bluuebunny/crossref_metadata_2025 huggingface dataset:
Strain and Host‐Cell Dependence of Vaccinia Virus Proteins
Authors: TSCHANNEN Roland , SCHÄFER Rolf
Published: July 2010

The neurotropic strain of vaccinia virus (WR) and the dermotropic strain of vaccinia virus (Elstree) which replicated on mouse L‐fibroblasts (a cell line of dermotropic origin) were highly purified, dissociated and chemically labelled by [125I]iodine. The proteins of the two virus strains were compared by two‐dimensional gel electrophoresis in the nanogram range, patterned after a modified procedure of O'Farrell. A total number of 113 protein spots were resolved in the radioautograms. Vaccinia virus Elstree contained five polypeptides with same molecular weights but different isoelectric points and three polypeptides with different molecular weights and different isoelectric points as compared with vaccinia virus WR. Vaccinia virus WR which replicated on rat adrenal pheochromocytoma PC12 (a cell line of neurotropic origin) contained four additional proteins as compared with the same strain which replicated on the mouse L‐fibroblasts.
Effect of High Pressure on the Heat Activation in vivo of Trehalase in the Spores of Phycomyces blakesleeanus
Authors: THEVELEIN Johan M. , VAN ASSCIIE Jozef A. , HEREMANS Karel
Published: July 2010

The effect of pressure on the heat activation in vivo of trehalase in the spores of Phrcomyces blukesleeonus has been investigated in order to obtain information about the molecular mechanism of the activation. For a protein comormational change directly induced in the enzyme by the heat treatment an upward shift with about 2–6 K/100 atm(1.013 × 105 Kpa to expected in the moderate high‐pressure region. On the other hand, for phospholipid phase transition causing the activation, a continuous upward shift with about 20 K/1000 atm is to be expected. For trehalase activation we find a contintttats upward shift of the activation temperature with about 5–9 K/1000 atm. The denaturation of trehalase, which occurs at slightly higher temperatures, is influenced by pressure completely as expected for a protein conformational change. File application of high pressure during spore heat activation makes it possible to break the dormancy of the spores without concomitant activation of trehalase.
Purification and Subunit Structure of Rat‐Liver Phosphoprotein Phosphatase whose Molecular Weight is 260000 by Gel Filtration (Phosphatase IB)
Authors: TAMURA Shinri , TSUIKI Shigeru
Published: July 2010

Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamara et al. (1980) Eur. J. Biochem. 104, 347–355] by earlier elution from DEAE‐cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase a. This enzyme was purified to apparent homogeneity by chromatography on DEAE‐cellulose, aminohexyl—Sepharose‐4B, histone‐Sepharose‐4B, protamine‐Sepharose‐4B and Sephadex G‐200.

The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from s 20° C, and Stokes' radius. Using historic phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one‐fifth that of phosphatase II.

Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely α, β and γ, whose molecular weights are 35000, 69000 and 58000, respectively. The α subunit is identical to the α subunit α phosphatase II. While the β subunit is also identical or similar to the β subunit of phosphatase II, the γ subunit appears to be unique to phosphatase IB.

When purified phosphatase IB was treated with 2‐mercaptoethanol at –20° C, the enzyme was dissociated to release the catalytically active α subunit. Along with this dissociation, there was a 7.4‐fold increase in phosphorylase phosphatase activity; but historic phosphatase activity increased only 1.6‐fold. The possible functions of the γ subunit are discussed in relation to this activation of enzyme.
The Xenopus Oocyte as a Surrogate Secretory System The Specificity of Protein Export
Authors: LANE Charles D. , COLMAN Alan , MOHUN Timothy , MORSER John , CHAMPION Janet , KOURIDES Ione , CRAIG Roger , HIGGINS Stephen , JAMES Tharappel Chacko , APPLEBAUM Shalom W. , OHLSSON Rolf I. , PAUCHA Eva , HOUGHTON Michael , MATTHEWS Jayne , MIFLIN Benjamin J.
Published: July 2010

Combining messenger RNA from one kind of secretory cell with the cytoplasm of another such cell can reveal the nature and specificity of protein export mechanisms. We show that messenger RNAs from secretory cells of chickens, rats, mice, frogs, guinea‐pigs, locusts and barley plants, when injected into Xenopus oocytes, direct the synthesis and export of proteins. Chicken ovalbumin, Xenopus albumin, mouse thyroid‐stimulating, hormone, locust vitellin and guinea‐pig milk proteins were identified using specific antibodies, whilst chicken lysozyme and ovomucoid, rat albumin, Xenopus vitellogenin and rat seminal vesicle basic proteins were identified provisionally from their molecular weights. Certain endogenous proteins are sequestered and secreted although most oocyte proteins arc not exported. Similarly the major polyoma viral protein and the simian virus 40 and polyoma tumour antigens are retained within the oocyte. Radioactive proteins exported by oocytes programmed with chicken oviduct or Xenopus liver RNA are not re‐exported in detectable amounts when injected into fresh oocytes, nor is there secretion of chicken oviduct or guinea‐pig mammary gland primary translation products prepared using wheat germ extracts. Thus the export of secretory proteins from oocytes cannot be explained by leakage and may require a cotranslational event. The secretory system of the oocyte is neither cell‐type nor species‐specific yet is highly selective. We suggest that the oocyte can be used as a general surrogate system for the study of gene expression, from transcription through translation to the final subcellular or extracellular destination of the processed protein.
Evidence for the Synthesis of a Somatomedin Similar to Insulin‐Like Growth Factor I by Chick Embryo Liver Cells
Authors: HASELBACHER Gisela K. , ANDRES Roger Y. , HUMBEL René E.
Published: July 2010

Chick embryo liver cells were incubated in serum‐free and hormone‐free medium and shown to produce a mitogen starting after the first day in culture. This mitogen was purified by gel filtration and ion‐exchange chromatography and identified by several assay systems as a somatomedin similar to human insulin‐like growth factor I (IGF‐I). This somatomedin stimulates thymidine incorporation into DNA of chick fibroblasts, it reacts in an IGF‐specific protein binding assay and it cross‐reacts immunologically with antibodies prepared against human IGF‐I, but not with those against IGF‐II. Its behaviour on ion‐exchange chromatography and on sodium dodecylsulfate/poly acrylamide gel electrophoresis is indistinguishable from that of authentic human IGF‐I. Concomitantly with the mitogenic activity, chick liver cells produce an IGF‐binding protein. Addition of purified human IGF‐I to the liver cells inhibits production of the binding protein. Chick liver cells slay thus be used to study synthesis and regulation of IGF and its binding protein.
Toxin‐Induced K+ Efflux through the Na+ Channel of Neuroblastoma Cells
Authors: JACQUES Yannick , ROMEY Georges , LAZDUNSKI Michel
Published: July 2010

Neurotoxins which modify the gating system of the Na+ channel in neuroblastoma cells and increase the initial rate of 22Na+. influx through this channel also give rise to the efflux of 86+ and 42K+. These effluxes are inhibited by tetrodotoxin and are dependent on the presence in the extracellular medium of cations permeable to the Na+ channel. These stimulated effluxes are not due to membrane depolarization or increases in the intracellular content of Na+ and Ca2+ which occur subsequent to the action of neurotoxins. The relationships of 22Na+ influx and 42+ (or 86Rb+) effluxes to both the concentration of neurotoxins and the concentration of external permeant cations strongly suggest that the open form of the Na+ channel stabilized by neurotoxins permits an efflux of K+ ions. Our results indicate that for the efflux of each K+ ion there is a corresponding influx of two Na+ ions into the Na+ channel.
Size of Vasopressin Receptors from Rat Liver and Kidney
Authors: GUILLON Gilles , COURAUD Pierre‐Olivier , BUTLEN Daniel , CANTAU Bernard , JARD Serge
Published: July 2010

Specific vasopressin receptors in rat liver membranes were recently characterized [Cantau et al.,Journal of Receptors Research, in the press]. They differ from the receptors characterized earlier in kidney membranes as regards coupling with adenylate cyclase and specificity towards vasopressin structural analogues [Rajerison et al. (1974)J. Biol. Chem. 249, 6390–6400; Butlen et al. (1978)Mol. Pharmacol. 14, 1006–1017]. The object of the present work was to see whether these differences reflect a difference in the molecular size of liver and kidney vasopressin receptors. For this purpose, rat liver and kidney membranes were incubated with [3H]vasopressin and solubilized with Triton X‐100 (0.3%). The properties of the macromolecular components of soluble extracts to which labelled vasopressin remained bound were observed to resemble those of the intact membrane receptors as regards binding reversibility at 30–37° C and sensitivity to guanyl nucleotides. The hydrodynamic parameters of the soluble hormone‐receptor complexes were estimated from Ultrogel column filtration experiments and from sucrose density gradient ultracentrifugation experiments. The following values were obtained for liver and kidney receptors respectively: Stokes' radii: 5.4 and 5.6 nm; standard sedimentation coefficients: 3.7 and 3.7 S; partial specific volumes: 0.75 and 0.78 ml · g−1molecular weight: 83000 and 80000. These results indicate that the marked functional differences between liver and kidney receptors are not accompanied by appreciable differences in molecular size.
Characterization of Cloned Complementary DNA Covering more than 6000 Nucleotides (97%) of Avian Vitellogenin mRNA
Authors: COZENS Peter J. , CATO Andrew C. B. , JOST Jean‐Pierre
Published: July 2010

The messenger RNA coding for chicken vitellogenin, a precursor of the egg‐yolk proteins lipoviteiiin and phosvitin, is synthesized in the liver following estrogen injection. This mRNA is 6600 nucleotides long. We have previously reported the cloning and preliminary characterization of some cDNA fragments representing portions of the vitellogenin mRNA [Biochem. Biophys. Acta, 606, 34–46 (1980)]. In this paper we report the full characterization of a larger series of such clones, representing almost the entire length of the mRNA, by restriction endonuclease mapping, R‐loop mapping, RNA‐DNA hybridization and by translation in vitro of the RNA which hybridizes to the cloned DNA. From the results we conclude that the chicken vitellogenin mRNA, unlike that of Xenopus laevis, does not vary in sequence over most of its length, although some variations in the cDNA sequences were detected particularly in clones derived from the 3′ terminus of the RNA. All sequence variants appear to be present in RNA prepared from single animals. The possible origins of these minor species are discussed. Furthermore, we describe a cDNA clone complementary to an mRNA which is about the same size as vitellogenin mRNA and which codes for an egg yolk protein antigenically related to lipovitellin. This mRNA is synthesized constitutively.
Characterization of 20‐S and 40‐S Non‐Polysomal Cytoplasmic Messenger Ribonucleoprotein Particles from Rat Liver
Authors: NORTHEMANN Wolfgang , SCHMELZER Elmon , HEINRICH Peter C.
Published: July 2010

Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low‐molecular‐weight RNA and protein components. 40‐S and 20‐S particles show very different RNA patterns. Four distinct low‐molecular‐weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40‐S mRNPs. The 20‐S mRNP particles contain one major low‐Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low‐Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low‐Mr RNAs found in nuclear RNP particles most of the low‐Mr RNA species present in 20‐S and 40‐S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low‐Mr RNA composition of 20‐S and 40‐S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117 000, 79 800, 76 700, 53 800, 43 900, 36 300 and several minor ones were found in both 20‐S and 40‐S mRNPs.

In a cell‐free system from wheat germs nether 20‐S nor 40‐S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol‐extracted RNA from 20‐S and 40‐S mRNPs stimulated total protein synthesis 16‐fold and 3‐fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro.
Synthesis and Processing of Semliki Forest Virus‐Specific Nonstructural Proteins in vivo and in vitro
Authors: LEHTOVAARA Päivi , ULMANEN Ismo , KÄÄRIÄINEN Leevi , KERÄNEN Sirkka , PHILIPSON Lennart
Published: July 2010

A large short‐lived virus‐specific nonstructural protein with an apparent molecular weight of about 250000 (nsp250) has been isolated from cells infected with the temperature‐sensitive mutants ts‐4 and ts‐6 of the Semliki Forest virus. nsp250 contained all peptides characteristic of the two previously identified nonstructural precursor proteins, nsp155 and nsp135, as revealed by limited proteolysis with Staphylococcus aureus V8 protease. Thus nsp250 is probably the translational product of the 5′ two‐thirds of the 42‐S RNA genome which codes for the virus‐specific nonstructural proteins.

A second viral nonstructural precursor protein, nsp220, was also characterized by peptide mapping. This protein contained all the peptides of nspl55, and several but not all of the peptides of asp 135. Some peptides were demonstrated which possibly are derived from ns60, the only nonstructural protein not yet isolated.

Small amounts of proteins with identical mobility to nsp250 and nsp220 were synthesized at 38°C in micrococcal‐nuclease‐treated rabbit reticulocyte lysate in response to virion 42‐S RNA from the ts‐6 mutant. The product of the wild‐type 42‐S RNA in vitro contained, in addition to nsp220 and nsp155, polypeptides which comigrated with ns86, ns72 and ns70, indicating processing of the translational product. The authenticity of nsp220, nsp155 and ns70 synthesized in vitro was confirmed by limited proteolysis with V8 protease.
Regulation of Glyoxysomal Enzymes during Germination of Cucumber
Authors: WEIR Elizabeth M. , RIEZMAN Howard , GRIENENBERGER Jean‐Michel , BECKER Wayne M. , LEAVER Christopher J.
Published: July 2010

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)‐rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X‐ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.
Effect of Freezing and Thawing on the Structure of Turnip Yellow Mosaic Virus
Authors: KATOUZIAN‐SAFADI Mehrnaz , FAVRE Alain , HAENNI Anne‐Lise
Published: July 2010

The uncoating of turnip yellow mosaic virus in vitro induced by freezing and thawing has been investigated using a variety of biochemical techniques including the aminoacylation capacity of the viral RNA and the ability of the RNA to stimulate protein synthesis, as well as physico‐chemical techniques such as sucrose gradient centrifugation and electron microscopy by negative staining. In particular a fluorescence test has been developed that can serve as a routine method to quantify the RNA liberated during the freeze‐thaw process. Escape of the viral RNA is a highly cooperative phenomenon: it depends critically on the virus concentration during freezing and thawing. Increasing the ionic strength or including foreign proteins diminish the escape of the RNA. The RNA is not damaged by this treatment and its liberation occurs without disruption of the viral capsid.
Purification, Some Properties and Amino‐Acid Sequences of Two Phospholipases A (CM‐II and CM‐III) from Naja naja kaouthia Venom
Authors: JOUBERT Francois J. , TALJAARD Nico
Published: July 2010

Two phospholipases A, CM‐II and CM‐III, were purified from the Siamese cobra (Naja raja kaouthia) venom, from Thailand, by gel filtration on Sephadex G‐50 followed by ion‐exchange chromatography on CM‐cellulose. They each comprise 119 amino acid residues including 14 halfcystine residues. The complete primary structures of the two phospholipases A have been elucidated and their sequences are 96% identical. The sequences, the invariant amino acid residues and the sequences around the active center (histidine‐48) of CM‐II and CM‐III resemble those from other snake venoms and also from mammalian pancreas. Further, the cysteine residues are in similar locations to those in phospholipase A of known structure so they are presumed to link similarly. The two phospholipases A from. N. n. kaouthia venom each contain a single histidine residue which is located at the active center (histidine‐48). The toxicities of the two enzymes differ among themselves, but are comparable to those encountered for phospholipases A from African cobra venoms.
Glutamate Dehydrogenase from Peas: Isolation, Quaternary Structure, and Influence of Cations on Activity
Authors: KINDT Rainer , PAHLICH Edwin , RASCHED Ihab
Published: July 2010

The most active multiple molecular form of glutamate dehydrogenase from pea seeds has been enriched 30000‐fold with a recovery of 60–70%. The preparation is homogenous as judged from gel electrophoresis of native and dodecylsulfate‐denatured enzyme and from analytical ultracentrifugation. Specific activities were 530 U/mg in the reductive amination and 90 U/mg in the oxidative deamination reaction. A sedimentation coefficient of s20, w= 10.49 S was determined. The specific volume and the molecular weight of the native enzyme were found to be v2= 0.759 cm3/g and Mr= 260000, respectively, by equilibrium sedimentation in H2O and 90%2H2O buffers. Dodecylsulfate electrophoresis of the denatured enzyme yielded a molecular weight of Mr= 44000 for the polypeptide chain. From our data we propose glutamate dehydrogenase from peas to be a hexamer. The hexameric structure is confirmed by the appearance of six electrophoretic bands after crosslinking with diimidates. Enzyme which was treated with Dowex A1 chelating resin was found to be almost completely inactive in the absence of divalent metal ions. From several metals tested, calcium was most efficient in reactivating the enzyme; half‐maximal activity was attained at about 5 μM calcium. In contrast to potassium ions, sodium ions were found to interfere with this regulatory mechanism by activating the enzyme at high concentrations.
Structural Studies on Two High‐Mobility‐Group Proteins from Calf Thymus, HMG‐14 and HMG‐20 (Ubiquitin), and Their Interaction with DNA
Authors: CARY Peter D. , KING Deborah S. , CRANE‐ROBINSON Colyn , BRADBURY E. Morton , RABBANI Azra , GOODWIN Graham H. , JOHNS Ernest W.
Published: July 2010

High mobility group (HMG) protein 14, which, like HMG‐17, has been implicated in the structure of ‘active chromatin’ is shown by 270‐MHz NMR and by circular dichroism to be in a disordered conformation in free solution. At low ionic strength protein HMG‐14 binds to DNA by weak attachment of the N‐terminal half of the molecule and is released by 0.3 M NaCl, the ionic strength at which the protein is extracted from chromatin. The protein HMG‐20 (ubiquitin), a constituent of the conjugate protein A24, is shown to be a highly stable compact globular protein that remains folded over a pH range of 1–13 and has a half‐denaturation temperature of 85°C when thermally denatured. Circular dichroism indicates 28% helix and 12%β sheet. Despite having 15% basic residues it binds only very weakly to DNA. A detailed study of the folding of ubiquitin has been made by a combination of several NMR approaches, including decoupling, nuclear Overhauser enhancement and titration. Several line assignments have been made and it is shown that, although the tyrosine and histidine are buried residues, they are not adjacent to one another nor are they close to either of the phenylalanines, of which at least one is also a buried residue.
Partial Purification of the α‐Tubulin and β‐Tubulin Messenger RNAs from Rat Brain
Authors: PORTIER Marie‐Madeleine , JEANTET Claude , GROS François
Published: July 2010

Poly(A)‐containing RNA from frozen adult rat brain were fractionated by centrifugation in a formamide/sucrose gradient. Individual fractions were used to program protein synthesis in vitro in a reticulocyte lysate. The cell‐free translation products were analyzed by two‐dimensional electrophoresis in polyacrylamide slab gels. We observed a heterodispersion of the mRNA translation activity coding for the β‐tubulin subunit which contrasts with a relatively homogeneous distribution of the α‐tubulin subunit mRNA. These last mRNA species are present in a peak which sediments near the 18‐S region of the gradient whereas the β‐tubulin mRNA activity is predominant in the fractions corresponding to the heaviest mRNA species.

When these heaviest RNAs were separated again by centrifugation in a second formamide/sucrose gradient, a poly(A)‐rich RNA population was obtained that was enriched in RNA for programming the β‐tubulin subunit. Analysis of the products whose synthesis in vitro was directed by this mRNA population revealed that β tubulin was the main protein formed, the ratio β/α being more than tenfold greater than in the products translated in vitro using total poly(A)‐rich RNA.
Fluorimetry Study of N‐(1‐Pyrenyl)iodoacetamide‐Labelled F‐Actin
Authors: KOUYAMA Tsutomu , MIHASHI Koshin
Published: July 2010

A fluorescent reagent, N‐(1‐pyrenyl)iodoacetamide, was conjugated to rabbit skeletal muscle actin at the site of the most reactive sulfhydryl group, and fluorescence characteristics (excitation and emission spectra, quantum yields, lifetimes) of the conjugate were investigated. Associated with polymerization of labelled G‐actin, the fluorescence intensity at 407 nm, after excitation at 365 nm, was enhanced by a factor of about 25. It was reduced to about 25% on the binding of heavy meromyosin (or subfragment 1). The results suggest that binding of heavy meromyosin to the protomer of F‐actin alters the local structure of the protomer towards a G‐actin‐like one.
Amino‐Acid Sequence of the L‐4 Light Chain of Chicken Skeletal‐Muscle Myosin
Authors: MAITA Tetsuo , UMEGANE Toshiyo , MATSUDA Genji
Published: July 2010

Tryptic and peptic peptides of the L‐4 light chain of chicken skeletal muscle myosin were isolated. The amino acid sequences of all the tryptic peptides were determined. The alignment of the tryptic peptides in the protein was deduced from their homology with the primary structure of the A2 (L‐4) light chain of rabbit skeletal muscle myosin. The compositions of the peptic peptides confirmed the alignment. Comparing the whole sequence of the L‐4 light chain thus established with that of the A2 light chain of rabbit skeletal muscle myosin, 24 amino acid substitutions, were recognized.
ADP‐Ribosylated Histone H1
Authors: BRAEUER Heinrich‐Christian , ADAMIETZ Peter , NELLESSEN Ulrich , HILZ Helmuth
Published: July 2010

Conjugates between histone H1 and (ADP‐ribose)n, formed in isolated nuclei of Ehrlich ascites tumor cells, were purified free of unmodified H1 using perchloric acid extraction, ion‐exchange and boronate‐cellulose chromatography. The isolated conjugates comprised 0.6% of the total protein‐bound ADP‐ribose residues, and about 1% of the histone H1 population.

Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining for protein or by fluorography of the [3H]ADP‐ribose residues. The neighboring bands appeared to differ from cach other by a single ADP‐ribose residue. In most preparations a mean chain length of 2–3 was found. Removal of the ADP‐ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher‐resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non‐ADP‐ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP‐ribosyl transfer.

Studies on the lability of the (ADP‐ribose)n protein linkage showed that about 20% of the ADP‐ribose was linked by an NH2OH/NaOH‐sensitive bond, 70% by an NH2OH‐resistant/NaOH‐sensitive bond, and the residual 10% apparently by an additional, NaOH‐resistant bond.

Cleavage of the (ADP‐ribose)n‐ histone H1 conjugates by N‐bromosuccinimide and gel eleetrophoretic analysis of the two fragments revealed that by far the most ADP‐ribose residues were linked (presumably at multiple sites) to the C‐terminal fragment. Furthermore, a large fraction of the conjugates carried ADP‐ribosyl groups exclusively either at the C‐terminal fragment or at the N‐terminal fragment.
Regulation of Protein Synthesis in Reticulocyte Lysates
Authors: BONANOU‐TZEDAKI Sophie A. , SOHI Maninder K. , ARNSTEIN Henry R. V.
Published: July 2010

Heating of a rabbit reticulocyte lysate at 44°C, in the presence of optimal concentrations of haemin, results in an inhibition of protein synthesis and in the appearance of an inhibitory activity in the postribosomal supernatant. These effects of supraoptimal heating are similar to those observed in lysates and supernatants incubated at physiological temperatures in the absence of haemin. In this paper we examined whether the haem‐regulated inhibitor produced by treating postribosomal supernatant with N‐ethylmaleimide and the inhibitor generated by heating at 44° C in the presence of haemin are the same molecular entity. Both inhibitors behaved similarly through a partial purification consisting of 50% (NH4)2SO4 precipitation, Sephadex G‐150 gel filtration and DEAE‐cellulose chromatography, and had the same pattern of polypeptides after polyacrylamide gel electrophoresis. However, when incubated with an antiserum to the haem‐regulated inhibitor the activity of the 44° C heated supernatant was not neutralized, whereas that of its more purified fractions was. This apparent contradiction was shown to be due to an interference of the immune serum assay by the levels of proinhibitor and haemoglobin present in the crude supernatant. Further experiments, with extensively diluted 44° C supernatants or with isolated proinhibitor subsequently heated, are consistent with the conclusion that both heating at supraoptimal temperatures and incubating in the absence of haem finally activate the same inhibitor.
Inhibition of Lactate Dehydrogenase Activity by Polymeric NAD Derivatives with Different NAD Densities
Authors: FURUKAWA Satoru , URABE Itaru , OKADA Hirosuke
Published: July 2010

Polymeric NAD derivatives with different NAD densities were prepared by the copolymerization of NAD N6‐[N‐(N‐acryloyl‐1‐methoxycarbonyl‐5‐aminopentyl)‐propionamide] with acrylamide in different molar ratios, from 4 × 10−4 to 1 × 10−2. The cofactor activities of the polymeric NAD derivatives for lactate, yeast alcohol and horse liver alcohol dehydrogenases decreased with the increase in the NAD density of the polymer, and the lactate dehydrogenase showed no activity for the polymeric NAD derivatives with NAD densities of 2 × 10−3 and 1 × 10−2 mol/mol acrylamide polymerized.

The polymeric NAD derivatives inhibited the activities of lactate and yeast alcohol dehydrogenases but did not inhibit those of horse liver alcohol and malate dehydrogenases. The polymeric NAD derivatives were competitive inhibitors with respect to NAD for the reaction of lactate dehydrogenase: the inhibition constant decreased from 2.2 μM to 0.02 μM with the increase in the NAD density frotn 4 × 10−4 to 1 × 10−2 mol/mol acrylamide polymerized.
Steady‐State Kinetic Studies on Benzylamine Oxidase from Pig Plasma
Authors: KELLY Iain D. , KNOWLES Peter F. , YADAV Kapil D. S. , BARDSLEY William G. , LEFF Paul , WAIGHT Roy D.
Published: July 2010

Steady‐state kinetic studies on the enzyme benzylamine oxidase from pig plasma are described. Eadie‐Hofstee plots with benzylamine as the varying substrate are non‐linear; examination of this data indicates that the observed effects are probably due to the amine substrate participating in at least two reactions with enzyme. Ammonia and imidazole modify the activity of the enzyme; under specified conditions of pH or modifier concentration, the effect on the activity can be either activation or inhibition. Eadie‐Hofstee plots of the data establish that the modifier also participates in at least three reactions with the enzyme. Eadie‐Hofstee plots at pH 9 with oxygen as the varying substrate are linear, which allows kinetic parameters to be determined. From studies on the effect of ammonia and imidazole on these parameters, information has been derived on how these modifiers affect component steps of the catalytic cycle.
2‐n‐Nonyl‐4‐hydroxy [3‐3H] quinoline N‐oxide: Interaction with the Mitochondrial Membrane
Authors: RICCIO Paolo , GÜNTHER Helmut , QUAGLIARIELLO Ernesto
Published: July 2010

Synthesized 2‐n‐nonyl‐4‐hydroxy[3‐3H]quinoline N‐oxide ([3H]NoHOQnO) is a powerful inhibitor of both NADH oxidase and succinate oxidase activities in submitochondrial particles from beef heart.

The binding of [3H]NoHOQnO to submitochondrial particles reveals one specific binding site, which is sensitive to antimycin, and a large amount of aspecific binding.

The content of [3H]NoHOQnO‐specific binding sites is related to the cytochrome b content in the particles.

The binding of [3H]NoHOQnO to the specific site causes the inhibition of electron transfer.

The relation between the inhibition of NADH oxidation and [3H]NoHOQnO‐specific binding is hyperbolic.
Ligand Binding to Catalase and Metmyoglobin
Authors: MILLAR Fiona , WRIGGLESWORTH John M. , NICHOLLS Peter
Published: July 2010

Spectroscopic measurements of the binding of the weak‐acid ligands formate, azide and cyanide to catalase indicate interaction of a primary haem‐binding site with the undissociated forms of the ligands between pH 5 and 8. A similar conclusion can be drawn from observations on the pH dependence of catalase activity in the presence of cyanide and formate.

Direct measurements of pH changes during catalase interaction with these ligands also indicate stoichiometric uptake of one proton per haem group upon addition of formate or azide at pH 6, but at greater pH values the proton uptake declines, suggesting the involvement of a secondary site in the liganding reaction. This secondary site can be occupied by formate and certain other anions inducing proton dissociation from the protein. The results also suggest a direct displacement of formate or azide from the primary haem site by cyanide.

Similar stoichiometric pH measurements on metmyoglobin indicate binding of the formate, azide and cyanide anions to a single (primary) site on the molecule.
Amino Acids and Glucose Utilization by Different Metabolic Pathways in Ascites‐Tumour Cells
Authors: LAZO Pedro A.
Published: July 2010

The utilization of amino acids and glucose by ascites tumour cells has been studied in order to elucidate which are their relative roles as energy substrates or building blocks for biosynthetic purposes, as well as the quantitative contribution of the different metabolic pathways involved.

Glucose is utilized at a rate of 1.1 μmol × min−1× g cells−1. 93% is transformed into lactate, 0.7% used by the pentose phosphate pathway. 1.5% by the tricarboxylic acid cycle and 2% is for lipid synthesis.

ATP production is derived: 78% from glucose conversion into lactate, 1% from glucose oxidation and 19% from glutamine oxidation.

Glucose starvation, in the presence of all amino acids, leads to a 70% decrease in the rate of protein synthesis, due to the drop in ATP levels.

Pentose phosphate pathway flux increases by 75% when glycolysing cells are incubated in the presence of all amino acids.

Pyruvate is decarboxylated at a rate of 66 nmol × min−1× g cells−1, 45‐80% of it is incorporated into lipids instead of being oxidized, depending on the incubation conditions.

Non‐essential amino acids (aspartate and glutamate) are oxidized at a low rate. Glutamine is oxidized at a rate 20‐times and 35‐times that of glucose and glutamate respectively. Glutamine can not replace glucose as the main energy source.

Leucine utilization, 28 nmol × min−1× g cells−1. is very high compared with normal cells, due to the high rate of lipid and protein synthesis. Its oxidation is similar to that of non‐tumoural cells.

Sterols account for 80% of the lipids synthesized either from leucine or glucose.
Effects of Antibiotics, N‐Acetylaminoacyl‐tRNA and Other Agents on the Elongation‐Factor‐Tu Dependent and Ribosome‐Dependent GTP Hydrolysis Promoted by 2′(3′)‐O‐l‐Phenylalanyladenosine
Authors: CAMPUZANO Sonsoles , MODOLELL Juan
Published: July 2010

GTP hydrolysis on elongation factor (EF) Tu · ribosome complexes has been assayed in the presence of 2′(3′)O‐l‐phenylalanyladenosine (AdoPhe). i.e. the 3′‐terminal portion of Phe‐tRNAPhe. Several requirements of the reaction have been characterized. Maximal activity is observed at 60‐120 mM NH4Cl and 5–15 mM magnesium acetate. The reaction requires the free sulflhydryl group of EF‐Tu normally implicated in aminoacyl‐tRNA binding. Intact EF‐Tu cannot be replaced by a large tryptic fragment of EF‐Tu (Mr 3000) that retains the ability to bind guanosine nucleotides.

The aminoglycoside antibiotics, neomycin C and several kanamycins and gentamicins, stimulate the AdoPhepromoted GTPase. Surprisingly, however, other closely related antibiotics, like neomycin B, paromomycin and ribostamycin, are ineffectual, thus indicating subtle differences in the actions of these antibiotics.

AcPhe‐tRNAPhe, bound to the ribosomal A‐site, stimulates the AdoPhe‐promoted GTPase, but this compound or AcTyr‐tRNATyr, present in unbound form, strongly inhibits the reaction. These results suggest that N‐blocked aminoacyl‐tRNAs form ternary complexes with EF‐Tu · GTP, which have not been previously detected because of their low stability.
Syntheses of l‐Tyrosine‐Related Amino Acids by Tyrosine Phenol‐lyase of Citrobacter intermedius
Authors: NAGASAWA Toru , UTAGAWA Takashi , GOTO Joji , KIM Chan‐Jo , TANI Yoshiki , KUMAGAI Hidehiko , YAMADA Hideaki
Published: July 2010

Degradation of tyrosine to phenol, pyruvate and ammonia by tyrosine phenol‐lyase from Citrobacter intermedius (formerly named Escherichia intermedia) is readily reversible at high concentrations of pyruvate and ammonia. Spectrophotometric studies indicate that ammonia is the first substrate which interacts with bound pyridoxal 5′‐phosphate. Kinetic results show that pyruvate is the second substrate bound, hence phenol must be the third. When an appropriate phenol derivative is substituted for phenol, the corresponding tyrosine analogue can be synthesized. 3‐Fluoro‐, 2‐fluoro‐, 3‐chloro‐, 2‐chloro‐, 3‐bromo‐, 2‐bromo‐, 2‐iodo‐, 3‐methyl‐, 2‐methyl and 2‐methoxy‐l‐tyrosines have been synthesized by this reaction. By using various phenol derivatives or tyrosine analogues as substrates, the substrate specificity of tyrosine phenol‐lyase is investigated, and the situation of its active site is discussed.
The Effect of Ligands on the Irreversible Inhibition of the NAD+‐Dependent Isocitrate Dehydrogenase from Ox Brain
Authors: WILLSON Vivian J. C. , TIPTON Keith F.
Published: July 2010

The effects of ligands on the irreversible inhibition of the NAD+‐dependent isocitrate dehydrogenase from ox brain were studied. Isocitrate in the presence of Mg2+ ions was found to protect against denaturation at 40°C and this protection was enhanced by ADP which also protected on its own. None of the substrates or the activator ADP afforded protection against inhibition by diethylpyrocarbonate. Inactivation by this compound and by elevated temperatures did not obey first‐order kinetics with respect to time. Inhibition by iodoacetate appeared to obey first‐order kinetics both with respect to time and to inhibitor concentration. Protection against inhibition was afforded by isocitrate, NAD+ and ADP. The dependence of the extent of protection on the concentration of the magnesium‐isocitrate complex was sigmoid at both pH 6.5 and pH 7.5 indicating the apparent homotropic cooperativity seen in initial‐rate kinetic experiments to be a reflection of cooperative binding of this substrate. ADP reduced the affinity of the enzyme for this substrate without affecting the degree of this cooperativity. The dependence of the extent of protection by ADP upon its concentration obeyed Michaelis‐Menten kinetics. The significance of these observations in terms of the kinetic and allosteric mechanism followed by this enzyme is discussed in the light of previous kinetic studies.
The Effect of Azastene, Cyanoketone and Trilostane upon Respiration and Cleavage of the Cholesterol Side Chain in Mitochondria from Bovine Adrenal Cortex
Authors: SHEARS Stephen B. , BOYD George S.
Published: July 2010

Mitochondria prepared from bovine adrenal cortex and incubated with ADP and phosphate respired at about 45% of the rate observed in the presence of an uncoupler of oxidative phosphorylation; there was, however, little inefficiency in the reactions involved in the phosphorylation of ADP.

Three inhibitors of the 3β‐hydroxysteroid dehydrogenase (azastene, cyanoketone and trilostane) were employed with a view to preventing pregnenolone metabolism and thus aiding the assay of cholesterol side‐chain cleavage. Freshly made solutions of these inhibitors did not modify mitochondria) respiratory rates, at concentrations of 10 μM. In contrast, solutions maintained at 0–4°C for one week subsequently inhibited the respiratory rate of uncoupled mitochondria. When fresh solutions of the inhibitors were used in the assays of cholesterol side‐chain cleavage, 10 μM azastene did not significantly inhibit pregnenolone metabolism. Cyanoketone and trilostane were both significant inhibitors of pregnenolone metabolism, but 10 ttM cyanoketone reduced the initial rate of cholesterol side‐chain cleavage by 50% in the presence of 10 mM malate, although this inhibition did not occur in the presence of 10 mM dl‐isocitrate. Thus trilostane may be the preferred inhibitor of the 3β‐hydroxysteroid dehydrogenase during studies of cholesterol side‐chain cleavage in vitro.
Isolation of a Second Rifamycin‐Binding from Escherichia coli by Affinity Chromatography
Authors: SEYDEL Friederike , HARTMANN Guido R. , LILL Helmut R.
Published: July 2010

When extracts of Escherichia coli are filtered through a Sepharose column containing covalently bound rifamycin a protein is bound which can be eluted either with a high concentration of urea or more specifically with low concentrations of rifamycins. Its Mr is 18000 ± 1000 in the presence of dodecylsulfate, in its absence 36000 ± 3000. The association constant of the protein for rifampicin is 2.4 ± 0.5 × 10−4 M with two binding sites per dimer as determined by equilibrium dialysis. Large amounts of this protein are released from the cells by an osmotic shock.
A Nuclear Mutant of Chlamydomonas reinhardtii Defective in Photosynthetic Photophosphorylation
Authors: PICCIONI Richard G. , CHUA Nam‐Hai , BENNOUN Pierre
Published: July 2010

Use of the Flagyl selection procedure [Schmidt et al. (1977) Proc. Natl Acad. Sci. USA, 74, 610‐614] led to the isolation of a nuclear mutant of Chlamydomonas reinhardtiidesignated thm‐24. This mutant displays normal electron transport rates in vitro, possesses high latent ATPase activity bound to the thylakoid membrane, but is incapable of photophosphorylation. Decay of the transmembrane potential, as indicated by the kinetics of the 520‐nm absorption change after illumination, is unusually slow and markedly biphasic. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of purified thylakoid membrane. shows mutant thm‐24 to be lacking a number of polypeptides including those previously designated 4.1, 4.2 and 8.1.

Treatment of purified thylakoid membranes of wild‐type and mutant algae, using the chloroform‐release procedure of Beechey et al. [(1975) Biochem. J. 748, 533–537] resulted in the removal of ATPase activity from each strain. In wild‐type cells, the ATPase activity was of heterogeneous enzymatic origin; fractionation of the chloroform‐release extracts by non‐denaturing polyacrylamide gel electrophoresis yielded three distinct bands displaying ATPase activity, designated ATPases I, II and III. In contrast, extracts from membranes of mutant thm‐24 yielded only one ATPase‐containing fraction, co‐migrating with ATPase I from wild‐type. Use of electrophoretic, immunological and enzymatic methods established a correspondence of the polypeptide subunits of ATPases II and III and those of spinach coupling factor, CF1. ATPase I from either algal strain was shown to be structurally distinct from higher plant CF1 and to C. reinhardtii ATPases II and III.
Affinity Labeling of Liver Alcohol Dehydrogenase
Authors: SYVERTSEN Christian , McKINLEY‐McKEE John S.
Published: July 2010

Both iodoacetic acid and (R, S)‐2‐bromo‐3‐(5‐imidazolyl)propionic acid (BrImPpOH) react with liver alcohol dehydrogenase in an affinity labelling mechanism between pH 6:1 and 10. 5. The buffer‐independent dissociation constants and the first‐order rate constants have been determined as a function of pH. With BrImPpOH a pKa close to 9 for the free enzyme is assigned to the zinc‐water ionization. The buffers used exerted a protective effect upon the inactivation of the enzyme by iodoacetic acid and BrImPpOH. Phosphate buffer showed a high degree of protection especially at lower pH, while zwitterionic buffers like Mes (4‐morpholineethanesulfonic acid), Pipes (1, 4piperazinediethanesulfonic acid), Epps [4‐(2‐hydroxyethyl)‐1‐piperazinepropanesulfonic acid] and Bicine [N, N‐bis(2‐hydroxyethyl)glycine] gave less protection to various degrees. An exception was Ches (cyclohexylaminoethanesulfonic acid) which had an anomalously high affinity for the iodoacetate binding site. The dissociation constants of the buffers were calculated for the case of inactivation by both iodoacetic acid and BrImPpOH.
Studies on the Mechanisms of Action of Proteolytic Enzymes Using Heavy Oxygen Exchange
Authors: ANTONOV Vladimir K. , GINODMAN Lev M. , RUMSH Lev D. , KAPITANNIKOV Yurii V. , BARSHEVSKAYA Tatyana N. , YAVASHEV Libert P. , GUROVA Aleksandra G. , VOLKOVA Ludmila I.
Published: July 2010

Transpeptidation reactions catalyzed by chymotrypsin, pepsin, leucine aminopeptidase and thermolysin have been studied in heavy oxygen water (H218O). The 18O incorporation into the peptide bond of transpeptidation products and into the non‐hydrolyzed substrate has been measured. The rates of 18O exchange in the carboxylic groups of N‐acetylphenylalanine and leucine, catalyzed by pepsin and leucine aminopeptidase, respectively, have also been determined. These rates have been compared with that of the exchange in the presence of amino compounds which reversibly form amide bonds with the above carboxyl‐containing substances. The data obtained show that, in contrast to chymotrypsin, other enzymes studied do not form ‘acyl‐enzymes’ but function by the mechanism of general‐base catalysis. In other words, their catalytically active groups promote the abstraction of a proton from the mater molecule, which attacks the susceptible bond of the substrate. The structure of intermediate compounds in this type of catalysis and the mechanism of the transpeptidation reaction are discussed.
Conversion of Free Lipoprotein to the Murein‐Bound Form
Authors: WENSINK Jan , WITHOLT Bernard
Published: July 2010

The time‐course of the conversion of free lipoprotein to the murein‐bound form was followed after pulsing growing cells of Escherichia coli for 2 min with [35S]methionine. The conversion of the free to the bound form was measured by two independent methods. First, the appearance of murein‐bound lipoprotein was determined in isolated murein sacculi after extraction with hot 4% sodium dodecylsulphate. Second, the ratio of various forms of the bound lipoprotein to the free lipoprotein was determined in outer membranes. Both methods indicated that the half‐maximal amount of bound lipoprotein was reached within one‐sixth of a generation time.
Regulation of Phosphorylation of Myosin Heavy Chain during the Chemotactic Response of Dictyostelium Cells
Authors: MALCHOW Dieter , BÖHME Roswita , RAHMSDORF Hans Jobst
Published: July 2010

Chemotactic stimulation of the cellular slime mould Dictyostelium discoideum by cyclic AMP increases the incorporation of 32P into a 210‐kDa polypeptide. This was determined in lysates prepared from stimulated and nonstimulated cells. Earlier results suggesting that the 210‐kDa polypeptide corresponds to myosin heavy chains [7] have now been confirmed.

We have investigated the sequence of events following chemotactic stimulation that lead to the change of myosin heavy‐chain phosphorylation.

Chemotactic stimulation did not activate myosin heavy chain kinase, but seemed to elicit the transient accumulation of dephosphorylated myosin heavy chains. Thus, the inefficient incorporation of 32P into myosin heavy chains in lysates of control cells seems to be due to the lack of dephosphorylated myosin heavy chains.

Using membranes of aggregation‐competent cells. we found that incorporation of 32P into myosin heavy chains was inhibited by 0. 1–1 mM Ca2+. The effect of calcium seems to be mediated by endogeneous calmodulin and was due to the inhibition of myosin kinase activity rather than to the activation of myosin phosphatase.

By the addition of calmodulin, the inhibition of myosin heavy‐chain phosphorylation was further enhanced.

Folic acid, an attractant of undifferentiated cells, also caused enhanced incorporation of 32P into myosin heavy chains, as determined in cell lysates.

The amount of 32P incorporated in response to cyclic AMP into myosin heavy chains increased during differentiation to the aggregation‐competent stage. Only a small fraction of the cell surface receptors had to be activated in order to elicit a maximal reaction.

Our results suggest that a phosphorylated form of myosin heavy chains prevails at the onset of the chemotactic response and that dephosphorylation begins within 5–10 s at 23°C.