bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide Sequence for a Novel Duck Alpha‐Globin Gene
Authors: PADDOCK Gary V. , GAUBATZ Jim
Published: July 2010

Nucleotide sequence analysis of a recombinant cDNA for a duck alpha‐globin gene indicates a gene with novel features and evolutionary history. The duck alpha‐globin double‐stranded cDNA, treated with S1 nuclease and tailed with poly(dC), was inserted into the PstI restriction endonuclease site of pBR322 tailed with poly(dG). Nucleotide sequence analysis of the resulting recombinant cDNA indicates that it contains the translated region and all of the 3′ untranslated region for an alpha‐globin gene. The sequence was determined by procedures designed especially for rapid analysis of pBR322 (PstI site) recombinant cDNAs. This duck globin has sequences related to the chicken alpha‐A globin in some regions but in other regions it is more closely related to another alpha globin found in anemic chickens.
Modification of the Spectral Properties of Cytochrome b in Mutants of Saccharomyces cerevisiae Resistant to 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethylurea
Authors: BRIQUET Michel , GOFFEAU André
Published: July 2010

The effects of five inhibitors of the cytochrome bc1 complex: 3‐(3.4‐dichlorophenyl)‐1,1‐dimethylurea (diuron), 2‐n‐heptyl‐4‐hydroxyqttinoline‐N‐oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuronresistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b.

The oxidation of NADH and of cytochronic b‐561 exhibits a similar resistance to diuron and HpHOQnO in Diu I and Diu2 mutants.

No extra reduction of cytochrome b‐561 and cytochrome b‐565 is observed in the presence of diuron and HpHOQnO.

Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b‐561 induced by concentrations of HpHOQnO 2‐3‐times higher than those required in the parental strain.

The spectral and respiratory effects of antimycin A, funiculosin and mucidin are generally similar in the diuron‐resistant mutants and in their parental strains. However a cross‐resistance between diuron and antimycin A is indicated in one Diu 2 mutant.

From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in tile central region of tile apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b‐565 and b‐561 as well as tile flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b‐561 is only slightly affected by the diu‐l and diu‐2 mutations.
Displacement of the Human Apoprotein A‐I by the Human Apoprotein A‐II from Complexes of (Apoprotein A‐I)‐Phosphatidylcholine‐Cholesterol
Authors: ROSSENEU Maryvonne , VAN TORNOUT Philippe , LIEVENS Marie‐José , ASSMANN Gerd
Published: July 2010

Reassembly experiments, involving isolated human apoproteins A‐I and A‐II and (dimyristoylglycerophosphocholine)‐cholesterol vesicles were performed with apoprotein mixtures at apoprotein A‐I/A‐II molar ratios varying between 0 and 3. The apoproteins were incubated at 24°C, 28°C and 32°C with either pure dimyristoylglycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 0, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after label ing the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A‐I and A‐II were quantified separately by immunonephelometry. The content of apoprotein A‐I was also monitored by measuring the intrinsic tryptophan fluorescence.

The results suggest that apoprotein A‐II has a greater affinity than apoprotein A‐I for the phospholipidcholesterol vesicles and that apoprotein A‐II is able to quantitatively displace apoprotein A‐I from the lipidprotein complexes. The content of apoprotein A‐II in the complexes increases proportionally to the concentration of apoprotein A‐II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A‐II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A‐I is displaced by 2 mol human apoprotein A‐II.
Effect of Funiculosin and Antimycin A on the Redox‐Driven H+‐Pumps in Mitochondria: on the Nature of ‘Leaks’
Authors: PIETROBON Daniela , AZZONE Giovanni F. , WALZ Dieter
Published: July 2010

The effect of antimycin A and funiculosin, two inhibitors which block electron transfer in the b‐c1 complex, on electron flow and electrochemical potential difference of H+ ions in mitochondria at static head (state 4) is investigated. In addition, the respiratory control ratio is determined as the ratio between uncoupler stimulated and static‐head electron flow. Malonate, a competitive inhibitor of succinic dehydrogenase, is used for comparison.

All three inhibitors cause an extensive depression of static‐head electron flow but only a limited decrease in the electrochemical potential difference of H+ ions. With the antimycin‐type of inhibitors, the respiratory control ratio slightly increases up to about 50% inhibition of electron flow and then steeply declines. With malonate, a strong decrease of the respiratory control ratio is observed in a concentration range where the electron flow is inhibited less than 10%.

It is shown that the data do not compiy with the generally accepted hypothesis of a leak conductance being regulated by the electrochemical potential difference of H+ ions. They can be interpreted in terms of not tightly coupled redox‐driven H+‐pumps. A non‐vanishing electron flow at static head then arises predominantly from molecular slipping in the pumps, and the (constant) leak conductance yields only a minor contribution.
Structure and Conformation of Human Pancreatic Carboxyl‐Ester Hydrolase
Authors: GUY Odette , LOMBARDO Dominique , BRAHMS J. Georges
Published: July 2010

Human pancreatic carboxyl‐ester hydrolase is a glycoprotein with a molecular weight of 100000 and a high content in carbohydrate, 20%. Sedimentation studies indicate that the molecule resembles an ellipsoid. The results of hydrodynamic and vacuum‐ultraviolet circular dichroism investigation allow one to propose a model of the carboxyl‐ester hydrolase three‐dimensional structure. The enzyme belongs to the ‘all β’ class of proteins; about 54–60% of its residues are in β‐sheets and in β‐turns, most probably forming the surface of an ellipsoid. A similarity with prealbumin structure is proposed and a comparison with structure of other pancreatic enzymes is presented.
Regulatory Properties of Phosphofructokinase 2 from Escherichia coli
Authors: KOTLARZ Denise , BUC Henri
Published: July 2010

Escherichia coli K12 contains two phosphofructokinases: phosphofructokinase 1, the most studied one, appears to behave as an allosteric enzyme, while phosphofructokinase 2 presents the features of a Michaelian enzyme.

We show in the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme.

The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6‐phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6‐phosphate but only one molecule of the product fructose 1,6‐bisphosphate.

Although both phosphofructokinases of E. coli K12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo.
Reconstitution of a Functional Acetylcholine Receptor
Authors: POPOT Jean‐Luc , CARTAUD Jean , CHANGEUX Jean‐Pierre
Published: July 2010

Highly purified plasma membrane fragments rich in acetylcholine (nicotinic) receptor were prepared fromTorpedo marmorataelectric organ, depleted at alkaline pH of the 43000‐Mr, protein and solubilized with sodium holate in the presence or absence of exogeneous lipids. The solubilized receptor (essentially in its 9‐S light form) was reinserted into artificial lipid vesicles in the course of detergent removal by two methods yielding reconstituted vesicles with mean diameters of about 40 nm and 100 nn respectively. The influence of the composition of the lipid and aqueous phases on the physical reinsertion of the receptor protein was studied by gel filtration. gradient centrifugation and electron microscopy. At low ionic strength, the α‐toxin binding sites were quantitatively reintegrated into vesicles from soy‐bean lipids, but not into egg phosphatidylcholine vesicles. Raising the ionic strength during detergent removal decreased receptor incorporation.

The permeability properties of the reconstituted vesicles were investigated by a Millipore filtration method using radioactive tracer ions. Passive equilibration of22Na+or134Cs+ions between internal and external compartments was slow and remained incomplete after several days. Addition of the agonists acetylcholine or carbamylcholine stimulated the exchange of cations which reached a plateau value within less than 15 s. These agomsts did not affect the rate of36Cl−exchange. The permeability response was totally blocked by snake α‐toxins. trimethisoquin, histrionicotoxin, and by preincubation with agonists (desensitization). It is concluded that these properties do not depend on the dimerization of the receptor protein into the 13‐S form nor on its association with the 43000‐Mrprotein and therefore appear to be intrinsic to the 9‐S form of the receptor.
Synthetic Monoacylphospholipids as Reactivators of the Calcium‐Dependent ATPase of Enzymatically Delipidated Sarcoplasmic Membranes
Authors: THE Rudy , HUSSEINI Hussein Shakib , HASSELBACH Wilhelm
Published: July 2010

The reactivating effect of synthetic analogues of natural lysophosphatidylcholines, acyldeoxyglycerophosphocholines, with acyl chain lengths between C10–C18 on enzymatically delipidated sarcoplasmic membranes has been analyzed.

The calcium‐dependent ATPase is fully restored by myristoyldeoxyglycerophosphocholine. The restoring effect of deoxyglycerophosphocholines declines when the length of the saturated acyl chain becomes shorter or longer.

In contrast to the weakly effective palmitoyldeoxyglycerophosphocholine, its cis‐9‐mono unsaturated analogue oleyldeoxyglycerophosphocholine proved to be a highly effective reactivating compound.

The transfer of the terminal phosphate residue of ATP to the transport protein and the phosphate exchange between ADP and ATP displays the same dependence on the acyl chain length of the deoxyglycerophosphocholines. In contrast to the ATPase activity the ATP‐supported phosphoryltransfer reactions can only partially be restored.

A significant restoration of the phosphorylation of the protein by inorganic phosphate could be achieved with none of the deoxyglycerophosphocholines.

Below 23°C the apparent activation energy of the calcium‐dependent ATPase increases with increasing chain length of the deoxyglycerophosphocholines while above 23°C the activation energies were identical for all restituted preparations.
Enzymic Synthesis of Juvenile Hormone in Locust Corpora Allata: Evidence for a Microsomal Cytochrome P‐450 Linked Methyl Farnesoate Epoxidase
Authors: FEYEREISEN Rene , PRATT Grahame E. , HAMNETT Anthony F.
Published: July 2010

Homogenates of corpora allata from adult Locusta migratoria in phosphate‐buffered EDTA have been analysed by sucrose‐density‐gradient centrifugation. Succinate‐cytochrome c reductase activity (mitochondrial) bands between d204 1.13–1.15, whereas NADPH‐cytochrome c reductase and NADPH–dependent methyl farnesoate 10, 11‐epoxidase activities band identically between d204 1.06–1.12. We conclude that the methyl farnesoate epoxidase is exclusively microsomal. Farnesoic acid O‐methyltransferase is an exclusively soluble enzyme which stoichiometrically transfers the S‐methyl group from S‐adenosylmethionine to farnesoic acid. No carboxyl esterase activity was found.

Isolated microsomes were used to obtain an apparent Km= 7.7 × 10−6 M for the epoxidase, although substrate solubility limits the rate to 0.5 V As expected, the product (juvenile hormone III) is chiral (10 R). The epoxidase is inhibited by excess NADP+ and oxidised cytochrome c, but neither inhibited nor synergised by NADH. NADH supports less than 10% of the NADPH rate of epoxidation. The epoxidase is inhibited by a carbon monoxide/oxygen atmosphere, half‐maximal inhibition occurring at a CO/O2 ratio of 4.0. This inhibition is reversed by white‐light irradiation.
Allotropism in Aspartyl‐tRNA Synthetase from Porcine Thyroid
Authors: VELLEKAMP Gary J. , KULL Fredrick J.
Published: July 2010

Three forms of aspartyl‐tRNA synthetase, PC‐1, PC‐2 and PC‐3 (based on their order of elution from phosphocellulose columns), were purified from porcine thyroid utilizing fractional pH precipitations and column chromatography. Two forms, PC‐1 and PC‐2, eluted identically from gel filtration columns and migrated the same electrophoretically and in glycerol density gradients. They were estimated to have sedimentation coefficients of 7 S and molecular weights of about 150000. Both PC‐1 and PC‐2 were equally inhibited by KCl. Their elution positions from phosphocellulose were unchanged upon rechromatography, indicating that they were nonequilibrium forms and they were present in post‐mitochondrial supernatants in roughly equal amounts. All three forms were present in the same ratios whether or not phenylmethylsulphonyl fluoride was present in homogenizing buffers.

PC‐3 had the characteristics of an aminoacyl‐tRNA synthetase complex. It contained aminoacyl‐tRNA synthetase activity for 16 of 18 amino acids tested and was about 4% low‐molecular‐weight RNA. The complex did not significantly penetrate 5% polyacrylamide gels, was excluded by Sephacryl S‐200 and had a sedimentation coefficient greater than 20 S indicating a particles mass in excess of 500000 daltons. In addition, the PC‐3 fraction contained a ribonuclease capable of degrading both aminoacyl‐tRNA and tRNA.

After storage, rechromatography of PC‐3 on phosphocellulose showed that some dissociation of the enzymes had taken place. Aspartyl‐tRNA synthetase activity was found in positions analogous to PC‐1 as well as PC‐3, and ribonuclease activity was found in two positions on the columns, one coincident with PC‐3 and one preceding the position of elution of PC‐1. Dissociation of the complex was also observed using glycerol density gradient centrifugation and by chromatography on Sepharose4B.

When present in the PC‐3 complex, the aspartyl‐tRNA synthetase activity and the ribonuclease activity were both stimulated by KC1. However, when dissociated to their free forms, the aspartyl‐tRNA synthetase activity was inhibited by KC1, whereas the ribonuclease remained stimulated. The effects on aspartyl‐tRNA synthetase, appeared to be potassium‐ion‐specific but were more pronounced in the presence of phosphate dianion than in chloride ion.

The differential effects of potassium ion on the free and complexed forms of aspartyl‐tRNA synthetase and the prescence of a ribonuclease in the aminoacyl‐tRNA complex that is active toward tRNA could have functional significance in the intact cell.
Latent Phosphorylase Phosphatases from Rat Liver: Relationship with the Heat‐Stable Inhibitory Protein
Authors: JETT Mary‐Frances , HERS Henri‐Géry
Published: July 2010

A high‐speed supernatant from rat liver contains at least two latent phosphorylase phosphatases the activities of which are revealed by treatment with ethanol, urea, mercaptoethanol or trypsin. This fraction also contains at least one protein which, after heating, inhibits to various degrees the activated form(s) of the two phosphatases. The two latent enzymes can be separated by cellulose‐phosphate chromatography and can be differentiated by their preferential activation by ethanol or trypsin and by their different sensitivity to the inhibitory protein after ethanol activation.

Activation of the latent phosphorylase phosphatases by ethanol, urea or mercaptoethanol is not accompanied by the destruction of the precursor of the inhibitory protein whereas activation by trypsin is. However, trypsin treatment of fractions previously activated by ethanol decreases their activity and also increases their sensitivity to the inhibitory protein in a way which is unrelated to the destruction of this inhibitor. Furthermore, some protein fractions, almost free of the precursor of the inhibitory protein can be readily activated by trypsin. It is concluded that the activation of the latent phosphorylase phosphatases is unrelated to the destruction of the inhibitory protein.
Glutaconate CoA‐Transferase from Acidaminococcus fermentans
Authors: BUCKEL Wolfgang , DORN Ulrike , SEMMLER Rosuitha
Published: July 2010

Glutaconate CoA‐transferase catalyses the transfer of CoAS− from acetyl‐CoA preferentially to (E)‐glutaconate, but glutarate, (R)‐2‐hydroxyglutarate, acrylate and propionate are also good acceptors. No reaction was observed with (Z)‐glutaconate and C4‐dicarboxdic acids.

The product of the reaction of acetyl‐CoA with (E)‐glutaconate is the 1‐isomer of glutaconyl‐CoA, i.e. the thiol ester is conjugated with the double bond. Other results indicate. however, that with (R)‐2‐hydroxyglutarate as substrate both possible isomers are generated.

Glutaconate CoA‐transferase was purified from cell‐free extracts of Acidaminococcus fermentans to apparent homogeneity and crystallized. The relative molecular mass of the enzyme is approximately 275000. It consists of two different polypeptide chains (Mr 32000 and 34000). On the catalytic pathway a thiolester is formed between CoASH and a carboxylate of the smaller polypeptide chain.

The structural and functional relationships between glutaconate CoA‐transferase and other CoA‐transferases are discussed.

Glutaconate CoA‐transferase is also present in other bacteria fermenting glutamate via hydroxyglutarate. Experiments with an antiserum against the enzyme indicate that the transferase is necessary for the decarboxylation of glutaconate but not for the dehydration of (R)‐2‐hydroxyglutarate.
Structure of 2′,3′‐Di‐O‐acyl‐α‐d‐glucopyranosyl‐(1 –> 2)‐d‐glyceric Acid, a New Glycolipid from Nocardia caviae
Authors: POMMIER Marie‐Thérèse , MICHEL Georges
Published: July 2010

The structure of a new giycolipid isolated from the acetone‐soluble lipids of a strain of Nocardia caviae has been determined. The water‐soluble moiety contains one mole of d‐glucose and one mole of d(–)‐glyceric acid; the lipid moiety is a mixture of myristic, palmitic and stearic acids with small amounts of oleic acid. The structure of the deacylated compound was determined by periodate oxidation, methylation and enzymatic degradation. The localization of fatty acid residues at 2′, 3′ on glucose was established by methylation and mass spectrometry. The structure was confirmed by 13C NMR spectrometry of the glycolipid and of the deacylated compound. This glycolipid is a 2′, 3′‐di‐O‐acyl‐α‐d‐glucopyranosyl‐(1 → 2)‐d‐(–)‐glyceric acid.
Distribution of Cyclic‐GMP‐Dependent Protein Kinase in Various Rat Tissues and Cell Lines Determined by a Sensitive and Specific Radioimmunoassay
Authors: WALTER Ulrich
Published: July 2010

Cyclic‐GMP‐dependent protein kinase purified from bovine lung was radioiodinated by the Bolton‐Hunter procedure yielding a specific radioactivity of 2200 Ci/mmol of enzyme. Using a specific precipitating rabbit antiserum to the cyclic‐GMP‐dependent protein kinase, a sensitive radioimmunoassay was developed which can detect 200 pg (1.33 fmol) of cyclic‐GMP‐dependent protein kinase. Immunoreactivity like that of cyclic‐GMP‐dependent protein kinase was detectable in extracts of all rat tissues tested, in extracts of cultured rat brain and heart cells, and in extracts of rat glioma (C6) and neuroblastoma × glioma hybrid cells. In extracts of several tissues and cell lines the presence of cyclic‐GMP‐dependent protein kinase was also demonstrated by a photoaffinity‐labeling procedure using 8‐azidoinosine 3′, 5′‐[32P]monophosphate. The results suggest that cyclic‐GMP‐dependent protein kinase is ubiquitously distributed although its level varies significantly from tissue to tissue and cell type to cell type. The results also support the hypothesis that cyclic‐GMP‐dependent protein kinase is involved in mediating some of the intracellular effects of those hormones, neurotransmitters and drugs which regulate the intracellular level of cyclic GMP.
Comparison of the NH2‐Terminal Sequences of Chick Type I Preprocollagen Chains Synthesized in an mRNA‐Dependent Reticulocyte Lysate
Authors: GRAVES Peter N. , OLSEN Bjørn R. , FIETZEK Peter P. , PROCKOP Darwin J. , MONSON Janet M.
Published: July 2010

RNA was extracted from 17‐day‐old chick embryo calvaria and translated by an mRNA‐dependent reticulocyte lysate. Procedures were developed for purifying intact proα1(I) and proα2 translation products from the lysate. These products were identified by comparing tryptic peptide elution patterns of the translation products with those obtained from secreted proα chains. Partial sequence data from the amino terminus of each translation product demonstrated that each chain begins with a sequence that is different from that of the corresponding proα chain, and that the two sequences are not the same. Also, the bacterial collagenase‐resistant peptide from the amino terminus of preproα1(I) had an apparent molecular weight which was 10000 greater than the peptide from proα1(I).
Identification of Transcription Initiation Sites for Bacterial RNA Polymerase and Eukaryotic RNA Polymerase B on the 5′ End of the Mouse β‐Globin Gene
Authors: CREPIN Michel , TRIADOU Patrick , LELONG Jean‐Claude , GROS François
Published: July 2010

Using a recombinant phage containing the mouse β‐globin gene with λgtWES bacteriophage DNA, transcription initiation sites for Escherichia coli RNA polymerase and calf thymus RNA polymerase B were mapped at the 5′ and 3′ ends of the mouse β‐globin gene. The bacterial enzyme was capable of initiating RNA synthesis at the 3′ end site located at about 700 residues from the 3′ end of the β‐globin restriction enzyme map. Initiation at this site was more efficient than initiation at the known early λ promotors (PL, PR). Calf thymus RNA polymerase B initiated transcription at the same sites as the bacterial enzyme but in this case maximum efficiency was at the 5′ end site as compared to the 3′ end site. Initiation of transcription occurs in the region of the d(T‐A‐T‐A‐A) sequence. Initiation efficiency at the 5′ end site, as probed by the maximum rate of transcription, was shown to depend partly upon the presence of the adjacent sequences upstream and downstream of the 5′ initiation site.
DNA Topoisomerase I from Mitochondria of Xenopus laevis Oocytes
Authors: BRUN Gilbert , VANNIER Patrick , SCOVASSI Ivana , CALLEN Jean‐Claude
Published: July 2010

Activity of DNA topoisomerase I has been characterized in extracts of mitochondria purified from Xenopus laevis oocytes. Several lines of evidence have been obtained for the intramitochondrial localization of the enzyme. The mitochondria‐associated activity of DNA topoisomerase I represents 1 % of the activity recovered from a total ovary population of oocytes. The enzyme has been purified by DEAE‐cellulose, phosphocellulose and double‐stranded DNA cellulose chromatography and its properties compared to those of its nuclear‐cytosolic counterpart purified by the same purification steps. Both enzymes appear to possess very similar if not identical physico‐chemical and catalytic properties. Neither enzyme shows DNA gyrase activity and they are both equally sensitive towards trypanocide drugs such as ethidium bromide and berenil. The mitochondrial enzyme is able to remove positive superhelical turns and possibly acts as a swivelase during replication of mitochondrial DNA. These results confirm the pioneering work of Fairfield et al. (1979, J. Biol. Chem. 254, 9352–9354) on the presence of a DNA topoisomerase I in mitochondria. However our results support the conclusion that in Xenopus laevis oocytes the mitochondrial and nuclear cytosolic DNA topoisomerase I are in all likelihood one and the same enzyme.
Ribosomal Protein S6 from Xenopus laevis Ovaries
Authors: KALTHOFF Holger , DARMER Dorothea , TOWBIN Harry , GORDON Julian , AMONS Reinout , MÖLEER Wim , RICHTER Dictmar
Published: July 2010

Ribosomal protein S6 from Xenopus laevis ovaries was prepared by ion‐exchange chromatography on phosphocellulose and gel filtration on Sephadex G‐75. The protein was identified as S6 from its position on two‐dimensional polyacrylamide gels and from its immunological cross‐reaction with monoclonal antibody raised against chicken liver S6, and from the fact that it is the major phosphorylated protein of the small subunit.

When oocytes were incubated with [32P]orthophosphate in the presence of progesterone. 32P incorporation of 40‐S ribosoniul proteins was stimulated about 10‐fold over controls without hormone. The bulk of the 32P radioactivity was incorporated into protein S6.
The 38000‐Mr Poly(A)‐Binding Protein of Non‐Polysomal Messenger Ribonucleoproteins of Cryptobiotic Gastrulae of Artemia salina
Authors: De HERDT Erik , PIOT Etienne , KONDO Masatoshi , SLEGERS Herman
Published: July 2010

The 38000‐Mr poly(A)‐binding protein has been purified to near homogeneity from non‐polysomal messenger ribonucleoprotein of Artemia salina [Slegers, H., De Herdt, E., and Kondo, M. (1981) Eur. J. Biochem. 117, 111‐120]. The protein consists of approximately 357 amino acids and is characterized by a high glycine content of 22.5% and the presence of dimethylarginine. From polynucleotide‐protein binding experiments a stoichiometry of 9–11 adenylate and 10–12 uridylate residues per protein molecule is calculated. The polypeptide is devoid of poly(A) polymerase and RNase activities. The poly(A)‐binding protein and the helix‐destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466–6472] have the same mobility in polyacrylamide/dodecylsulphate gel electrophoresis and exhibit a comparable amino acid composition and protein‐polynucleotide stoichiometry. Based on the length of poly(A) sequences of mRNA and from protein‐poly(A) binding experiments, a repetitive binding of the 38000‐Mr protein on the poly(A) sequence is demonstrated. The 38000‐Mr protein of cytoplasmic and membrane‐bound non‐polysomal messenger ribonucleoproteins is also compared.
Molecular Cloning of the 8000‐Base Thyroglobulin Structural Gene
Authors: CHRISTOPHE Daniel , MERCKEN Luc , BROCAS Huguette , POHL Viviane , VASSART Gilbert
Published: July 2010

Bovine tliyroglobulin mRNA was reverse‐transcribed into full‐length double‐stranded cDNA. The existence of three HindIII restriction endonuclease sites in the 8000‐base tliyroglobulin structural gene had allowed the easy cloning of the two internal HindIII fragments [Cliristophe et al. (1980) Eur. J. Biochem. 111, 419‐423], In the present study, the central portion of the structural gene was cloned in Escherickia coii as two individual recombinant plasmids containing 2000‐base‐pair and 4700‐base‐pair segments located respectively 5′ and 3′ relative to the unique BamHI site of the cDNA. BumHI linkers were added to the double‐stranded cDNA and, following restrictions with HindIII. selective cloning of the 5′ (2600‐base‐pair) and 3′ (1000‐base‐pair) terminal HindIII fragments was achieved by inserting them between the HindIII and BamHI sites of the plasmid pBR322. Partial sequencing of the 1000‐base‐pair 3′‐terminal fragment demonstrated the presence of an A‐A‐U‐A‐A‐A sequence in the inRNA 14 bases upstream from a poIy(A) tract corresponding to the 3′ end of the mRNA.

Together, the four clones represent about 99%of the thyroglobulin structural gene and provide the starting material for the determination of thyroglobulin primary structure.
Transport of the Precursor for Rat‐Liver Ornithine Carbamoyltransferase into Mitochondria in vitro
Authors: MORITA Tetsuo , MIURA Satoshi , MORI Masataka , TATIBANA Masamiti
Published: July 2010

Transport of the precursor form of rat liver ornithine carbanioyltransferase was studied in a cell‐free reconstitution system. The precursor. synthesized in vitro. was converted to an apparently mature form of the enzyme by incubation with a mitochondria1 preparation of rat liver. On further purification of the preparation by isopycnic sucrose gradient centrifugation, the processing activity cosedimented with mitochondria. The processed pro‐duct was no longer susceptible to externally added proteases.

The activity responsible for the conversion of the precursor to the mature form disappeared when the mitochondria were disrupted by digitonin treatment. sonication or freeze‐thawing. The inactivation apparently pardleled the leakage of the marker enzyme of the intermembrane space from the mitochondria.

Processing of the precursor by intact mitocliondria was strongly inhibited by both 0.1 mM dinitroplienol and 1 μM carbonylcyanide p‐trifluoromethoxyphenylhydrazone. However. neither KCN, NaN3. antimycin A nor oligomycin had any marked effect on the processing.

Mitochondria from rat spleen and ascites hepatonin cells, which lack ornithine carbamoyltransferase. were able to take up the enzyme precursor and to process it to the mature form of the enzyme. Mouse liver kind kidney mitochondria also took up and processed the rat precursor.

In pulse and pulse‐chase experiments with isolated rat hepatocytes. the labeled precursor first appeared in the cytosol and then disappeared with a half‐life of less than 3 min. Dinitrophenol and carbonylcyanide p‐trifluoromethoxyphenylhydrazone failed to inhibit the disappearance of the precursor from the cytosol.

The results indicate that the ornithine carbamoyltransferase precursor is transported into isolated mitochondria in association with the proteolytic processing to the mature enzyme. The transport‐processing system requires mitochondria of high integrity. It was sensitive to uncouplers but not much to respiratory inhibitors. and may be common to more than one mitochondria1 enzyme precursor.
Studies on Blood‐Groups AEnzymes.1 and A2
Authors: SCHENKEL‐BRUNNER Helmut
Published: July 2010

Blood‐group O and A2 erythrocytes were treated with the A1‐gene‐dependent and A2‐gene‐dependent N‐acetylgalactosaminyl transferases in the presence of UDP‐N‐acetyl[14C]galactosamine. Although the transfer of N‐acetyl[14C]galactosarnine with A2 transferase was slower than with A1 enzyme, group O as well as A2 cells became agglutinable by anti‐A1 reagents when incubated with both transferases. Fractionation of the labelled erythrocyte stroma into glycoprotein and glycolipid components showed an approximately equal distribution pattern of radioactivity in all experiments. Likewise, when the short‐chain glycolipids and polyglycosylceramides isolated from the labelled stroma were further analyzed by thin‐layer chromatography, no major differences were detected in the chromatographic profiles of O and A2 cells when treated with either transferase. These observations indicate that (a) the blood‐group‐H‐type oligosaccharide chains of A2 cells may be similar to those of group‐O cells and (b) the serological differences between A1 and A2 cells are likely to be due to a lower density of A‐antigenic sites on A2 cells.
Pig Heart [35S]Thiophosphoryl Pyruvate Dehydrogenase Complexes
Authors: TONKS Nicholas K. , KEARNS Anne , RANDLE Pshilip J.
Published: July 2010

Thiophosphorylation of pig heart pyruvate dehydrogenase complex with atp [35S] (adenosine 5′[γ‐thio]‐ triphosphate) was analogous to phosphorylation with [γ‐32p]ATP except that thiophosphorylation of sites 2 and 3 was more rapid. With pyruvate dehydrogenase phosphatase the rate of dethiophosphorylation was 0.5% of that of dephosphorylation. Thiophospghorylation of sites 2 and 3 in complex phosphorylated i n site educed the rate of dephosphorylation of site 1.
Bromopyruvate as an Affinity Label for Baker's Yeast Flavocytochrome b2
Authors: ALLIEL Patrick M. , GUIARD Bernard , GHRIR Rachid , BECAM Anne‐Marie , LEDERER Florence
Published: July 2010

It was previously reported that bromopyruvate behaves as an active‐site‐directed reagent for flavocytochrome b2 [Mulet and Lederer (1980) Eue. J. Biochcwi. 73, 443–447], but that some unspecific labeling also took place [Alliel, Mulet, and Lederer (1980) Eur. J. Biochem. 105, 343–351], In this work, radioactive peptides were purified after labeling the enzyme with bromo[2‐14C]pyruvate. Direct proteolysis of the labeled enzyme led to a multiplicity of labeled peptides, due to incomplete proteolysis. Four of them were characterized, corresponding to two unique cysteine residues. Cyanogen bromide cleavage of the labeled protein, followed by enzymatic digestion, led to the isolation of peptides corresponding to four cysteines, including the two previously identified ones. Comparison of the specific radioactivity of the various labeled peptides lead us to the conclusion that the active‐site cysteine must be the one present in the 85‐residue cyanogen bromide peptide αCB3. The sequence around that cysteine is Ala‐Ser‐Cys‐Ser‐Pro‐Gln‐Gln‐Ile‐Ile‐Glu‐Ala‐Ala‐.
Kinetic Equivalence of the Subunits of Liver Alcohol Dehydrogenase
Authors: ANDERSON Pia , PETTERSSON Gösta
Published: July 2010

The kinetics of two‐substrate enzyme reactions proceeding by an ordered ternary‐complex mechanism have been analyzed theoretically with respect to the precise magnitude of amplitudes of the two slowest transients governing such reactions under single‐turnover conditions. Relationships derived have been applied to the liver alcohol dehydrogenase system for detailed interpretation of amplitudes of the biphasic absorbance changes associated with the enzymic reduction of aromatic aldehydes in catalytic‐suicide experiments.

Amplitudes of the slow phase 328–330‐nm absorbance changes occurring during the enzymic reduction of various para‐substituted benzaldehydes by NADH or NAD2H correspond to 6–26% of the total absorbance change under conditions of substrate and coenzyme saturation. Saturation values of amplitudes determined in the present and previous investigations agree within 2 % of the total absorbance change with those calculated theoretically for an ordered mechanism of enzyme action at equivalent sites. In particular, amplitudes observed exhibit the expected sensitivity to electronic substituent and deuterium isotope effects.

Enzyme recycling, aldehyde binding, and alcohol desorption provide negligible contributions to the 328–330‐nm absorbance changes observed during aldehyde reduction in the presence of 20 mM pyrazole. The maximum slow phase amplitude contribution provided by pyrazole binding corresponds to 5–6% of the total absorbance change. Additional slow phase absorbance changes persisting at saturating concentrations of coenzyme and substrate reflect incomplete accumulation of the enzymic intermediate formed through hydride transfer in the rapid reaction phase. This intermediate contains no detectable amounts (less than 2 %,) of NADH, but exhibits spectral properties indistinguishable from those of enzyme. NAD+. alcohol complexes examined.

It has been concluded in recent publications that an ordered ternary‐complex mechanism involving equivalent sites is insufficient to account for the transient‐state kinetics of liver alcohol dehydrogenase catalysis. These conclusions are shown to be based on an inadequate interpretation of the observations made. Results reported up to date provide no evidence whatsoever for the kinetic significance of subunit interactions leading to a non‐equivalence of the enzymic sites. Subunit interactions resulting in a “half‐site7rdquo; reactivity of the enzyme are definitely not at hand.
Differences in the Binding of Thyroid Hormones and Indoles by Rat α1‐Fetoprotein and Serum Albumin
Authors: HERVÉ Françoise , GRIGOROVA Anne‐Marie , RAJKOWSKI Krzysztof , CITTANOVA Nicole
Published: July 2010

The transport of small molecules in the blood, normally assured by serum albumin in the adult. is not well known in the fetus since the albumin concentration is low in fetal serum and inversely related to the α1‐fetoprotein concentration.

In order to investigate whether rat α1‐fetoprotein might be a fetal counterpart to albumin. the binding properties of these two proteins have been compared with respect to a series of molecules of biological importance, especially during fetal development: thyroid hormones and indole analogues.

Though high‐affinity binding of thyroxine was found with both rat α1‐fetoprotein and albumin, a significant difference in the number of binding sites for this hormone was found with the two protcins. Further, while rat serum albumin strongly bound L‐tryptophan and indolyl‐3‐acetic acid (Ka∼ 105 M−1), rat α1‐fetoprotein did not bind any of the indoles tested.

These results are discussed with respect to the physiological and pharmacological significance of the transport role of these proteins.
Purification and Characterisation of a Small DNA‐Binding Protein, HB, from Bacillus globigii
Authors: IMBER Roldnd , BÄCHINGER Hdnspetei , BICKLE Thomas A.
Published: July 2010

We have purified a small, heat‐stable DNA‐binding protein from Bacillus glohigii: HB protein. The protein binds cooperatively to double‐stranded DNA and the DNA‐protein complexes are destabilised by the presence of moderate (0.1 M) levels of monovalent cations or by low levels (10 mM) of Mg2+. We have also purified two small DNA‐binding proteins from Eschericlzia coli, NS1 and NS2 [Suryanarayana, T. and Subranianian, A. R. (1978) Biochim. Biophys. Acta, 520, 342‐357], also known as HU protein [Rouviére‐Yaniv, J. and Gros, F. (1975) Proc. Nut1 Acad. Sci. USA, 72, 3428‐3432] that have been extensively studied by others. We have prepared antibodies against all three proteins and have shown that all three are, by immunological criteria, related.
Binding of 21 Thiol Reagents to Human Hemoglobin in Solution and in Intact Cells
Authors: GAREL Marie Claude , BEUZARD Yves , THILLET Joëlle , DOMENGET Chantal , MARTIN Josiane , GALACTEROS Frédéric , ROSA Jean
Published: July 2010

The reactivity of the cysteine‐β93 residue of human hemoglobin was investigated in order to define the optimal structure of potential antisickling agents. The properties of 21 thiol reagents were compared with regard to (a) their binding rate to hemoglobin in solution and within intact cells; (b) the modification of the oxygen dissociation curve of intact cells and (c) the effect on methemoglobin formation in solution or within intact cells. The results show the very different behaviors of these reagents.
Preferential Utilization of Phosphorylated 40‐S Ribosomal Subunits during Initiation Complex Formation
Authors: DUNCAN Roger , McCONKEY Edwin H.
Published: July 2010

HeLa cells grown to a high density in spinner culture contain little or no phosphorus associated with ribosomal protein S6. When cells are transferred to fresh medium containing 10% calf serum, S6 becomes rapidly and multiply phosphorylated. Ribosomal proteins were extracted from subpolysome and polysome fractions, displayed on two‐dimensional gels, and the distribution of phosphorylated S6 was quantified. Polysonial ribosomes have a higher percentage of phosphorylated S6 than subpolysomes at all times after transfer and the difference becomes more pronounced as the extent of phosphorylation increases. This difference cannot be explained by preferential phosphorylation of polysomal ribosomes, since kinase activity is equally distributed between polysomes and subpolysomes. Likewise, preferential dephosphorylation or subpolysomal ribosomes during cell fractionation does not occur. We interpret our results to mean that phosphorylated 40‐s subunits form initiation complexes more efficiently than non‐phosphorylated 40‐S subunits
The Characteristics of Some Components of the Fatty Acid Synthetase System in the Plastids from the Mesocarp of Avocado (Persea americana) Fruit
Authors: CAUGHEY Isaac , KEKWICK Roy G. O.
Published: July 2010

Preparations of NADH‐specific and NADPH‐specific, 3‐oxoacyl‐[acyl‐carrier‐protein] reductase enzymes (EC 1.1.1.100), enoyl‐[acyl‐carrier‐protein] reductase (EC‐ 1.3.1.9) and [acyl‐carrier‐protein] malonyltransferase (EC 2.3.1.39) have been purified from preparations of avocado mesocarp plastids and characterized. The enzymes are quite similar in molecular and kinetic characteristics to analogous enzymes known in Escherichia coliand Euglena and are clearly components of a type‐II fatty acid synthetase system.
Occurrence of a Catabolic l‐Serine (l‐Threonine) Deaminase in Saccharomyces cerevisiae
Authors: RAMOS Fernando , WIAME Jean‐Marie
Published: July 2010

Saccharomyces cerevisiae mutants lacking the anabolic l‐threonine deaminase, the ilvl−mutants, have been found to exhibit a normal ability to grow, without auxotrophy towards isoleucine, on l‐threonine or l‐serine as only nitrogen nutrient. Starting from a strain carrying a ilvl−mutation, which also impairs the ability to utilize l‐serine, has been denominated cha−, for ‘catabolism of hydroxyamino acids’ and was found to result in the lack of a catabolic l‐serine (l‐threonine) deaminase.

This enzyme which, unlike the anabolic threonine deaminase, is more active towards serine that towards threonine, differs from the latter enzyme by a number of biochemical and regulatory properties. Whereas the anabolic enzyme is an allosteric enzyme sensitive to feedback inhibition by isoleucine, the catabolic enzyme exhibits Michaelian kinetics; no control of its activity has been detected. Its synthesis is induced by l‐serine and l‐threonine.

These two enzymes, which thus can be easily differentiated by means of their regulations, display a limited ability to compensate for one another's absence and appear to play clearly distinct roles under normal physiological conditions.
Mitochondrial Adenylate Kinase from Chicken Liver
Authors: WATANABE Kiyotaka , KUBO Shuichiro
Published: July 2010

Mitochondrial adenylate kinase has been purified 5400‐fold chicken liver extract in an overall yield of 36%. The purified enzyme has a specific activity of 810 U/mg, a molecular weight of 28000, and the following amino acid composition:21 aspartic acid or asparagines, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 16 proline, 22 glycine, 22 alanine, 15 valine, 6 methionine, 11 isoleucine, 29 leucine, 5 tryosine, 7 phenylalanine, 16 lysine, disulfide bond and one sulfhydryl group. The disulfide bond is related to the active conformation of the enzyme, whereas the sulfhydryl group does not contribute to the enzyme activity. The sulfhydryl group is easily oxidized in the presence of Cu2+ resulting in the formation of dimmer with about one half of the specific activity of the monomer. The enzyme is similar to porcine heart mitochondrial adenylate kinase in antigenicity but different from chicken cytosolic adenylate kinase.

mitochondrial adenylate kinase was synthesized in the mRNA‐dependent rabbit reticulocyte lysate system programmed with total chicken liver RNA. The mobility in sodium dodecylsulfate gel electrophoresis of the product obtained in vitro was the same as that of the purified mitochondrial aadenylate kinase. This evidence indicates that the mitochondrial adenylate kinase is synthesized as a polupeptide with a molecular weight indistinguishable from that of the mature protein.
Properties of Histidine Decarboxylase from Rat Gastric Mucosa
Authors: SAVANY Alain , CRONENBERGER Lucien
Published: July 2010

The properties of specific histidine decarboxylase from purified rat gastric mucosa preparations were studied. The kinetic parameters were pH dependent: the apparentKmvalue varied inversely with pH; the maximum reaction velocity was reached at pH .6; the optimum pH was related to substrate concentration. The enzyme was unstable below pH 5.5. the effect of temperature was investigated and the enzyme activity was optimum near 56°C. the thermal inactivation of the enzyme showed the presence of several active forms displaying distinct thermostabilities. The effect of coenzyme and substrate on heat stability was established. A small amount of pyridoxal phosphate was required for maximum enzyme activity, and theKmwas low. The cofactor appeared to be tightly bound to the apoenzyme; nevertheless there was a fraction more easily resolved by dialysis. With high pyridoxal phosphate concentrations non‐competitive inhibition occurred. Histamine inhibited the enzyme at high concentrations, the inhibition being competitive with respect to the substrate. No metal ion was required for enzyme activity; the enzyme was inhibited by sulfhydryl reagents and heavy metal ions, and also by high concentrations of reducing agents. The tryptophan residue of the holenenzyme seemed to be essential for the catalytic process.
Purification and Characterisation of a Pore Protein of the Outer Mitochondrial Membrane from Neurospora crassa
Authors: FREITAG Helmut , NEUPERT Walter , BENZ Roland
Published: July 2010

The major protein of the outer mitochondrial membrane of Neurospora was purified. On dodecylsulfate‐containing gels it displayed a single bend with an apparent molecular weight of 31000. reconstitution experiments with artifical lipid bilayers showed that this protein forms pores. Pore conductance was dependent on the voltage across the membrane. The protein inserted into the membrane in an oriented fashion, the membrane current being dependent on the sign of the voltage. Single pore conductance was 5nS, suggesting a diameter of 2nm of the open pore. This mitochondrial protein shows a number of similarities to the outer membrane porins of gram‐negative bacteria.
Two Aspects of DNA Polymorphism and Microheterogeneity:
Authors: PULLMAN Bernard , LAVERY Richard , PULLMAN Alberte
Published: July 2010

Polymorphism and microheterogeneity of DNA appear today as being capable of having important biological consequences. This paper presents a synthetic view of two major properties of the main known forms of DNA (A, B, alternating B, C, D and Z), namely their molecular electrostatic potentials and stcric accessibilities. Variations in these properties are explained in terms of the conformational changes involved and deductions are drawn concerning their influence on the interactive properties of DNA with external agents.
Endonucleases in Yeast Mitochondria
Authors: FOURY Françoise
Published: July 2010

An apurinic endonuclease activity has been characterized in yeast mitochondria. It is dependent on Mg2+, stimulated by about 50% in the presence of 50 mM NaCl and inhibited at higher NaCl concentrations. It is located in the inner mitochondrial membrane and requires high concentrations of detergent (1.5–3%) Triton X‐100) to be extracted. The same treatment extracts several other endonuclease activiriey: the two Mg2+‐ dependent endonuclease activities cleaving double‐stranded DNA at pH 7.5 and 5.4 respectively, the ethidium‐bromide‐stimulated endonuclease activity, the endonuclease activity cleaving single‐stranded DNA at pH 7.5 [Jacquenmin‐Sablon et al. (1979) Biochemistry, 18, 119–127], and a manganese‐stimulated deoxyribonucleasc activity cleaving double‐stranded DNA at pH 7.5 which has been discovered during the present work. Another endonuclease activity cleaving double‐stranded DNA at pH 7.5 in the presence of Mg2+, slightly stimulated by low NaCl concentrations and inhibited by ethidium bromide is extracted from the membrane pellet remaining after the treatment with 1.5% Triton X‐100 by a second treatment with 1.5% Triton X‐100 plus 1 M KCI. The presence in the mitochondrial membrane of this apurinic endonuclease activity indicates that, like nuclear and prokaryotic DNA, yeast mitochondrial DNA is also subject to specialized repair systems.