005

We have studied qualitative and quantitative changes of several parameters during chloroplast development in Spirodela oligorhiza (duckweed). On a dry weight basis, the amount of protein increases from 2.5 % (w/w) in dark‐grown to 7.8 % (w/w) in light‐grown fronds. At the same time the amount of starch drops from 50 % to 27 % (w/w).Using an immunochemical quantification method we have found that during greening of etiolated plants the amount of all subunits of the ATPase complex per frond increases 10‐fold, whereas the level of the subunits of ribulose‐1,5‐biphosphate carboxylase increases 50‐fold. Cytochrome f was found to be present in dark‐grown Spirodela and the amount of this polypeptide per frond increases about 30‐fold. The concentration of a polypeptide that possibly represents a cytochrome b6 subunit increases about 10‐fold upon greening.The molar ratio of the CF1‐β and CF1‐γ subunits of the ATPase complex varies over 2–3, while in all stages of chloroplast development studied the molar ratio of the carboxylase subunits is about 1. As these values are in agreement with the stoichiometrical amounts in the native protein complexes, we conclude that the synthesis of CF1‐β and CF1‐γ, as well as the synthesis of the large and small carboxylase subunits, are strictly coordinated during chloroplast biogenesis in Spirodela oligorhiza.
Supercoiling and the mechanism of restriction endonucleases
Authors: SNOUNOU Georges , MALCOLM Alan D. B. 
Published: March 2005

We have used topoisomerase I in the presence of netropsin and ethidium bromide to generate DNA molecules of varying superhelical density.
Digestion by endonuclease EcoRI is sensitive to supercoiling, being maximal for the relaxed form. Endonucleases AvaI and BamHI, by contrast, are relatively unaffected.
The results are interpreted in terms of the base composition of the DNA in the vicinity of these sites. dA + dT‐rich regions are more susceptible to deformation than are dG + dC‐rich ones.
Analysis of the rates of disappearance of linear molecules confirms a two‐step mechanism for EcoRI cleavage but suggests that BamHI and AvaI cleave both strands simultaneously.
Purification and characterization of 3′‐phosphoadenylylsulfate: N‐desulfoheparan sulfate sulfotransferase from arterial tissue
Authors: GÖHLER Dieter , NIEMANN Reinhard , BUDDECKE Eckhart 
Published: March 2005

A 3′‐phosphoadenylsulfate: N‐desulfoheparan sulfate sulfotransferase (EC 2.8.2.12) was purified 450‐fold from the microsomal fraction of calf arterial tissue and separated from 3′‐phosphoadenylylsulfate: chondroitin sulfotransferase (EC 2.8.2.5) activity.The enzyme has optimal activity at neutral pH, requires divalent cations (Mn2+, Mg2+, Ca2+) for maximal activity and exhibits specificity towards N‐desulfoheparan sulfate, N,O‐desulfoheparan sulfate and oligosaccharides derived therefrom. N,O‐desulfoheparan sulfate tetrasaccharides serve as acceptor substrates only if the nonreducing terminus is occupied by glucuronic acid (not iduronic acid).
The N,O‐desulfoheparan sulfate sulfotransferase transfers [35S]sulfate from 3′‐phosphoadenylyl[35S]sulfate to the 2‐amino groups and to the 6‐hydroxy groups of glucosamine units of the acceptor substrates. The ratio of N/O‐sulfation ranged between 3:1 and 2:1.
O‐[35S]Sulfated unsaturated disaccharides were obtained from enzymatically labelled [35S]N‐desulfoheparan sulfate by heparitinase degradation and subsequent deamination. Evidence for the O‐sulfation at C‐6 of the glucosamine units was provided by isolation of anhydromannose [35S]monosulfate, which was formed from uronosylanhydromannose [35S]monosulfate by β‐glucuronidase treatment.
An N‐desulfo‐N‐[1‐14C]acetylheparan sulfate deacetylase activity was copurified with the N‐desulfoheparan sulfate sulfotransferase.
Detection of type‐2 casein kinase and its endogenous substrates in the components of the microsomal fraction of rat liver
Authors: MEGGIO Flavio , BRUNATI Anna Maria , DONELLA‐DEANA Arianna , PINNA Lorenzo A. 
Published: March 2005

Rat liver type‐2 casein kinase‐TS (Ck‐TS) is unevenly distributed among the different components of the rat liver post‐mitochondrial particulate fraction: while it accounts for the whole casein kinase activity of protein‐glycogen particles, it is absent in the microsomal membranes (exhibiting exclusively casein kinase activity of type‐1) and coexists with type‐1 casein kinase in ribosomes.At least seven proteins whose phosphorylation is promoted by Ck‐TS have been detected in these fractions. The only important target of Ck‐TS in protein‐glycogen particles is a rather heterogeneous protein of Mr around 85000 which has been identified as glycogen synthase. Two polypeptides of Mr about 16000, possibly identifiable with acidic proteins P1 and P2, and an unidentified protein of Mr about 35000 are the main ribosomal substrates of Ck‐TS. Three protein bands of Mr 52000, 79000 and 91000 are also very efficiently phosphorylated whenever the microsomal membranes, devoid of intrinsic casein kinase‐2, are incubated with cytosolic Ck‐TS. These membrane‐bound radiolabeled proteins require deoxycholate for their solubilization: they are very acidic, according to their high affinity for DEAE‐cellulose, and give rise to partially superimposable [32P]peptide maps, suggesting extensive homologies among them. They also exhibit a low affinity Ca2+ binding activity comparable to that of calsequestrin.
The variable relation between myosin light‐chain phosphorylation and actin‐activated ATPase activity in chicken gizzard smooth muscle
Authors: MERKEL Linda , MEISHERI Kaushik D. , PFITZER Gabriele 
Published: March 2005

Using pure myosin light‐chain kinase and myosin from chicken gizzard and rabbit skeletal actin, we have established the relationship between myosin phosphorylation and actin‐activated ATPase activity by varying the concentration of Ca2+ or calmodulin (CaM). By increasing Ca2+ or CaM the extent of myosin phosphorylation increased from 10% to about 100%, the requirements for half‐maximal activation being 0.17 μM Ca2+ and 1 nM CaM. ATPase was activated only when a phosphorylation ‘threshold’ of about 60 % was surpassed. By increasing phosphorylation from 60% to 100%, ATPase activity was further stimulated, the relationship between myosin phosphorylation and ATPase activity being curvilinear. Addition of 1 μM tropomyosin lowered the Ca2+ requirement for half‐maximal ATPase activity in the prescence of 0.05 μM CaM from 0.4 μM to 0.2 μM but did not affect the maximal ATPAse activity. In addition, the CaM requirement of ATPase activity was also lowered by gizzard tropomyosin at 9 μM Ca2+. The Ca2+/CaM requirement for myosin phosphorylation remained unaffected by tropomyosin.Thus, tropomyosin altered the relationship between myosin phosphorylation and ATPase activity: the phosphorylation ‘threshold’ was lowered from 60% to about 10%, hence causing a leftward shift of the relationship which becomes similar to that in the case of native actomyosin.
Glutathione conjugation by isolated lung cells and the isolated, perfused lung
Authors: DAWSON Janet R. , VÄHÄKANGAS Kirsi , JERNSTRÖM Bengt , MOLDÉUS Peter 
Published: March 2005

Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1‐chloro‐2,4‐dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH‐transferase activity.Conjugation of 1‐chloro‐2,4‐dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium.The results indicate the ability of rat lung to utilize extracellular GSH.
Nuclear‐magnetic‐resonance investigation of 15N‐labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin
Authors: FRANKEN Hans‐Dieter , RÜTERJANS Heinz , MÜLLER Franz 
Published: March 2005

Flavin derivatives, enriched with 15N (≊95%) at the four nitrogen atoms of the isoalloxazine ring, have been investigated in the oxidized and the two‐electron reduced state by the 15N nuclear magnetic resonance technique. The measurements were conducted with aqueous and chloroform solutions of flavin. A comparison of the chemical shifts of the N(1) and N(5) atoms of oxidized flavin in the two solvents revealed that these atoms are sensitive indicators for possible hydrogen‐bridge formation to these atoms. The N(5) atom of oxidized flavin resonates at low field and shifts about 300 ppm upfield upon reduction.A pKa of 6.8 was determined from pH‐dependent 15N NMR measurements of the two‐electron reduced flavin molecule. In addition it is also shown that reduced flavin in aqueous solution possesses a more coplanar structure than in chloroform solution.The 15N chemical shifts of flavin bound to Megasphaera elsdenii apoflavodoxin indicate that various hydrogen bridges are formed between the prosthetic group and the apoprotein. Especially the N(1) atom of the prosthetic group in the oxidized state seems to form a strong hydrogen bond with the apoprotein. In the reduced state the prosthetic group is bound in the anionic form and possesses an almost coplanar structure. These results are in agreement with published crystallographic data on the related flavodoxin from Clostridium M P.Where possible 15N‐1H, 15N‐15N and 13C‐15N coupling constants were determined. Some of the coupling constants are useful parameters for the elucidation of the planarity of free and protein‐bound flavin and for the evaluation of the interaction between flavin and apoprotein.Spin‐lattice relaxation measurements show that the relaxation of the 15N(3)H group of flavin is predominantly determined by dipole‐dipole interaction. The calculated rotational correlation times of flavin in two different solvents were determined and are in good agreement with published results.
Glutathione transport across hepatocyte plasma membranes
Authors: INOUE Masayasu , KINNE Rolf , TRAN Thao , ARIAS Irwin M. 
Published: March 2005

Transport of reduced glutathione (GSH) and its derivatives was studied in rat‐liver sinusoidal plasma‐membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited transport of GSH into an osmotically active intravesicular space. Equilibrium uptake of vesicle‐associated GSH was similar to that of free ligands which can be trapped by the intravesicular space of sinusoidal membrane samples. Kinetic analysis of the transport process revealed that the sinusoidal membrane vesicles have high‐affinity and low‐affinity GSH transport systems; the former has an apparent Km of 0.34 mM for GSH and V of 1.4 nmol × mg protein−1× 20 s−1, and the latter has an apparent Km of 3.3 mM and V of 3.9 nmol × mg protein−1× 20 s−1. Both Km values are lower than intrahepatic GSH levels, indicating that GSH transport across the sinusoidal membrane occurs via carrier‐mediated mechanism and argues simple diffusion mechanism. The presence of oxidized glutathione (GSSG) or S‐benzylglutathione inhibited GSH transport by the vesicles; the low‐affinity transport system was inhibited more markedly than the high‐affinity transport system. This suggests that these glutathione derivatives interacted preferentially with the low‐affinity transport system for GSH. S‐Dinitrophenylglutathione was also transported by the vesicles by a process which was inhibited by GSH and GSSG. The vesicles also transported GSSG, and this transport was markedly inhibited by S‐benzylglutathione or GSH.The transport systems in sinusoidal plasma membranes may function in vivo in translocating GSH and its derivatives from hepatocytes into plasma and play an important role in inter‐organ metabolism of these compounds.
Synthesis of pigment‐binding protein in toluene‐treated Rhodopseudomonas capsulata and in cell‐free systems
Authors: DIERSTEIN Roland 
Published: March 2005

Pigment‐binding protein of the facultatively phototrophic bacterium Rhodospeudomonas capsulata could be selectively synthesized in toluene‐treated cells as well as in homologous and heterologous cell‐free translation systems by isolated polysomes. It is shown that the pigment‐binding polypeptides of the light‐harvesting complexes are encoded by messenger RNA of extreme longevity. The dependence of their synthesis on the concomitant synthesis of tetrapyrroles was demonstrated in the toluene‐treated cells.The large Mr‐28000 polypeptide of the reaction center and the Mr‐10000 pigment‐binding polypeptide of the light‐harvesting complex II were found to be synthesized by free (water‐soluble) polysomes without a cleavable ‘leader’ or ‘signal’ peptide [reviewed by W. Wickner (1979) Annu. Rev. Biochem. 48, 23–45]. The Mr‐10000 polypeptide, as synthesized in vitro, was studied in more detail. Unlike the membrane‐assembled polypeptide in vivo it was insoluble in an organic solvent mixture (chloroform/methanol 1:1, v/v). After detergent denaturation in the presence of membrane isolated from the organism it became organic‐solvent‐soluble. Obviously the polypeptide could be induced to assume alternative conformations in which its apolar residues were either exposed to the solvent or buried within. These findings, in agreement with Wickner's hypothesis, indicate that the Mr‐10000 polypeptide may enter the lipid bilayer by a ‘membrane‐triggered’ conformational change.
Identification of an essential carboxylate group at the active site of lacZβ‐galactosidase from Escherichia coli
Authors: HERRCHEN Monika , LEGLER Günter 
Published: March 2005

[3H] Conduritol C cis‐epoxide (1,2‐anhydro‐epi‐inositol, I) was synthesized as an active‐site‐directed inhibitor for lacZβ‐galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH4 of the p‐benzoquinone‐derived precursor for I.Complete loss of β‐galactosidase activity occurred on incorporation of 4 mol I/mol β‐galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH < 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo‐inositol. The stereochemistry of the expoxide reaction (trans‐diaxial ring opening) is thus the same as for β‐glucosidases with the corresponding epoxides.The binding site for I was identified as Glu‐461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the N‐terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified γ‐carboxyl group of its N‐terminal Glu‐461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions.The participation of the carboxylate of Glu‐461 in catalysis is discussed.
Studies on reconstituted partially purified glycerophosphate acyltransferase from Escherichia coli
Authors: KESSELS Joop M. M. , OUSEN Harry , VAN DEN BOSCH Henk 
Published: March 2005

Solubilized glycerophosphate acyltransferase from Escherichia coli was reconstituted in small unilamellar vesicles consisting of phosphatidylcholine/phosphatidylglycerol in a molar ratio of 4:1. Glycerol 3‐phosphate, trapped inside these vesicles, cannot be acylated by the enzyme upon addition of extra‐vesicular palmitoyl‐CoA. Thus, substrate‐binding sites and active sites are asymmetrically oriented in the model membrane.When up to 10mol/100mol lysophosphatidic acid was incorporated in the vesicles a decrease in glycerophosphate acyltransferase activity is observed at amounts exceeding 1 mol% lysophosphatidate. Similar experiments, using lysophosphatidylcholine and phosphatidic acid, suggest the decrease to result from an increase in negative surface charge. Reconstituted glycerophosphate acyltransferase exhibits a preference for palmitoyl‐CoA over oleoyl‐CoA. This preference increases considerably at elevated temperatures. The glycerophosphate acyltransferase could, therefore, participate in the temperature‐dependent changes in the fatty acid composition of the phospholipids in E. coli.