bluuebunny/crossref_metadata_2025 huggingface dataset:
13C Nuclear Magnetic Resonance and Circular Dichroism Studies of the Tuftsin Conformation in Water
Authors: SIEMION Ignacy Z. , LISOWSKI Marek , KONOPIŃSKA Danuta , NAWROCKA Eleonora
Published: March 2005

13C‐NMR and circular dichroic (CD) spectra of tuftsin and its analogue, are discussed in connection with our hypothesis that the β‐turn is the biologically active conformation of tuftsin. The changes in CD spectra evoked by an increase in pH are interpreted as a demonstration of the increasing amount of β‐turn conformers in solution. Configurational changes in successive residues of tuftsin showed that residues 2 and 3 of the peptide chain are important for the tuftsiri conformation.
Complex Reaction Pathway of Aryl β‐Xyloside Degradation by β‐Xylanase of Cryptococcus albidus
Authors: BIELY Peter , VRŠANSKÁ Maria , KRÁTKÝA Zdeněk
Published: March 2005

The extracellular endo‐1,4‐β‐xylanc se of the yeast Cryptococcus albidus catalyzes degradation of aryl β‐xylosides by other reactions than simple hydrolytic cleavage. Liberation of phenol or β‐nitrophenol from the corresponding β‐xylosides is accompanied by formation of xylose oligosaccharides and only small amounts of xylose. With the aid of phenyl β‐[U‐14C]xyloside synthesized from [U‐14C]xylose, it was established that the reaction followed a complex pattern with the rate of phenyl β‐xyloside digestion and appearance of various products varying markedly with time. The reaction involves multiple transglycosylic reaction leading first to phenyl glycosides of xylooligosaccharides, which are subsequently hydrolyzed mainly to xylobiose and xylotriose.

At concentrations of phenyl β‐xyloside lower than 100 mM the reaction exhibited a significant lag phase, which was followed by period during which the rate of the degradation of the substrate could be determined. The rate showed a strong sigmoidal dependence on phcnyl‐β‐xyloside concentration. The lag phase could be eliminated and the initial rate accelerated by addition of xylose oligosaccharides, which are hydrolyzed by β‐xylanase. After disappearance of the added oligosaccharides, the reaction transitioNatly ceased and then resumed again at a rate comparable to the control without added oligosaccharides. It is proposed that β‐xylanase utilizes for degradation of phenyl β‐xyloside two reaction pathways differing in the nature of glycosyl donors.
p‐Toluenesulfonyl Chloride as an Activating Agent of Agarose for the Preparation of Immobilized Affinity Ligands and Proteins
Authors: NILSSON Kurt , MOSBACH Klaus
Published: March 2005

A number of biomolecules were coupled covalently by nucleophilic displacement to agarose preparations substituted with tosyl groups. In one series of experiments N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate and N6‐(6‐aminohexyl)adenosine 2′,5′‐bisphosphatewere bound by their terminal amino groups to the polysaccharide support. It could be shown that from a mixture of lactate and 6‐phosphogluconate dehydrogenase the immobilized monophosphate showed bio‐affinity only for NAD+‐dependent lactate dehydrogenase, whereas the immobilized bisphosphate showed affinity only for the NADP+‐dependent 6‐phosphogluconate dehydrogenase. Furthermore, the immobilized monophosphate (5 μmol/g wet gel) was applied for the single‐step purification of lactate dehydrogenase from crude beef heart extract.

To demonstrate the immobilization of proteins, soybean trypsin inhibitor (75 mg/g dry support) was immobilized to tosylated agarose, tested as affinity chromatography material and shown to bind 60 mg trypsin/g dry gel. Horseradish peroxidase and horse liver alcohol dehydrogenase were used as model enzymes. Although no optimization had been attempted, the former (approximately 70 mg/g dry support) had a coupling yield of approximately 18% with a specific activity (relative to soluble enzyme) of approximately 10%, whereas approximately 60% of alcohol dehydrogenase was coupled (approximately 100 mg/g dry support) with a specific activity of approximately 25%.
A Novel Chiral Microenvironmental Probe at the Active Site of Trypsin
Authors: NAKAYAMA Hitoshi , TANIZAWA Kazutaka , KANAOKA Yuichi , WITKOP Bernhard
Published: March 2005

p‐Amidinophenyl esters of an enantiomeric pair of N‐(2,4‐dinitrophenyl)alanine (N2Ph‐Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the d‐isomer are 1/2.5 of those for the l‐isomer. Slow rates of deacylation of the two substrates made it possible to prepare the pair of enantiomeric acyl‐trypsins. Circular dichroic (CD) spectra of the purified acyl‐trypsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p‐amidinophenol was added, not only was the deacylation rate of N2Ph‐dAla‐trypsin noticeably increased, but also the transient CD spectrum of the enzyme derivative changed markedly in comparison with that of the l‐derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments.
The Subunit Positions within RNA Polymerase Holoenzyme Determined by Triangulation of Centre‐to‐Centre Distances
Authors: STÖCKEL Peter , MAY Roland , STRELL Irmtraud , CEJKA Zdenka , HOPPE Walter , HEUMANN Hermann , ZILLIG Wolfram , CRESPI Henry L.
Published: March 2005

The complete ‘centre‐of‐subunit structure’ of the multisubunit enzyme DNA‐dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre‐to‐centre distances d between the core subunits α2, β and β′ presented in the preceding paper, the values of d between initiation factor σ and α2 (8.4 ± 1.6 nm), β (4.4 ± 2.2 nm) and β′ (10.7 ± 1.5 nm) were derived from R of σ (4.1 ± 0.3 nm) in situ and of the pairs α2‐σ (6.1 ± 0.4 nm), β‐σ (5.6 ± 0.3 nm) and β‐σ (7.5 ± 0.4 nm) within the holoenzyme (α2ββ′σ).

The structural parameters of the subunits within their molecular complex are accessible for neutron small‐angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of ‘hydrogenated’ molecular parts and extended exposure times because of weak scattering effects. The overall shape of σ bound to core enzyme (α2ββ′) proved to be identical (within experimental resolution) with σ in the isolated state measured recently by X‐ray small‐angle scattering. The refined shape of isolated σ was reduced to an ellipsoid which was orientated with respect to the core structure (α2‐β‐β′) in a ‘space‐filling’ way around the position of the a centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three‐dimensional model.
Receptor‐Specific Large‐Scale Purification of Cholera Toxin on Silica Beads Derivatized with LysoGM1 Ganglioside
Authors: TAYOT Jean‐Louis , HOLMGREN Jan , SVENNERHOLM Lars , LINDBLAD Marianne , TARDY Michel
Published: March 2005

A receptor‐specific affinity chromatographic method for large‐scale purification of cholera toxin is described. The receptor ganglioside for cholera toxin, GM1, is hydrolysed to lysoGM1 which is then covalently coupled, via stabilized Schiff's bases, to porous silica beads (Spherosil) onto which a layer of DEAE‐dextran has been adsorbed and cross‐linked before coupling. Columns of these Spherosil‐DEAE‐dextran‐lysoGM1 beads, in contrast to particles derivatized with lysoGA1, bound the cholera toxin of Vibrio cholerae culture filtrates, after which the toxin could be eluted with the aid of an acid citrate buffer (pH 2.8).

The toxin‐binding capacity was directly proportional to the amount of lysoGM1 in the column: 2.3 mg/μmol lysoGM1. The yield of purified toxin after acid elution and pH neutralization was essentially quantitative (83–107%).

The affinity‐purified toxin contained less than 5% impurities, but consisted of a mixture of predominantly intact holotoxin and B subunit protomer which could readily be separated by gel filtration on Sephadex G‐100.

Scaling up of the technique was possible: a 1 kg column enabled us to treat 1000‐l cultures of V. cholerae and thus to isolate 20 g of cholera toxin per cycle.
Blood‐Group A and B Determinants are Located in Different Polyglycosyl Peptides Isolated from Human Erythrocytes of Blood‐Group AB
Authors: VIITALA Juha , KARHI Kimmo K. , GAHMBERG Carl G. , FINNE Jukka , JÄRNEFELT Johan , MYLLYLÄ Gunnar , KRUSIUS Tom
Published: March 2005

The distribution of blood‐group A and B determinants was studied by isolating blood‐group ABH‐active polyglycosyl peptides from delipidated human blood‐group AB erythrocyte membranes after extensive digestion with pronase followed by chromatography on Bandeiraea simplicifolia I (BsI) lectin coupled to Sepharose. 20% of the polyglycosyl peptides were bound to Bs I lectin. The glycopeptides bound were further fractionated using the blood‐group‐A‐specific lectin from Vicia cracca (Vc). Approximately half of these were bound to the Vc lectin. The glycopeptides, which were bound to the Vc column, were not bound to the blood‐group‐B‐specific isolectin from B. simplicifolia (BsIB4) whereas the Vc‐unbound glycopeptides readily bound. The results indicate that in the polyglycosyl peptides isolated from AB erythrocytes A and B determinants are located in different carbohydrate chains.

The polyglycosyl peptides, which did not bind to BsI lectin, were composed on the average of 30 monosaccharide units and those that bound contained on the average 55 monosaccharide units. The sugar composition was similar in both fractions except that N‐acetylgalactosamine was found only in the BsI‐bound glycopeptides. The substitution patterns of the monosaccharides were quite similar in both fractions except 2,3‐O‐linked galactose, which was enriched 7.5‐fold in the BsI‐bound glycopeptides and 3,6‐O‐linked galactose, which was also enriched in the BsI‐bound glycopeptides suggesting that these have a more branched structure than the BsI‐unbound glycopeptides.

Glycopeptides derived from bands 3 and 4.5 were prepared from A1B‐blood‐group erythrocyte membranes and fractionated as above. 25% of the glycopeptides were bound to BsI‐lectin from both samples. 70% of the BsI‐bound material from band 3 was bound to Vc lectin and 60% from band 4.5. The results indicate heterogeneity in the glycosylation of these bands.
Induction of Metallothionein in HeLa Cells by Dexamethasone and Zinc
Authors: KARIN Michael , HERSCHMAN Harvey R.
Published: March 2005

Metallothioneins are induced by both Zn2+ and dexamethasone in HeLa cells grown in serum‐free medium. Dexamethasone is able to induce metallothionein synthesis in HeLa cells in virtually zincfree medium ([Zn2+] = 0.1 μM). The presence of dexamethasone does not shift the dose/response curve for metallothionein induction by Zn2+, further indicating that the two inducers work through independent mechanisms.

Dexamethasone stimulates Zn2+ uptake 1.7‐fold over 24 h. However, there is no increase in Zn2+ uptake during the first 4 h. In contrast, metallothionein synthesis in response to either Zn2+ or dexamethasone is clearly observable within 4 h of exposure to either inducer. The increased intracellular 65Zn2+ content observed at 24 h is completely accounted for by the increased level of metal bound to metallothionein.

In a continuous labeling experiment the rate of synthesis of metallothionein reached a steady state after about 4 h, in response to either inducer. The lag period was identical for both dexamethasone and Zn2+, with similarly shaped induction curves. Induction by dexamethasone, but not by Zn2+, was inhibited by progesterone. Zn2+ and dexamethasone appear to induce metallothionein synthesis in HeLa cells by mechanisms independent of one another.
Relationship between the Rate of Citrulline Synthesis and Bulk Changes in the Intramitochondrial ATP/ADP Ratio in Rat‐Liver Mitochondria
Authors: WANDERS Ron J. A. , VAN WOERKOM George M. , NOOTEBOOM Ron F. , MEIJER Alfred J. , TAGER Joseph M.
Published: March 2005

The relationship between intramitochondrial and extramitochondrial ATP‐utilizing systems and the intramitochondrial ATP/ADP ratio was studied in isolated rat‐liver mitochondria. Citrulline synthesis was used as an intramitochondrial ATP‐utilizing system, and glucose‐6‐phosphate synthesis as an extramitochondrial ATP‐utilizing system. The intramitochondrial ATP/ADP ratio was manipulated in three ways: with succinate and different concentrations of malonate and/or hexokinase; with 2‐oxoglutarate (plus oligomycin) and different concentrations of hexokinase; and with added ATP in uncoupled mitochondria (oligomycin present).

Under all conditions used, citrulline synthesis was strictly correlated with the bulk intramitochondrial ATP/ADP ratio.

The curve relating citrulline synthesis and intramitochondrial ATP/ADP was shifted towards lower ATP/ADP ratios when the activity of carbamoyl‐phosphate synthetase was enhanced by increasing the mitochondrial content of N‐acetylglutamate.

It is concluded that under the experimental conditions used the intramitochondrial adenine nucleotides behave as a homogeneous pool.
Amino Acid Utilisation in Isolated Hepatocytes from Rainbow Trout
Authors: FRENCH Christopher J. , MOMMSEN Thomas P. , HOCHACHKA Peter W.
Published: March 2005

The utilisation (conversion to CO2 and/or glucose) of a series of amino acids by isolated trout hepatocytes was investigated and compared to the utilisation of lactate and palmitate. In fed fish, several amino acids (alanine, serine, asparagine and glycine) and lactate produced CO2 at considerably higher rates than palmitate. During starvation plus exercise, the rate of CO2 production from palmitate increased while that from lactate and most of the amino acids decreased.

Gluconeogenesis from amino acids in fed fish was lower than from lactate. Serine and asparagine were the most effective substrates: alanine gave lower rates of incorporation. During prolonged starvation plus exercise, the rates of gluconeogenesis from amino acids increased twofold and, simultaneously, there was a corresponding increase in phosphoenolpyruvate carboxykinase activity in liver.

It is concluded that several amino acids (dietary or released from muscle protein) are potentially major oxidative substrates in trout. In addition, amino acids appear to have the capability to maintain supplies of glucose during a period of prolonged starvation and exercise. No evidence could be found to support the contention that alanine is the most important glucogenic amino acid.
The Influence of Topology and Glycosylation on the Fate of Heterologous Secretory Proteins Made in Xenopus Oocytes
Authors: COLMAN Alan , LANE Charles D. , CRAIG Roger , BOULTON Anthony , MOHUN Tim , MORSER John
Published: March 2005

Secretory proteins made in Xenopus laevis oocytes under the direction of heterologous messenger RNA are modified, topologically segregated and exported. Thus the oocyte may serve as a useful surrogate secretory system and we have studied some of the factors governing access to the export pathway. Unglycosylated chicken ovalbumin, synthesized and trapped in the cytosol, is not secreted but glycosylated ovalbumin, found sequestered within vesicles, is exported from oocytes. However, ovalbumin, which is transferred across the endoplasmic reticulum in the presence of tunicamycin and which is indistinguishable by immunoprecipitation, by two‐dimensional gel electrophoresis and by concanavalin‐A—Sepharose binding from the cytosolic form, is still secreted. Guinea‐pig milk proteins and human interferon are also exported from tunicamycin‐treated frog cells. These observations demonstrate that access to the endoplasmic reticulum but not glycosylation is a mandatory intermediate step in secretion. and emphasize the advantages of the oocyte as a surrogate system for the study of the later events in the gene expression pathway.
Identification of Different Forms of the Murein‐Bound Lipoprotein Found in Isolated Outer Membranes of Escherichia coli
Authors: WENSINK Jan , WITHOLT Bernard
Published: March 2005

The identification of the free and murein‐bound forms of theEscherichia coli lipoprotein on dodecylsulphate‐polyacrylamide gels was systematically investigated by analyzing the low‐molecular‐weight proteins (Mr < 20000) of both cytoplasmic and outer membranes.

The free form of the lipoprotein was identified on 15% polyacrylamide gels as the fastest migrating component (Mr= 7200–7500) of isolated outer membranes; it could be separated from a small cytoplasmic membrane protein (Mr= 6500) which was probably identical to the dicyclohexylcarbodiimide binding proteolipid of the membrane‐bound ATPase.

Lysozyme treatment of both outer membranes and murein sacculi failed to convert the murein‐bound lipoprotein into a fragment of uniform size; instead the bound form appeared as a series of bands consisting of lipoprotein bound to one, two,… eight murein subunits. The composition of this ladder depended on the method used to isolate outer membranes.

Beside these lipoprotein bands the outer membrane contained two other proteins, III and V; the relation of these proteins to previously described proteins is discussed.
Precursor Proteins Are Intermediates in vivo, in the Synthesis of Two Major Outer Membrane Proteins, the OmpA and OmpF Proteins, of Escherichia coli K12
Authors: CROWLESMITH Ian , GAMON Konrad , HENNING Ulf
Published: March 2005

The OmpA and OmpF proteins are major outer membrane proteins ofEscherichia coli K12. Their precursors, the pro‐OmpA and pro‐OmpF proteins, have been detected in vivo in pulse‐labelling experiments carried out with [35S]methionine at 25°C. When the pulse was at 37°C, however, no precursors were detected. The pulse‐labelled precursors were processed rapidly and quantitatively into mature protein at 25°C. The apparent half‐life of the pro‐OmpF protein was estimated to be 30 s, and the pro‐OmpA protein may be processed even faster. In short pulses (10 s) the precursors of both proteins were the predominant labelled species, indicating that at 25°C processing does not start until chain elongation of the precursor is almost, if not entirely, complete. When French press lysates of cells pulse‐labelled for 10 s were subjected to sucrose gradient centrifugation to separate the inner and outer membranes, both precursors comigrated with the inner membrane.
Appearance of Elongation Factor Tu in the Outer Mernbrane of Sucrose‐Dependent Spectinomycin‐Resistant Mutants ofEscherichia coli
Authors: DOMBOU Munehiko , BHIDE Shobhana V. , MIZUSHIMA Shoji
Published: March 2005

When sucrose‐dependent spectinomycin‐resistant (Sucd‐Spcr) mutants ofEscherichia coli were grown in the absence of sucrose, a new protein appeared in the membrane fraction insoluble in Triton X‐100. The protein had a hydrophobic nature. However, unlike other outer membrane proteins the new protein was extracted with sodium dodecyl sarcosinate. The new protein was found to be identical with elongation factor Tu (EF‐Tu), as judged from the electrophoretic mobility in three different gel systems, coprecipitation with the antiserum against EF‐Tu, the profiles of peptide fragments produced with three different proteases and analyses of N‐terminal and C‐terminal amino acids. This membrane EFTu accounted for 5–10% of total cell EF‐Tu. When spheroplasts were pretreated with trypsin, EF‐Tu in the outer membrane disappeared. Incubation of cytosol EF‐Tu with the outer membrane did not result in the binding of EF‐Tu to the membrane. These results indicate that the appearance of EF‐Tu in the outer membrane is not due to artificial binding during membrane preparation. It is suggested that the ribosomal alteration resulted in dislocation of the cytosol protein into the outer membrane.
Effect of NADH on the pKa of Zinc‐Bound Water in Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , LINDSTRÖM Anders , OLDÉN Bertil , PETTERSSON Gösta
Published: March 2005

Equilibrium constants for coenzyme binding to liver alcohol dehydrogenase have been determined over the pH range 10–12 by pH‐jump stop‐flow techniques. The binding of NADH or NAD+ requires the protonated form of an ionizing group (distinct from zinc‐bound water) with a pKa of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group with a pKa of 11.2. The binding of trans‐N,N‐dimethylaminocinnamaldehyde to the enzyme · NADH complex is prevented by ionization of the latter group.

It is concluded from these results that the pka‐11.2‐dependence of NADH binding most likely derives from ionization of the water molecule bound at the catalytic zinc ion of the enzyme subunit. The pKa, value of 11.2 thus assigned to zinc‐bound water in the enzyme · NADH complex appears to be typical for an aquo ligand in the inner‐sphere ligand field provided by the zinc‐binding amino acid residues in liver alcohol dehydrogenase. This means that the pKa of metal‐bound water in zinc‐containing enzymes can be assumed to correlate primarily with the number of negatively charged protein ligands coordinated by the active‐site zinc ion.
5‐Aminolevulinic Acid Dehydratase
Authors: SEEHRA Jasbir S. , JORDAN Peter M.
Published: March 2005

5‐Aminolevulinic acid dehydratase from bovine liver has been shown to be inactivated by 5‐halolevulinic acids and 3‐halolevulinic acids.

The substrate, 5‐aminolevulinic acid, protects the enzyme from modification by 5‐halolevulinic acids.

Using tritiated chlorolevulinic acids, it was shown that four of the subunits in the octameric enzyme are preferentialiy modified.

The susceptible enzyme group modified is an–SH group of a reactive cysteine at or near the active site.

Oxidized enzyme is not affected by either 5‐chlorolevulinic acid or 3‐chlorolevulinic acid.

Evidence is presented which suggests that 5‐chlorolevulinic acid is acting as an active‐site‐directed reagent.
On the Mechanism of Interaction of N‐Acetylphenylalanyl‐tRNAPhe with Ribosomes of Escherichia coli
Authors: IVANOV Yuri V. , GRAJEVSKAJA Rosa A. , SAMINSKY Eugen M.
Published: March 2005

The tetranucleotide TpΨpCpGp acts as a specific inhibitor of the rate of AcPhe‐tRNAPhe binding in the ribosomal P site. This effect is observed both in the presence and in the absence of poly(U).

In the absence of poly(U) antibiotics tetracycline and puromycin also decrease the rate of AcPhe‐tRNAPhe binding. Some inhibition by tetracycline is observed with poly(U).

All these inhibitors are known to be ligands of the ribosomal A site, and their influence on the P site binding can be most naturally explained by the suggestion that AcPhe‐tRNAPhe enters the ribosome via the A site, forms there an intermediate complex, and spontaneous translocation into the P site follows. In the presence of poly(U) arguments in favour of this hypothesis are much weaker, but the same sequence of events is possible.
Binding of 4‐methylumbelliferyl β‐d‐galactopyranoside to Momordica charantia Lectin
Authors: KHAN M. Islam , MAZUMDER Tripti , PAIN Debkumar , GAUR Namita , SUROLIA Avadhesha
Published: March 2005

The binding of 4‐methylumbelliferyl β‐d‐galactopyranoside (MeUmb‐Galp), to Mormordica charantia lectin was studied by equilibrium dialysis and quenching of ligand fluorescence. The fluorescence of MeUmb‐Galp decreases as a function of solvent polarity. On binding to M. charantia lectin, its fluorescence was nearly 100% quenched, showing that the binding of the glycoside takes place in a hydrophobic environment. The binding of the fluorescent sugar was saccharide‐specific as evidenced by reversal of MeUmb‐Galp fluorescence quenching by lactose. The association constant is independent of the experimental method used and at 25° C the value is (1.96 ± 0.05) × 104 M−1. The number of binding sites as determined by equilibrium dialysis and fluorescence quenching agree very well with each other; n being equal to 1.98 ± 0.02. The ka value for the glycoside was also determined by competition studies employing reversal of fluorescence quenching of MeUmb‐Galp by lactose. The value of Ka obtained for lactose is 1.21 × 104 M−1 at 30° C. The internal consistency of the association constant and number of binding site values at low and high saturation indicates the absence of additional subsite on M. charantia lectin. The thermodynamic parameters do not differ greatly with change in temperature: the values of −ΔH° and −ΔS° are equal to 30 ± 0.63 kJ mol−1 and 21 ± 0.3 J mol−1 K−1 respectively in the range of 15–35° C indicating that the binding of M. charantia lectin to saccharide is exothermic in nature.
On the Activation of Fructose‐1,6‐bisphosphatase of Spinach Chloroplasts and the Regulation of the Calvin Cycle
Authors: PRADEL Jacques , SOULIÉ Jean‐Michel , BUC Jean , MEUNIER Jean‐Claude , RICARD Jacques
Published: March 2005

Activation by light of spinach fructose‐1,6‐bisphosphatase is mimicked by dithiothreitol. This process of activation by dithiothreitol implies the specific reduction of two disulfide bridges and a conformation change of the enzyme that makes eight sulfhydryl groups available. The activated enzyme has an apparent allosteric kinetic behavior with respect to both fructose‐1,6‐bisphosphate and magnesium.
5′‐Terminal Nucleotide Sequence of the Messenger RNA Coding for Bovine Corticotropin/β‐Lipotropin Precursor
Authors: INOUE Akira , NAKAMURA Masahiro , NAKANISHI Shigetada , HIDAKA Soh , MIURA Kin‐ichiro , NUMA Shosaku
Published: March 2005

The complete 5′‐terminal nucleotide sequence of the mRNA coding for the bovine common precursor of corticotropin and β‐lipotropin has been determined. The 5′‐32P‐labelled, 21‐nucleotides‐long, single‐stranded DNA fragment complementary to a portion of the 5′‐noncoding region of the mRNA was prepared from a cDNA clone and elongated by reverse transcriptase reaction with the mRNA as template. The DNA transcript formed was sequenced by the procedure of Maxam and Gilbert, and the resultant sequence was cross‐checked by two‐dimensional electrophoretic analysis of the partial alkaline digest of the 5′‐32P‐labelled mRNA. The 5′‐terminal nucleotide residue was determined by two‐dimensional thin‐layer chromatography of the complete hydrolysis product of the 5′‐32P‐labelled mRNA. The nucleotide sequenced determined, which partially overlaps the known sequence of the cloned cDNA, reveals the complete 5′‐terminal sequence of the mRNA. This, in conjunction with our previous data, defines the complete primary structure of the mRNA. The mRNA is composed of 1098 nucleotides, including an unusually long 5′‐noncoding sequence of 128 nucleotides. The presence of a ‘cap’ structure at the 5′ terminus of the mRNA is suggested. The 5′‐terminal 48 nucleotide residues of the mRNA are extremely purine‐rich, having an A + G content of 83%, whereas all pyrimidine‐rich segments are located downstream from there. Because the 5′‐noncoding region of the mRNA contains three segments of potential secondary structure which partially overlap, it can exist in a number of alternative base‐pairing configurations. However, its interaction with the 3′‐terminal segment of 18‐S rRNA at the site of maximal complementarity would fix the mRNA configuration in such a way as to bring the possible site of ribosome binding near the initiation codon.
Structure de la région heptose du lipopolysaccharide de Escherichia coli K12 CR34
Authors: BLACHE Denis , BRUNETEAU Maud , MICHEL et Georges
Published: March 2005

Structure of the Heptose Region of the Lipopolysaccharide from Escherichia coli K12 CR34

The heptose region of the lipopolysaccharide of Escherichia coli K12 CR34 was studied. The Glucose linked to the heptose II was found to be substituted by a d‐galactose and the linear chain of the core polysaccharide has two (1→3) linked heptoses. The heptose II is substituted by a lateral (1→7) linked heptose III and the heptose I is linked in (1→5) to 2‐deoxy‐d‐manno‐octulosonic acid. The three sugars of the linear chain, heptose I, heptose II and glucose are substituted by phosphate, pyrophosphate or pyrophosphorylethanolamine group linked to C‐4 hydroxyl groups. However, in some polysaccharidic chains one or two substituting groups may be absent. This result may explain the heterogeneity in the length of the core polysaccharidic chains.
Turnover of the Major Polypeptides of 40‐S Monomer Particles
Authors: IVANOVA Emilia , PIRONCHEVA Ganka , DJONDJUROV Lalju
Published: March 2005

The pulse‐chase experiments with Friend erythroleukemia cells designed to reveal the metabolic properties of the protein complex of 40‐S particles showed that the major polypeptides of this complex turn over with half‐lives between 19 h and 206 h. The main conclusion from the experiments is that the complex does not degrade as a single unit. Since the individual polypeptides forming the complex live much longer than hnRNA, and in addition degrade at a different rate, we considered the following two modes of degradation as most likely. (1) The complex might not be subjected to a profound degradation at the end of the processing of associated pre‐mRNA. In this case it should exist as a long‐lived recyclable mosaic of metabolically differing polypeptides whose replacement takes place at a specific rate. (2) Alternatively, the protein complex might be completely degraded at the end of processing, but in a way that liberates free individual polypeptides available for recycling. The further experiments indicate that the 37000‐Mr, 34000‐Mr and 32000‐Mr core proteins in isolated 40‐S particles and in particles associated with a nuclear fraction released from chromatin after micrococcal nuclease digestion degrade at different rates. These experiments suggest the existence of at least a metabolic heterogeneity among the population of nuclear particles carring pre‐mRNA.
Actin Typing on Total Cellular Extracts
Authors: VANDEKERCKHOVE Joël , WEBER Klaus
Published: March 2005

Based on the finding that the amino‐terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein‐chemical procedure for actin typing. The method is performed on non‐radioactively labelled cells and tissues and six actins can be identified unambiguously in warm‐blooded vertebrates. The method is quantitative and gives directly the ratio of the different actins present in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino‐terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic actin present in Schneider L‐2 Drosophila melanogaster cells. Although the method is currently used routinely on 105 cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity.
Variable Ca2+ Transport: Phosphoprotein Ratios in the Early Part of the GTP‐Driven Calcium‐Transport Reaction of the Sarcoplasmic Reticulum
Authors: FASSOLD Elisabeth , VON CHAK Dorothee , HASSELBACH Wilhelm
Published: March 2005

Initial Ca2+ transport and phosphoprotein formation of the sarcoplasmic reticulum‐membrane with GTP were investigated in a comparative study. While saturation of the high‐affinity sites for Ca2+ binding and transporting as well as for GTP binding on the external surface of the membrane resulted in Ca2+ transport and phosphoprotein formation in a molar ratio of 2, the variation of the concentrations of the two reactants yielded ratios between 1.7 and 5.7. The ratios varied with a similar dependence on the concentrations of Ca2+ and GTP, except at 500 μM Ca2+, if the reaction was started ty Ca2+ instead of GTP but the overall rates decreased. 1 mM dl‐propranolol in the preincubation medium selectively inhibited Ca2+ transport but had no effect on initial phosphoprotein formation. These observations indicate that: (a) phosphorylation of one enzyme molecule induces Ca2+ transport by a variable but limited number of neighbouring molecules, (b) not all Ca2+ bound is essential for phosphorylation but can be transported in parallel, (c) Ca2+ bound to low‐affinity sites occupied at 500 pμM Ca2+ in the reaction medium is also transported initially, (d) the accessibility of the high‐affinity Ca2+ binding sites for dl‐propranolol differs, (e) dl‐propranolol interacts with Ca2+ binding and transporting sites only in that conformation of the enzyme that can be phosphorylated by the nucleotide.
Correlated Effluxes of Adenine Nucleotides, Mg2+ and Ca2+ Induced in Rat‐Liver Mitochondria by External Ca2+ and Phosphate
Authors: ZOCCARATO Franco , RUGOLO Michela , SILIPRANDI Dagmar , SILIPRANDI Noris
Published: March 2005

The presence of inorganic phosphate and Ca2+ in the external medium induces a closely parallel efflux of both endogenous adenine nucleotides and Mg2+ from rat liver mitochondria. These effluxes are (a) pH‐dependent and inhibited by uncouplers, respiration inhibitors and external Mg2+; (b) completely prevented by bongkrekate, but stimulated by atractylate.

ATP, ADP or AMP each inhibit the release of Mg2+ promoted by Ca2+ and phosphate: however, in the presence of oligomycin and P1,P5‐di(adenosine‐5′)‐pentaphosphate (an inhibitor of adenylate kinase) only ADP is effective. Also the release of accumulated Ca2+ observed when ∼ 50% Mg2+ is discharged is retarded by bongkrekate and added Mg2+ whereas it is accelerated by atractylate.

All adenine nucleotides have a significant effect in retarding the efflux of accumulated Ca2+ but, in the presence of oligomycin and P1,P5‐di(adenosine‐5′)‐pentaphosphate only ADP is active.

From these results we conclude that effluxes of Mg2+, Ca2+ and adenine nucleotide from rat liver mitochondria induced by external phosphate are interconnected and regulated by external ADP and Mg2+ levels.
The Effects of pH and Redox Potential on the Hydrogen Production Activity of the Hydrogenase from Megasphaera elsdenii
Authors: VAN DIJK Cees , VEEGER Cees
Published: March 2005

The effects of temperature on the ionization constant (pK′) and apparent midpoint potential (EB) of the unprotonated species of Megasphaera elsdenii flavodoxin hydroquinone shows that, above 15° C, ΔpK′· K−1=− 9.7 × 10−3 and ΔEB= K−1=− 0.6 mV. The effects of pH and redox potential on the hydrogen production activity with fixed concentrations of methyl viologen semiquinone (0.3 mM; artificial donor) and M. elsdenii flavodoxin hydroquinone (50 μM; natural donor) show that with decreasing pH the activity increases.

Irrespective of the pH and electron donor, at increasing redox potential, a redox‐potential‐independent production activity is followed by a redox‐potential‐dependent production activity. This redox‐potential‐dependent behaviour of the hydrogen production activity represents an n= 2‐type of redox titration curve with an ‘apparent midpoint potential’ which corresponds with the potential of the hydrogen electrode at that pH. The effect of pH on the manometrically determined hydrogen production activity (direct) is in good agreement with that determined spectrophotometrically (indirect; see preceding paper), with both electron donors tested.

In contrast to predictions from the models for hydrogenase activity [van Dijk et al. (1980) Eur. J. Biochem. 102, 317–330], a double‐reciprocal plot of the kinetic data for M. elsdenii flavodoxin hydroquinone at pH 5.5 is non‐linear. A slightly adapted kinetic model based on a similar mathematical formulation of its rate equation, to explain the effects of redox potential, proton and electron (donor) concentration on the hydrogenase activity is proposed. This model also explains, on it theoretical basis, the effects of pH and redox potential on the hydrogen production activity.

The effect of pH on the hydrogen oxidation activity with methyl viologen and benzyl viologen as electron acceptors shows for both dyes an optimum at pH 9.7. The ratio of the activities with both viologens is constant over the pH range tested.
Binding of Ribosomes to Linear and Circular Forms of the 5′‐Terminal Leader Fragment of Tobacco‐Mosaic‐Virus RNA
Authors: KONARSKA Maria , FILIPOWICZ Witold , DUMDEY Horst , GROSS Hans J.
Published: March 2005

The sequence of the 5′‐terminal leader fragment preceding the AUG codon in the RNA of tobacco mosaic virus (TMV), tomato strain, SPS isolate, has been determined. This RNA, similarly to the RNAs of the U1 and Dahlemense strains of TMV [Kukla et al. (1979) Eur. J. Biochem. 98, 61–66] has the 7‐methylguanosine(5′)triphospho(5′)guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues.

The RNase‐T1‐resistant 73‐nucleotide‐long leader fragment of TMV RNA from the SPS isolate was assayed for its ability to interact with eukaryotic and prokaryotic ribosomes. The linear fragment, labelled either at its 5′ or 3′ end, efficiently formed disome initiation complexes when incubated with wheat‐germ protein‐synthesis extract. In contrast to its linear counterpart, the circular covalently closed RNA leader fragment, obtained in a reaction catalysed by T4 RNA ligase, was unable to interact with wheat germ ribosomes. Both kinds of leader fragment bound equally well to Escherichia coli 70‐S ribosomes.

The results offer further support to the notion that in eukaryotic initiation the free 5′ end (either capped or uncapped) is required for mRNA interaction with ribosomes. Furthermore, they suggest that both ribosomes found in disome initiation complexes with the TMV RNA leader fragment enter the mRNA sequentially via the free 5′ terminus.
Mechanisms for Albumin‐Mediated Membrane Damage
Authors: DRAINAS Denis , HARVEY Elizabeth , LAWRENCE Anthony J. , THOMAS Adedapo
Published: March 2005

Bee venom phospholipase A2 causes a small increase in the sublytic leakiness of rabbit erythrocytes; which is strongly inhibited by lysolecithin and potentiated by albumin.

Albumin extracts long‐chain fatty acids from the erythrocyte membrane by a process which is faster than diffusion‐determined efflux of fatty acid and during this process it greatly increases the sublytic leakiness of the membrane. In contrast, albumin rapidly terminates sublytic responses to lysolecithin.

These actions of albumin are abolished only when albumin is preloaded with four molecules of oleic acid per protein molecule.

A minor proportion of the response of phospholipase‐A2‐treated cells to albumin is due to accumulated fatty acid products; the combination of active enzyme and accumulated fatty acids is much more effective than either alone. Membrane partially depleted of phospholipids by enzyme attack is not highly leaky.

Albumin may, in part, act by removing lysolecitihin, the inhibitory reaction product. A model is proposed in which susceptibility to phospholipase A2 attack is not uniform on the cell surface and is used to explain the inhibitory action of lysolecithin and the high sensitivity of the membrane in the presence of albumin to damage by newly released fatty acids.
A New Procedure for the Simultaneous Large‐Scale Purification of Bacteriophage‐T4‐Induced Polynucleotide Kinase, DNA Ligase, RNA Ligase and DNA Polymerase
Authors: DOLGANOV Gregory M. , CHESTUKHIN Arsenii V. , SHEMYAKIN Michael F.
Published: March 2005

A procedure for simultaneous large‐scale purification of the bacteriophage‐T4‐induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non‐permissive Escherichia coli B‐23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essentially lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.
Large‐Scale Purification and Some Properties of Malate Synthase from Baker's Yeast
Authors: DURCHSCHLAG Helmut , BIEDERMANN Gunthard , EGGERER Hermann
Published: March 2005

Malate synthase from baker's yeast (5 kg) was purified 400–500‐fold to homogeneity. About 50–200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell‐free crude extracts. The enzyme, pl = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies.

Sedimentation and diffusion coefficients were determined: . The molecular weight of the synthase was found to be 175000 ± 10000 and 180000 ± 10000 by sedimentation/diffusion and by high‐speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180000 ± 10000.

The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass.

The binding of acetyl‐coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism.

The intermolecular isotopic effect, kH : k2H= 1.4, was determined with acetyl‐coenzyme A and [2H3]acetylcoenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H,14C]acetyl‐coenzyme A is also presented.
5‐Aminolevulinic Acid Dehydratase
Authors: SEEHRA Jasbir S. , GORE Michael G. , CHAUDHRY Abdul G. , JORDAN Peter M.
Published: March 2005

The thiophilic reagent 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2) reacts with four sulphydryl groups in native 5‐aminolevulinic acid dehydratase from bovine liver (groups I, II, III and IV). All four of these groups exhibit various degrees of half‐site reactivity. Groups I and II are highly reactive and their rates of reaction with Nbs2 have been investigated using stopped‐flow analysis. The reaction of these groups with Nbs2 results in the formation of an intramolecular disulphide bond which may be reduced with dithioerythritol to regenerate the free sulphydryl groups. Groups I and II appear to be at, or near, the catalytic site whereas group III is involved in the maintenance of conformation in the native enzyme.
Segregation of Specific Classes of Messenger RNA into Free and Membrane‐Bound Polysomes
Authors: ADESNIK Milton , MASCHIO Frank
Published: March 2005

Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane bound polysomes contains a diverse set of messengers with a minimum of 500–2000 diferent species although approximately 5–8 messengers may constitute 25–30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA‐bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane‐bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These finding, are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes.
Studies on the Mechanism of Action of the ATP‐Dependent DNAase from Alcaligenes faecalis
Authors: YOUNG Douglas B. , ROSAMOND John
Published: March 2005

An ATP‐dependent DNAase has been purified to homogeneity from extracts of Alcaligenes faecalis, and has been shown to couple the degradation of DNA to the hydrolysis of ATP. Enzyme activity also requires divalent ions, with Mn2+, Mg2+ and Co2+ being effective cofactors for both DNAase and ATPase activities. We have studied the intermediates formed by the enzyme during the degradation of duplex DNA with each of these cofactors using sedimentation velocity, binding to nitrocellulose filters and sensitivity to a nuclease specific for single‐stranded DNA.

With Mn2+ or Co2+, the enzyme acts processively to produce mostly acid‐soluble material and acid‐insoluble single‐strand fragments up to 400‐nucleotides long. However, with Mg2+ present, the enzyme produces intermediates comprising a duplex region with one or more single‐strand tails, while little acid‐soluble oligonucleotide is formed. From these results, we propose a model to describe the mechanism by which the ATP‐dependent DNAase from A. faecalis degrades duplex DNA.
5′‐Methylthioadenosine Nucleosidase
Authors: GURANOWSKI Andrzej B. , CHIANG Peter K. , CANTONI Giulio L.
Published: March 2005

5′‐Methylthioadenosine nucleosidase (EC 3.2.2.9), the enzyme which catalyzes hydrolytic cleavage of 5′‐methylthioadenosine with the formation of adenine and 5′‐methylthioribose, has been purified to homogeneity from Lupinus luteus seeds. The nucleosidase has a native molecular weight of 62000 and consists of two identical subunits, as judged by gel filtration and dodecylsulfate/polyacrylamide gel electrophoresis. The nucleosidase exhibits highest specificity towards the natural substrate with a Km of 4.1 × 10−7 M for 5′‐methylthioadenosine. It does not cleave adenine from S‐adenosylhomocysteine. Among the synthetic analogs of 5′‐methylthioadenosinc tested, eleven compounds appear to be able to substitute as substrates. Furthermore, the enzyme can liberate hypoxanthinine from six inosyl (deaminated) derivatives obtained by enzymatic deamination of 5′‐methylthioadenosine and its synthetic analogs. The Km for 5′‐methylthioinosine is 55 μM, and the maximal velocity about 50‐times lower than for 5′‐methylthioadenosine. The reaction catalyzed by the nueleosidase can be inhibited by adenine (Ki= 11 μM), 3‐deazaadenine (Ki= 19 μM), and 9‐erythro(2‐hydroxyl‐3‐nonyl)adenine (Ki= 37 μM).
A Mechanism of l‐Canaline Toxicity
Authors: ROSENTHAL Gerald A.
Published: March 2005

l‐Canaline, a highly toxic structural analogue of l‐ornithine, reacted with pyridoxal phosphate to form a stable, ninhydrin‐positive complex. NMR analysis revealed the involvement of a Schiff's base in complex formation. Interaction of l‐canaline with l‐tyrosine decarboxylase, a known B6‐containing enzyme, curtailed significantly enzyme‐mediated decarboxylation. Spectral scans provided evidence that the loss in catalytic activity is associated with the reaction of canaline with the pyridoxal phosphate moiety of the enzyme. Thus, the marked antimetabolic properties of canaline are due in part to its ability to form a Schiff's‐base‐containing complex with the B6 moiety of the enzyme.