bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of the Physical State of Lipopolysaccharides in the Interaction with Complement
Authors: GALANOS Chris , LÜDERITZ Otto
Published: March 2005

Lipopolysaccharides interact with complement only when they are present in a state of high aggregation with a high apparent molecular weight. Lipopolysaccharides in uniform salt forms prepared by electrodialysis and neutralization with different bases exhibited distinct differences in their anticomplementary activity which correlated with differences in their sedimentation coefficients.

Conversion of smooth (S) form lipopolysaccharides into the low‐molecular‐weight triethylamine form completely abolished their anti‐complementary activity while conversion into the highmolecular‐weight sodium form increased their activity. In contrast, a similar treatment of highly defective Re and Rd rough (R) form lipopolysaccharides had no effect on their ability to interact with complement. Both the triethylamine and sodium forms were strongly anti‐complementary despite large differences in their molecular weight. This was found to be due to the property of R lipopolysaccharides to reaggregate into a large‐molecular‐weight form through absorption of Mg2+ and Ca2+ present in the guinea pig serum used as complement source. Defective lipopolysaccharides derived from the Ra and Rb classes showed only negligible anti‐complementary activity which did not increase by conversion into salt forms with high molecular weight.
Enzymic Unwinding of DNA
Authors: ABDEL‐MONEM Mahmoud , DÜRWALD Hildegard , HOFFMANN‐BERLING Hartmut
Published: March 2005

The DNA‐stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA · RNA hybrid duplexes and DNA · DNA partial duplexes prepared by polymerization on fd phage single‐stranded DNA template. The enzyme was found to denature these duplexes in an ATP‐dependent reaction, without delectably degrading. EDTA, an inhibitor of the Mg2+ ‐requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme‐DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single‐stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double‐stranded parts separating base‐paired strands by processive, zipper‐like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme.
Calorimetric Study on Human Erythrocyte Glycolysis
Authors: MINAKAMI Shigeki , de VERDIER Carl‐Henric
Published: March 2005

The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization.

The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and monoiodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3‐bisphosphoglycerate.

When inosine, dihydroxyacetone or d‐glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed.

The value for heat production in red cells in the above‐mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway.
Unequivalence between Hemoglobin Subunits
Authors: CASSOLY Robert
Published: March 2005

The effects of inositol hexakisphosphate on the visible absorption spectrum of liganded valency hybrids α2COβ2+H2O, α2+H20β2CO and α2COβ2+CN, α2+CNβ2co (where αCO, α+H2O, α+CN and βCO, β+H2O, β+CN represent the α and β chain of hemoglobin, respectively, in their carbon monoxide, aquomet and eyanomet forms) have been examined in an attempt to determine if their α and β chains are equally sensitive to the action of the anion.

The difference spectra induced by inositol hexakisphosphate, are different for the two chains. The β+H2O subunit contributes chiefly to the absorbance changes, rather than α+H2O. In contrast, it is the αCO chain which is more sensitive to inositol hexakisphosphate than βCO. The question ofwhether such a selective response would also occur in aquomethemoglobin and carboxyhemoglobin is discussed. Criteria of changes in the quaternary structure of hybrids has been studied by measuring the effects of inositol hexakisphosphate on their ultraviolet absorption spectrum.
Co‐operative Ionisation of Aspartic‐Acid‐158 and Histidine‐159 in Papain
Authors: BENDALL M. Robin , LOWE Gordon
Published: March 2005

The chemical shift of the single resonance in the 19F nuclear magnetic resonance spectrum of papain which has been irreversibly inhibited by 3‐bromo‐1,1,1‐trifluoropropanone, exhibits pH‐dependence. The fluorescence intensity of this papain derivative shows pH‐dependence on two groups which exhibit co‐operative ionisation. This co‐operative behaviour is probably a function of the probe since the fluorescence intensity of S‐ethane‐thio‐papain is dependent on a single ionisation constant, whereas that of S‐(2‐hydroxyethane)‐thio‐papain is dependent on two ionisable groups again acting co‐operatively. The 1,1,1‐trifluoroketone probe will be hydrated in aqueous solution and would be capable of hydrogen bonding with the protein. The two groups detected are considered to be aspartic‐acid‐158 and histidine‐159. The co‐operative ionisation of these groups in substrate hydrolysis is discussed.
Isolation and Characterization of Iron‐Binding Proteins from Rat Intestinal Mucosa
Authors: HUEBERS Helmut , HUEBERS Eiko , RUMMEL Walter , CRICHTON Robert R.
Published: March 2005

Two iron‐binding proteins were isolated from rat intestinal mucosa. From determination of their molecular weights, their electrophoretic and iron‐binding properties it was established that one was a mucosal ferritin and the other a mucosal transferrin. The mucosal ferritin is compared in its molecular weight, isoelectric point, amino acid composition and tryptic peptide pattern with the ferritins of rat spleen and liver. All three ferritins are distinctly different from one another. In addition the iron content of mucosal ferritin was found to be much lower than that of liver and spleen ferritins.

Mucosal transferrin was separated into two components by isoelectric focusing, as was plasma transferrin. The plasma and mucosal transferrins differ in their isoelectric points and in their amino acid compositions. Differences were also found in vitro in the iron‐binding of mucosal transferrin as compared with plasma transferrin. The role of these mucosal proteins in the absorption of iron is briefly discussed.
Functional Identity of the Monomeric and Multiple Forms of Elongation‐Factor 1 from Krebs‐II Mouse‐Ascites‐Tumor Cells
Authors: GRASMUK Hans , NOLAN Robert D. , DREWS Jürgen
Published: March 2005

Highly purified 3H‐labelled elongation factor 1 (EF‐1) from Krebs II mouse ascites tumour cells was separated into biologically active monomeric and aggregate forms of the enzyme by either gradient centrifugation or gel filtration. When corrected for their content of inactive enzyme both forms of the factor were found to he equally active whether tested in the binding or synthesis reaction. The only form of the enzyme found bound to ribosomes was the monomer; it was therefore concluded that the aggregate form of the enzyme must first dissociate before it reacts with the ribosome. The stoichiometry of the aminoacyl‐tRNA binding reaction to ribosomes in the presence of.guanosine nucleotides was also studied. It was found that one molecule of aminoacyl‐tRNA and of Guo‐5′‐P2‐CH2‐P is bound per molecule of EF‐1 bound to the ribosome. Following interaction with and release from, the ribosomes, EF‐1 was found to be predominantly monomeric.
The Amino‐Acid Sequences of the α‐Crystallin A Chains of Red Kangaroo and Virginia Opossum
Authors: DE JONG Wilfried W. , TERWINDT Eugénie C.
Published: March 2005

The amino acid sequence of the A chain of the eye lens protein α‐srystallin from the red kangaroo (Macropus rufus) was completely determined by manual Edman degradation of tryptic, thermolytic and cyanogen bromide peptides. The sequence of the α‐crystallin A chain from the Virginia opossum (Didelphis marsupialis) was deduced from amino acid analyses and partial Edman degradation of peptides. The 173‐residue A chains of kangaroo and opossum differ in six positions, whereas comparison with the bovine α‐crystallin A chain reveals 17 and 22 substitutions, respectively. Most substitutions occur in the COOH‐terminal part of the chain.
Possible Phospholipid Precursor for Phosphatidylserine in Rat Heart
Authors: KISS Zoltán
Published: March 2005

The incorporation of 32P‐labelled inorganic phosphate into phospholipids of rat heart slices was investigated in pulse‐chase experiments. The comparison of the specific activities of individual phospholipids suggested that phosphatidic acid can be 32P precursor for phosphatidylserine. Studies with l‐serine revealed the full calcium dependence of phosphatidylserine formation in heart homogenates. Chasing experiments with [1,2‐14C]ethanolamine and [methyl‐14C]choline proved the precursor role of phosphatidylethanolamine and phosphatidylcholine and indicated a base exchange mechanism. Among phospholipid phosphatidic acid was the most potent in stimulating phosphatidylserine formation in homogenates. The conclusion was reached that phosphatidic acid can also be regarded as precursor for phosphatidylserine.
Isolation and Characterization of Rat‐Lens Messenger RNAs
Authors: COHEN Louis H. , SMITS Dimphy P. E. M. , BLOEMENDAL Hans
Published: March 2005

Messenger RNA has been isolated from rat lens tissue. The mRNA species which codes for the A2 chain of α‐crystallin, revealed the same extremely high sedimentation value (14S) as the corresponding messenger from calf lens. However, it has been shown that in rat lens the 14‐S messenger preparation directs the synthesis of an additional α‐crystallin chain, designated as αX with. an. approximate molecular weight of 24000. For comparative purpose crystallin synthesis has also been studied as well in cultured rat lens cells as in the rat lens cell‐free system
Gel Isoelectric Focusing of Human‐Serum Transferrin
Authors: HOVANESSIAN Ara G. , AWDEH Zuhayr L.
Published: March 2005

Analysis of human serum transferrin on gel isoelectric focusing resolved this iron‐transport protein into two iron‐containing components which were identified by the use of radioactively labelled iron and iron‐specific stain. These two components were found to be monoferric transferrin (Fe‐transferrin) and diferric transferrin (Fe2‐transferrin) with isoelectric points of 5.6 and 5.2 respectively. The estimation of the relative proportions of these two components in serum did not correspond to the calculated theoretical ratio based on random binding of iron to the two binding sites of transferrin. However, the analysis of partially resatured apotransferrin gave a ratio corresponding to a random distribution of iron. The significance of these results is discussed.
Chemical Modification of the Tryptophan Residues of Wheat‐Germ Agglutinin
Authors: PRIVAT Jean‐Paul , LOTAN Reuben , BOUCHARD Philippe , SHARON Nathan , MONSIGNY Michel
Published: March 2005

The oxidation of the tryptophan residues of wheat germ agglutinin by N‐bromosuccinimide was investigated under non‐denaturing and denaturing conditions. All three tryptophan residues present in wheat germ agglutinin subunit (molecular weight 18000) could be modified in 0.1 M acetic acid 8 M urea, pH 3.9. One of the residues failed, however, to react with N‐bromosuccinimide when the modifications in 0.1 M citrate buffer, pH 6.0.

Tryptophan fluorescence of the protein was quenched concomitantly with the oxidation of two tryptophan residues even when the modification was carried out in acetic acid urea. After oxidation of two tryptophan residues per subunit of wheat germ agglutinin, only 15% of the original tryptophan fluorescence remained; upon excitation at 280 nm, tyrosine fluorescence entered at 305 rim could be resolved. The results suggest that there are only two emitters in the protein and that the third tryptophan residue is buried in the native protein and can be modified only in acetic acid urea. This tryptophan residue is quenched in the native protein.

Saturation of wheat germ agglutinin with tri‐N‐acctylchitotriose did not protect the tryptophan residues from oxidation by N‐bromosuccinimide. Under these conditions, however, the reactivity of the tryptophan residues towards N‐bromosuccinimide was reduced and a higher concentration of' the reagent was required to achieve the same extent of oxidation as in the absence of the saccharide.

Oxidation of one tryptophan residue per subunit in acetic acid urea led to almost complete loss (97%) of hemagglutinating activity, a 3.5‐fold decrease in the affinity constant for tri‐N‐acetylchitotriose and loss of ability of the subunits (s020.w.= 2.0S) to reassociate to the native dimer (s020.w= 3.5 S) after dialysis against a non‐denaturing buffer.

No significant changes in the circular dichroism spectrum of wheat germ agglutinin were observed after oxidation of the three tryptophan residues, suggesting that no gross conformational changes occurred.

The steric relationships between the fluorescent tryptophan residues of wheat germ agglutinin and saccharides are discussed.
Nanosecond‐Pulse Fluorimetry of Wheat‐Germ Agglutinin (Lectin)
Authors: PRIVAT Jean‐Paul , WAHL Philippe , MONSIGNY Michel , AUCHET Jean‐Claude
Published: March 2005

Nanosecond‐pulse fluorimetry of wheat germ agglutinin is analyzed as a function of both excitation and emission wavelengths.

When excited at 280 nm, wheat germ agglutinin fluorescence exhibited three lifetimes: one corresponding to the tyrosine residues as a whole and two others corresponding to the tryptophyl emission.

The tyrosine contribution to the emission spectrum deduced from this method was in good agreement with that reported previously in steady‐state fluorescence experiments [Privat, J. P. and Monsigny. M. (1975) Eur. J. Biochem. 60, 555–567].

The fluorescence decay of each tryptophan residue was not a single exponential function when wheat germ agglutinin was excited at 295 nm. This could he related to the microenvironment of the indole chromophores in the protein. The comparison of the quantum yield and of average lifetime showed that some tryptophan residues were completely quenched.

Energy transfer from tyrosines to tryptophan residues previously detected in steady‐state fluorescence was also revealed by fluorescence decay measurements. Comparison of both methods showed that an important part of transfers occurred with a very fast rate equal to or greater than 1010s−1.

Both lifetimes and the ratio of the short and the long‐lived component were found dependent on tri‐N‐acetylchitotriose binding.
The Metabolism of O‐Acyl‐N‐acylneuraminic Acids
Authors: CORFIELD Anthony P. , Clarice FERREORA DO AMARAL , WEMBER Margret , SCHAUER Roland
Published: March 2005

The enzymic synthesis of 4‐O‐acetyl‐N‐acetylneuraminic acid, 4‐O‐acetyl‐N‐glycolylneuraminic acid, 4‐O‐glycolyl‐N‐acetylneuraminic acid, 9‐O‐acetyl‐N‐acetylneuraminic acid and 9‐O‐acetyl‐N‐glycolylneuraminic acid is shown using radioactive precursors with surviving slices, membrane fractions or particle‐free homogenates from bovine and equine submandibular glands.

Acetyl‐CoA: N‐acetylneuraminate‐9(or 7)‐O‐acetyltransferase activity was found in a microsome fraction and in the cytosol of bovine submandibular glands. The properties of the membrane‐bound enzyme acting on endogenous, glycoprotein‐bound N‐acetyl‐ and N‐glycolylneuraminic acids were compared with those of the soluble enzyme, O‐acetylating exogenous, non‐glycosidically bound N‐acetyl‐ and N‐glycolylneuraminic acids.

A rapid, radioactive assay for the membrane‐bound enzyme activity is described. The enzyme activity shows an optimum at pH 7 and has a Km for acetyl‐CoA of 0.1 mM. The enzyme is inhibited by p‐chloromercuribenzoate and iodoacetate. Divalent cations, EDTA and glutathione have no influence on its activity while CoA proved to be a competitive inhibitor with a K1 of 0.56 mM.

The soluble enzyme activity, assayed using a radioactive procedure, shows Km values of 0.01 mM, 0.5 mM and 0.39 mM for acetyl‐CoA, N‐acetylneuraminic acid and N‐glycolylneuraminic acid respectively. The general properties are similar to those found for the membrane‐bound enzyme, except that membrane‐bound activity is stable for longer on storage at 4 °C.

Acetyl‐CoA, acyl‐CoA and CoA concentrations of 33 nmol, 65 nmol and 106 nmol/g wet tissue respectively are found in fresh bovine submandibular glands.

The occurrence of the CMP‐glycosides of N‐acetylneuraminic acid, 9‐O‐acetyl‐N‐acetylneuraminic acid and N‐glycolylneuraminic acid in bovine submanibular glands is demonstrated.

The results are discussed in relation to the general metabolism of acylneuraminic acids.
Effects of Lipolytic and Antilipolytic Drugs on Metabolism of Adenosine 3′: 5′‐Monophosphate in Brown Adipose Tissue of Cold Acclimated Rats
Authors: BERTIN Roseline , PORTET René
Published: March 2005

The intracellular level of adenosine 3′:5′‐monophosphate (cyclic AMP) and its stimulation in vitro by norepinephrine were studied in brown and white adipocytes from rats adapted to constant or fluctuating cold. Cold acclimatization had no effect on the basal cyclic AMP intracellular content in both tissues, but the level in brown adipocytes was four‐fold higher than in the white ones. Addition of norepinephrine in the incubation medium doubled the cyclic AMP content of white adipocytes from control or fluctuating‐cold‐adapted rats, and enhanced four‐fold in constant‐cold‐adapted rats. In brown adipocytes norepinephrine increased cyclic AMP levels in the first two groups, but had no effects in constant‐cold‐adapted rats. In the two tissues of control and fluctuating‐cold‐adapted rats the norepinephrine action was increased by phentolamine and decreased by propranolol. The lack of response to norepinephrine of brown adipocytes from constant cold‐adapted rats was not due to the predominance of the α component of hormone receptors. Antilipolytic drugs (nicotinic acid, insulin and prostaglandin E2) inhibited the action of norepinephrine on white adipocytes; only prostaglandin E2 had an effect on brown ones.
Uridine – Cytidine Kinase from Foetal and Adult Rat Liver
Authors: FULCHIGNONI‐LATAUD Marie‐C. , TUILIE Monique , ROUX Jeanne‐M.
Published: March 2005

Partial purification of uridine‐cytidine kinase (EC 2.7.1.48) from foetal rat liver by chromatography on DEAE‐cellulose gives two active fractions. The first in order of elution was identified as a form specific for foetal liver. It was purified 300‐fold. The second fraction was common to foetal, and adult rat liver and spleen and was purified 20‐fold. The foetal fraction, of the enzyme was found to be heat‐sensitive and protected against inactivation by PO3−4 anions. The two isolated forms have different apparent Km for uridine, respectively 410 μM for the foetal form and 52 μM for the adult form.
The Effects of Temperature and Some Inhibitors an the Carnitine Exchange System of Heart Mitochondria
Authors: RAMSAY Rona R. , TUBBS Philip K.
Published: March 2005

11‐Trimethylamino‐undecanoyl‐l‐carnitine (K; = 56 μM) and 6‐trimethylaminohexanoyl‐l‐carnitine (K1, = 1.3 mM) are competitive inhibitors of the ox heart mitochondrial carnitine exchange system. The latter is itself a poor substrate, with a V about 0.7 of that for carnitine. These compounds have been'used as ‘stop inhibitors’ in a study of the effect of temperature an the carnitine exchangc.

The carnitineout↔ H carnitinein exchange has a temperature coefficient (Q10) of 8.5 and an activation energy of 176 kJ/mol which is constant from 0 °‐18 °C. The Km for L‐carnitine (5.3 mM) is also constant over this temperature range.

The slow efflux (leak) of mitochondrial L.‐[14C]carnitine in the absence of external substrate has a similar temperature dependence and is also inhibited by 11‐trimethylamino‐undecanoyl‐L‐carnitine. lt is probably mediated by the same system as the very much Taster exchange with external carnitine
Differentiation in Avian Keratinocytes
Authors: WALKER Ian D. , ROGERS George E.
Published: March 2005

An investigation to determine the number of proteins comprising the keratin of the chick down feather was carried out using anion‐exchange chromatography of the carboxymethylated derivatives of reduced feather proteins, followed by examination of discrete protein fractions by polyacrylamide gel electrophoresis at pH 9.5 and pH 2.7 and by amino acid analysis. Twenty two different proteinswere identified by these techniques and it is likely that at least nineteen are closely related to one another in terms of primary structure. The heterogenteity observed was not the result of incomplete alkylation or of non‐spceific reactions during the carboxymethylation procedure nor did it arise during the procedures used to separate or analyse the carboxymethylated keratin.
Role of DNA · RNA Hybrids in Eukaryotes Purification of Two Ribonucleases H from Yeast Cells
Authors: WYERS Françoise , SENTENAC André , FROMAGEOT Pierre
Published: March 2005

Two ribonucleases H, which specifically degrade RNA in RNA‐DNA hybrid structures, have been extensively purified from extracts of Saccharomyces corevisiae. Ribonuclease H1 can be obtained in high yield by a rapid purification procedure mainly involving ammonium sulfate fractionation and phosphocellulose chromatography. The enzyme preparation was shown to be homogeneous by acrylamide gel electrophoresis with sodium dodecylsulfate. Ribonuclease H2 was also obtained in an essentially homogeneous form by phosphocellulose and Amberlite chromatography. About 60 mg of ribonuclease H1 and 2 mg of ribonuclease H2 were obtained from 100 g of wet cells. Using T7 DNA · RNA, hybrids, the specific activity of ribonuclease H2 was approximately 10‐fold higher than that of ribonuclease H1.
Purification and Characterization of Microsomal and Lysosomal β‐Glucuronidase from Rat Liver by Use of Immunoaffinity Chromatography
Authors: HIMENO Masaru , NISHIMURA Yukio , TSUJI Hiroshi , KATO Keitaro
Published: March 2005

Rat liver β‐glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were pinked about 9500‐fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland β‐glucuronidase to Nepharose 2B and isoelectric focusing.

The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal β‐glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal β‐glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal β‐glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal β‐glucuronidase revealed that the both enzymes had the same or quite similar antigenic determinants.
Serine Transhydroxymethylase from Rabbit Liver
Authors: BOSSA Francesco , BARRA Donatella , MARTINI Filippo , SCHIRCH La Verne , FASELLA Paolo
Published: March 2005

The amino acid sequence of′ the coenzyme‐binding site of serine transhydroxymethylase from rabbit liver has been determined. After reduction with NaBi14 and aminoethylation, a first sample of enzyme was digested with thermolysin and a single phosphopyridoxyl peptide was isolated.

A second sample of similarly treated enzyme was digested with chymotrypsin and three phosphopyridoxyl peptides clearly originating from a unique coenzyme‐binding site were isolated. Sequence analysis of these peptides indicate the following structure: Val‐Val‐Thr‐Thr‐Thr‐His‐Lys (Pxy) ‐Thr‐Leu. Sequence homologies of the active site of various pyridoxalphosphate enzymes are discussed in terms of a possible catalytic role and of evolution of this class of proteins.
A Study of Calf‐Thymus Histone H2B Using 13C Magnetic Resonance
Authors: TANCREDI Teodorico , TEMUSSI Piero A. , PAOLILLO Livio , TRIVELLONE Enrico , CRANE‐ROBINSON Colyn , BRADBURY E. Morton
Published: March 2005

Calf thymus histone H2B has been studied by means of 13C magnetic resonance. Most sidechain resonances have been assigned. Analysis of 13C peak intensities by a simple computer‐aided method proves that 13C magnetic resonance spectroscopy can he used to monitor aggregation phenomena more accurately than 1H magnetic resonance or other physico‐chemical methods.

Re‐examination of 1H spectral simulations shows substantial agreement with 13C results. It is concluded that in 0.1 M added NaCI the H2B aggregates include the chain segment from 50 (± 5)to 108 (± 5).
DNA Blockade by Rifampicin‐Inactivated Escherichia coli RNA Polymerase, and Its Amelioration by a Specific Mutation
Authors: HAYWARD Richard S.
Published: March 2005

Partially diploid strains of Escherichia coli containing both rifampicin‐sensitive and rifampicin‐resistant RNA polymerase are, in general, sensitive to the drug: of the two copies of the rpoB gene present in such strains, that which codes for sensitive enzyme is dominant.

RNA polymerase purified from a normal sensitive strain of E. coli, and inactivated by rifampicin, can ‘blockade’ bacteriophage T7 DNA in vitro, inhibiting its transcription by drug‐resistant enzyme molecules.

A mutation, rcs‐40, reverses the normal dominance relationship in vivo, without detectably affecting the concentrations of resistant and sensitive RNA polymerase in the diploid cell. I show that rcs‐40 is closely linked to the rpoB gene which codes for the rifampicin‐sensitive enzyme.

Rifampicin‐sensitive RNA polymerase purified from E. coli rcs‐40, although indistinguishable from the normal enzyme by many criteria, is significantly less efficient in the production of drug‐dependent DNA blockade.
Rat b/b Anemia: Translation of Normal and Anemic Globin mRNA in Wheat‐Germ Cell‐Free System
Authors: CRKVENJAKOV Radomir , ĆUŠIĆ Slavko , IVANOVIĆ Ivan , GLIŠIN Vladimir
Published: March 2005

Globin mRNAs were isolated from circulating reticulocytes both from rats carrying a homozygous, recessive mutation causing a severe thalassemia‐like syndrome and from normal rats. After first identifying the rat globin chains as alpha or beta chains, the translational products primed by both polysomal and nonpolysomal mRNAs in wheat germ 30 000 × g supernatant were analyzed: the ratio of alpha to beta globin mRNAs found in polysomes isolated from mutant rats is identical to the ratio of their products synthesized in vivo while the ratio of these mRNAs is quite different in the nonpolysomal fraction, the latter being enriched in alpha globin mRNA. No difference is found in the ratio of alpha and beta globin mRNAs in the polysomal and nonpolysomal RNA isolated from normal rats, both being identical to the ratio of their products synthesized in vivo. One third of the total amount of mRNA found in mutant cells is not in polysomes as compared to only 6% for the mRNA from normal lysates. These results suggest that a translational control mechanism is involved although the decreased globin synthesis in b/b anemia can not be fully accounted for by its operation.
Studies on the Influence of Fatty Acids on Pyruvate Dehydrogenase Interconversion in Rat‐Liver Mitochondria
Authors: WALAJTYS‐RODE Elżbieta I.
Published: March 2005

The effect of fatty acids on the interconversion of pyruvate dehydrogenase between its active (nonphosphorylated) and inactive (phosphorylated) forms was measured in rat liver mitochondria respiring in state 3 with pyruvate plus malate and 2‐oxoglutarate plus malate and during state 4 to state 3 transition in the presence of different substrates. The content of intramitochondrial adenine nucleotides was determined in the parallel experiments.

Decrease of the intramitochondrial ATP/ADP ratio with propionate and its increase with palmitoyl‐l‐carnitine in state 3 is accompanied by a shift of the steady‐state of the pyruvate dehydrogenase system towards the active or the inactive form, respectively.

Transition from the high energy state (state 4) to the active respiration (state 3) in mitochondria oxidizing 2‐oxoglutarate or palmitoyl‐l‐carnitine causes an increase of the amount of the active form of pyruvate dehydrogenase due to the decrease of ATP/ADP ratio in the matrix.

No change in ATP/ADP ratio can be observed in the presence of octanoate in mitochondria oxidizing pyruvate or 2‐oxoglutarate in state 3 or during state 4 to state 3 transition. Simultaneously, no significant change in phosphorylation state of pyruvate dehydrogenase occurs and a low amount of the enzyme in the active form is present with octanoate or octanoate plus 2‐oxoglutarate. Pyruvate abolishes this effect of octanoate and shifts the steady‐state of pyruvate dehydrogenase system towards the active form.

These results indicate that fatty acids influence the interconversion of pyruvate dehydrogenase mainly by changing intramitochondrial ATP/ADP ratio. However, the comparison of the steady‐state level of the pyruvate dehydrogenase system in the presence of different substrates in various metabolic conditions provides some evidence that accumulation of acetyl‐CoA and high level of NADH may promote the phosphorylation of pyruvate dehydrogenase.

Pyruvate exerts its protective effect against phosphorylation of pyruvate dehydrogenase in the presence of fatty acids of short, medium or long chain in a manner which depends on its concentration. It is suggested that in isolated mitochondria pyruvate counteracts the effect of acetyl‐CoA and NADH on pyruvate dehydrogenase kinase.
Enzyme‐Linked Sandwich Immunoassay of Macromolecular Antigens Using the Rabbit Antibody‐Coupled Glass Rod as a Solid Phase
Authors: HAMAGUCHI Yoshitaka , KATO Kanefusa , FUKUI Hideo , SHIRAKAWA Isao , OKAWA Sachiyo , ISHIKAWA Eiji , KOBAYASHI Keiko , KATUNUMA Nobuhiko
Published: March 2005

A highly sensitive sandwich immunoassay of macromolecular antigens using the rabbit antibody Fab′—β‐d‐galactosidase complex and the rabbit antibody immunoglobulin‐G‐coupled glass rod as a solid phase is described. The Fab′ fragments of rabbit antibody IgG are conjugated with β‐d‐galactosidase from Escherichia coli using N,N′‐o‐phenylenedimaleimide. Rabbit antibody IgG is coupled to the aminoalkylsilyl glass rods (3 mm in diameter and 5 mm in length) using glutaraldehyde. A wide range of the concentrations of rabbit IgG fraction (20–2000 μg/ml) is effective for coupling, and the amount of rabbit immunoglobulin G coupled can be controlled. The smallest amounts of ornithine σ‐aminotransferase from rat liver, human immunoglobulin G and 2,4‐dinitrophenyl human immunoglobulin G that can be determined are 0.03, 0.3 and 0.04 fmol, respectively. The sensitivity of the assay for these antigens is affected mainly by the non‐specific binding of the complexes to the solid phase and the ability of antigen molecules, adsorbed on the solid phase, to bind specifically the complexes. The assay with the rabbit antibody immunoglobulin‐G‐coupled glass rods is simpler and more sensitive than that with the rabbit antibody immunoglobulin‐G‐coupled Sepharose 4B.
Purification of the Membrane‐Bound dd‐Carboxypeptidase of the Unstable Spheroplast L‐Form of Proteus mirabilis by Affinity Chromatography
Authors: MARTIN Hans Herbert , SCHILF Wolfgang , MASKOS Christian
Published: March 2005

Membrane‐bound dd‐carboxypeptidase of the unstable L‐form of Proteus mirabilis was solubilized by the non‐ionic detergent Genapol X‐100 and purified to protein homogeneity by affinity chromatography on ampicillin bound to succinyl‐aminododecyl‐cellulose. The purified enzyme with a molecular weight of 43000 is inhibited non‐competitively by penicillin G and carbenicillin, indicating a function of the penicillins as allosteric inhibitors. Sensitivity of the enzyme to penicillins is only moderate with a Ki of 1 μM for penicillin G. Breakdown of the enzyme‐inhibitor complex EI with different penicillins occurs rapidly with reappearance of active dd‐carboxypeptidase. The half‐life of EI with penicillin G is 5.5 min at 30 °C and 3.5 min at 37 °C, 10–1000‐fold shorter than EI half‐lives of dd‐carboxypeptidases in several other bacteria. The low stability of the enzyme‐inhibitor complex and the moderate penicillin sensitivity appear to be the basis for the continued activity of dd‐carboxypeptidase during growth of the L‐form and synthesis of peptidoglycan in the presence of high concentrations of penicillin.
An Endonuclease from Mouse Cells Specific for Single‐Stranded DNA
Authors: OTTO Bernd , KNIPPERS Rolf
Published: March 2005

An endonuclease with a molecular weight of about 70000 (5–6 S) was extensively purified from mouse ascites cells. The enzyme specifically attacks single‐stranded DNA which is degraded mainly to oligonucleotides, with 5–10 residues. Supercoiled covalently closed circular phage DNA is converted to the linear relaxed form. The enzyme activity is highly sensitive to salt and can be stimulated by reagents lowering the dielectric constant of the buffer such as dimethylsulfoxide and glycerol.
Interactions of Yeast tRNAPhe with Ribosomes from Yeast and Escherichia coli
Authors: ROBERTSON James M. , KAHAN Michael , WINTERMEYER Wolfgang , ZACHAU Hans G.
Published: March 2005

The interaction of ethidium‐labeled tRNAPhe from yeast with ribosomes from yeast and Escherichia coli was studied by steady‐state measurements of fluorescence intensity and polarization. The ethidium label was covalently inserted into either the anticodon or the dihydrouridine loop of the tRNA. The codon‐independent formation of a tRNA. ribosome complex led to only a moderate increase of the observed fluorescence polarization indicating a considerable internal mobility of the labeled parts of the tRNA molecule in the ribosome complex. When the ribosome complex was formed in the presence of poly(U), the probes both in the dihydrouridine loop and in the anticodon loop were strongly immobilized, the latter exhibiting a substantial increase in fluorescence intensity. A smaller intensity change was observed when E. coli ribosomes were used, although the extent of immobilization was found to be similar in this case. Competition experiments with non‐labeled tRNAPhe showed that the labeled tRNAPheEtd was readily released from the complex with yeast ribosomes when poly(U) was absent, whereas in the presence of poly(U) it was bound practically irreversibly. The finding that the mobility of a probe in the dihydrouridine loop is affected by the codon‐anticodon interaction on the ribosome suggests a conformational change of the ribosome‐bound tRNA which may involve opening of the tertiary structure interactions between the dihydrouridine and the TΦC loop.
Synthesis of Stereospecifically Deuterated Phenylalanines and Determination of Their Configuration
Authors: BARTL Knut , CAVALAR Christiane , KREBS Traute , RIPP Engelbert , RÉTEY János , HULL William E. , GÜNTHER Helmut , SIMON Helmut
Published: March 2005

Starting from trans‐cinnamic acid a chiral (−)3‐phenyl‐[2,3‐2H]propionic acid has been synthesized using Clostridium kluyveri cells as catalyst.

The chiral dideuterated acid has been converted by chemical methods to a mixture of (2R) and (2S)‐phenyl[2,3‐2H]‐alanine.

By means of 1H nuclear magnetic resonance spectroscopy and the action of D and L‐amino‐acid oxidase the configuration of the phenylalanine has been shown to be (2R, 3S) and (2S, 3S), respectively. The labelled phenylalanine is thus sterically and isotopically homogeneous at position 3 but heterogeneous at position 2.
The Euglena gracilis Chloroplast Genome:
Authors: KOPECKA Helena , CROUSE Edwin J. , STUTZ Erhard
Published: March 2005

Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells.

Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4–7 × 106 and G + C‐rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients.

Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G + C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R. EcoRI, endo R. HindII, endo R. HindIII, endo R. HindII + III, endo R. HpaI, endo R. HpaII and endo R. HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions.

With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions.

Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns.

Digestion of total DNA with the endo R·HaeIII yielded 51–52 fragments separated in the gels in a total of 36 bands of which 11–12 bands were composed of 2–3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 × 106 or 95% of the genome (92 × 106).

Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr 92 × 106). These cistrons are located on seven different types of endo R. HaeIII fragments. The hybridising fragments are preferentially found in the G + C‐rich subfraction and in bands which are composed of 2–3 fragments.
The Roles of c‐Type Cytochromes in Algal Photosynthesis
Authors: WOOD Paul M.
Published: March 2005

A membrane‐bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas, Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome f, with α‐band maxima near 554 nm, very asymmetrical β‐bands, and γ‐band maxima at 421 nm. The cytochrome from Anacystis had α and γ‐bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as +350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c‐552, and was unaffected by Euglena cytochrome c‐552 antiserum. A very fast rate of electron transfer occurred between this cytochrome purified from Euglena and cytochrome c‐552. The roles of the membrane‐bound and soluble c‐type cytochromes in algal photosynthesis are discussed, and it is recommended that the name cytochrome f should be reserved for the membrane‐bound cytochrome (to emphasize its affinity with higher plant cytochrome f), while the soluble one should be named by its α‐band (c‐552, c‐553, etc.) to make clear its distinctness from higher plant cytochrome f and homology with mitochondrial cytochrome c.
Prolyl 3‐Hydroxylase: Partial Characterization of the Enzyme from Rat Kidney Cortex
Authors: RISTELI Juha , TRYGGVASON Karl , KIVIRIKKO Kari I.
Published: March 2005

The formation of 3‐hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3‐hydroxyproline was observed with all these substrates and the formation of 3‐hydroxyproline ranged up to seven residues per pro‐α‐chain. The highest rate of 3‐hydroxylation took place at 20 °C and the reaction required Fe2+, O2, 2‐oxoglutarate and ascorbate. The formation of 3‐hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple‐helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3‐hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4‐hydroxylase or by poly(L‐proline) when these substances were used in concentrations which clearly inhibited 4‐hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4‐hydroxylase did not synthesize any 3‐hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3‐hydroxylase and prolyl 4‐hydroxylase are separate enzymes.
Physicochemical Properties of T4 Polynucleotide Kinase
Authors: LILLEHAUG Johan R.
Published: March 2005

Some physicochemical properties of T4 polynucleotide kinase (EC 2.7.1.78) have been studied. The enzyme is an oligomer of one polypeptide chain. The molecular weight of the monomer is 33000, as determined from the amino acid analysis. Phenylalanine is the N‐terminal amino acid. Each monomer contains two −SH groups, one exposed and one more buried. Circular dichroic spectra suggest a high content of α‐helical structure, 45 – 55%. Excitation at 280 nm gave a strong emission fluorescence spectrum with a maximum centering at 340 nm.

Sedimentation studies suggested the enzymically active form to be a tetramer. High ionic strength (0.1 M KCl), spermine, and the substrates ATP and thymidine 3′‐monophosphate were found to be essential factors in order to stabilize the protein in an oligomeric structure. The association constants for ATP, thymidine 3′‐monphosphate, and Pi were determined fluorimetrically to be 7.9 × 105, 4.8 × 105, and 7.2 × 102 M−1 respectively.
Thyrotropin Stimulation of the Phosphorylation of Serine in the N‐Terminal Region of Thyroid H1 Histones
Authors: LAMY Françoise , LECOCQ Raymond , DUMONT Jaques E.
Published: March 2005

The analysis of thyroid nuclear proteins by polyacrylamide gel electrophoresis has demonstrated that thyrotropin and dibutyryl adenosine 3′:5′‐monophosphate stimulate specifically the phosphorylation of H1 histones in an intact cell system.

This effect does not require new protein synthesis and implicates the phosphorylation of serine residue(s) situated in the N‐terminal part of H1 histones.
An Investigation of Mistranslation in vivo Induced by Streptomycin by an Examination of the Susceptibility of Abnormal Proteins to Degradation
Authors: HEWITT Jane , KOGUT Margot
Published: March 2005

Proteolysis rates in vivo were measured in Escherichia coli cultures during treatment with dihydrostreptomycin and under various other conditions. Dihydrostreptomycin treatment caused an increase in the proteolysis rate, compared to untreated controls. The proteolytic system in vivo responsible for the elevated proteolysis in the early stages of dihydrostreptomycin treatment, or that during canavanine and puromycin treatment, were not inhibited by addition of phenylmethanesulphonyl fluoride. This agent did inhibit proteolysis rates in cultures whose growth was inhibited by starvation, or had been completely stopped by dihydrostreptomycin. It seems, therefore, that the extremely high proteolysis rates in cultures at this stage of dihydrostreptomycin treatment were due to the action of two protease systems: the one concerned with the breakdown of abnormal proteins, and the other concerned with normal protein turnover and active during a non‐specific decline of growth.

The proteolytic rate at complete growth inhibition brought about by dihydrostreptomycin was intermediate between those induced by canavanine and puromycin at the same stage of treatment. This indicated a similar hierarchy in the extent and nature of abnormality in the proteins synthesised under these conditions. The relationship between the abnormality of proteins induced by dihydrostreptomycin and the importance of this in the antibiotic mechanism is discussed.
Cathepsin L
Authors: KIRSCHKE Heidrun , LANGER Jürgen , WIEDERANDERS Bernd , ANSORGE Siegfried , BOHLEY Peter
Published: March 2005

Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM‐Sephadex.

Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide‐gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23000 – 24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8 – 6.1 ascertained by analytical isoelectric focusing.

Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6 – 7. In contrast to cathepsin B1, its capability of hydrolyzing N‐substituted derivatives of arginine is low and it does not split esters.

Greatest activity is obtained close to pH 5.0 with 70 – 90% of maximal activity at pH 4.0 and pH 6.0 and 30–40% at pH 7.0.

The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl‐lysine. Leupeptin acts as a pseudo‐irreversible inhibitor.

The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep‐frozen state.
Snake Venom Toxins
Authors: JOUBERT François J.
Published: March 2005

Three toxins (9B, 11 and 12A) were purified from the venom of Hemachtus haemachatus as described previously. Whereas toxin 11 and 12A comprise 61 amino acid residues, toxin 9B contains 63 residues. All three toxins are cross‐linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The properties of the toxins were compared with those of the cytotoxin group. The toxicities, the sequences and some of the invariant residues of toxin 11 and 12A resemble the corresponding properties of the cytotoxin group. However their immunochemical properties indicate that they are distinct from both the cytotoxin and neurotoxin groups. The sequence of toxin 9B shows that it is related to the cytotoxins, but its toxicity is much lower than those encountered among members of this group.
Structural Studies on Periodate‐Oxidized Chicken Ovomucoid
Authors: KRYSTEVA Milka Assenova , DOBREV Ivan Dochev
Published: March 2005

About 50% of the carbohydrate moiety of ovomucoid was destroyed by periodate oxidation. The oxidation was carried out for 6 h or 24 h. The data obtained showed that in the carbohydrate chain 2–5 glucosamines and 1–2 neutral sugar residues were decomposed with the consumption of 16 mol and 29 mol of periodate respectively. Periodate oxidation slightly changed the inhibitory activity of the ovomucoid, but altered its spectral properties. An increase of the absorption maximum at 278 nm was noted, as well as a tendency for normalization of phenolic ionization and an increase of the re‐lative fluorescence. The reactivity of tyrosine residues towards tetranitromethane is also changed. It was suggested that even in native ovomucoid the tyrosines could be regarded as ‘dissolved’ in the ‘carbohydrate solvent’. This contact could be achieved by the hydrogen bonds in the formation of which the NHCOCH3 groups of the glucosamine residues play an essential role. Peroxidate oxidation seems to lead to an alteration of the nature of the ‘sugar solvent’ and disturbs the conformation of the sugar chain.