bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of the Hydrophobic Binding Site of Phosphatidylcholine Exchange Protein with Photosensitive Phosphatidylcholine
Authors: MOONEN Peter , HAAGSMAN Henk P. , VAN DEENEN Laurens L. M. , WIRTZ Karel W. A.
Published: March 2005

1‐Acyl‐2‐{7‐(4‐azido‐2‐nitrophenoxy)‐[1‐14C]heptanoyl}‐sn‐glycero‐3‐phosphocholine was synthesized in order to study the lipid‐binding site of the phosphatidylcholine exchange protein from bovine liver. Photosensitive phosphatidylcholine was incorporated into the protein by incubation with vesicles of this phosphatidylcholine derivative. The lipid‐protein complex was separated from the vesicles by chromatography on Biogel A‐0.5m. Photolysis of the complex by irradiation with light of a high pressure mercury lamp at a wavelength above 340 nm generated the highly reactive nitrene. Sodium dodecyl sulfate gel electrophoresis of the photolysed complex indicated that 30% of the endogenous 14C‐labeled phosphatidylcholine was covalently linked to the protein. Peptides were isolated after digestion of the photolysed complex with protease from Staphylococcus aurem and trypsin. It was determined that the 2‐acyl chain of the phosphatidylcholine molecule was linked to the peptide segment ‐Gly‐Ser‐Lys‐Val‐Phe‐Met‐Tyr‐Tyr‐. This segment was part of a protease peptide of about 65 residues of which the sequence was determined by Edman degradation for the first 38 residues. This peptide contains a cluster of apolar residues ‐Val‐Phe‐Met‐Tyr‐Tyr‐Phe with an extremely high hydrophobicity index and with a predicted β sheet conformation. It is concluded that this hydrophobic cluster forms part of the binding site.
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.
Interaction of Non‐histone Proteins with DNA and Chromatin from Drosophila and Mouse Cells
Authors: DASTUGUE Bernard , CREPIN Michel
Published: March 2005

The specificity of the binding of purified non‐histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non‐histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non‐histone protein confirmed the interpretation that some protein. DNA complexes were specific. Protein · DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non‐histone protein was bound to homologous or heterologous DNA respectively. Purified non‐histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non‐histone proteins and purified non‐histone proteins added in excess. DNA‐bound and chromatin‐bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA‐bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA‐bound non‐histone proteins in the regulation of gene expression.
Uptake and Conversion of the Antibiotic Albomycin by Escherichia coli K‐12
Authors: HARTMANN Anton , FIEDLER Hans‐Peter , BRAUN Volkmar
Published: March 2005

The antibiotic albomycin is transported into cells of Escherichia coli K‐12 by the same uptake system as the iron‐supplying ferrichrome complex. The iron‐complexing hydroxamate moieties of albomycin and ferrichrome are structurally similar. During the phase of rapid iron uptake the chelators were not found in the cells. In order to understand the antibiotic activity of albomycin, it was labeled in the hydroxamate with tritium and in the presumed antibiotically active area with radioactive sulfur. While the tritium label was not retained by the cells, part of the sulfur label was taken up and concentrated 500‐fold within the cell. The sulfur was not incorporated into proteins or nucleic acids since it was recovered as a low molecular weight component. Gel filtration on Bio‐Gel P‐2 revealed one tritium‐labeled and two sulfur‐labeled cleavage products in the incubation medium. We conclude that albomycin is actively transported via its ferrichrome‐like portion into the cells and that the growth‐inhibitory moiety is released by hydrolysis intracellularly and remains there.
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).
Mechanistic and Stereochemical Studies on the Glycine Reductase of Clostridium sticklandii
Authors: BARNARD Graham F. , AKHTAR Muhammad
Published: March 2005

Clostridial glycine reductase multienzyme complex which catalyses the reaction: Glycine + ADP + Pi+ 2H → Acetate + ATP + NH3 was solubilised and fractionated essentially according to the method of Stadtman [T. C. Stadtman 1970 Methods Enzymol. 17A, 956–966] into two components: protein A and “glycine reductase” fraction. A reconstituted system obtained by combining the two components in the presence of dithiothreitol catalysed the conversion of glycine into acetate concomitant with the phosphorylation of ADP to ATP. Using the reconstitued system, in which the unwanted enzymic activity catalyzing an exchange of the α hydrogen atoms of glycine with the protons of the medium had been greatly reduced, it was found that the conversion of (2RS)‐[2‐14C, 2‐3H1]glycine (3H/14C = 7.16) into acetate (3H/14C = 7.03) was attended by the retention of both the C‐2 hydrogen atoms of glycine. Conversion of (2S)‐[2‐2H1, 2‐3H1]glycine and (2R)‐[2‐2H1, 2‐3H1]glycine by the reconstituted system gave (2S)‐acetate and (2R)‐acetate respectively showing that the reductive deamination of glycine occurs through an inversion of configuration. The cumulative information available on the glycine reductase reaction is embodied in a hypothetical mechanism of action for the enzyme.
Tubulin‐Associated Nucleoside Diphosphokinase
Authors: JACOBS Michael , HUITOREL Philippe
Published: March 2005

Microtubule protein, prepared by cycles of polymerisation and dissociation, contained a nucleoside diphosphokinase (NDP kinase) activity (EC 2.7.4.6). This activity was not intrinsic to the tubulin dimer or the so‐called microtubule‐associated proteins. The NDP kinase had the following properties. (1) The enzyme existed in a low‐molecular‐weight form and in association with the complex of microtubule‐associated proteins and tubulin (i.e. multimeric tubulin). (2) The low‐molecular‐weight species was also formed by dissociation of multimeric tubulin by salt or by removal of microtubule‐associated proteins on phosphocellulose. (3) GDP bound to the exchangeable site of multimeric tubulin and also GDP derived from the E site of the tubulin dimer was a substrate for the NDP kinase. (4) The NDP kinase showed a 7‐fold increase in activity during ATP‐dependent microtubule assembly. On the basis of these properties, it is proposed that microtubule protein contains an NDP kinase specifically associated with tubulin and its functions.
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.
Characterization of Bovine Anterior‐Lens‐Capsule Basement‐Membrane Collagen
Authors: SCHWARTZ David , CHIN‐QUEE Trevor , VEIS Arthur
Published: March 2005

Basement membrane collagen was extracted sequentially from bovine anterior lens capsules by repeated pepsinization at very low enzyme‐to‐collagen ratios. The earliest extracts, derived from 1–7‐days pepsinization at 4°C showed that a substantial fraction of basement membrane collagen material did not enter a 6% or 3% sodium dodecylsulfate/polyacrylamide gel. The material that did penetrate the gel ran as discrete bands with molecular weights between 220000 and 140000. The relative proportions of these components could be controlled solely by the duration of pepsinization. Furthermore, as pepsinization was continued or as the quantity of pepsin or the extraction temperature was increased basement membrane collagen peptides with molecular weights between 115000 and 30000 were produced, with little material not entering the gel. The extracts containing collagen chains with Mr > 130000 were precipitable from acetic acid solution at NaCl concentrations of 0.3–0.6 M and could undergo a thermal gelation at pH 7.03. At or near the transition to the lower‐molecular‐weight species (Mr < 130000), the precipitability at low NaCl concentrations and thermal gelation properties were lost. Correlation of these properties with amino acid composition and with previous electron microscopic studies suggest that strong attractive interactions between pepsin‐susceptible molecular end regions are responsible for the salt precipitability and gelation. These end region interactions may also play a role in inhibiting D‐periodic striated fibril formation in basement membrane structures in vivo.
Short‐Term Effect of Ammonium Chloride on Nitrogen Fixation by Azotobacter vinelandii and by Bacteroids of Rhizobium leguminosarum
Authors: LAANE Colja , KRONE Willy , KONINGS Wil , HAAKER Huub , VEEGER Cees
Published: March 2005

Evidence is presented that the direct depressing effect of ammonium chloride on nitrogen fixation by Azotobacter vinelandii is due to inhibition of the electron transport system to nitrogenase. Furthermore, we were able to confirm the observation [Houwaard, F. (1979) Appl. Environ. Microbiol. in the press] that ammonium chloride has no short‐term effect on nitrogen fixation by isolated bacteroids of Rhizobium leguminosarum.

By means of the flow dialysis technique it could be demonstrated that in A. vinelandii ammonium is taken up as a cation in response to the ΔΨ and that uptake of ammonium specifically inhibits the flow of reducing equivalents to nitrogenase by lowering the ΔΨ across the cytoplasmic membrane. In A. vinelandii, like in bacteroids, the generation of reducing equivalents at a potential low enough to reduce nitrogenase was found to be extremely sensitive towards changes in ΔΨ. At ΔΨ values less than 80 mV, interior negative, no such reducing equivalents are generated, while at a ΔΨ value of 110 mV nitrogenase is supplied optimally with reducing equivalents. The nature of the ammonium transport system in A. vinelandii and its significance as a regulator for the rapid ‘switch off/switch on’ of nitrogenase activity is discussed.

Bacteroids of R. leguminosarum did not accumulate ammonium and no effect of ammonium on ΔΨ was observed. On the contrary, it could be demonstrated that bacteroids excrete ammonium in response to the ΔpH.
Studies on the Intracellular Distribution of Sindbis Messenger RNA in Infected Chick‐Embryo Fibroblasts
Authors: BONATTI Stefano , CERASUOLO Alfonso , CANCEDDA Ranieri , BORGESE Nica , MELDOLESI Jacopo
Published: March 2005

Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron microscopy and cell fractionation.

Electron microscopy of infected and non‐infected cells revealed that infection produced a disaggregation of polyribosomes into monomers. Apart from this observation most cells appeared well preserved, and no degranulation of the rough endoplasmic reticulum was visible.

Analysis of postnuclear supernatants by sucrose density gradients showed that no change in the relative proportions of free and membrane‐bound ribosomes was produced by infection. Approximately 30% of the ribosomes and 50% of the viral RNA were found to be associated with membranes. Of the membrane‐associated viral RNA, 70% was recovered as 26‐S RNA.

Similar results were obtained with fibroblasts infected by the temperature‐sensitive Sindbis mutant ts2, which is defective in the co‐translational processing of the viral gene products at the non‐permissive temperature.

Sucrose gradient analysis of membrane‐bound polyribosomes solubilized by detergent indicated that as much as 50% of the membrane‐associated viral 26‐S RNA is not integrated into polyribosomes and that most of the ribosomes are present as monomers or ribosomal subunits.

Treatment with puromycin of living cells or of isolated membrane fractions under a variety of ionic conditions revealed that the viral RNA‐membrane linkage is insensitive to puromycin but sensitive to high concentrations of monovalent ions. The bulk of the membrane‐bound ribosomes were detached by high salt and recovered as ribosomal subunits on sucrose gradients.

These results are consistent with the idea that in chick embryo fibroblasts infected with Sindbis virus only a small percentage of the ribosomes are engaged in protein synthesis, and that the Sindbis messenger RNA may attach to endoplasmic reticulum membranes through a ribosome‐independent, salt‐sensitive link.
Assembly Map of the 50‐S Subunit from Escherichia coli Ribosomes, Covering the Proteins Present in the First Reconstitution Intermediate Particle
Authors: ROTH Hans E. , NIERHAUS Knud H.
Published: March 2005

Highly purified proteins and 23‐S RNA from the 50‐S subunit of Escherichia coli ribosomes were used to study the assembly dependences of the early assembly proteins. The proteins under observation and the RNA were incubated at 4 mM Mg2+ and 44 °C, the unbound proteins were separated by sucrose gradient centrifugation, the RNA · protein complex was precipitated with trichloroacetic acid, and the complex‐bound proteins were identified by means of sodium dodecyl‐sulfate gel electrophoresis. A systematic analysis led to the establishment of an assembly map including 17 proteins which represent the protein moiety of the first reconstitution intermediate particle.
Presence of RNA Methylases in Intracisternal A Particles Purified from a Mouse Plasma Cell Tumor
Authors: CHEPELINSKY Ana Berta , GANTT Raymond , WIVEL Nelson
Published: March 2005

Intracisternal A particles share several biochemical properties with RNA tumor viruses, including 70‐S RNA, reverse transcriptase, and a group‐specific protein. In addition they form by budding from cytoplasmic membranes. It has been shown previously that various purified RNA tumor viruses contain N2‐guanine RNA methylase; purified A particles were tested to determine if they contained this activity as well. The results showed that instead of a single methylase, six methylases were associated with intracisternal A particles: a cytidine and a uridine methylase, two N2‐guanine monomethylases, an N2,N2‐dimethylguanine methylase and an adenine methylase. These enzymes were indistinguishable from the cellular enzymes when Escherichia coli tRNAPhe was used as an acceptor RNA and three were shown to have the identical specificity, i.e. N2‐guanine methylases for positions 10 and 34 and methylation of adenine in position 73 of the tRNAphe. The specific activity of the core‐associated RNA methylases increased approximately twofold over the methylases of the intact particles, indicating that these methylases were associated with the internal structure and not simply adherent to the envelope surface. This suggests that these cellular enzymes are packaged within intracisternal A particles during synthesis. Thus, intracisternal A particles are similar to the oncornaviruses in that reverse transcriptase is the only known enzyme not of cellular origin, but they appear to contain cellular methylases not found in other RNA tumor viruses.
A Substance Effecting Differentiation in Streptomyces griseus
Authors: BIRÓ Sandor , BÉKÉSI István , VITÁLIS Sándor , SZABÓ Gábor
Published: March 2005

A conidium‐producing variant of Streptomyces griseus, strain 45‐H, produces a substance, factor C, which is capable of inducing conidium formation in the hyphae of a conidium‐non‐producing mutant, strain 52‐1. Factor C can be determined quantitatively on the basis of this biological effect. The biologically active substance can be purified by ion‐exchange chromatography on cellulose phosphate combined with affinity chromatography on DNA‐agarose. The purified substance is concentrated at least 1700 times. The molecular weight of factor C, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, is about 34500. On determining the amino acid composition of factor C 60% of the amino acids were found to be hydrophobic.
Properties of Galactosyltransferase‐Enriched Vesicles of Golgi Membranes from Lactating‐Rat Mammary Gland
Authors: KUHN Nicholas J. , WOODING F. B. Peter , WHITE Adrian
Published: March 2005

Lactose was synthesised within the lumen of purified Golgi membrane vesicles, prepared from lactating rat mammary gland, from externally added glucose and UDP‐galactose. An apparent Km of 1.5 mM was shown towards glucose at anomeric equilibrium, but only β‐glucose was utilised. Two apparent Km values, about 17 μM and 112 μM, were shown towards UDP‐galactose. 5‐d‐Thioglucose, 6‐deoxy‐d‐glucose and 6‐deoxy‐6‐chloro‐d‐glucose were alternative substrates, acquiring α‐lactalbumin dependence when the vesicles were lysed with detergent. Substrates independent of α‐lactalbumin, or inhibited by it, included a wide range of, N‐acylated glucosamines as well as phenyl‐β‐glucoside. The galactosylation of these by vesicle preparations could be ascribed to a proportion of leaky vesicles. Suitably low concentrations of Triton X‐100 activated lactose synthesis by intact vesicles, indicating the membrane as a rate‐limiting feature of the system.
A Simple Procedure for Covalent Immobilization of NADH in a Soluble and Enzymically Active Form
Authors: FULLER Carl W. , RUBIN Joyce Rachel , BRIGHT Harold J.
Published: March 2005

Using a new and simplified technique for covalent immobilization of adenine nucleotides [Fuller, C. W. and Bright, H. J., J. Biol. Chem. 252, 6631 (1977)], we have prepared immobilized NADH using two water‐soluble polymers and tested these preparations for activity with several dehydrogenases. The first polymer used, having a weight‐average molecular weight of about 350000, is a copolymer of methacrylyl choline and the epoxide‐containing monomer 3‐[4‐(2,3‐epoxypropoxy)butoxy]‐2‐hydroxypropyl acrylate. Immobilization of NADH onto this copolymer was accomplished in three steps, namely, alkylation of NAD at N‐1, reduction of the nicotinamide moiety with dithionite and Dimroth rearrangement of the alkyl linkage from the N‐1 to the C‐6 amino position. The second copolymer tested was a copolymer of the same epoxide‐containing monomer and N‐methacrylyl‐2‐glucosamine. Using this copolymer, immobilization of NADH through the adenine C‐6 amino position was accomplished in a single step.

Measurements of the steady‐state kinetics of five dehydrogenases at pH 7 and pH 9 showed that, on the average, V and Km values obtained with the immobilized NADH were, respectively, about one third and twice those found for the free coenzyme. In general, when compared with the free coenzyme, the immobilized NADH had greater relative enzymic reactivity at pH 9 than at pH 7.

The simplicity of this method, the general enzymic reactivity, and the ability to be recycled enzymically suggest that this immobilized NADH may be useful in creating enzyme reactors for synthetic, analytical and other purposes.
Chicken‐Gizzard Actin: Polymerization and Stability
Authors: STRZELECKA‐GOLASZEWSKA Hanna , PRÓCHNIEWICZ Ewa , NOWAK Ewa , ZMORZYÑSKI S. , DRABIKOWSKI W.
Published: March 2005

Preparations of chicken gizzard actin obtained from acetone‐dried muscle powders prepared with various methods developed for skeletal muscle contain variable amounts of a β‐actinin‐like protein. This contamination is minimized if the procedure of muscle powder preparation includes washing with EDTA solution, and can be completely removed by gel filtration of G‐actin on Sephadex G‐100. The presence of β‐actinin activity manifests itself in an increased rate of actin polymerization, low filament lengths resulting in low reduced viscosity and enhanced ATP‐splitting activity of actin polymer, and instability of the polymer in the absence of free ATP. Gizzard actin purified on a Sephadex G‐100 column does not differ from rabbit skeletal muscle actin in its polymerization properties. The distinct property of gizzard actin is the instability of its G form in the absence of added Ca2+, indicating that the affinity of this cation for the single high‐affinity site in gizzard actin is lower than in skeletal muscle actin.
Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli
Authors: BIELLMANN Jean‐François , EID Pierre , HIRTH Christian , JÖRNVALL Hans
Published: March 2005

The substrate binding site of aspartate‐β‐semialdehyde dehydrogenase from Escherichia coli was studied by affinity labeling with l‐2‐amino‐4‐oxo‐5‐chloropentanoic acid. The substrate analogue irreversibly inactivates the enzyme with pseudo‐first‐order kinetics and with a half‐of‐the‐sites reactivity. The substrate aspartate β‐semialdehyde protects the enzyme against the inactivation. A single group is labeled at the active site and is concluded to be the side‐chain of a histidine residue. The amino acid sequence around the active site residue was established from a peptic digest of the labeled enzyme: Phe‐Val‐Gly‐Gly‐Asp‐(modified residue)‐Thr‐Val‐Ser.
Studies on Interactions between Immobilized Lysine Residues and Oligomers of Thymidylic and Deoxyadenylic Acids
Authors: SCHOTT Herbert , ECKSTEIN Heiner
Published: March 2005

Two groups of crosslinked polyacrylic gels with immobilized lysine and lysine peptides (Lys)5 and (Lys)5‐Pro have been used as models for the chromatographic investigation of lysine‐peptide–oligonucleotide interactions. One group carries carboxylic groups in addition to the peptide residues in the gel matrix; the other gel type contains no such carboxylic groups in the gel matrix.

Nucleotides of the series (dT)2–5, p(dT)1–4, p(dT)1–4p, (dA)2–5, p(dA)1–5 and p(dA)1–4p were chromatographed on these gels under various conditions in an aqueous buffer. On the gels of the first group the nucleotides were retarded only slighly, the positive charges of the ɛ‐amino groups being compensated partially or totally by the negatively charged carboxyl groups of the polymer matrix. On the gels of the second group, however, the oligonucleotides underwent specific interactions. These interactions were based primarily upon electrostatic forces between the positively charged ɛ‐amino groups of the immobilized peptides and the negatively charged phosphate groups of the oligonucleotides.

Our results indicate that, in addition to the electrostatic interaction, the conformation plays a crucial role. We explain the selectivity of the interaction with a conformation‐fit mechanism. The origin of this mechanism, which creates specific interactions from unspecific forces, is discussed.
Phosphoenolpyruvate Carboxykinase in Cultured Foetal Hepatocytes from the Rat
Authors: STEELE John G. , MCGRATH Malcolm C. , YEOH George C. T. , OLIVER Ivan T.
Published: March 2005

The involvement of glucocorticoids and insulin in the induction of cytosolic Ppyruvate carboxykinase was studied in cultured hepatocytes derived from 19‐day foetal rat liver. Dexamethasone did not increase Ppyruvate carboxykinase activity, but had a synergistic effect on the induction of the enzyme by N6,O2′‐dibutyryladenosine 3′,5′‐monophosphate (Bt2cAMP). Insulin partially inhibited the induction of Ppyruvate carboxykinase by glucagon, epinephrine or Bt2cAMP. The inhibitory effect of insulin was dose‐dependent: significant inhibition was obtained with 0.1 nM insulin. However, the effect of insulin was independent of the dose of glucagon or epinephrine added to the culture medium.

The ontogeny of inducibility of Ppyruvate carboxykinase by Bt2cAMP or hormones was tested in foetal hepatocytes after one day of culture. Ppyruvate carboxykinase activity could be induced by epinephrine, glucagon or Bt2cAMP in hepatocytes isolated from day‐19, day‐16 or day‐14 foetuses. Similarly, hepatocytes isolated from day‐12 foetal liver were competent to respond to the hormones and Bt2AMP by the accumulation of Ppyruvate carboxykinase. Binding proteins for adenosine 3′,5′‐monophosphate (cyclic AMP) were demonstrated in the cytosol from hepatocytes isolated from day‐14 and day‐19 foetal liver.

The results are discussed in terms of the acquisition during hepatocyte differentiation of the competence to respond to hormonal signals, and the role of hormones in the induction of hepatic Ppyruvate carboxykinase at birth.
Glutathione Metabolism in Relation to the Amino‐Acid Permeation Systems of the Yeast Saccharomyces cerevisiae
Authors: PENNINCKX Michel , JASPERS Charles , WIAME Jean‐Marie
Published: March 2005

A careful enzyme specificity analysis has revealed the presence of a typical γ‐glutamyltranspeptidase in the yeast Saccharomyces cerevisiae. The enzyme cellular level is low in the presence of NH+4 as a sole nitrogen source and rises when individual amino acids are used as nitrogen sources. The γ‐glutamyltranspeptidase appears to be repressed by NH+4 and escapes to the regulatory circuits under the control of glutamine and the glutamate‐dehydrogenase · NH+4 complex. The transpeptidase cellular level is unaffected in mutants which have lost the general amino acid permease and specific systems for l‐arginine and l‐lysine. In contrast, a low enzyme level is observed when growing an apf mutant on urea; this mutant is most probably affected in a common element shared by all the amino acid permeation systems. Urea appears to be a nitrogen source which promotes a high transpeptidase level in the wild‐type strain.

The reported data are discussed in the light of the current theories about the intervention of glutathione metabolism in the translocation of amino acids.
Subcytoplasmic Distribution of Thyroglobulin mRNA in Normal Sheep Thyroid
Authors: CHABAUD Odile , CHEBATH Judith , JACQUET Michel
Published: March 2005

The thyroglobulin 33‐S mRNA was isolated from sheep thyroid total polysomes. The 33‐S RNA, twice purified on a 1% sodium dodecylsulfate/sucrose gradient, was 30‐fold enriched in thyroglobulin messenger activity and was estimated as 50% pure by its messenger activity and 80% pure by the electrophoretic profile. It was used as template for complementary DNA synthesis and hybridized up to 85% of the DNA copy with pseudo‐first‐order kinetics. Back‐hybridization kinetics showed that the purified mRNA corresponds to a major kinetic component with a base sequence complexity of 10000 nucleotides as determined by comparison to globin mRNA. Cross‐reactivity of [3H]cDNA with liver RNA is less than 10%. Restriction endonuclease digestion of [3H]cDNA yielded a discrete band pattern.

The distribution of thyroglobulin mRNA among free polysomes, membrane‐bound polysomes and extrapolysomal pools was analyzed using hybridization to the specific [3H]cDNA probe. Free particles were recovered in the supernatant and membrane‐bound particles in the pellet after a brief centrifugation of detergent‐free homogenate (5 min at 27000 ×g: procedure A; 12 min at 130000 ×g: procedure B) with precautions taken to avoid cross‐contamination. Using procedure A, 80% of thyroglobulin mRNA sequences were found in the membrane‐bound fraction. Using procedure B, where contamination of free particles by membrane‐bound particles was avoided by high‐speed initial centrifugation and further isolation through a discontinuous sucrose gradient, 95–98% of thyroglobulin mRNA sequences were recovered in membrane‐bound polysomes. In total polysomes, 89% of thyroglobulin mRNA sequences were in the polysomal area and shifted to ribosomal subunits after EDTA treatment.
Identification of the Blood‐Group ABH‐Active Glycoprotein Components of Human Erythrocyte Membrane
Authors: FINNE Jukka
Published: March 2005

The blood‐group ABH‐active components of the erythrocyte membrane were identified by incubation of polyacrylamide gels after electrophoresis with radio‐iodinated blood‐group‐specific lectins. The anti‐A and anti‐B lectin of Bandeiraea simplicifolia bound to components from A1, AB and B erythrocytes but not A2 or O erythrocytes, whereas the opposite was observed for the anti‐H lectin of Lotus tetragonolobus. Both lectins revealed as major blood‐group‐active components band 3 and the region of band 4.5, which was resolved into at least two components. Smaller amounts of the lectins were bound to the low‐molecular‐weight region and the dye front, which contained the poly(glycosyl)ceramides. In addition to these components, the B. simplicifolia lectin also bound to the sialoglycoprotein bands 1 and 2 (stained with periodic acid/Schiff). The distribution of bound radioactivity, as determined with this lectin was: Band 3, 22–25%; HIO4/Schiff band‐1, 10–15%; band 4.5, 34–37%; HIO4/Schiff band‐2, 12–15%; and the poly(glycosyl)ceramide region, 14–17%. The different lectin‐binding properties indicate a structural difference between the antigenic determinants of the sialoglycoproteins and of the other blood‐group‐active components. It is suggested that the antigenic determinants in the sialoglycoproteins are of the alkali‐labile type previously characterized, and that the carbohydrate chains of bands 3 and 4.5 correspond to the previously isolated alkali‐stable poly(glycosyl)peptides. The detection of two different groups of glycoproteins could thus explain the controversy concerning the nature of the blood‐group ABH antigens in glycoproteins of the erythrocyte membrane.
The Role of Calcium and Guanosine 3′: 5′‐Monophosphate in the Action of Acetylcholine on Thyroid Metabolism
Authors: DECOSTER Christine , MOCKEL Jean , VAN SANDE Jacqueline , UNGER Jacques , DUMONT Jacques E.
Published: March 2005

The role of calcium and guanosine 3′: 5′‐monophosphate (cyclic GMP) in the regulation of thyroid metabolism has been investigated in dog thyroid slices. Carbamoylcholine enhanced glucose carbon‐1 oxidation, protein iodination, cyclic GMP accumulation and decreased thyrotropin‐induced adenosine 3′:5′‐monophosphate (cyclic AMP) accumulation and iodine secretion; it did not affect protein synthesis. The effects of carbamoylcholine were reproduced under various experimental conditions by supplementary calcium in the medium, ouabain, and in media in which Na+ had been replaced by choline chloride. They were inhibited by lanthanum. These results further support the hypothesis that free intracellular Ca2+ is the intracellular signal for carbamoylcholine effects and suggest that a Na+‐gradient‐driven Ca2+ extrusion mechanism operates in the thyroid cell. Mn2+ reproduced the effect of Ca2+ on glucose oxidation, protein iodination and cyclic GMP accumulation in Ca2+‐depleted slices and medium, and thus mimicked some intracellular effects of Ca2+. On the other hand Mn2+ inhibited the carbamoylcholine effect on thyrotropin‐induced thyroid secretion and cyclic AMP accumulation, and Ca2+ inhibited the Mn2+‐induced cyclic GMP accumulation. This suggests that the two ions compete for the same channel. Similarly Mn2+ inhibited calcium effects in the presence of ionophore A23187. Procaine inhibited protein iodination under all conditions suggesting a primary effect; it also inhibited all carbamoylcholine and ouabain actions. However the drug did not inhibit the effects of choline chloride and its action was reversed by raising carbamoylcholine but not Ca2+ concentration; it is therefore doubtful that procaine acts by blocking Ca2+ channels. In media without added Ca2+, Mn2+ increased cyclic GMP accumulation but did not decrease thyrotropin‐induced cyclic AMP accumulation or iodine secretion, which suggests that cyclic GMP cannot be the sole mediator of the latter two effects of carbamoylcholine.
The Peroxidatic Reaction Catalyzed by Horse Liver Alcohol Dehydrogenase
Authors: FAVILLA Roberto , MAZZINI Alberto , FAVA Adriano , CAVATORTA Paolo
Published: March 2005

The main results obtained during the investigation of the peroxidatic reaction between NAD+ and H2O2, catalyzed by horse liver alcohol dehydrogenase, are here presented.

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the reaction can be followed spectrophotometrically around 300 nm. It consists of a biphasic pattern: the faster step reflects the enzyme‐catalyzed formation of a NAD+ derivative, here called ‘compound I’ (ɛ300= 15000 M−1 cm−1), from the reagents NAD+ and H2O2. The slower step is enzyme independent and consists of at least two distinct processes, each of them leading independently to the disappearance of the intermediate compound I. These two processes are competitive and conditions can be selected such that either one or the other occurs preferentially: one of them leads to the formation of NADX (ɛ300= 23000 M−1 cm−1), the final stable product of the overall reaction, which can be recovered quantitatively only if catalase is added soon after the end of the faster enzymatic step. The second process of the slow step is due to further reaction of compound I with excess H2O2 and the final product Y can be recovered almost quantitatively only if catalase is not added after the fast step.

A careful re‐examination of the stoichiometry of the reaction unequivocally indicated that one molecule of NAD+ is converted to compound I per molecule of H2O2 reacted at the enzyme active site during the fast step.

One proton has been found to be released per mole of NAD+ converted to compound I during the enzymatic fast step. This very significant observation allowed us to deduce the complete stoichiometric balance of the overall reaction.

An initial activation of the peroxidatic activity of horse liver alcohol dehydrogenase, followed by a slow progressive decrease of the same activity, was observed when the enzyme was preincubated with 1 mM EDTA. This behaviour has been interpreted as being due to the essential role played by the active‐site zinc ion in catalysing the peroxidatic reaction.

The peroxidatic reaction has been found to be catalyzed also by yeast alcohol dehydrogenase, thus suggesting a possible physiological meaning to this general ability of alcohol dehydrogenases.
The Structure of Sea‐Urchin‐Sperm Histone φ1 (H1) in Chromatin and in Free Solution
Authors: PUIGDOMENECH Pere , PALAU Jaume , CRANE‐ROBINSON Colyn
Published: March 2005

The lysine‐rich H1‐type histone φ1 from the sperm of the sea urchin Arbacia lixula has been subjected to tryptic digestion in free solution at high ionic strength. Tw trypsin‐resistant peptides Gφ1 and LGφ1 have been isolated, comprising 81 and 93 amino acids respectively, i.e. about a third of the intact histone. Circular dichroism and 270‐MHz proton NMR have been used to demonstrate that the smaller peptide Gφ1 contains all the secondary and tertiary structure of intact histone φ1. It is concluded that the resistant peptides represent a compact folded portion of the histone φ1 chain, whilst the remaining two‐thirds is disordered in free solution. Trypsin digestion of Arbacia lixula nuclei, under conditions where histone φ1 remains tightly bound to the chromatin, leads to the same resistant peptide Gφ1. From this it is concluded that in chromatin the chain region corresponding to Gφ1 is likewise folded and inaccessible to trypsin, whilst the remaining exposed residues are located periferally and probably in an extended conformation. The rather unusual H1‐type histone φ1 from sea urchin sperm thus has a three‐domain structure like calf thymus H1 and chicken erythrocyte H5. The present data emphasise the fact that this three‐domain structure exists in chromatin and is not merely a free‐solution artefact.
Interpretation of the Mössbauer Spectra of the High‐Potential Iron Protein from Chromatium
Authors: MIDDLETON Peter , DICKSON Dominic P. E. , JOHNSON Charles E. , RUSH James D.
Published: March 2005

The Mössbauer spectra of both reduced and oxidized high‐potential iron protein (Hipip) from Chromatium have been analysed using computer fits to theoretical spectra derived from a spin Hamiltonian. Fits to spectra obtained over a range of temperatures between 4.2 and 195 K and in applied magnetic fields up to 10.0 T lead to a consistent set of hyperfine parameters.

These results are interpreted in terms of a model of the four‐iron four‐sulphide active centre which is consistent with its electronic and magnetic properties in both redox states. In the model for the reduced centre all four iron atoms have essentially the same valence, intermediate between ferric and ferrous, with the spins being coupled antiferromagnetically to give the centre zero net spin. The oxidized centre has one less electron which at low temperatures appears to have come predominantly from one pair of iron atoms which thus become ferric with the other pair remaining substantially unchanged. It is clear from the Mössbauer hyperfine parameters obtained from the computer fits to the low‐temperature spectra that a larger magnetic moment is associated with the ferric/ferrous pair of iron atoms than with the ferric pair of iron atoms. This also explains the g values with an average of greater than 2 which are observed in electron paramagnetic resonance (EPR) measurements. At higher temperatures the differences between the electron charge density at the different iron atoms in the oxidized centre appear to become smeared out.
Characterization of Horse Thyroid Cyclic Nucleotide Phosphodiesterases
Authors: ERNEUX Christophe , COUCHIE Dominique , DUMONT Jacques E.
Published: March 2005

Cyclic nucleotide phosphodiesterases have been characterized in crude soluble extract (100000 ×g supernatant) of horse thyroid. DEAE‐cellulose ion‐exchange chromatography at pH 7.4 followed by ultracentrifugation on sucrose‐density gradients separated the phosphodiesterases into three enzymatic activities: (a) a phosphodiesterase hydrolyzing both cyclic GMP (guanosine 3′,5′‐mono‐phosphate) and cyclic AMP (adenosine 3′,5′‐monophosphate), more efficient for cyclic GMP at micromolar levels, activated by the calcium‐dependent regulator or calmodulin; it is called the calmodulin phosphodiesterase; (b) a phosphodiesterase hydrolyzing cyclic GMP more efficiently than cyclic AMP, presenting positive cooperativity for both substrates and cyclic GMP activation of cyclic AMP hydrolysis; it is called the cyclic‐GMP‐activated phosphodiesterase; (c) a cyclic‐AMP‐specific phosphodiesterase not activated by cyclic GMP or calmodulin. Ultracentrifugation of crude horse thyroid preparation (100000 ×g supernatant) on linear sucrose density gradients (5 to 20%) resolved two peaks of activity. The first one (3.6 S) was cyclic‐AMP‐specific. The second one (6.6 S) hydrolyzed both substrates but was 2.5‐times more active against cyclic GMP than against cyclic AMP. Ultracentrifugation of separated DEAE‐cellulose peaks of activity purified the enzymes from contaminating activities: (a) a high molecular weight form (6.6 S) was specifically activated by calmodulin and was separated from the cyclic AMP enzyme (3.6 S); (b) the cyclic‐GMP‐activated phosphodiesterase prepared by anion‐exchange chromatography at pH 7.4 or 6 was still contaminated by the cyclic AMP enzyme (3.6 S) as shown by ultracentrifugation or gel filtration. After separation of the latter enzyme, cyclic GMP specifically activated one single non‐specific enzyme that sedimented at 6.2 S and whose stimulation factor was increased. The data are consistent with the presence in the thyroid of a mixture of three different phosphodiesterases; fractions containing two or three of these enzymes exhibited the properties of the sum of these activities; the enzymes were not interconvertible.
Polystyrene‐Adsorbed Gangliosides for Investigation of the Structure of the Tetanus‐Toxin Receptor
Authors: HOLMGREN Jan , ELWING Hans , FREDMAN Pam , SVENNERHOLM Lars
Published: March 2005

This paper describes the elaboration, using cholera toxin and GM ganglioside as a model system, of versatile, sensitive methods allowing simple determinations of the specific binding of protein ligands to polystyrene‐adsorbed gangliosides, and, secondly, the application of these methods to the tentative identification of the receptor structure for tetanus toxin.

The adsorption characteristics and the ligand‐binding properties of the adsorbed gangliosides were determined, and the specificity and sensitivity of the various ligand‐detection methods (radio‐isotope; enzyme‐linked antibody or water‐vapour condensation) defined. The adsorption did not vary with the type of ganglioside and was found to be linearly proportional to the square root of the incubation concentration and to the log of the incubation time. No difference in adsorption was seen between temperatures of 4 to 37 deg;C. As analyzed with cholera toxin, the polystyrene‐adsorbed ganglioside was structurally specific in binding; i.e. only GM1 was active while after hydrolysis by Vibrio cholerae sialidase also GD1a, GD1b and GT1b, for example, had the same activity as GM1. The affinity of cholera toxin for polystyrene‐adsorbed GM1 was similar to that for viable cells (Ka= 1.2 nM−1).

Tetanus toxin was found to bind specifically to gangliosides of the G1b series (GD1b, GT1b and GQ1b) and though with less affinity, also to GT1a, GM1, GD1a and GD3. The binding affinity of tetanus toxin for the G1b gangliosides was less than that of cholera toxin for GM1. The studies showed that tetanus toxin has specific binding affinity for tetraose‐gangliosides with a disialosyl group linked to the inner galactose. It is conceivable that the natural binding structure for the tetanus toxin has the end sequence Galβ1 → 3Ga1NAcβ1 → 4Ga1(2← 8αNeuAc2 ← 3αNeuAc) which occurs in the ganglioside G1b series and possibly also in the oligosaccharide chains of certain cell membrane glycoproteins.