bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Characterization of Presynaptically Acting Neurotoxins from the Venom of Bungarus Snakes
Authors: ABE Teruo , ALEMÀ Stefano , MILEDI Ricardo 
Published: March 2005

1. Five presynaptic toxins have been isolated in pure from from the venom of Bungarus multicinctus and Bungarus caeruleus and named β1, β2, β3, β4, and β‐ceruleotoxin.2. They differ in electrophoretic mobility and amino acid composition, while all have the same molecular weight (22000) and are composed of two subunits of molecular weight 9000 and 12000.3. The toxins have phospholipase A activity when assayed with both natural and synthetic phospholipids, and this activity requires the presence of Ca2+ ions.4. β‐Bungarotoxin (β3) binds 1 mol of Ca2+ per mol of protein and this binding induces a conformational change as detected by fluorescence measurements in the presence of the dye 8‐anilino‐1‐naphthalene sulfonic acid.5. The phospholipase activity of all the toxins is lost when a critical histidine residue is modified with p‐bromophenancyl bromide.6. As a result of the modification the lethality of the toxins is greatly reduced.7. Native toxin causes a rapid decrease in amplitude of end‐plate potentials, followed by a transient increase and subsequent decrease, until transmitter release is completely abolished. The modified toxin still causes the early decrease in release but toxin action does not progress to complete block.8. The rate of blockage of transmitter release by native toxin is reduced in the presence of modified toxin.9. It is concluded that phospholipase activity plays an important role in the action of this class of toxins at the neuromuscular junction.
Glutathione Reductase from Human Erythrocytes
Authors: KROHNE‐EHRICH Gisela , SCHIRMER R. Heiner , UNTUCHT‐GRAU Renate 
Published: March 2005

1. From 90 l erythrocytes 200 mg glutathione reductase was purified in four steps with an overall yield of 40%. The specific activity rose from 3.71 U glutathione reductase per g hemoglobin in the erythrocytes to 235 U/mg protein in the isolated enzyme. The purification procedure which was completed in 5 days, included (a) denaturation of hemoglobin by a mixture of butan‐1‐ol and chloroform [Scott, E. M. (1976) Prep. Biochem. 6, 147–152], (b) precipitation of glutathione reductase with acetone, (c) extraction of the precipitate and fractionation of the extract with ammonium sulfate, (d) affinity chromatography on adenosine‐2′,5′‐bisphosphate–Sepharose 4B.2. The 280/460 nm absorbance ratio was found to be 5.9 for the purified enzyme; all other studies on physical and chemical properties of the enzyme confirmed the results of Worthington and Rosemeyer [Eur. J. Biochem. 67, 231–238 (1976) and earlier papers].3. The new purification procedure provided crystals suitable for X‐ray diffraction analysis at high resolution. Furthermore sufficient amounts for sequencing the enzyme were isolated. As a first step in the determination of the protein's primary structure, CNBr‐produced fragments of the carboxymethylated enzyme were fractionated.4. A chain segment containing the catalytic disulfide of the native protein was isolated. Its sequence was determined to be . An identical sequence is present in yeast glutathione reductase, and lipoamide dehydrogenases contain an active site peptide with very similar sequence [Williams, C. H. Jr(1976) The Enzymes 13, 89–173]. Thus the long‐recognized mechanistic similarities between glutathione reductases and lipoamide dehydrogenases are reinforced by sequence similarities around the two redox‐active cysteine residues. The sequenced regions are, however, too short to allow speculations on phylogenetic relationships within this category of proteins.
Studies by Small‐Angle X‐Ray Scattering of the Quaternary Structure of the β‐Haemocyanin of Helix pomatia
Authors: BERGER Johann , PILZ Ingrid , WITTERS Raphaël , LONTIE René 
Published: March 2005

Helix pomatiaβ‐haemocyanin was studied in solution by small‐angle X‐ray scattering. The following molecular parameters were determined: molecular weight = 9.02 × 106, volume = 14000 nm3, radius of gyration = 18.4 nm, radius of the spherical subunits = 2.5 ± 0.2 nm.With these data, and with information of dissociation products in a former paper, a model of the molecule was built whose theoretical scattering curve showed good agreement with the experimental one. The model consists of 160 spherical subunits of a radius of 2.5 nm; 12 rings each built up of 10 spheres form the outer wall of a hollow cylinder; 20 subunits are situated at the inner side of each end.
Separation of Saccharomyces cerevisiae tRNAs on Two‐Dimensional Polyacrylamide Gels as Applied to Investigations on the Mutational Alterations of tRNA that Produce Nonsense Suppressors
Authors: PIPER Peter W. , WASSERSTEIN Marilyn 
Published: March 2005

Nonsense suppressors of the yeast Saccharomyces cerevisiae have been found that cause the translational suppression of UAA (ochre) or UAG (amber) nonsense mutants of iso‐1‐cytochrome c by inserting tyrosine, serine or leucine at the site of the nonsense codon. This report describes the fractionation of tyrosine, serine and leucine tRNAs from yeast on two‐dimensional polyacrylamide gels, as well as a partial characterisation of these species. There is only one tyrosine tRNA in yeast, whilst the serine and leucine tRNAs identified in this study can between them decode all the serine and leucine codons which differ in only one base from either UAA or UAG. We did not characterise those serine and leucine tRNAs whose cognate codons differ in more than one base from the amber or ochre codon.Separation of the tRNA of individual yeast strains on two‐dimensional gels can be used in conjunction with RNA fingerprinting techniques to investigate whether the amber or ochre suppressor mutations of yeast determine structural changes in the tyrosine, serine or leucine tRNAs. This approach has already allowed an elucidation of the tRNATyr structural change determined by tyrosine‐inserting amber suppressor mutations at the SUP5 locus. Although it has, to date, only been successfully applied to the sequencing of yeast amber suppressors, it has also a potential application in structural studies of ochre suppressors. One of the leucine tRNAs analysed in this study contains an unidentified modified nucleotide in its anticodon, a minor component of interest as it may also be present in the anticodon of yeast ochre suppressor tRNAs and cause the restrictive recognition of UAA but not UAG codons. The possible anticodon structures of yeast ochre‐suppressing tRNAs are discussed in the light of present knowledge of codon‐anticodon pairing.
Inhibition of Fructose‐1,6‐bisphosphate Aldolase from Rabbit Muscle and Bacillus stearothermophilus
Authors: LEWIS Dennis J. , LOWE Gordon 
Published: March 2005

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose‐1,6‐bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed.The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt(II) fructose‐1,6‐bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki= 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time‐dependant and the association and dissociation constants have been estimated.Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose‐1,6‐bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active‐site lysine residue.A new condensation assay for fructose‐1,6‐bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.
Differences in the Modulations of the Soluble or Plasma‐Membrane‐Bound 5′‐Nucleotidase
Authors: LELIEVRE Lionel , ZACHOWSKI Alain , MAGET‐DANA Régine , AUBRY Jacques , JONKMAN‐BARK Gitty 
Published: March 2005

The treatment of plasma membranes by a French pressure cell in sucrose medium devoid of detergents solubilized 20% of the total protein and 95–100% of 5′‐nucleotidase activity. The soluble enzyme was 40–90‐fold purified by centrifugation in a sucrose gradient with a 10–20% yield with respect to the original lysate.The purified fraction retained the same high specificity for 5′‐AMP (Km= 20 μM) as in the plasma membranes and was enriched in sphingomyelin. Whereas 5′‐AMP at high concentration inhibited the membrane‐bound enzyme, it had no effect on the solubilized form. The soluble enzyme was stimulated by 2 × 10−13–2 × 10−15 g concanavalin A without any inhibition with higher doses of lectin. The plasma‐membrane‐bound stimulated and inhibited 5′‐nucleotidase was modulated by concanavalin A concentrations higher than 0.1 μg. Inhibition of the activity of the soluble enzyme by antiphosphorylcholine antibodies was not observed with membranes. The regulation of 5′‐nucleotidase activity in plasma membranes might be associated with a supramolecular organization.
Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from Torpedo marmorata Electric Organ
Authors: SOBEL André , WEBER Michel , CHANGEUX Jean‐Pierre 
Published: March 2005

A method is described for the large‐scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol α‐toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor‐rich membrane fragments is composed of only one class of membrane fragments covered with 8‐nm rosettes identified as acetylcholine receptor molecules.This preparation is used as a starting material for the detergent solubilization and the large‐scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X‐100 extract of receptor‐rich membranes done in the presence of 2‐mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy.Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecylsulfate reveals three bands of apparent molecular weights 40000 ± 1000, 50000 ± 2000 and 66000 ± 2000, the two heaviest ones being present in significantly lower amounts than the 40000‐Mr one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the 40000‐Mr band to the 50000 and 66000‐Mr ones.
Effects of Chloroquine and Colchicine on the Degradation of Chyle Cholesteryl Ester and Phospholipids in vivo
Authors: FLORÉN Claes‐Henrik , NORDGREN Hans , NILSSON Åke 
Published: March 2005

1. Both chloroquine and cholchicine given intraperitoneally delayed the hydrolysis of chyle cholesteryl ester after the uptake by the liver. The cholesteryl ester radioactivity per mg protein of hepatocytes isolated from the drug‐treated rats was 97.2 and 96.5%, respectively, of that in the intact liver. The decrease could be accounted for by hydrolysis of cholesteryl ester during cell isolation. Most of the unhydrolyzed cholesteryl ester was thus intracellular or firmly bound to the cells. Also in perfused rat livers chloroquine inhibited the hydrolysis of chyle cholesteryl ester that had been taken up in vivo.2. After subcellular fractionation of livers from chloroquine‐treated rats, that had been injected with cholesterol‐labelled chyle 60 min earlier, 55% of the radioactive cholesteryl ester was in the large granule fractions (mitochondrial plus light mitochondrial pellet). The 600 ×g and 100000 ×g pellets contained a higher proportion of the unesterified radioactive cholesterol. Similar findings were made with colchicine‐treated animals.3. 60 min after injection of chyle labelled with 32P and [3H]cholesterol only 6% of the injected [32P]phospholipid radioactivity was in the liver in normal rats and 7.3% and 7.8% in the liver of rats treated with chloroquine and colchicine, respectively. Whereas the cholesteryl ester radioactivity in the large granule fractions in rats treated with chloroquine and colchicine was increased by 2.4 and 3.1 times, there was no such increase in the 32P radioactivity of any of the phospholipid classes.4. Three conclusions are drawn. Chylomicron remnant cholesteryl esters are likely to be interiorized by the hepatocytes before their degradation. The data are compatible with a role of the acid cholesterol esterase in their hydrolysis. There was no positive evidence that any significant part of the chylomicron phospholipids are taken up by the liver and metabolized by lysosomal enzymes.
Metabolism of 2‐Deoxy‐D‐galactose in Liver Induces Phosphate and Uridylate Trapping
Authors: STARLING James J. , KEPPLER Dietrich O. R. 
Published: March 2005

The metabolites of 2‐deoxy‐D‐galactose were quantitatively determined in liver and in various tissues of the rat. After intraperitoneal administration of 2‐deoxy‐D‐[1‐14C]galactose in a dose of 1 mmol/kg the liver was the predominant organ metabolizing this galactose analog. 2‐Deoxy‐D‐galactose 1‐phosphate and UDP‐2‐deoxy‐D‐galactose were the major hepatic metabolites and after 3 h comprised 78% and 20% of the total radioactivity in liver, respectively; UDP‐2‐deoxy‐D‐glucose amounted to 0.7%. Liver was the only tissue in which substantial formation of UDP‐2‐deoxy‐D‐hexoses was observed.The synthesis of UDP‐2‐deoxy‐D‐galactose was shown to be catalyzed by UDP‐glucose:galactose‐1‐phosphate uridylyltransferase. The low affinity of this enzyme for 2‐deoxy‐D‐galactose 1‐phosphate explained the accumulation of this substrate analog to levels as high as 14 mmol/kg liver. Enzymatic analysis of alkaline ethanol extracts showed a marked fall of the hepatic Pi content during the period of rapid 2‐deoxy‐D‐galactose 1‐phosphate formation. The trapping of Pi was associated with a loss of ATP and a lowering of the adenylate energy charge.The formation of UDP‐2‐deoxy‐D‐galactose occurred only after high levels of 2‐deoxy‐D‐galactose 1‐phosphate had been attained. The limited epimerization of UDP‐2‐deoxy‐D‐galactose to UDP‐2‐deoxy‐D‐glucose led to an accumulation of the former deoxy‐sugar nucleotide. The trapping of uridylate in the form of UDP‐2‐deoxy‐D‐galactose was accompanied by a transient decrease in the hepatic contents of UTP, UDP‐glucose, and UDP‐galactose. The administration of 2‐deoxy‐D‐galactose together with uridine or orotate completely prevented the depression of UTP.The accumulation of 2‐deoxy‐D‐galactose 1‐phosphate and the associated depression of Pi, in the absence of UTP deficiency, represent a state analogous to that induced by galactose in uridylyltransferase‐deficient cells and tissues and may serve as a biochemical model in the study of the cellular injury in galactosemia.
The Distribution of Lipids in the Protoplast Membranes of Bacillus subtilis
Authors: BISHOP David G. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M. 
Published: March 2005

The distribution of phospholipids in the protoplast membrane of Bacillus subtilis has been studied by comparing the effects of treatment with phospholipase C or trinitrobenzenesulphonic acid on intact protoplasts or membranes derived from them. After 2 h treatment of protoplasts at 37°C with phospholipase C (B. cereus), 45–50% of the membrane phospholipid has been hydrolyzed. including 90% of the phosphatidylethanolamine and 70–80% of the lysylphosphatidylglycerol. but no lysis of the protoplasts can be detected. Complete hydrolysis of phosphatidylethanolamine and lysylphosphatidylglycerol by phospholipase C is obtained in isolated membranes at a rate greater than in intact protoplasts. The rate and extent of hydrolysis by phospholipase C in both protoplasts and membranes is strongly temperature‐dependent and part of this effect is ascribed to phase separations in the membrane lipid reducing accessibility of the substrates.About 60% of the aminophospholipids in intact protoplasts are labelled by exposure to trinitrobenzenesulphonic acid and this level increases to only 70% in isolated membranes. It is concluded that the introduction of the bulky head group into both protein and lipid during trinitrobenzenesulphonic acid labelling precludes the reaction proceeding to completion.The results do not permit an unequivocal distribution of the phospholipids between the inner and outer halves of the bilayer. It appears however that at least 60% of the phosphatidylethanolamine is located in the outer monolayer, and under conditions where lysylphosphatidylglycerol is accumulated, that at least 60% of this lipid is also in the outer monolayer. Furthermore, the membrane appears to contain the capacity for transbilayer movement (flip‐flop) of at least the aminophospholipids and this capacity is expressed when the composition of the outer half of the bilayer is modified by treatment with phospholipase C.
An Analysis of the Contracted Sheath Structure of Bacteriophage Mu
Authors: CREMERS Alfons F. M. , SCHEPMAN A. Marius H. , VISSER Marja P. , MELLEMA Jan E. 
Published: March 2005

Polymers of contracted sheath particles of bacteriophage Mu were imaged by the negativestaining technique and the electron images were analyzed by optical and digital methods.By means of the three‐dimensional reconstruction technique of DeRosier and Klug [Nature (Lond.) 217, 130–134 (1968)] an averaged density map of the sheath structure at a resolution of about 2.0 nm was derived. The sheath is known to consist of one type of protein with a molecular weight of about 52000 [Admiraal and Mellema, J. Ultrastr. Res. 56, 48–64(1976)].The interpretation of the map has given information about packing and shape of the protein subunits. One way to describe the structure is by a set of annular rings with 6‐fold symmetry. The height of these rings is about 1.8 nm and successive rings in the structure change by about 33° in azimuth. The protein subunit which occupies more than one ring in the polymer, is elongated. The long axis of the protein subunit is at an angle of about 20° with the plane normal to the polymer axis. These data are discussed in relation to changes in the sheath molecules upon contraction.
6‐Aminohexanoic Acid Cyclic Dimer Hydrolase. A New Cyclic Amide Hydrolase Produced by Acromobacter guttatus KI72
Authors: KINOSHITA Shinichi , NEGORO Seiji , MURAMATSU Minoru , BISARIA V. S. , SAWADA Shujo , OKADA Hirosuke 
Published: March 2005

6‐Aminohexanoic acid cyclic dimer hydrolase produced by Achromobacter guttatus cells grown on 6‐aminohexanoic acid cyclic dimer (1,8‐diazacyclotetradecane‐2,9‐dione) was purified until it was homogeneous according to polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, Sephadex G‐200 column chromatography, and analytical centrifugation. The following characteristics of the purified enzyme were observed.(1) The molecular weight of the enzyme, estimated to be 110 000 by column chromatography on Sephadex G‐200 and 100 000 by sedimentative equilibrium, together with the minimum molecular weight of 55 000 obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicated it is a dimeric enzyme. The sedimentation constant (s20) was estimated to be 5.7 S from the sedimentation velocity.(2) The enzyme had an optimum pH of 7.3 when 6‐aminohexanoic acid cyclic dimer was used as substrate. It was stable in a pH range between 5.5 to 8.5, and 50% and 100% of activity was lost after 10 min of heating at 45°C and 50°C respectively. The optimum temperature for activity was 33°C. The Michaelis‐Menten constant (Km) was 6 mM toward the dimer, and the turnover number was 8 s−1 assuming a molecular weight of 100 000.(3) This enzyme was strongly inhibited by 1 μM diisoprpylphosphofluoridate and 10 μM p‐chloromercuribenzoate but scarcely inhibited by 100 mM ethylenediaminetetraacetic acid. The enzyme inhibited by p‐chloromercuribenzoate could be reactivated by 2‐mercaptoethanol.(4) This enzyme was only active on 6‐aminohexanoic acid cyclic dimer to form 6‐aminohexanoyl‐6‐aminohexanoic acid. It was inactive on 6‐aminohexanoic acid oligomers (degree of polymerization 2 to 6), various cyclic amides, cyclic diamides, amides, oligopeptides, and casein.(5) The absorption coefficient (A1%280 nm) of the enzyme was 19.4. The enzyme was composed of 500 amino acid residues including 3 cysteines; from this the molecular weight was calculated to be 103 200 as a dimer.(6) 6‐Aminohexanoic acid cyclic dimer hydrolase was classified as a new cyclic amide hydrolase.
Effect of Borate on the Catalytic Activities of Muscle Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: WOLNY Marian 
Published: March 2005

The effect of borate on glyceraldehyde‐3‐phosphate dehydrogenase from human, pig and rabbit muscle was studied. At lower concentration of borate only the dehydrogenase activity is inhibited, reversibly and competitively against NAD. At concentration of borate above 6 mM the plots of 1/vversus borate concentration become nonlinear and the inhibition is extended to the esterase and acetylphosphatase activities. In certain conditions a time‐dependent inactivation and reactivation was observed. The direct interaction between borate (if present at concentration of at least 6 mM) and glyceraldehyde‐3‐phosphate dehydrogenase is postulated, the possible site of the reaction being the histidine residue(s).The esterase activity of the human muscle enzyme and the effect of borate on it are different from the other mammalian enzymes.
Thyrotropin Stimulation of a Thyroid Contractile Protein Phosphorylation
Authors: LAMY Françoise , LECOCQ Raymond , HAUMONT Michèle , DUMONT Jacques E. 
Published: March 2005

Contractile proteins were isolated from preincubated dog thyroid slices by a procedure identical to the one used for the preparatio of muscle actomyosin. At least two of these proteins (molecular weight = 26000 and 15000) were phosphorylated in the intact cell system. After one hour of incubation of the slices with thyrotropin (10 mU/ml) and [32P]phosphate, the specific activity of the 26000‐Mr protein was increased by a factor of three while no significant change in specific activity was observed in the 15000‐Mr contractile protein. This effect of thyrotropin was already observed after 30 min of action, was elicited at hormone concentrations of the same order as those required to induce secretion and has been reproduced by dibutyryl adenosine 3′:5′‐monophosphate.Cycloheximide (0.35 mM) which almost totally inhibited protein synthesis in thyroid slices. did not affect the thyrotropinstimulation of the phosphorylation of the 26000‐Mr protein.The phosphorylation of serine residue(s) has been demonstrated in this protein but the presence of radioactive phosphothreonine could not be detected.On the basis of its molecular weight, the 26000‐Mr protein could be similar to the inhibitory component of muscle troponin complex (TN‐I).