bluuebunny/crossref_metadata_2025 huggingface dataset:
The Association of the Serotonin‐Sensitive Aryl Acylamidase with Acetylcholinesterase in the Monkey Brain
Authors: OOMMEN Anna , BALASUBRAMANIAN Aiylam S.
Published: March 2005

The serotonin‐sensitive aryl acylamidase was partially purified from monkey brain. The aryl acylamidase activity was inhibited by serotonin (Ki= 0.425 mM) and tryptamine (Ki= 3.6 mM) but not by a number of other amines. It was also inhibited by acetylcholine (Ki= 2 mM) and its analogues and homologues. The relationship of aryl acylamidase to acetylcholinesterase was examined. The ratios of specific activities of aryl acylamidase and acetylcholinesterase in the different steps of purification were approximately constant and the percentage recoveries of both enzyme activities were comparable. Elution profiles of both enzyme activities from concanavalin‐A‐Sepharose, Sephadex G‐200 and DEAE‐Sephadex A‐25 columns were similar. Both enzyme activities migrated in a similar fashion on gel electrophoresis in different percentage gels for different time intervals. Both enzymes showed similar distribution in the various anatomical regions and in the different subcellular fractions of monkey brain.

Eserine and neostigmine, potent competitive inhibitors of acetylcholinesterase also potently inhibited aryl acylamidase in a non‐competitive manner. Inhibition of both enzymes was 100% at 10 μM of both the inhibitors. Tetraisopropylpyrophosphoramide, a selective inhibitor of pseudo‐cholinesterase, did not inhibit either the brain acetylcholinesterase or aryl acylamidase at 10μM. Serotonin inhibited acetylcholinesterase only at concentrations above 10 mM. Dixon plots of one inhibitor in the presence of additional inhibitors indicated that serotonin, butyrylcholine, acetylcholine, neostigmine and eserine (all non‐competitive inhibitors of aryl acylamidase) acted at the same site as inhibitors of aryl acylamidase.

The serotonin‐insensitive aryl acylamidase of monkey liver was also insensitive to acetylcholine and eserine and was not found associated with acetylcholinesterase [A. Oommen and A. S. Balasubramanian (1978) Biochem. Pharmacol. 27, 891–895]. Erythrocyte membrane known to contain true cholinesterase had aryl acylamidase activity sensitive to serotonin, acetylcholine and eserine. All these considerations suggest that the serotonin‐sensitive aryl acylamidase activity is a property of true cholinesterase. Although some of the experimental results would suggest that in the monkey brain the two activities may be associated with the same protein with two different active sites further experiments are needed to confirm this.
Yeast Argininosuccinate Synthetase
Authors: HILGER François , SIMON Jean‐Paul , STALON Victor
Published: March 2005

Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228000 as determined by gel sieving. It is composed of identical subunits of Mr 49000 as shown by gel electrophoresis.

The quaternary structure as determined by cross‐linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric.

The saturation functions by citrulline and aspartate are hyperbolic; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and asparate; it is increased by argininosuccinate and arginine.

Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead‐end inhibition studies comply with a rapid‐equilibrium random model, except for the interconversion of the central quaternary complexes; the different kinetic constants have been established on this basis.

Yeast arginonosuccinate synthetase has a double metabolic function: anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well‐adjusted activity for both roles and to an economic and efficient utilization of ATP.
The Biosynthesis of Ribulose Bisphosphate Carboxylase
Authors: BARRACLOUGH Roger , ELLIS R. John
Published: March 2005

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5 h. Analysis of the products of incorporation by one‐dimensional and two‐dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme. ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2‐(4‐methyl‐2,6‐dinitroanilino)‐N‐methylpropionamide stops incorporation into the small subuniit, but large subunit, continues to be made for at least 4 h. For accurate estimates of incoroporation into the large subunit, it is essential to use two‐dimensional gel electrophoresis, because the large subunit region on one‐dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are discussed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.
Crayfish Carboxypeptidase
Authors: ZWILLING Robert , JAKOB Frank , BAUER Heinrich , NEURATH Hans , ENFIELD David L.
Published: March 2005

In an effort to trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase has been isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3‐phenyllactate and by 6‐aminohexanoate. The pH optimum of activity is about pH 6.5, and the isoelectric point, pH 4.0. Inhibition by typical metal chelating agents (i.e. ethylenediamine tetraacetate and 1,10‐phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypeptidase is a zinc metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecylsulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino‐terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.
Quaternary Structure of Higher Plant Glyceraldehyde‐3‐Phosphate Dehydrogenases
Authors: CERFF Rüdiger
Published: March 2005

NAD(P)+‐induced changes in the aggregational state of prepurified NADP‐linked glyceraldehyde‐3‐phosphate dehydrogenase (EC 1.2.1.13) were used to isolate the enzyme from Spinacia oleracea, Pisum sativum and Hordeum vulgare. Each of the three plant species contains two separate isoenzymes. Isoenzyme 1 (fast moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 55–70% saturation. It shows two separate subunits in dodecylsulfate gels, which are probably arranged as A2B2 in the native enzyme molecule. Isoenzyme 2 (slow moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 70–95%. It contains a single subunit of the same Mr as subunit A in isoenzyme 1 and is apparently a tetramer (A4). The molecular weights of subunits A/B for spinach, peas and barley were determined as 38000/40000, 38000/42000 and 36000/39000 respectively.

The NAD‐specific glyceraldehyde‐3‐phosphate dehydrogenase (EC 1.2.1.12) was purified from Spinacia oleracea and Pisum sativum by affinity chromatography on blue Sepharose CL‐6B. The enzyme from both plant species is shown to be a tetramer of subunits with Mr 39000.

The present findings contrast with heterogeneous results obtained previously by other authors. These results suggested that there are considerable interspecific differences in the quaternary structure of glyceraldehyde‐3‐phosphate dehydrogenases from higher plants.
Fast Kinetic Studies on the Allosteric Interactions between Acetylcholine Receptor and Local Anesthetic Binding Sites
Authors: HEIDMANN Thierry , CHANGEUX Jean‐Pierre
Published: March 2005

Preincubation of receptor‐rich membrane fragments from Torpedo marmorata with tertiary amine local anesthetics and several toxins such as histrionicotoxin, crotoxin and cerulotoxin, modifies the amplitude and time course of the relaxation processes monitored upon rapid mixing of the membrane fragments with the fluorescent agonist, Dns‐C6‐Cho. In particular, the amplitude of the rapid relaxation process, which is proportional to the fraction of acetylcholine receptor sites in a high‐affinity state, increases; accordingly, the rate constant of the ‘slow’ and ‘intermediate’ relaxation processes also increases up to ten times (except with histrionicotoxin) whereas in a higher range of local anesthetic concentrations the rate constant of the ‘rapid’ relaxation processes decreases. The data are accounted for by a two‐state model of the acetylcholine regulator, assuming distinct binding sites for cholinergic agonists and local anesthetics and allosteric interactions between these two classes of sites; local anesthetics stabilize the regulator in a high‐affinity state for agonists even in the absence of agonist, and modify the rate constants for the interconversions between the low‐affinity and high‐affinity states. The model accounts for the ‘slow’ fluorescence increase monitored upon addition of local anesthetics to a suspension of receptor‐rich membranes supplemented with trace amounts of Dns‐C6‐Cho. The effect of local anesthetics on the apparent rate constant of the ‘rapid’ relaxation process can be accounted for on the basis of an additional low‐affinity binding of local anesthetics to the acetylcholine receptor site. Finally the increase of the apparent rate constant of the ‘intermediate’ relaxation process can be simply accounted for by assuming the existence of a third state, corresponding to the ‘active’ state, to which local anesthetics bind and block ionic transport.
Localisation of an Endonuclease Specific for Double‐Stranded RNA within the Nucleolus and Its Implication in Processing Ribosomal Transcripts
Authors: GRUMMT Ingrid , HALL Stanton H. , CROUCH Robert J.
Published: March 2005

Nucleoli of both chick embryos and mouse Ehrlich ascites cells contain an enzymatic activity that is very similar to RNase DII, an enzyme isolated from total chick embryos for its ability to degrade double‐stranded RNA. The enzyme can be extracted by low salt/EDTA from nucleoli and is associated with pre‐ribosomal 80‐S and 55‐S particles. Under ionic conditions which are inhibitory for the nucleolytic activity the transcript in vitro of nucleoli is not processed and sediments around 45 S. Under salt conditions which are optimal for the nucleolar enzyme the nucleolar transcripts are cleaved to distinct intermediate‐sized moleculues. Addition of the chicken RNase DII or RNase III to the nucleolar transcription system results in a similar shift of the chain length of the RNA molecules.

It is concluded that a nucleolar RNase recognizing double‐stranded regions in the pre‐ribosomal RNA is involved in the maturation of ribosomal RNA.
Metal Ion/Buffer Interactions
Authors: FISCHER Beda E. , HÄRING Ulrich K. , TRIBOLET Roger , SIGEL Helmut
Published: March 2005

The interaction of 2‐amino‐2(hydroxymethyl)‐1,3‐propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I= 0.1 or 1.0 M, KNO3, 25°C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4− as a second ligand were also investigated and it is shown that in the presence of Tris, mixed‐ligand complexes of the type M(ATP)(Tris)2− are formed. The values for Δlog Km, where Δlog Km= log KM(ATP)TrisM(ATP)− log KM(Tris)'M are all negative, thus indicating that the interaction of Tris with M(ATP)2minus; is somewhat less pronounced than with M2+. However, it should be noted that even in mixed‐ligand system complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2minus; and the ternary M(ATP)(Tris)2− complexes are discussed. The participation of a Trishydroxo group in complex formation is, at least for the M(Tris)2− species, quite evident.
Chemical Modification of Taipoxin and the Consequences for Phospholipase Activity, Pathophysiology, and Inhibition of High‐Affinity Choline Uptake
Authors: FOHLMAN Jan , EAKER David , DOWDALL Michael J. , LÜLLMANN‐RAUCH Renate , SJÖDIN Torgny , LEANDER Stefan
Published: March 2005

Treatment of taipoxin with p‐bromophenacyl bromide resulted in modification of single histidine residues in the α and β subunits. The modification decreased the neurotoxicity (lethality) 350‐fold, but the inhibitory action on high‐affinity choline transport was reduced only threefold. The phospholipase activity and Ca2+‐association constants for taipoxin and its subunits were determined. A model for the neurotoxicity of taipoxin indicates the α subunit as the ultimate cause of the disruption of synaptic transmission.
The Use of Biochemical Solid‐Phase Techniques in the Study of Alcohol Dehydrogenase
Authors: ANDERSSON Lars , MOSBACH Klaus
Published: March 2005

The EE and SS isozymes of horse liver alcohol dehydrogenase have been immobilized separately to weakly CNBr‐activated Sepharose 4B. The resulting immobilized dimeric preparations lost practically all of their activity after treatment with 6 M urea. However, enzyme activity was regenerated by allowing the urea‐treated Sepharose‐bound alcohol dehydrogenase to interact specifically with either soluble subunits of dissociated horse liver alcohol dehydrogenase or soluble dimeric enzyme. The regeneration of steroid activity in the immobilized preparations after treatment of the bound S subunits with soluble E subunits seems to show that true reassociation of the enzyme had taken place on the solid phase, since only isozymes with an S‐polypeptide chain are active when using 5β‐dihydrotestosterone as substrate. The results presented in this paper indicate that immobilized single subunits of horse liver alcohol dehydrogenase are inactive and that dimer formation is a prerequisite for the enzymic activity.
The Effect of Dephosphorylation on the Properties of a Helix‐Destabilizing Protein from Meiotic Cells and Its Partial Reversal by a Protein Kinase
Authors: HOTTA Yasuo , STERN Herbert
Published: March 2005

Properties of the helix‐destabilizing protein from Lilium meiotic cells, ‘R‐protein’, have been examined after treating it either with alkaline phosphatase or with two types of protein kinase. Dephosphorylation with the phosphatase increases binding capacity for single‐strand DNA, but abolishes specificity of binding. Dephosphorylated R‐protein binds equally to single and double‐strand DNA. The capacity to facilitate denaturation or renaturation of DNA is also abolished by the treatment, but cooperativity characteristics are unaffected. The consequences of protein kinase treatment of native or dephosphorylated R‐protein depend upon the origin of the kinase. Heterologous cyclic‐AMP‐dependent protein kinase cannot reverse the effects of dephosphorylation. However, it abolishes the binding affinity of either native or dephosphorylated R‐protein for DNA. A protein kinase isolated from meiotic cells has no effect on the native protein, but it does restore all native properties tested to the dephosphorylated form after phosphorylating approximately two residues/molecule of protein.
Respiratory Nitrate Reductase: Its Localization in the Cytoplasmic Membrane of Klebsiella aerogenes and Bacillus licheniformis
Authors: WIENTJES Frans B. , KOLK Arend H. J. , NANNINGA Nanne , Van 'T RIET Jan
Published: March 2005

The sidedness of the respiratory nitrate reductase in the cytoplasmic membrane of Bacillus licheniformis and Klebsiella aerogenes was studied by indirect immunofluorescence and by lactoperoxidase‐catalyzed iodination. It was shown that the two subunits (Mr 150000 and 57000, respectively) of nitrate reductase of B. licheniformis are localized on the cytoplasmic side of the membrane, whereas the K. aerogenes enzyme is a transmembrane protein. The different localization of nitrate reductase in the membranes of these organisms may be related to their different rôle in oxidative phosphorylation.
Induction of the Phosphoenolpyruvate: Hexose Phosphotransferase System Associated with Relative Anaerobiosis in an Obligate Aerobe
Authors: PELLICCIONE Nicholas , JAFFIN Barry , SOBEL Mark E. , KRULWICH Terry A.
Published: March 2005

Arthrobacter pyridinolis possesses alternative transport systems for d‐fructose: a respiration‐coupled transport system whereby d‐fructose transport occurs with concomitant oxidation of l‐malate, and a phosphoenolpyruvate: d‐fructose phosphotransferase system. Studies of d‐fructose uptake by whole cells in the presence and absence of cyanide demonstrate that respiration‐coupled transport is used almost exclusively during the first half of logarithmic growth, after which it accounts for only 15–20% of d‐fructose uptake. Phosphotransferase levels are low during log phase, peak during late log, and then slowly decline. In a mutant of A. pyridinolis which requires δ‐amino‐levulinic acid for growth, the growth rate, cell cytochrome content, and activity of the respiration‐coupled transport system increased with increasing concentrations of δ‐aminolevulinic acid up to 50 μg/ml. By contrast, phosphotransferase activity was highest in cells grown on limiting δ‐aminolevulinic acid. l‐Malate, which stimulates respiration‐coupled transport, repressed the phosphotransferase system. The respiratory activity and the ability to release CO2 from internalized d‐fructose was consistently low in d‐fructose‐grown cells. A cyanide‐resistant cytochrome, tentatively identified as cytochrome d, appeared in the late exponential phase of growth. Isocitrate lyase activity, required for aerobic growth of this organism, declined markedly during the late exponential phase. Thus the phosphotransferase system is maximally induced, in this obligate aerobe, under conditions of relative anaerobiosis during which metabolism is primarily fermentative.
Effects of Phospholipase A2 and Albumin on the Calcium‐Dependent ATPase and the Lipid Composition of Sarcoplasmic Membranes
Authors: SWOBODA Gertrude , FRITZSCHE Joachim , HASSELBACH Wilhelm
Published: March 2005

The calcium‐dependent ATPase activity of phospholipase‐A2‐digested sarcoplasmic vesicles decreases concomitantly with the contents of residual lysophospholipids and fatty acids when increasing albumin concentrations are applied.

Delipidated albumin preferentially removes unsaturated fatty acids and lysophosphatidylcholine. A complete removal of the phospholipids by albumin does not occur.

The membrane‐bound lysophospholipids were analysed with respect to type of phospholipid, plasmalogen content and fatty acid chains by means of thin‐layer chromatography and gas chromatography.

While the fatty acid composition of the lysophospholipids is independent of the degree of delipidation, the composition of the residual free fatty acids is found to change with the albumin concentration.

Reactivation of the Ca2+‐ATPase by oleate leads to reasonable activities at room temperature as long as a minimum of about 30 lysophospholipid moleucules per ATPase is left. The course of the residual Ca2+‐ATPase activity with the degree of delipidation is related to the presence of unsaturated fatty acids.

No specific role of either sphingomyelin or the plasmalogens has been found.
Dual Localization of Long‐Chain Acyl‐CoA Hydrolase in Rat Liver: One in the Microsomes and One in the Mitochondrial Matrix
Authors: BERGE Rolf Kristian , FARSTAD Mikael
Published: March 2005

Subcellular fractionation studies of rat liver localized the activity of palmitoyl‐l‐carnitine hydroalse to the microsomal fraction whereas palmitoyl‐CoA hydrolase activity was found both in the microsomal fraction and in mitochondria.

An unusual biphasic saturation curve for palmitoyl‐CoA was observed when intact mitochondria were used, possibly reflecting the existence of an intramitochondrial hydrolase activity. Disruption of the mitochondrial structure doubled the palmitoyl‐CoA hydrolysis. Discontinuous sucrose gradient centrifugation and digitonin of rat liver mitochondria demonstrated that a palmitoyl‐CoA hydrolase was associated with the matrix fraction.

Pure matrix and microsomal fractions showed that the two hydrolase activities were differently affected by the presence of divalent cations. Both the specific activity and the saturation concentration of palmitoyl‐CoA were higher for the microsomal enzyme than for the matrix‐associated enzyme.
Energetics of the Thermal Transitions of RNA 1 and RNA 4 of Alfalfa‐Mosaic Virus in the Presence and Absence of Coat Protein
Authors: HINZ Hans‐Jürgen , SRINIVASA Sri , JASPARS E. M. J.
Published: March 2005

Transition enthalpies of one genomic RNA, RNA 1. and the subgenomic RNA 4 of alfalfa mosaic virus were determined by scanning microcalorimetry in the absence and presence of various amounts of coat protein in a buffer containing 10 mM NaH2PO4, 1 mM EDTA, 1mM NaH3, pH 7.0. Analogous measurements were performed with the top and bottom components of alfalfa mosaic virus in the same buffer and with the coat protein in water. A comparison of the various enthalpies showed that the energetics of the protein–nucleic‐acid interaction are different for RNA 1 and RNA 4. The thermally induced transition of the coat protein in water is characterized by an enthalpy of 418 ± 67 kJ (100 ± 16 kcal) mol−1 at 38.9°C and a heat capacity change of approximately 6.7 kJ (1.6 kcal) mol−1 K−1. The unfolding pattern of the native bottom component of alfalfa mosaic virus, which consists of RNA 1 encapsulated by 240 coat protein molecules, is similar to that of the protein alone in exhibiting a single sharp heat capacity maximum and a heat capacity increase after the transition. The corresponding transition enthalpy is 523 ± 42 kJ (125 ± 10 kcal) · (mol protein)−1, the heat capacity change amounts to approximately 8 ± 1.7 kJ (1.9 ± 0.4 kcal) · (mol protein)−1, · K−1. An analogous transition curve results from unfolding of native top component fragments of alfalfa mosaic virus, which comprise two RNA 4 molecules per 132 protein molecules. The enthalpies involved in the unfolding process are 489 ± 42 kJ (117 ± 10 kcal) · (mol protein)−1, the heat capacity change 9.6 ± 2.5 kJ (2.3 + 0.6 kcal) · (mol protein)−1· K−1. The noticeable differences to the transition of the pure protein are the transition temperatures of 49.5°C and 47.9°C for the bottom and top components, respectively. The transition enthalpies of all protein · nucleic‐acid complexes are smaller than the sum of the transition enthalpies of the single components. This finding has been used to estimate enthalpies of interaction per mole of protein. The characteristic feature of these values is the fact that they all are positive, which indicates a decrease of the internal energy of the system on protein–nucleic‐acid interaction.
Protein—Non‐ionic Detergent Interaction
Authors: WASYLEWSKI Zygmunt , KOZIK Andrzej
Published: March 2005

The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 °C and 25 °C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglucosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non‐ionic ligands at 7°C and about 15 and 13 sites at 25°C for octyl and decyl glucosides respectively. The values for standard free energy changes −ΔG°, were calculated from the intrinsic association constants. Fourier‐transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure.
The Complete Amino‐Acid Sequence of a Trout‐Testis Non‐histone Protein, H6, Localized in a Subset of Nucleosomes and Its Similarity to Calf‐Thymus Non‐histone Proteins HMG‐14 and HMG‐17
Authors: WATSON David C. , WONG Norman C. W. , DIXON Gordon H.
Published: March 2005

The complete amino acid sequence of a basic non‐histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636–5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion‐exchange chromatography on carboxymethyl‐cellulose (CM‐52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89‐residue calf thymus HMG‐17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461–468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810–2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883–898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].
Globin mRNA Precursor
Authors: AGLIANÓ Anna Maria , NACCI Aldo , REYMOND Christophe , APPLEBY David , SPOHR Georges
Published: March 2005

The globin mRNA sequences present in duck erythroblast nuclei appear under non‐denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes.

Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA.

To find out whether hydrogen‐bonded structures occur in situ or arise after RNA extraction. nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double‐stranded nucleic acids, which were visualised by electron microscopy. It appears that, after cross‐linking, a fraction of the globin mRNA sequences present in nuclei is associated with high‐molecular‐weight hnRNA molecules by a link which is resistant to stringent denaturing conditions. In particular globin mRNA sequences are found associated with a band of 0.9×106 molecular weight approximately.

It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.
Amino Acid Sequence Studies of Horseradish Peroxidase
Authors: WELINDER Karen Gjesing
Published: March 2005

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a hemin prosthetic group, 2 Ca2+and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side‐chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3, Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidaseCis close to 44000.

Cyanogen bromide fragments of reduced and carboxymethylated apo‐peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine‐containing tryptic fragments of apo‐peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions.

The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion.
The Influence of Ribonucleoside Triphosphates, and Other Factors, on the Formation of Very‐Salt‐Stable RNA‐Polymerase · su+III‐tRNA(tRNATyr)‐Promoter Complexes
Authors: DEBENHAM Paul
Published: March 2005

The formation of a stable RNA‐polymerase · su+III‐tRNA‐promoter complex was found to require sigma factor and the incorporation of ribonucleoside triphosphates which match the 5′ sequence of the su+III tRNA transcript. This complex, stable to at least 2 M KCl, can be retained on a Millipore filter. Its formation closely parallels the extent of transcription obtained from the su+III tRNA promoter in response both to increasing ionic strength and to temperature during incubation of RNA polymerase with the DNA. The RNA‐polymerase · DNA complex retained during this assay therefore appears to relate directly to that formed during promoter‐directed transcription. The formation of RNA‐polymerase · su+III‐tRNA‐promoter complexes is sensitive to the presence of ppGpp.
Synthesis of Non‐globin Proteins in Rabbit‐Erythroid Cells
Authors: THIELE Bernd J. , BELKNER Jutta , ANDREE Hartwig , RAPOPORT Samuel M. , RAPOPORT Tom A.
Published: March 2005

Peripheral rabbit reticulocytes synthesize at least 30 non‐globin proteins. One of them is identified as a characteristic lipoxygenase on the basis of its molecular weight, its immunological properties and its behaviour on an ion‐exchange column. The enzyme is not produced in bone marrow cells. The synthesis of the lipoxygenase in peripheral blood cells commences on the 3rd day of a bleeding anaemia, increases up to the 5th day and stays constant thereafter at least up to the 14th day. It is concluded that the appearence of the lipoxygenase, which plays a key role in the degradation of mitochondria in the course of maturation of reticulocytes to erythrocytes, is regulated at the translational level.
Purification and Properties of Sheep‐Liver Aldehyde Dehydrogenases
Authors: MACGIBBON Alistair K. H. , MOTION Rosemary L. , CROW Kathryn E. , BUCKLEY Paul D. , BLACKWELL Leonard F.
Published: March 2005

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with a specific activity of 380 nmol NADH min−1 mg−1.

An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions.

On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6‐fold enhancement of nucleotide fluorescence. The protein fluorescence (λexcit= 290 nm, λemission= 340 nm) is quenched on the binding of NAD+ and NADH.

The enhancement of nucleotide fluorescence on the binding of NADH has been utilised to determine the dissociation constant for the enzyme · NADH complex (Kd= 1.2 ± 0.2 μM). A Hill plot of the data gave a straight line with a slope of 1.0 ± 0.3 indicating the absence of co‐operative effects.

Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecyl‐sulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme.

Molecular weights were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212000 ± 8000 and 205000 respectively. Each enzyme consisted of four subunits with molecular weight of 53000 ± 2000.

Properties of the cytoplasmic and mitochondria1 aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases.
Peptide Bond Formation Stimulated by Protein Synthesis Factor EF‐P Depends on the Aminoacyl Moiety of the Acceptor
Authors: GLICK Bernard R. , CHLÁDEK Stanislav , GANOZA M. Clelia
Published: March 2005

Elongation factor EF‐P is a soluble protein that stimulates peptide bond synthesis catalyzed by the 50‐S ribosomal subunit. This factor was previously identified and characterized based on its ability to promote the synthesis of formylmethionine‐puromycin. In the present work, we tested the ability of EF‐P to promote peptide bond synthesis between ribosome‐bound fMet‐tRNA and several analogues of the 3′ terminus of aminoacyl‐tRNA, i.e. the cytidylyl(3′‐5′)‐[2′(3′)‐O‐l‐aminoacyladenosines]. EF‐P promoted synthesis to the greatest extent with certain acceptors which were otherwise inefficient in the peptidyl transferase reaction. This activity of EF‐P could not be replaced by the other soluble proteins known to be involved in polypeptide synthesis, such as EF‐Tu, EF‐Ts and EF‐G. One role of EF‐P in protein synthesis may be to allow peptide bond synthesis to occur more efficiently with some aminoacyl‐tRNAs that are poor acceptors for the ribosomal peptidyl transferase.
Purification and Properties of a Histone Acetyltransferase from Artemia salina, Highly Efficient with H1 Histone
Authors: CANO Amparo , PSTAÑA Angel
Published: March 2005

An histone acetyltransferase has been purified from nuclei of 40‐h‐old Artemia salina larvae. The enzyme is very unstable at 0°C, requires free − SH groups for activity and is rapidly inactivated at 40°C. The optimal pH for activity is 8.5 and the activity is half inhibited by millimolar concentrations of Mn2+, Ca2+ or Mg2+ or decimolar concentrations of Na+ and K+. The molecular weight of the enzyme, determined by gel filtration chromatography, changed with the ionic strength of the medium (280000 in 10 mM Tris · HCl, 170000 in 0.2 M KCl). The very‐lysine‐rich histone H1 is a better substrate acceptor than the arginine‐rich histones H3 or H4. Under proper conditions, the enzyme can modify all the internal lysyl residues in histones H1 and H4. The acetylation of H1 is inhibited when all the other histone fractions are present in the assay mixture.
Hydrodynamic Parameters of the Detergent‐Solubilised Hydrogenase from Paracoccus denitrificans
Authors: SIM Edith , SIM Robert B.
Published: March 2005

The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X‐100. The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O. The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X‐100, are 0.73 and 0.74ml. g−1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X‐100. The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent. The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X‐100 compared to 6.7 nm in the absence of detergent. The apparent molecular weight therefore increases from 242500 to 466000 on removal of detergent. In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63000. The enzyme therefore behaves as a non‐covalently linked tetramer in the presence of Triton X‐100. Removal of Triton X‐100 results in association of tetramers to form octamers.
Generation of High δɛ in Respiring Submitochondrial Particles by Steady‐State Accumulation of Oxidized N,N,N′,N′‐Tetramethyl‐p‐phenylenediamine
Authors: SAGI‐EISENBERG Ronit , GUTMAN Menachem
Published: March 2005

Oxidation of N,N,N′,N′‐tetramethyl‐p‐phenylenediame [Ph(NMe2)2] plus ascorbate by inside out submitochondrial particles is associated with accumulation of the oxidized form [Ph(NMe2)2+] in the inner space of the vesicles. The concentration of [Ph(NMe2)2+]in, can exceed by 5–6‐fold the concentration of Ph(NMe2)2 added to the reaction mixture.

The steady‐state concentration of [Ph(NMe2)2+]in, is a function of the rate of Ph(NMe2)2 influx driven by concentration gradient, the rate of Ph(NMe2)2+ oxidation, determined by its concentration, and Ph(NMe2)2+ efflux. The last rate is determined by Ph(NMe2)2+ concentration gradient and the membrane potential component (δɛ) of the proton‐motive force. Collapse of δɛ by permeant anions, ionophores or carbonylcyanide p‐trifluoromethoxyphenylhydrazone, builds up the internal concentration of [Ph(NMe2)2+]in till the concentration gradient can support an efflux identical to the rate of Ph(NMe2)2+ formation (equivalent to the rate of respiration). This relationship is analyzed according to Nernst‐Planck equation and δɛ calculated by this method amounts to 100–120 mV (positive inside). This value is corroborated by equilibrium dialysis. The measured membrane potential is built by the positive charge of Ph(NMe2)2+itself plus that of accumulated H+. Still, the contribution of Ph(NMe2)2+ is about half of the total electric force.

At high Ph(NMe2)2 concentrations (0.5–5 mM), substantial electron flux through cytochrome oxidase bypasses cytochrome c and is carried by direct interaction of Ph(NMe2)2 with the terminal oxidase.
Binding of Semliki Forest Virus and Its Spike Glycoproteins to Cells
Authors: FRIES Erik , HELENIUS Ari
Published: March 2005

We have studied the binding of the Semliki Forest virus and its isolated spike glycoproteins, in the form of water‐soluble octameric complexes, to various cells at 5°C. The number of viruses bound per cell increased strongly with increasing free concentrations of virus up to about 0.2 nM. At higher concentrations smaller increases in binding were observed but saturation was not achieved. The number of viruses bound at a given free concentration was widely different for different cells. For some cells the binding of the virus was maximal at pH 6.8 with little decrease at lower pH, for other cells it was maximal around pH 6.0.

The spike protein complexes were used at 100 times higher molar concentrations than the virus. The binding increased strongly with increasing free concentrations up to about 50 nM and saturation was obtained at higher concentrations. Up to 1.3 × 106 spike protein complexes could be bound per cell but great variation could be seen between different cell types. For all cells maximal binding was found below pH 6.0.

Together with earlier observations, our results suggest that the virus can bind to a cell by two different modes. Around neutral pH the virus binds to specific gycoproteins and at low pH un‐specifically to the lipids of the plasma membrane. The possible physiological roles of these two types of binding are discussed.
Nucleotide Sequence of Three Isoaccepting Lysine tRNAs from Rabbit Liver and SV40‐Transformed Mouse Fibroblasts
Authors: RABA Manfred , LIMBURG Klaus , BURGHAGEN Margaret , KATZE Jon R. , SIMSEK Mehmet , HECKMAN Joyce E. , RAJBHANDARY Uttam L. , GROSS Hans J.
Published: March 2005

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a ‘new’ lysine tRNA, which is practically absent in non‐dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the ‘new’ lysine tRNA isolated from SV40‐transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows.

The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2′‐O‐methyl‐ribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C‐U‐U is followed by N‐threonyl‐adenosine (t6A). tRNA3Lys has the anticodon S‐U‐U and contains two highly modified thionucleosides, S (shown to be 2‐thio‐5‐carboxymethyl‐uridine methyl ester) and a further modified derivative of t6A (2‐methyl‐thio‐MN6‐threonyl‐adenosine) on the 3′ side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors.

Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRN2Lys with anticodon C‐U‐U recognized A‐A‐G only, whereas tRNA3Lys, which contains thionucleotides in and next to the anticodon, decodes both lysine codons A‐A‐G and A‐A‐A, but with a preference for A‐A‐A. In a globin‐mRNA‐translating cell‐free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that α‐globin and β‐globin mRNAs contain more A‐A‐G than A‐A‐A codons for lysine.

A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the ‘new’ tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not to be t6A. In addition we have found U, T and Ψ besides Tm as the first nucleoside in loop IV.
Changes in the Frequency and Diversity of Messenger RNA Populations in the Course of Myogenic Differentiation
Authors: LEIBOVITCH Marie‐Pierre , LEIBOVITCH Serge‐Alexandre , HAREL Jacques , KRUH Jacques
Published: March 2005

Complementary DNAs (cDNAs) were synthesized from polyadenylated RNAs of myoblasts and myotubes and used to analyze changes in the sequence complexity and frequency distribution of messenger RNAs during myogenesis in vitro. cDNA · polyadenylated‐RNA hybridization kinetics show the presence of messenger RNA sequences specific for myotubes in fully differentiated muscle cultures. These sequences are accumulated just prior to fusion, as was shown by hybridizations of myotube cDNA and total cytoplasmic RNAs from cells at different stages of differentiation. The myotube cDNA can be enriched 10‐fold in myotube‐specific RNA species by a hybridization with cytoplasmic RNAs from myoblasts and subsequent removal of these hybridized sequences by hydroxyapatite.
Photo‐Induced Protein‐RNA Cross‐Linking in Mammalian 60‐S Ribosomal Subunits
Authors: BUISSON Monique , REBOUD Anne‐Marie , MARION Marie‐Jeanne , REBOUD Jean‐Paul
Published: March 2005

Rat liver 60‐S ribosomal subunits were submitted to increasing doses of radiation (253.7 nm), at 4°C and 25°C, as previously reported for 40‐S subunits. The existence of protein‐RNA cross‐linking was demonstrated by two methods. The first consisted in the separation of protein‐RNA complex; the second was indirect, and took into account alteration either in the electrophoretic mobility of cross‐linked proteins or the separability of 28‐S RNA in a 4 M urea/3 M LiCl buffer. The peptide synthetase activity and the sedimentation characteristics of the particles irradiated at 4°C were well preserved, but at 25°C the large subunits were progressively inactivated and unfolded for doses higher than 2 × 1018 quanta. The dose‐dependent variations of protein cross‐linkage determined by two‐dimensional gel electrophoresis allowed us to distinguish those proteins which reacted at the lowest doses with a first‐order reaction from those which cross‐linked to RNA after a subtle modification of the subunit structure. At 25°C, all proteins became low‐dose reactive. The curve obtained for 28‐S RNA cross‐linkage was similar to that of the total protein moiety, while those obtained for the 5‐S and 5.8‐S RNA (which were parallel) suggest a lower reactivity of these RNAs. As a general rule, proteins from the large subunits were more reactive to RNA than those from the small subunits. This could indicate differences in the organisation of the two subunits.
Enzymic Synthesis of Lignin Precursors
Authors: WYRAMBIK Dorothea , GRISEBACH Hans
Published: March 2005

Isoenzyme 2 of cinnamyl‐alcohol dehydrogenase from soybean suspension cultures was purified about 3800‐fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+‐agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40000 ± 500.

The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the non‐chelating 7,8‐benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10‐phenanthroline could be partially reversed by addition of Zn2+. 1,10‐Phenanthroline and 7,8‐benzoquinoline are non‐competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme.

In steady‐state kinetics inhibition patterns were obtained which are consistent with an ordered bi‐bi mechanism in which NADP(H) is the first substrate to bind and the last product released.

The cinnamyl‐alcohol dehydrogenase belongs to the A‐specific dehydrogenases and removes the pro‐R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.
Properties of the F0F1 ATPase Complex from Rhodospirillum rubrum Chromatophores, Solubilized by Triton X‐100
Authors: SCHNEIDER Erwin , MÜLLER Hans‐Werner , RITTINGHAUS Klaus , THIELE Volker , SCHWULÉRA Udo , DOSE Klaus
Published: March 2005

A cold‐stable oligomycin‐sensitive F0F1 ATPase complex from chromatophores of Rhodospirillum rubrum FR 1 was solubilized by Triton X‐100 and purified by gel filtration.

The F0F1 complex is resolved by sodium dodecyl sulfate electrophoresis into 14 polypeptides with approximate molecular weights in the range of 58000–6800; five of these polypeptides are derived from the F1 moiety of the complex which carries the catalytic centers of the enzyme.

The purified F0F1 complex is homogeneous according to analytical ultracentrifugation and isoelectric focusing.

The molecular weight as determined by gel filtration is about 480000 ± 30000. so20,w is 1.45 ± 0.1 S and the pI is 5.4.

The amino acid composition of the F0F1 complex is compared with the data obtained for the F1 moiety of the enzyme.

Quantitative data on the sensitivity to N,N′‐dicyclohexyl‐carbodiimide as well as kinetic parameters, regarding substrate specificity and dependence of ATPase activity on divalent cations, are reported.