bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Binding of Myosin Subfragment One Species to Immobilized ADP, and Actin: the Influence of the Alkali Light Chains
Authors: WINSTANLEY Monica A. , SMALL David A. P. , TRAYER Ian P. 
Published: March 2005

Isolated myosin head regions (subfragments 1) have been prepared from a number of muscle types, and studied by affinity chromatography upon immobilized ADP and actin matrices.It has been shown that the two alkali light chain isoenzymes of subfragment 1 specific for a given skeletal muscle can be fractionated; therefore, these procedures provide essentially simple and rapid methods for the preparation of myosin head populations which are homogeneous with regard to their light chain content, and readily amenable to further study.Fractionation of slow twitch skeletal subfragment 1 species upon immobilized actin extends previous findings with fast twitch species [Winstanley, Trayer and Trayer (1977) FEBS Lett.77, 239–2421. In each case the heads containing the less anodic alkali light chain bind more tightly to the actin matrix. These results are supported by kinetic data which show differences only in the apparent Kmfor actin between the two acto‐subfragment 1 MgATPase. Comparison between the subfragment 1 species from different muscle types indicates a correlation between the strength of the interaction between subfragment 1 and immobilized actin and the myosin ATPase activity and contractile properties of the muscle. Tighter actin binding is associated with the low‐ATPase‐activity myosins characteristic of slow twitch skeletal and cardiac muscles.A possible relationship is suggested between the amino acid composition of the alkali light chain present, and the actin binding properties of the myosin head.
Structure of Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii
Authors: VOORDOUW Gerrit , VEEGER Cees , VAN BREEMEN Jan F. L. , VAN BRUGGEN Ernst F. J. 
Published: March 2005

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy.2. At high pH (8.5–9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris‐Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient 520,w= 15 S and its Stokes' radius Rs= 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 ± 2) × 103 it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit (‘unimer’) from which the polymeric form of transhydrogenase is constructed.3. Gel filtration and sucrose gradient centrifugation studies of cell‐free extracts from A. vinelandii show the unimer to be the predominant active species.
Mouse‐Liver Glutathione Reductase
Authors: LÓPEZ‐BAREA Juan , LEE Chi‐Yu 
Published: March 2005

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8‐(6‐aminohexyl)‐amino‐2′‐phospho‐adenosine diphosphoribose and N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate‐Sepharose columns. A facile procedure for the synthesis of 8‐(6‐aminohexyl)‐amino‐2′‐phospho‐adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr, 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a ‘branched’ mechanism.The enzyme was stabilized against thermal inactivation at 80°C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto‐inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.
Purification and Characterization of a Protein Factor that Reverses the Inhibition of Protein Synthesis by the Heme‐Regulated Translational Inhibitor in Rabbit Reticulocyte Lysates
Authors: AMESZ Hans , GOUMANS Hans , HAUBRICH‐MORREE Thea , VOORMA Harry O. , BENNE Rob 
Published: March 2005

We have purified and partially characterized a supernatant factor which reverses the effect of the heme‐regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti‐inhibitor restores protein synthesis activity in heme‐deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme.The factor has no effect on the phosphorylation of eIF‐2 by the inhibitor nor on any reaction carried out with purified initiation factors.The anti‐inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF‐2. A possible mechanism of action is discussed.
Protein · Nucleic‐Acid Reaction Kinetics
Authors: GIACOMONI Paolo U. 
Published: March 2005

This paper presents methods developed in order to analyze experimental results concerning the binding of Escherichia coli DNA‐dependent RNA polymerase to DNA at high and at low DNA concentrations, using the filter retention assay.The basic hypotheses, under which the mathematical expressions for describing the kinetics of binding are derived, are as follows. (a) At low DNA concentration: equivalence and independence of the specific binding sites; first‐order dependence of the binding reaction on both DNA and protein concentration. (b) At high DNA concentration: equivalence and independence of the nonspecific binding sites; no direct transfer or one‐dimensional sliding of the protein along the DNA.Comparison between theoretical predictions and experimental results at high DNA concentration will alow one to determine the relative value of the rates of binding of RNA polymerase to different promoters (between 1 and 2 in T5 DNA).Binding experiments performed at low DNA concentration are reported in this paper: these results and the analysis which is reported allow one to determine the value of the rate constant of formation of non‐filterable complexes for the system fd DNA (replicative form) · RNA‐polymerase (ka= 3.3 × 108 M−1 s−1 in 0.1 M NaCl, 0.01 M MgCl2).
Flavanone Synthase from Petroselinum hortense
Authors: KREUZALER Fritz , RAGG Hermann , HELLER Werner , TESCH Rudolf , WITT Irene , Dietrich HAMMER  , HAHLBROCK Klaus 
Published: March 2005

Flavanone synthase from irradiated cell suspension cultures of parsley was purified to apparent homogeneity. Molecular weights of about 77000 for the enzyme and about 42000 for the subunits were determined respectively by sedimentation‐equilibrium measurements and disc‐gel electrophoresis in the presence of dodecyl sulfate. A specific antiserum was prepared for the enzyme and was used in an assay for flavanone synthase mRNA activity in partially purified RNA preparations. The apparent molecular size of flavanone synthase mRNA was estimated by sucrose gradient centrifugation and gel electrophoresis under partially denaturing conditions. Values of about 17 S and Mr= 0.62 × 106 were obtained. The fractionation patterns suggested that flavanone synthase mRNA was homogeneous in size. All together, the results support the idea that the enzyme is composed of two subunits which are probably identical.Amino acid analysis and a microbial assay were carried out to test the possible occurrence of cysteamine, β‐alanine, and pantothenate in the enzyme. The results were negative, indicating the absence of pantetheine or a similar residue. The possible similarity in mechanism between flavanone synthase and 3‐oxoacyl‐(acyl carrier protein) synthase is discussed.
Enzymology of Ubiquinone‐Utilizing Electron Transfer Complexes in Nonionic Detergent
Authors: WEISS Hanns , WINGFIELD Paul 
Published: March 2005

The enzymology of isolated succinate: ubiquinone reductase and ubiquinone: cytochrome c reductase in nonionic detergents (alkyl polyoxyethylene derivatives) was studied.In the membrane the two multiprotein complexes and their hydrophobic substrates ubiquinone and dihydroubiquinone, are embedded in a common lipid bilayer. In detergent solutions the complexes are each inserted into micelles. Detergent micelles also serve as a solvent for the complexes hydrophobic substrates. As a consequence the isolated complexes are in a discontinuous phase with respect to their hydrophobic substrates and with respect to each other.Three types of assays were used. Firstly, single enzyme assays in which the hydrophobic substrates had to transfer from free micelles to the complex‐bound micelles in order for enzymic reactions to occur. Secondly, assays in which the enzymic reactions were coupled to auxiliary non‐enzymic reactions which rapidly converted the hydrophobic products back into substrates within the complex‐bound micelle. Dichloroindophenol was used for the oxidation of dihydroubiquinone and dihydroduroquinone for the reduction of ubiquinone. Thirdly, assays in which the succinate: ubiquinone reductase reaction was coupled with the ubiquinone: cytochrome c reductase reaction.With the first type of assay, the kinetics of the substrate transfer reaction was dependent upon the type of detergent. In detergents with small polyoxyethylene head groups the transfer reactions were rate‐limiting, and in detergents with large polyoxyethylene head groups the transfer reactions were fast and the enzymic reactions were rate‐limiting. With the second type of assay, the substrate concentration within the complex‐bound micelle was controlled by the equilibrium position of the substrate transfer reaction. Half‐maximal activity was obtained when the hydrophobic substrate concentration was half the total micelle concentration. This indicated that the equilibrium constant of the substrate transfer reaction was equal to one. With the third type of assay, detergent micelles containing the hydrophobic product/substrate couple functioned as substrate carriers between the two complexes. The reaction rates depended on the kinetics of the substrate transfer reaction. Direct complex‐complex interaction did not occur.
A Molecular Order in the Synthesis and Degradation of Glycogen in the Liver
Authors: DEVOS Pierre , HERS Henri‐Géry 
Published: March 2005

When fasted rats or mice were refed, liver glycogen was synthesised at a linear rate of approximately 1%/h for a period of about 6 h. The administration of a radioactive precursor, usually [14C]galactose, allowed pulse labelling of the glycogen formed at various times after the initiation of refeeding. Soon after the administration of the label, the radioactive glucosyl units were preferentially located on the outer chains of glycogen. An almost equal distribution between the inner and outer chains was attained after a time which increased with increasing amounts of preexisting glycogen. Later on, the radioactive molecules were not further enlarged although the mass of glycogen increased threefold.The degradation of the glycogen that had been labelled at various times after initiation of refeeding was induced either in vivo in anesthetized rats, or in vitro in isolated hepatocytes and in a cell‐free system made up of the enzyme‐glycogen complex as isolated by concanavalin A. Under all of these conditions, the radioactive units that were incorporated last were liberated first and vice versa. This ordered degradation could not be explained by the initial removal of outer chains. In the concanavalin‐A‐bound glycogen, the ordered degradation was much less apparent when the preparation had been submitted to high‐speed centrifugation, vigorous homogenization, ultrasonic treatment or exposure to detergents.
Isolation and Purification of a Neurodepressing Hormone from the Eyestalk of Procambarus bouvieri (Ortmann)
Authors: HUBERMAN Alberto , ARECHIGA Hugo , CIMET Avivah , DE LA ROSA Jorge , ARAMBURO Carlos 
Published: March 2005

1. A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann).2. Purification was achieved by gel filtration on Sephadex G‐25 and G‐15, and preparative paper electrophoresis at four pH values (1.8, 3.6, 6.0 and 10.0).3. The recovery of hormone activity was 85% and the specific activity was 4.0 × 104 times that of the starting material.4. The hormone is a thermostable peptide of approximately 1200 molecular weight and composed of neutral amino acids.5. No N‐terminal group could be found. From its electrophoretic behavior it is concluded that the C‐terminal group is also blocked.
Cell‐Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa
Authors: ZIMMERMAN Richard , PALUCH Ulrich , SPRINZL Matthias , NEUPERT Walter 
Published: March 2005

ADP/ATP carrier protein was synthesized in heterologous cell‐free systems programmed with Neurospora poly(A)‐containing RNA and homologous cell‐free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl‐tRNA (fMet‐tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant.The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence.
The Primary Structure of the Ovine β‐Caseins
Authors: RICHARDSON Barry C. , MERCIER Jean‐Claude 
Published: March 2005

Ovine whole casein contains 2 multiphosphorylated β‐casein components designated as β1 and β2‐caseins. The complete sequence of β1‐casein and the partial sequence of β2‐casein have been determined from the intact proteins and from the peptides isolated from cyanogen bromide and tryptic digests. The ovine β1 and β2‐caseins have the same polypeptide chain and appear to differ only in that they contain 6 and 5 phosphates respectively. The amino acid composition of ovine β‐casein can be written as: Asp4, Asn4, Thr10, ThrP1, Ser9, SerP5, Glu19, Gln21, Pro34, Gly5, Ala4, Val21, Met6, IIe9, Leu22, Tyr3, Phe9, Trp1, Lys12, His5, Arg3. Compared to bovine β‐casein A2, which is made up of 209 residues, ovine β1‐casein has a deletion of 2 residues (either Pro‐179–Tyr‐180 or Tyr‐180–Pro‐181) and 20 largely conservative amino acid substitutions. Although 20% of the substitutions involve proline residues, the proline contents of ovine β1 and bovine β2‐caseins are very similar, around 16%. The average hydrophobicity, calculated according to Bigelow, is 5.51 kJ/residue, which is similar to that calculated for bovine β‐casein A2.The cluster of 4 phosphorylated serine residues and the highly charged nature of the amino terminal region observed for bovine β‐casein are conserved in the ovine β‐caseins. The substitution from IIe‐12 (bovine) to Thr‐12 (ovine) results in a new phosphorylation site, according to the phosphorylation code proposed for caseins. This site is only partially phosphorylated hence the occurrence of both β1 and β2‐caseins in ovine milk.
Apolipoprotein A‐IV: a Protein Occurring in Human Mesenteric Lymph Chylomicrons and Free in Plasma
Authors: UTERMANN Gerd , BEISIEGEL Ulrike 
Published: March 2005

1. Human mesenteric lymph chylomicrons were isolated from chylous ascites fluid by ultracentrifugation and agarose/gel chromatography and their apoprotein composition was analysed by dodecylsulfate/polyacrylamide gel electrophoresis, analytical isoelectric focusing and immunochemically. Major components of mesenteric lymph chylomicrons were apoprotein A‐I, proteins of Mr < 15000 including the C‐group apoproteins and a protein of Mr 46000. Minor components were apoprotein E and a protein of Mr∼ 200000 (B‐like protein). This apoprotein composition was qualitatively identical with that of chylomicrons from intestinal lymph of the rat, but was distinctly different from plasma chylomicrons of humans with fasting chylomicronaemia.2. The protein of Mr∼ 46000 has been isolated by preparative dodecylsulfate/polyacrylamide gel electrophoresis from human and rat lymph chylomicrons and was compared to a protein of identical Mr present in rat high‐density lipoproteins (apolipoprotein A‐IV) and in the ϱ < 1.006 g/ml serum lipoprotein fraction of individual humans with alimentary hypertriglyceridaemia. In both species the 46000‐Mr proteins isolated from lymph and serum were identical according to amino acid composition and isoelectric point in 6 M urea. The human proteins from both sources were also immunologically identical. The similarities in the molecular properties of the human apolipoprotein and rat apolipoprotein A‐IV indicate that these proteins are homologous.3. Plasma levels of human apolipoprotein A‐IV determined by electroimmunodiffusion were 14.15 ± 3.66 mg/100 ml (n= 59), but > 90% of the protein was unassociated with the major lipoprotein fractions.It is concluded, that apolipoprotein A‐IV is a main protein component of human lymph chylomicrons, that is removed from the particles in the plasma compartment.
Conversion of a Mitochondrial Precursor Polypeptide into Subunit 1 of Cytochrome Oxidase in the mi‐3 Mutant of Neurospora crassa
Authors: WERNER Sigurd , BERTRAND Helmut 
Published: March 2005

1. The cytochrome‐aa3‐deficient mi‐3 cytoplasmic mutant of Neurospora crassa synthesizes a mitochondrial translation product which crossreacts with antibodies specific to subunit 1 of cytochrome oxidase. The immunoprecipitated polypeptide migrates more slowly during gel electrophoresis than the authentic 41000‐Mr subunit 1 of the wild‐type enzyme. An apparent molecular weight of about 45000 was estimated for the mutant product.2. Radioactive labelling experiments in vivo show that the crossreacting material found in the mutant is relatively stable and does not form complexes with other subunits of the oxidase.3. After induction of a functional cytochrome oxidase in the mutant cells with antimycin A, the 45000‐Mr polypeptide is converted to a 41000‐Mr component, which exhibits the same electrophoretic mobility as subunit 1 of the oxidase. Pulse‐chase labelling kinetics reveal a typical precursor product relationship.4. The converted polypeptide becomes assembled with other enzyme subunits to form a protein complex which has the immunological characteristics of cytochrome oxidase.A possible physiological role of the post‐translational processing of the mitochondrially synthesized component is discussed.
Role of the Mitochondrial Anion Transporters in the Regulation of Ammoniagenesis in Renal Cortex Mitochondria of the Rabbit and Rat
Authors: CHEEMA‐DHADLI Surinder , HALPERIN Mitchell L. 
Published: March 2005

The purpose of this study was to investigate factors which may regulate ammoniagenesis in the kidney cortex. Emphasis was placed on the segment of the pathway by which the carbons derived from glutamine must exit from the mitochondrion. These pathways were compared in the rat with high rates of ammoniagenesis and the rabbit which has a low rate of ammoniagenesis. The dicarboxylate transporter, which is essential for ammoniagenesis, has a maximum velocity which was much lower in the rabbit. The malate concentration required for half‐maximal rates of transport was 14 nmol/mg mitochondrial protein and similar in both species. There was no effect of chronic metabolic acidosis on dicarboxylate transporter activity. The tricarboxylate transporter activity with phosphoenol pyruvate as substrate also had a low activity in the rabbit kidney‐cortex mitochondria.The maximum velocity of phosphate dependent glutaminase, glutamate dehydrogenase and phosphoenolpyruvate carboxykinase were all much greater than the maximal rate of ammoniagenesis observed in vivo in the rabbit. Therefore, the low rates of ammoniagenesis and the failure to adapt to acidosis in the rabbit are best explained by factors influencing the dicarboxylate transporter.
Macromolecular Complexes of Aminoacyl‐tRNA Synthetases from Eukaryotes
Authors: KELLERMANN Odile , BREVET Annie , TONETTI Hubert , WALLER Jean‐Pierrc 
Published: March 2005

Starting from homogenates of sheep liver, extensive co‐purification of seven aminoacyl‐tRNA synthetases to high specific activities was achieved by a three‐step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose‐bound tRNA.The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 106 estimated by gel filtration on 6% agarose. It contains aminoacyl‐tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co‐elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex.The enzyme composition of the high‐molecular‐weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156–167).
Effects of Apramycin, a Novel Aminoglycoside Antibiotic on Bacterial Protein Synthesis
Authors: PERZYNSKI Stanislaw , CANNON Michael , CUNDLIFFE Eric , CHAHWALA Suresh B. , DAVIES Julian 
Published: March 2005

1. The novel aminoglycoside antibiotic apramycin is shown to be a potent inhibitor of protein synthesis in bacteria both invivo and invitro.2. In cell‐free systems from Escherichia coli programmed with poly(U), apramycin induces translation errors, as assayed by incorporation of leucine, isoleucine and serine, although this effect occurs only to a limited extent.3. Apramycin inhibits the translocation step of protein synthesis both invivo, in protoplasts of Bacillus megaterium, and invitro, in cell‐free systems from E. coli. It is proposed that this is the primary inhibitory effect of the drug.