bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigenic Determinants of Murein Lipoprotein and Its Exposure at the Surface of Enterobacteriaceae
Authors: BRAUN Volkmar , BOSCH Valerie , KLUMPP Ernst Rudolf , NEFF Ingrid , MAYER Hubert , SCHLECHT Siegfried
Published: March 2005

Murein lipoprotein from the outer membrane of Escherichia coli could be fixed to erythrocytes without pretreatment of the erythrocytes. Passive hemagglutination or immune hemolysis could thus be used as sensitive assays to determine antibodies against lipoprotein. In rabbit antisera prepared against whole cells of E. coli, Sulmonellu, Arizona and Shigellu antibodies against lipoprotein were present. The respective titers were lowest in encapsulated smooth strains and highest in rough mutants. Antisera against deep rough mutants showed even higher anti‐lipoprotein titers than anti‐R‐lipopolysaccharide titers. Correspondingly, absorption of lipoprotein antibodies with enterobacterial strains was most pronounced with deep rough mutants and lowest with smooth strains. Lipoprotein becomes increasingly an immunogen as well as an antigen the more sugar residues are missing in the lipopolysaccharide on the cell surface. In wild‐type cells lipoprotein is buried in the outer membrane; its exposure in mutant cells is related to defects at the cell surface.

Antisera were prepared in rabbits against erythrocytes coated with various lipoprotein samples which differ at the C‐terminal end of the polypeptide chain. Antibodies against lipoprotein containing two attached muropeptides (called lipoprotein I) are directed against the muropeptide attachment region. Antibodies against lipoprotein ending with lysine‐55 (lipoprotein II) were directed specifically against this terminal lysine residue. No serological reaction was observable after splitting off this lysine residue by carboxypeptidase B. Remarkably this antiserum against lipoprotein II neither reacted with the free lipoprotein ending with lysine‐58 (lipoprotein IV) nor with the muropeptide containing lipoprotein I. Antisera against the lipoprotein lacking lysine‐55 (lipoprotein III) reacted with all lipoprotein samples as did the antibodies in antisera prepared against whole heat‐killed cells, thus indicating a common binding site located in the polypeptide chain towards the N‐terminal end. No indication of an antigenic reactivity of the co‐valently linked lipoid was obtained.

Lipoprotein treated with boiling 4% sodium dodecylsulfate and acetone reacted strongly with antisera against the native lipoprotein. The recovery of the antigenic sites demonstrates the remarkable stability of the native conformation.

The lipoprotein is a new common antigen for closely related Enterobacteriaceae. Lipoprotein antisera might be useful for characterization of outer membrane mutants of Enterobacreriaceue.
Purification of the mRNAs for Ewe αs‐Casein and β‐Casein by Immunoprecipitation of Polysomes
Authors: HOUDEBINE Louis‐Marie , GAYE Pierre
Published: March 2005

Specific polysomes synthesizing s‐casein and β‐casein were immunoprecipitated from total polysomes of the lactating ewe mammary gland. The polysome anti‐casein complex was immunoprecipitated by anti‐immunoglobulins. 22%, 32% and 10% of polysomes were immunoprecipitated with saturating amounts of anti‐αs‐casein, anti‐β‐caseinr and anti‐ℋ‐casein respectively. PolyU)‐Sepharose chromatography of the immunoprecipitated RNAs permitted the isolation of the corresponding polyA)‐containing RNA which migrated as one major band in polyacrylamide gel electrophoresis. As judged by the contamination with the messenger activity for one of the other caseins, the purity of the mRNA for αs‐casein as well as for β‐casein was estimated to be between 75 % and 80 %.
Conformational Changes Associated with Transient Activation of Phosphorylase in Glycogen Particles
Authors: BUSBY Stephen J. W. , GADIAN David G. , GRIFFITHS John R. , RADDA George K. , RICHARDS Rex E.
Published: March 2005

Calcium‐dependent transient phosphorylation of phosphorylase b has been monitored in a rabbit muscle glycogen particle fraction. Using a phosphorus nuclear magnetic resonance assay, the changes in concentrations of small phosphate‐containing metabolites associated with this event have been measured. In addition, the conformation of phosphorylase has been monitored during transient activation by observing changes in the electron spin resonance signal from added spin‐labelled phosphorylase.

The transient activation was associated with a loss of glucose 6‐phosphate from phosphorylase b; newly formed phosphorylase a binds the nucleotides ADP, AMP or IMP. Because of the fast interconversion of these nucleotides the species bound to phosphorylase a change throughout the process.

Lowering the [Mg2+]: [Ca2+] ratio during transient activation causes accumulation of ADP. Electron spin resonance data from spin‐labelled phosphorylase shows that, under these conditions, ADP binding to phosphorylase a is potentiated.

Calcium‐dependent activation in the glycogen particle fraction is compared to the activation of phosphorylase in vivo.
Influence of Chain Length and Unsaturation on the Effects of Fatty Acids on Phosphoglyceride Biosynthesis in Isolated Rat and Pig Hepatocytes
Authors: ÅKESSON Björn , SUNDLER Roger , NILSSON Åke
Published: March 2005

Hepatocytes isolated from rat or pig by collagenase perfusion were incubated with [3H]glycerol and different albumin‐bound fatty acids. Among C22 fatty acids docosahexaenoic acid stimulated phosphatidylethanolamine synthesis in rat hepatocytes most effectively. Addition of docosahexaenoic acid plus either palmitic or stearic acid resulted almost in the same stimulation whereas combinations of this acid with lauric or myristic acid had no effect. Lauric acid and myristic acid alone inhibited phosphatidylethanolamine synthesis. The chain length specificity for monoenoic fatty acids was similar, the hexadecenoic and octadecenoic acids (both cis and trans) being most stimulatory. The addition of 0.2 mM ethanolamine markedly stimulated phosphatidylethanolamine synthesis, but most effects of fatty acids were similar in its presence or absence.

The effect of fatty acids on phosphatidylcholine and total lipid synthesis were often similar but clearly deviated from those on phosphatidylethanolamine synthesis.

In isolated pig hepatocytes the effects of fatty acids were similar to those in the rat. The distribution of [3H]glycerol among molecular species of glycerolipids changed drastically upon fatty acid addition.

The different effects on phosphatidylethanolamine and phosphatidylcholine synthesis are most probably due to differences in the substrate specificities of ethanolaminephosphotransferase and cholinephosphotransferase.
Lysis of Yeast Cell Walls Lytic β‐(1→6)‐Glucanase from Bacillus circulans WL‐12
Authors: ROMBOUTS Frank M. , PHAFF Herman J.
Published: March 2005

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL‐12 produces wall‐lytic enzymes in addition to non‐lytic β‐(1→3)‐and β‐(1→6)‐glucanases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic β‐(1→6)‐glucanase and two lytic β‐(1→3)‐glucanases. The lytic β‐(1→6)‐glucanase was further purified by chromatography over diethylamino‐ethyl‐agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went through a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls was attributed to a debranching of the alkali‐insoluble β‐(1→3)‐glucan in the wall. Low molecular weight products from yeast cell walls included gentiotriose, gentiobiose and glucose but β‐(1→3)‐linked oligosaccharides were not detected. The lytic β‐(1→6)‐glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis‐Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro‐equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement.

The lytic β‐(1→6)‐glucanase differs in essence from the non‐lytic β‐(1→3)‐glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.
Nuclease S1 Cleavage and the Primary Structure of Mitochondrial DNA
Authors: KOCH Jürgen , BRUHN Andreas
Published: March 2005

The single‐strand‐specific nuclease S1 from Aspergillus oryzae rapidly converts superhelical mitochondrial DNA (African Green Monkey cells, Vero ATCC; CCL 81) into nicked circular DNA. These nicked mitochondrial DNA molecules contain two nicks, one in each strand. The phosphodiester backbones are cleaved during this reaction at or near sites that are alkali‐labile.

In a second slow reaction the circular mitochondrial DNA is converted into a linear duplex DNA. Permutation tests indicate that this linear DNA represents a nonpermutated collection of DNA molecules.

These results suggest that two of the alkali‐labile sites in the phosphodiester backbones of the mitochondrial chromosome are closely spaced on opposite strands and at specific positions.
Algal Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: O'BRIEN Michael J. , POWLS Roy
Published: March 2005

Two enzymes with glyceraldehyde‐3‐phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion‐exchange chromatography and gel filtration.

The D‐enzyme has a molecular weight of 550000 and a VNADH: VNADPH ratio of 16 whereas the T‐enzyme has a molecular weight of 140000 and a VNADH: VNADPH ratio of 0.15.

The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption.
l‐α‐Hydroxyacid Oxidase Isozymes
Authors: DULEY John A. , HOLMES Roger S.
Published: March 2005

l‐α‐Hydroxyacid oxidase isozymes from rat liver (A isozyme) and kidney (B isozyme) have been isolated in a high state of purity with specific activities of 61 and 14.7 microkatals per gram protein respectively. The subunit molecular weights determined by sodium dodecylsulphate polyacrylamide gel electrophoresis were 40000 ± 3000; the mouse A and B isozymes were also partially purified and their subunit molecular weights shown to be 37000.

The total amino acid contents of the two rat isozymes were very similar and had identical tyrosine (9 residues per 40000) and tryptophan (5 per 40000) contents. No free suiphydryl groups were found following suiphydryl titration experiments using the purified native rat isozymes. Following denaturation with sodium dodecylsulphate, 2 (A subunit) and 3 (B subunit) suiphydryl residues were titratable. The apoenzyme form of rat isozyme B exhibited 2 of the 3 titratable suiphydryl groups in the absence of sodium dodecylsulphate which suggests an involvement of these residues in coenzyme binding. Flavin mononucleotide content was estimated from spectral studies to be I residue per subunit (40000 molecular weight) of native enzyme. Following storage in ammonium sulphate at 4°C for several months, flavin mononucleotide was irreversibly removed such that the rat A and B isozymes contained 0.3 and 0.7 residue of coenzyme per subunit. This accounts for the more pronounced loss of specific activity following storage for the A isozyme than the B isozyme as well as the comparatively high minimal molecular weights based upon flavin mononucleotide values previously reported by other workers.

This evidence for chemical homology together with the previously reported immunochemical crossreactivity for these isozymes provide evidence that the A and B polypeptides are products of recently duplicated genes during evolution.
Purification and Properties of Cyclopentanone Oxygenase of Pseudomonas NCIB 9872
Authors: GRIFFIN Martin , TRUDGILL Peter W.
Published: March 2005

Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40‐fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis.

The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three or four subunits of molecular weight 54000–58000.

The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein.

The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixedfunction oxygenases with external electron donors. The oxygenated product has been identified as 1‐oxa‐2‐oxocyclohexane (5‐valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2‐lactonizing) (EC 1.14.13.–).

Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p‐hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent.

The enzyme has a pH optimum of 7.7. The Km for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits.

Anaerobic reduction of enzyme‐bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments.

Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated.
Temperature‐Dependent Change in Rate‐Limiting Step of the Magnesium‐Stimulated ITPase of Myosin
Authors: HOZUMI Tetsu
Published: March 2005

The effects of temperature on Mg‐ITPase activity of heavy meromyosin and myosin sub‐fragment 1 were measured in 0.1 M KCl. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.e. one mol per two mol of myosin active sites, at 20 °C. However, it was almost zero mol below 8 °C. Effects of KCl concentration and pH on ITPase activity of heavy meromyosin at 20 °C were different from those below 8 °C, suggesting that the rate‐limiting step in the Mg‐ITP hydrolysis of myosin depends on temperature.

The effect of temperature on the actin activation of heavy meromyosin Mg‐ITPase was analyzed by measuring the temperature dependence of double‐reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at lower temperature.

The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule.
Fluorescence of Proteins in 6‐M Guanidine Hydrochloride
Authors: PAJOT Patrick
Published: March 2005

To determine the tryptophan content in proteins, an analytical ultraviolet fluorescence method is proposed based on making uniform the environment of aromatic chromophores in 6–7 M guanidine hydrochloride.

The fluorescence intensity scale is calibrated using standard solutions of free tryptophan. A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins.

This method is particularly suited to proteins carrying groups absorbing in the 290–370‐nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2‐mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods. It is less time‐consuming than techniques requiring hydrolysis or chemical reactions.
Studies on the Mechanism of Site I Energy Conservation
Authors: OHNISHI Tomoko
Published: March 2005

Of the several iron‐sulfur centers detected in the site I segment of the respiratory chain, centers N‐2 and N‐1a alone exhibit apparent phosphate‐potential dependent half‐reduction potentials, indicating their possible involvement in energy conservation.

At high phosphate potential, the apparent half‐reduction potential of center N‐2 shifts positively by 125 ± 20 mV, while center N‐1a shifts negatively by approximately 60 mV.

The redox state of individual iron‐sulfur centers in various metabolic states was analyzed. Center N‐2 is highly reduced (> 90%) in “state 4” mitochondria, while center N‐1a stays mostly oxidized.

In a submitochondrial system, ATP addition induced reduction of center N‐2 if the Eh of the suspension was poised from the high potential side of site I using the succinate/fumarate couple. In contrast, center N‐2 was oxidized upon energization, if the Eh of the system was poised from the low potential side using the NADH/NAD couple.

Based on these redox behaviors of center N‐2, a tentative hypothesis of site I energy transduction was proposed.
Plastocyanin : Possible Significance of Quaternary Structure
Authors: SIEGELMAN Mark H. , RASCHED Ihab R. , KUNERT Karl‐Josef. , KRONECK Peter , BÖGER Peter
Published: March 2005

Some properties of the blue copper protein plastocyanin from the green alga Scenedesmus have been investigated and compared with that from spinach, including amino acid composition, isoelectric point and copper content. The protein from Scenedesmus contains two, that from spinach four copper atoms per molecular weight of 40000.

A combination of sodium dodecylsulfate/polyacrylamide gel electrophoresis and quantification of sulfhydryl groups indicates a strong preference for a species composed of 4 polypeptide chains of identical amino acid composition representing the enzymically active entity. Due to various treatments the subunits of both plastocyanins are detected as either monomer species alone or as monomer and dimer in a molar ratio of 2 : 1 on sodium dodecylsulfate gels.

The four ‐SH groups per molecule are of different reactivity: two ‐SH groups can be detected after destruction of the chromophore; two more (forming an S‐S bridge in the dimer) become evident after appropriate reduction.

A KCN treatment for production of apoprotein is reported and the use of electrodialysis to improve incomplete apoprotein formation. These studies lend support to the proposal of a quaternary structure.

Apoproteins were subjected to dodecylsulfate gel analysis, which proved to be an effective means of estimating both the extent of apoprotein formation and its reconstitution to the holoprotein.
The Incorporation of Newly Synthesized RNA into Nuclear Ribonucleoprotein Particles after Oestrogen Administration to Immature Rats
Authors: KNOWLER John T.
Published: March 2005

Ribonucleoprotein particles, known as informofers or heterogeneous nuclear ribonucleoprotein particles (hnRNA · protein), have been extracted from rat uterine nuclei and found to have properties similar to those characterized from other tissues.

Incorporation of RNA precursors into the RNA of these particles is stimulated up to 8‐fold by oestrogen administration to rats.

When uteri are dissected from oestrogen‐treated rats and incubated in vitro with radioactive RNA precursor, only the RNA in the ribonucleoprotein particles is synthesized at a rate faster than can be accounted for by increase in the uptake of precursor. This contrasts with previous studies where both hormone treatment and incorporation of radioactive precursor were performed in vivo and the synthesis of all RNA species was stimulated [Knowler, J. T. and Smellie, R. M. S. (1971) Biochem. J. 125, 605‐614; Knowler, J. T. and Smellie, R. M. S. (1973) Biochem. J. 131, 689–697].
Structural Properties of Escherichia coli RNA Polymerase Subunits
Authors: LOWE Peter A. , MALCOLM Alan D. B.
Published: March 2005

The surface of the RNA‐polymerase ‐ DNA complex possesses an exposed polypeptide loop.

Proteinases with differing specificities (trypsin, chymotrypsin, subtilisin and clostripain) preferentially cleave the exposed region.

The cleaved polypeptide is reassembled into RNA polymerase by renaturation from a solvent which promotes a random coil conformation.

Isolated β subunit has a proteolytically resistant nucleus of approximately 70000 molecular weight. This resistant polypeptide may be generated by trypsin, chymotrypsin, subtilisin or clostripain.

Isolated α subunits are comparatively resistant to proteolysis.

Although of similar molecular weights β and β′ appear to have unrelated primary sequences and markedly different conformations in free solution.

Digestion of the β subunit may be blocked by formation of the α2β subassembly.

Evidence is presented suggesting that β in the intact enzyme (α2ββ′) possesses the exposed polypeptide loop.
Role of Calcium Ions in the Thermostability of Thermolysin and Bacillus subtilis var. amylosacchariticus Neutral Protease
Authors: TAJIMA Masahiro , URABE Itaru , YUTANI Katsuhide , OKADA Hirosuke
Published: March 2005

The stabilizing effect of calcium ions on thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease has been investigated. Calcium and zinc ions were removed from the proteases by gel filtration over Sephadex G‐25 equilibrated with metal chelating agents. Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures. Removal of calcium ions caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (ΔH*) and entropy (ΔS*) for heat inactivation. There was little difference in stability between thermolysin containing 0.3 g‐atom fmol and B. subtilis neutral protease containing 1.4 g‐atoms/ mol.

Calcium binding isotherms of the proteases were obtained by equilibrium gel chromatography with various concentrations of free calcium ions. Thermolysin had four independent calcium binding sites with an identical intrinsic binding constant (K) of 2.0 104 M−1. B. subtilis neutral protease had four independent sites. The K value for three sites was 1.1 105 M−1 and the binding constant for the other site was 1.5 103 M−1. There was little difference in total free energy change for calcium binding between these proteases.

From these results it is concluded that the stabilizing effect of calcium on these enzymes is almost equal, and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.
Transglycosylic Reactions of Nucleotides of Deoxy Sugars
Authors: ZEMEK Jiři. , STRMEŇ Ján. , KULČÁR Štefan. , BAUER Štefan.
Published: March 2005

The biosynthesis of α, α′‐trehalose was investigated in a reaction catalyzed with yeast UDP‐Glc:glucose‐6‐phosphate 1‐glucosyl transferase. It was revealed that the hydroxyls at positions C‐2, C‐3, C‐4 and C‐6 are not essential for preserving its substrate properties in this reaction. The affinity of the enzyme and the rate of hexose incorporation into trehalose decreases in following sequence: UDP‐glucose, UDP‐2‐deoxy‐d‐glucose, UDP‐6‐deoxy‐d‐glucose, UDP‐4‐deoxy‐d‐glucose and UDP3‐deoxy‐d‐glucose.
Haptoglobin‐Haemoglobin Interaction
Authors: LAVIALLE Francoise , ROGARD Monique , ALFSEN Annette
Published: March 2005

The formation of two different complexes when haptoglobin (Hp) and haemoglobin (Hb) are mixed in a 1:1 molar ratio is demonstrated by isoelectrofocusing. In these two complexes, the affinity of Hp for Hb is shown to be different, since Hb can be displaced only from one of the complexes, by a further addition of Hp. This is confirmed by a quantitative study of the reaction stoichiometry, when [Hp]/[Hb] = 1 and [Hp]/[Hb] > 1, which allows an evaluation of the amount of each complex formed.

All these data cannot be explained other than by the existence of two forms of Hp molecule and a reaction scheme which fits these experiments, is proposed.
Phosphatidic Acid Metabolism in Rat Liver Microsomes
Authors: TZUR Ruth , SHAPIRO Benyamin
Published: March 2005

Rat liver microsomes contain phosphatidate phosphatases which split phosphatidic acid into inorganic phosphate and diacylglycerol and a system of phospholipases and lipases, which split phosphatidic acid into free fatty acids, glycerol and inorganic phosphate.

In the presence of ATP, CoA and [1‐14C]palmitate, part of the monoacyl‐sn‐glycerol 3‐phosphate formed by phospholipase action is reesterified, yielding radioactive phosphatidic acid. The sum of di‐ and triacylglycerols formed from phosphatidic acid in the presence of ATP and CoA exceeded the amount of diacylglycerol fromed in their absence.

The yield of neutral lipids from sn‐glycerol 3‐phosphate and monoacyl‐sn‐glycerol 3‐phosphate markedly exceeded that from phosphatidic acid.

Comparison of the yields of di‐ and triacylglycerols from glycerol‐labelled and fatty‐acid‐labelled phosphatidic acid was used to establish the extent of deacylation and reacylation. About 60% of the diacylglycerol was formed by direct dephosphorylation. The triacylglycerols, on the other hand, were formed almost exclusively from recycled phosphatidic acid.
Studies on the Role of Uncharged tRNA in Pleiotypic. Response of Animal Cells
Authors: GRUMMT Friedrich , GRUMMT Ingrid
Published: March 2005

Experiment were carried out to assess the physiological significance of the charging level of tRNA. Histidinol, a competitive inhibitor of charging of tRNAHis, was used to induce uncharged tRNA in mammalian cells. It is demonstrated that both in the presence of histidinol and under histidine depletion about 40% of the tRNAHis is uncharged. Concomitant with this appearance of uncharged tRNA (a) the pools of GTP and ATP are decreased rapidly by 25–30 %; (b) the synthesis of both protein and ribosomal RNA is inhibited, whereas that of nucleoplasmic RNA is not affected; (c) the uptake of 2‐deoxyglucose, phosphate, Ca 2+; uridine and adenosine is inhibited; and (d) the growth of 3T6 fibroblasts is arrested. It is suggested that the appearence of uncharged tRNA is one of the earliest events occurring under conditions of amino acid starvation, which in turn causes the various metabolic changes observed.
Dissociation Kinetics of Complexes between the Antibiotic Rifamycin and DNA‐Dependent RNA Polymerase from Escherichia coli
Authors: STENDFR Walter , SCHEIT Karl Heinz
Published: March 2005

Phosphocellulose chromatography was employed to measure the binding of 3‐(2‐[14C]acetamidoethyl)‐thiorifamvcin(abbreviated[14C]AcNHEtS‐Rif) to RNA polymerase core enzyme. The technique yielded a correct value for the stoichiometry of interaction. The same method was successfully applied to an investigation of the dissociation kinetics of the [14C]AcNHEtS‐Rif · core‐polymerase complex. We observed biphasic dissociation kinetics not in agreement with the existence of one single first‐order dissociation step. Assuming two independent dissociation reactions, rate constants have been evaluated differing roughly by 10‐fold (1.7 × 10−3 s−1 and 1.5 × 10−4 s−1 at 25 °C). The ratio of the amplitudes of the biphasic dissociation kinetics changed with temperature. The kinetic data are interpreted as indirect evidence for the existence of two enzyme species differing in their dynamic properties with respect to the binding of the antibiotic rifamycin. Our data furthermore lend support to the assumption that the two enzyme forms are in equilibrium. The observed sigmoidal dependence of the dissociation rate on temperature could indicate a conformational transition of RNA core polymerase with a transition midpoint at around 20 °C. The dye, rose bengal, was found to be as effective as AcNHEtS‐Rif itself as a competing agent. The dissociation kinetics of the [14C]AcNHEtS‐Rif · core‐polymerase complex in the presence of an excess of the dye rose bengal were found to be very similar to those measured in the presence of AcNHEtS‐Rif. This could mean that rose bengal and AcNHEtS‐Rif compete for the same site at RNA core polymerase. The dissociation of the ternary complex [14C]AcNHEtS‐Rif core‐polymerase · poly[d(A‐T)] was followed by gel filtration. Up to the extent of dissociation measured, the reaction appeared to follow first‐order kinetics. The dissociation rate constant was calculated to be 1.7 × 10−4 s−1. Experiments to determine the effect of the antibiotic streptolydigin on the dissociation kinetics of the [14C]AcNHEtS‐Rif. corepolymerase complex have not led to conclusive results.
Elongation Factor 1 from Artemia salina Properties and Disaggregation of the Enzyme
Authors: NOMBELA Cesar , REDFIELD Betty , OCHOA Severn , WEISSBACH Herbert
Published: March 2005

Elongation factor 1 has been purified from undeveloped embryos of Artemia salina. The purified enzyme appears to be an aggregate (molecular weight ∼ 200000) which on sodium dodecylsulfate gels shows the presence of two major protein bands whose estimated molecular weights are 52000 and 47000. Lipid material appears to be associated with the purified protein. In aminoacyl‐tRNA binding to ribosomes, there is only a limited turnover of the enzyme, but the protein acts catalytically in amino acid polymerization. The enzyme is disaggregated by a partially purified phospholipase C preparation, elastase and under certain conditions, by guanosine nucleotides. The significance of these results is discussed with respect to the overall role of elongation faction I in aminoacyl‐tRNA binding to ribosomes.
Enzymic Unwinding of DNA
Authors: ABDEL‐MONEM Mahmoud , HOFFMANN‐BERLING Hartmut
Published: March 2005

Evidence from various sources in the literature suggests that, in connection with DNA, ATP dephosphorylation can be used to provide energy for mechanical effects. Starting from this concept we have studied a novel DNA‐dependent ATPase purified to 90% homogeneity from Escherichia coli. The enzyme has a peptide weight near 180000 and, in high salt, is a monomeric, probably highly anisometric molecule. In salt‐free buffer, where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km 0.27 mM) and dephosphorylated at the γ‐position at a maximal rate near 104 molecules per enzyme monomer per min at 35 °C. A requirement for divalent cation is best satisfied by Mg2+ or Ca2+ and the requirement for DNA best by the single‐stranded, circular DNA of phages øX174 (Km 62 nM nucleotide) and fd indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein, øX DNA is not accepted but, once in contact with the DNA, the enzyme is little inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the recognition of DNA.

The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB‐product‐associated ATPase or the ATPase activity found in DNA polymerise III holoenzyme under appropriate conditions, and it is certainly not identical with a DNA‐dependent ATPase of molecular weight 69000 from E. coli which has recently been purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, have failed.
Fluorescent Guanosine‐Nucleotide. Analogs Suitable for Photoaffinity‐Labeling Experiments
Authors: WIEGAND Georg , KALEJA Rudolf
Published: March 2005

The synthesis and properties of strongly fluorescent derivative of inosine and its nucleotides are described. Reaction of 2‐chloro‐inosinic acid with sodium azide leads to a product bearing the tetrazole ring between position 2 and 3. Methylation at N1 was effected with dimethyl sulfate. The corresponding nucleosides and their 5′‐triphosphates were also prepared. Only the non‐methylated series is at equilibrium with small concentrations of their azido forms, and can be photolyzed by wavelengths above 300 nm. Both series are strongly fluorescent, their emission and excitation fluorescence spectra, as well as their quantum yields were measured. The nucleoside 5′‐triphosphates ate able to initiate and sustain polymerization of porcine brain tubulin.
Wheatgerm Hexokinase (LII): Fluorimetric Measurement of the Binding of Substrates and Products
Authors: HIGGINS Trevor J. C. , EASTERBY John S.
Published: March 2005

The change in intrinsic fluorescence observed when wheatgerm hexokinase combines with its substrates or products has been investigated. The dissociation constants for the enzyme · ligand complexes have been evaluated and found to be equal to their respective Michaelis constants, and confirm that fructose is the preferred hexose substrate. Both hexoses and nucleotides can bind independently to the enzyme and the data are consistent with previous proposals that conformation changes in the enzyme may accompany the random binding of substrates.
Interconversion between 17β‐Hydroxy‐5α‐androstan‐3‐one (5α‐Dihydrotestosterone) and 5α‐Androstane‐3α,17 β‐diol in Rat Kidney: Heterogeneity of 3α‐Hydroxysteroid Oxidoreductases
Authors: VFRHOEVEN Guido , HEYNS Walter , DE MOOR Pieter
Published: March 2005

3α‐Hydroxysteroid oxidoreductases catalyzing the interconversion between 17β‐hydroxy‐5‐α‐androstan‐3‐one (5α‐dihydrotestosterone) and 5α‐androstane‐3α,17β‐diol (3α‐androstanediol) have been studied in rat kidney. Three enzymes can be distinguished: a soluble NADPH‐dependent oxidoreductase, a microsomal NADPH‐dependent enzyme and a microsomal NADH‐linked enzyme. Traces of the microsomal enzymes are consistently observed in the 108000 × g supernatant. Studies on crude preparations reveal that these enzymes differ not only in subcellular localization and cofactor requirement, but also in optimum pH, kinetic characteristics, sensitivity to potential steroidal inhibitors and sensitivity to detergents, ionic strength and temperature. Moreover, salient sex differences exist in the activity of all three kidney enzymes. The soluble NADPH‐dependent enzyme is more active in female rats whereas both microsomal enzymes are considerably more active in male animals. The microsomal NADH‐dependent oxidoreductase displays favorable characteristics to catalyze the 3α‐dehydrogenation of 3α‐androstanediol. Evidence is presented that it is mainly this enzyme that enables the kidney to use 3α‐androstanediol as an efficient precursor for the local formation of 5α‐dihydrotestosterone.
Protein Phosphorylation of the Smooth and Rough Endoplasmic Reticulum in Normal and Neoplastic Liver of the Rat
Authors: SHARMA Rameshwar K. , McLAUGHLIN Charles A. , PITOT Henry C.
Published: March 2005

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3′:5′‐monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of the rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane‐phosphorylated proteins by gel electrophoresis showed 5 major bands having estimated molecular weights of 155000, 62000, 50000, 46000 and 43000, whereas only 3 major phosphorylated bands with estimated molecular weights of 62000, 50000 and 43000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.
Identification of the 30‐S Ribosomal Proteins at the Decoding Site by Affinity Labelling with a Reactive Oligonucleotide
Authors: LÜHRMANN Reinhard , GASSEN Hans Günter , STÖFFLER Georg
Published: March 2005

Proteins S18 and S1 were identified by immunological and electrophoretic techniques as constituents of the 30‐S ribosomal decoding site by the use of G‐U‐U‐i[3H]acn5U (where i[3H]acn5U =5‐iodo[3H]acetylaminouridine) as a chemical affinity label. S12 was modified to a minor extent and mainly wheel assays were carried out in the absence of tRNA

Evidence for the specificity of the covalent binding of the oligonucleotide probe is given by the following experiments: G‐U‐U‐iacn5U bound twice as much Val‐tRNA as compared to G‐U‐U‐U. The covalent binding was strongly inhibited by prebound poly(U)‐Phe‐tRNA. ‘Programmed’ ribosomes, i.e. ribosomes which contained only covalent bound oligonucleotide, stimulated the binding of Val‐tRNA.

Val‐tRNA bound to G‐U‐U‐iacn5U labelled ribosomes could not be transferred to puromycin. This might indicate that binding of the oligonucleotide occurred at the aminoacyl site.

80–90% of the radioactivity was found in the 30‐S subunit and only the protein moiety of the ribosome was modified.

Unambiguous identification of the labelled proteins was achieved by immunological techniques and revealed proteins S1 and S18 as constituents of the 30‐S ribosomal decoding site.
Activation Parameters of the Adenosine Triphosphatase of Micrococcus lysodeikticus A Comparison of the Soluble and Membrane‐Bound Forms of the Enzyme
Authors: AYALA Juan , NIETO Manuel , CARREIRA Josè , MUÑOZ Emilio
Published: March 2005

The Arrhenius plots for the membrane‐bound ATPase and its soluble form purified from Micrococcus Iysodeikticus, presented discontinuities near 30°C at pH 7.5. Glycerol‐containing lipids were not responsible for these discontinuities. The values of the enthalpies of activation were 12 (soluble) and 22 (membrane‐bound) kcal/mol (50.2 and 92.0 kJ/mol) above 30°C and 42 (soluble) and 29 (membrane‐bound) kcal/mot (175.7 and 121.3 kJ/mol) below that temperature.

The results suggested that both molecular forms of the ATPase were able to adopt at least two different structures, above and below the critical temperature. Of the two, only the hightemperature structure seemed to be enzymically active. In the case of lipid‐dependent ATPases, such as the Escherichia coli enzyme, the transition between both enzyme structures probably occurred with simultaneous “melting” of their lipid microenvironment.
Determination of the Amino‐Acid Sequence of the Ribosomal Protein S8 of Escherichia coli
Authors: STADLER Herbert , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

The primary structure of protein S8 from the 30‐S ribosomal subunit of Escherichia coli was determined mainly by automatic Edman degradation using a modified Beckman protein sequenator and the solid‐phase sequenator of Laursen. The complete sequence, containing 109 amino acids, was derived by analysing peptides from tryptic, chymotryptic, thermolysin, staphylococcal protease and cyanogen bromide digestion of the protein. The amino acid composition was found to be (aspartic acid)6, (asparagine)3, (threonine)5, (serine)s, (glutamic acid)7, (glutamine)6, (proline)s, (glycine)6, (alanine)11, (valine)9, (methionine)4, (isoleucine)7, (leucine)9, (tyrosine)3, (phenylalanine)3, (lysine)11, (arginine)8, (cysteine)1. S8 is a basic protein and binds to the 16‐S RNA; knowledge of its sequence is necessary for a detailed study of its interaction with the ribosomal RNA.
A Study of the Interaction of 4‐Bromomercuriocinnamic Acid with α‐Chymotrypsin by 79Br and 81Br Pulsed Nuclear‐Magnetic Resonance
Authors: GARNETT Michael W. , HALSTEAD Thomas K. , HOARE David G.
Published: March 2005

Br− has been used as a nuclear magnetic resonance (NMR) probe to study the reversible association of α‐chymotrypsin and an Hg‐labelled substrate (4‐bromomercuriocinnamic acid, BrHgCin) which rapidly exchanges Br‐. T1 was measured for 79Br and 81Br, using a pulse spectrometer.

Values of the parameters that determineTl, Obs in aqueous solutions of KBr (pH = 5.5) containing α‐chymotrypsin and BrHgCin are reported. It is found that the rate of Br exchange is diffusion‐limited and faster than the rate of reorientation of the BrHgCin‐α‐chymotrypsin complex.

The rate constant for the formation of the covalent BrHgCin‐α‐chymotrypsin complex determined by this technique agrees well with previously published data. The rapid rate of Br exchange with the complex, however, is incompatible with the side chain of BrHgCin being entirely buried in a nonpolar pocket on the enzyme but compatible with the side chain being exposed to the solution. The contribution to the NMR signal from the non‐covalent complex is negligible.
Binding of a Metyrapone Spin Label to Microsomal Cytochrome P‐450
Authors: RUF Hans Heinrich , NASTAINCZYK Wolfgang
Published: March 2005

Probing of the active site of microsomal cytochrome P‐450 was carried out with a spin label derived from 2‐methyl‐1,2‐bis(3‐pyridyl)‐1‐propanone (metyrapone). Its optical binding spectra to cytochrome P‐450 resemble the spectra with metyrapone. The electron paramagnetic resonance spectrum of the Spin label in microsomes indicates binding with strong immobilization. Since the apparent optical and EPR binding constants agree very well (Ks ∼ 2 10−5 M), and metyrapone is found to displace the spin label, we conclude, that the spin label binds to the active site of cytochrome P‐450. Addition of detergents or an increase in temperature mobilizes the bound spin label slightly.

The EPR signal accounts for only 60% of the bound spin label due to its dipolar interaction with the low‐spin ferric heme of cytochrome P‐450. From this finding, the distance between nitroxide and iron is evaluated to be 11 Å. This supports the model that one of the pyridine nitrogens of metyrapone is coordinated to the iron of cytochrome P‐450. The bound spin label shows virtually no interaction with ferricya'nide as if the active site of cytochrome P‐450 is a hydrophobic pocket not accessible to ions.
Cooperative Effects of Initiation Factors and fMet‐Trna in the Formation of the 40‐S Initiation Complex
Authors: VAN DER HOFSTAD Gerard A. J. M. , FOEKENS John A. , VAN DEN ELSEN Peter J. , VOORMA Harry O.
Published: March 2005

In this paper the mode of action of IF‐1 in 40‐S initiation complex formation was studied with MS 2 RNA as messenger. Using initiation factors IF‐2 and IF‐3 labeled in vitro it appeared that IF‐1 did not influence the binding of these factors in the absence of fMet‐tRNA. However, in the presence of fMet‐tRNA it was found that the enhancement of the fMet‐tRNA binding by IF‐1 was accompanied with an equimolar increase in binding of IF‐2. Moreover, it appeared that also in absence of IF‐1, fMet‐tRNA binding is coupled with an equimolar enhancement of the IF‐2 binding, which suggests the existence of a preribosomal complex between IF‐2 and fMet‐tRNA. The apparent Km values for both the binding of fMet‐tRNA and IF‐2 to 30‐S subunits were determined and appeared to be equal, which makes a functioning of such a preribosomal complex in protein initiation very likely. The participation of GTP in this complex will be discussed. Functions of IF‐1 in dissociation and recycling of IF‐2, described by others, and the stimulation on the 30‐S subunit level might well be explained as pleiotropic effects of one basic action of IF‐1, i.e. a conformational change of 30‐S subunits.
Bacillus subtilis Phage φ 29 Characterization of Gene Products and Functions
Authors: CARRASCOSA José L. , CAMACHO Ana , MORENO Felipe , JIMÉNEZ Fernando , JIMÉNEZ Fernando , VIÑUELA Eladio , SALAS Margarita
Published: March 2005

A total of 22 φ 29‐induced proteins have been resolved by slab gel electrophoresis; two of these proteins are the precursor and product fragment, respectively, in the synthesis of the neck appendage protein of the phage. The protein products of 10 out of the 17 cistrons detected in the genome of phage φ 29 have been identified. Mutants in two other cistrons fail to synthesize two proteins. Mutants in six genes to not synthesize phage DNA. A cistron, probably involved in the final lysis of the infected bacteria, has been found. Mutants in this gene give place, under restrictive conditions, to delayed lysis and produce, after artificial lysis, a burst size similar or higher than that obtained after wild‐type phage infection.
Glycopeptides from Bovine Liver Basement Membrane and Plasma Membrane
Authors: GIBBONS Richard A. , DIXON Stephen N. , SELLWOOD Richard
Published: March 2005

Proteolytic digests of liver plasma‐cell membranes from the cow were fractionated to yield two homogeneous glycopeptides and a third preparation about 92% pure. The composition of the two homogeneous glycopeptides made it clear that they were derived from basement membrane material rather than the plasma membrane. Ruminants are unusual in having large amounts of basement membrane in the liver while other animals generally have little or none.

Both basement‐membrane‐derived glycopeptides contained a glucosyl galactosyl disaccharide linked to hydroxylysine, the smaller one contained no other sugar structure but the larger one contained in addition an acidic heterosaccharide, the two chains probably being linked separately to the same molecule. Smith degradation and β‐elimination operations show that this heterosaccharide has an inner structure containing mannose and hexosamine, with the sugars galactose, N‐glycollyl‐neuraminic acid and fucose situated more peripherally. The amino‐acid‐hetero‐saccharide linkage is alkali stable.

The third glycopeptide, which may be plasma‐membrane‐derived, differs from the heterosaccharide saccharide described above in that it contains no glucose and contains some O‐seryl and O‐threonyl amino‐acid‐sugar linkage. It, too, has a periodate‐resistant structure of hexosamine and mannose.
Reconstitution of Chemically Synthesized Ribooligonucleotides with Naturally Occurring tRNA Fragments
Authors: OHTSUKA Eiko , NISHIKAWA Satoshi , IKEHARA Mono , TAKEMURA Shosuke
Published: March 2005

Chemically synthesized yeast tRNA terminal fragments were reconstituted with natural tRNA fragments which were obtained by partial digestion with RNase T1. The synthetic 3′‐nonanucleotide (I) accepted alanine (3 % with respect to the intact tRNA) when combined with a 4‐fold excess of the natural 5′‐quarter and the chemically synthesized hexanucleotide (II) stimulated the aminoacylation of the natural 3′‐half molecule.
DNA‐Dependent RNA Polymerase C from Xenopus laevis Ovaries Ability to Transcribe Intact Double‐Stranded DNA
Authors: LONG Eric , DINA Dino , CRIPPA Marco
Published: March 2005

DNA‐dependent RNA polymerase C, partially purified from Xenopus laevis ovaries, has been resolved by DEAE‐Sephadex chromatography in two forms, eluting at 0.2 M and 0.3 M ammonium sulfate, respectively. Both are sensitive to high concentrations of α‐amanitin (200 μg/ml). Their ionic strength dependence and divalent cation requirements are indistinguishable. Quantitatively, RNA polymerase C represents the major form of RNA polymerase activity solubilized from the ovaries.

Both RNA polymerases C are able to transcribe efficiently either high‐molecular‐weight Xenopus DNA or intact adenovirus DNA, as compared to nicked DNA. In contrast, RNA polymerase A has little activity on an intact DNA template. The salt dependence of the RNA polymerases C activity is different on the two kinds of template. Nicked DNA is efficiently transcribed up to a salt concentration of 100 mM ammonium sulfate. On intact DNA, optimal transcription is obtained at 40 mM ammonium sulfate and is inhibited by higher salt concentrations.