bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross‐linking of Histone H1 in Chromatin
Authors: THOMAS Jean O. , KHABAZA Andrew J. A.
Published: July 2010

Arrays of neighbouring histone H1 molecules are present in rat liver chromatin, and can be cross‐linked to each other by bisimidoesters. The product, poly(H1), can be extracted from crosslinked chromatin in high yield with 5% perchloric acid and thus distinguished from cross‐linked oligomers of the core histones. On analysis in dodecyl sulphate/polyacrylamide gels poly(H1) gives a striking pattern of alternating strong and weak bands which have been shown, by the use of cleavable cross‐linked reagents and two‐dimensional gel electrophoresis, to arise respectively from H1 homopolymers and H1 polymers linked to the nucleosome core histones.

H1‐H1 proximities as measured by cross‐linking exist both at low ionic strength where the nucleosome filament is extended, and at higher ionic strengths at which it is folded into a 30‐nm‐diameter fibre, probably in the form of a solenoid, although some additional H1‐H1 contacts seem to occur in the folded form. A similar pattern of H1‐H1 cross‐linking is observed for nucleosome oligomers too short to form a complete turn of a solenoid. The lack of any strong dependence of the H1 cross‐linking pattern on ionic strength, and the results for short oligomers, suggest that in a soleniud the major H1‐H1 interactions are lateral (i.e. they occur between neighbouring nucleosomes along a turn) although additional vertical contacts (between successive turns) are not excluded. For a short nucleosome oligomer containing n nucleosomes, the number of H1 molecules that may be cross‐linked into an array at low ionic strength (∼ 15 mM) is n, consistent with the presence of one H1 per nucleosome.

H1‐H1 cross‐linking occurs only within the chromatin framework; when cross‐linking is carried out in 0.5 M NaCl so that H1 is dissociated from chromatin, no poly(H1) is formed. When H1 which has been dissociated in 0.5 M NaCl is allowed to reassociate by gradual lowering of the ionic strength, the poiy(H1) is identical with that in native chromatin, suggesting faithful rebinding of H1 molecules. H1‐H1 proximities do not seem to be mediated by the presence of bound high‐mobility‐group non‐histone proteins because the poly(H1) pattern persists when cross‐linking is carried out at 365 mM ionic strength, when these high‐mobility‐group proteins are dissociated from chromatin.

The H1 cross‐linking pattern described here could prove to be a useful assay for the native arrangement of H1 molecules in reconstituted chromatin and for changes in H1‐H1 contacts that might result from cell‐cycle modifications (e.g. phosphorylation) of H1. It may not prove a good assay for higher‐of der structure because of the relative insensitivity to ionic strength of the poly(H1) pattern as studied here. A detailed analysis will be necessary to determine whether there are any subtle differences in the interactions between H1 molecules in the folded and extended nucleosome filament.
Non‐specific Lanosterol and Hopanoid Biosynthesis by a Cell‐Free System from the Bacterium Methylococcus capsulatus
Authors: ROHMER Michel , BOUVIER Pierrette , OURISSON Guy
Published: July 2010

A cell‐free system from the bacterium Methylocoecus capsulatus was incubated with [12‐3H]squalene; diploptene and diplopterol, normally present in the bacterium, were labelled.

The same cell‐free system was incubated with (RS)‐2,3‐epoxy‐2,3‐dihydro‐[3‐3H]squalene. Several radioactive 3‐hydroxytriterpenes were purified. Lanosterol, which is normally present in this bacterium, was found labelled as well as 3‐epilanosterol. In addition, radioactive 3α‐hydroxy and 3β‐hydroxydiploptene were formed.

These data may be explained by the coexistence of two cyclases in M. capsulatus: a squalene/hopane cyclase and a squalene epoxide/lanosterol cyclase. The squalene cyclase exhibits the same lack of substrate specificity as those of Acetobacter pasteurianum and Tetrahymena pyriformis, i. e. in addition to its normal substrate squalene, it can cyclize the two enantiomers of squalene epoxide into 3‐hydroxyhopanoids.

The presence of a squalene epoxide/lanosterol cyclase activity, which was suspected in view of the unique 3β‐hydroxy 4α‐methyl steroids of M. capsulatus, was demonstrated by the labelling of lanosterol. More surprisingly 3‐epilanosterol was also present and labelled. We showed that this does not derive from lanosterol by isomerization via a 3‐oxo compound. Therefore the squalene epoxide cyclase of M. capsulatus, like the one of eukaryotes cyclizes the (3S) enantiomer of squalene epoxide into lanosterol. But it is definitely less substrate‐specific as it can also cyclize the (3R) enantiomer into 3‐epilanosterol.
Bovine Kidney Pyruvate Dehydrogenase Complex
Authors: KRESZE Georg‐B. , RONFT Hedwig
Published: July 2010

Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (Mr 26000) and a fragment with apparent Mr of 45000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately.

A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of Mr 26000 and another fragment of apparent Mr 45000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of Mr 36000 or lower.

The enzyme‐bound lipoyl residues of the pyruvate dehydrogenase complex have been labelled covalently by incubation with [2‐14C]pyruvate. After treatment of this [14C]acetyl‐enzyme with papain, elastase, or trypsin, radioactivity was associated exclusively with the 45000‐Mr and 36000‐Mr fragments but not with the active 26000‐Mr fragment.

It is concluded that the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of two dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center foi‐transacylation whereas the other possesses the enzyme‐bound lipoyl residues.
Microtubule‐Associated‐Protein MAP1 is not Implicated in the Polymerization of Microtubules
Authors: VILLASANTE Alfredo , de la TORRE Javier , MANSO‐MARTINEZ Rafael , AVILA Jesús
Published: July 2010

Addition of DNA to microtubule protein solutions results in a decrease of the amount of the high‐molecular‐weight microtubule‐associated proteins (MAP1 and MAP2) available for polymerization. Complete removal of MAP1 from microtubules requires lower concentrations of DNA compared to MAP2. Removal of MAP1 from microtubules has little or no effect on microtubule polymerization (nucleation and propagation). In contrast, removal of MAP2 produces a marked decrease in microtubule polymerization.

Incubation of microtubular protein with DNA results in complete disappearance of the 30‐S ring‐shaped oligomers at concentrations of DNA which are not markedly inhibitory of polymerization.
Identification and Characterization of the Translationally Repressed Cytoplasmic Globin Messenger‐Ribonucleoprotein Particles from Duck Erythroblasts
Authors: VINCENT Alain , CIVELLI Olivier , MAUNDRELL Kinsey , SCHERRER Klaus
Published: July 2010

Globin messenger ribonucleoprotein (mRNP) particles which have been isolated from duck erythroblast post‐polyribosomal supernatant are translationally inactive in vivo and in vitro but contain translatable mRNA active after deproteinisation. They were characterized following pruification by successive sucrose gradient sedimentation in a buffer containing 0.05 M KCI. The complex, which sediments homogeneously at about 20 S, has a density of 1.39 g/cm3 and thus consists of four parts protein to one part RNA; 40% of this RNA is globin mRNA and no other mRNA could be detected.

Sedimentation of the purified globin mRNP on sucrose gradients in 0.5 M KCl produced four components while polyacrylamide gel electrophoresis in non‐denaturing conditions and in the presence of EDTA resulted in the separation of three components. Hybridization to globin cDNA and translation in vitro of the RNA extracted from these subparticles revealed the existence of two core parcticles containing globin mRNA with nominal sedimentation coefficients of 13 S and 16 S.

Analysis of the protein components of the isolated sub‐complexes by dodecyl sulfate and bidimensional gel electrophoresis indicated a very characteristic protein composition for each of these complexes. The 16‐S and 13‐S globin mRNPs differed essentially by the presence in the 13‐S mRNP only of a group of major polypeptides. Of the other two sub‐complexes, one consisted of 90% small RNA in the 4‐S range; the second sedimented ahead of the globin mRNP core particles at about 19 S and consisted of a very characteristic set of about 14 polypeptides. The polyrisohomal 73000‐Mr poly(A)‐binding protein was not detected in the purified free globin mRNP although the PIRNA in the untranslatable particle is polyadenylated.

The presence in the cytoplasm of duck erythroblasts of two forms of untranslated globin messenger ribonucleoprotein particles, distinct in their protein composition from polyribosomal globin mRNP, suggests that they may have a specific role in the regulation of translation of globin mRNA.
Translation of Zea mays Endosperm Sucrose‐Synthase mRNA in vitro
Authors: WÖSTEMEYER Johannes , BEHRENS Ute , MERCKELBACH Armin , MÜLLER Markus , STARLINGER Peter
Published: July 2010

mRNA of 23‐day‐old maize endosperm was translated both in wheat germ extracts and rabbit reticulocyte lysates. A protein with an apparent molecular weight of 88000 comigrates in dodecylsulfate/polyacrylamide electrophoresis with sucrose synthase. This protein is precipitated with an antiserum against sucrose synthase and shows the same protease digestion pattern as the enzyme. It is not synthesized with mRNA extracted from sh/sh mutant kernels lacking sucrose synthase. By these criteria, the protein is the translation product in vitro of sucrose synthase mRNA.

The separation of mRNA in methylmercury‐hydroxide‐agarose gels and subsequent translation indicates a length of sucrose synthase mRNA of 2800 nucleotides which is compatible with the coding length necessary for a protein with a molecular weight of 88000 plus untranslated sequences.
Isolation of Two Hydrogenase Activities in Chromatium
Authors: LLAMA Maria J. , SERRA Juan L. , RAO K. Krishna , HALL David O.
Published: July 2010

Kinetic, chromatographic and electrophoretic studies of Chromatium hydrogenase show the existence in vitro of two different activities (I and II). The two hydrogenases exhibit different kinetic parameters and properties. Using reduced methyl viologen, Km and [S]0.5 values of about 20 μM and 360 μM were calculated for the hydrogenases I and II, respectively. Hill plots revealed that hydrogenase I followed classical hyperbolic (Michaelis‐Menten) kinetics. However, a Hill coefficient (h= 0.68) indicating non‐hyperbolic kinetics could be shown for hydrogenase II. After several purification steps hydrogenase II still showed kinetics typical of the action of either (a) two enzymes each of which shows Michaelis‐Menten kinetics but with different substrate affinities or (b) only one enzyme which shows apparent negative cooperative regulation. The molecular weights of the hydrogenases were about 37000 (I) and 55000 (11) when determined by gel filtration. Sodium dodecyl sulphate; polyacrylamide gel electrophoresis revealed that both enzymes give a coincidental single protein band with the same relative mobility indicating a molecular weight of 31000. Both hydrogenases were able to catalyse the reversible activation of H2 in the presence of artificial electron carriers but with different rates, hydrogenase II being much more active in the H2‐uptake mode. The kinetic properties and molecular weight of hydrogenase II are partially modified by high ionic strength resulting in an increased substrate affinity and Hill coefficient and thus resembling hydrogenase I. These results are interpreted as due to the existence in vitro of monomeric and dimeric forms of Chrontatium hydrogenase.
Spectroscopic Properties of Light‐Chain Derivatives of Murine MOPC‐315 Immunoglobulin A
Authors: ZIDOVETZKI Raphael , FARVER Ole , PECHT Israel
Published: July 2010

Three light‐chain derivatives of the homogeneous IgA, secreted by the mouse myeloma MOPC‐315, were studied employing circular dichroism and thermal‐perturbation spectroscopy: (a) the light‐chain dimer with intact native inter‐chain disulfide bond, L2.cov (b) the light‐chain dimer with this bond reduced and alkylated, LL2.ncov and (c) the dimer of only the variable regions of the light chains, (VL)2. Comparison of the well resolved circular dichroism spectra of these derivatives allowed the assignments of the bands above 290 nm to the following chromophores: Trp‐35L and Trp‐91L in the variable domains, and Trp‐148L Trp‐185L and the disulfide of Cys‐214L in the constant domains. The differences in the spectral characteristics of L2.cov as compared to those of L2.ncov and (VL)2 illustrate the significant influence of the disulfide bridge on the conformation of the L2.cov. Pronounced differences are found between these light‐chain derivatives and light chain‐heavy chain associates, namely the intact protein M‐315 and its Fv fragment.

The comparison between the CD spectra of the free and the hapten‐bound L2.cov, L2.ncov and (VL)2 directly demonstrates the existence of the conformational transitions in these proteins induced by hapten binding.
A Native‐Like Intermediate on the Ribonuclease A Folding Pathway
Authors: SCHMID Franz X.
Published: July 2010

Unfolded ribonuclease A consists of a mixture of fast‐folding (UF) and slow‐folding species (US); US folds slowly because it contains non‐native proline isomers. The folding kinetics of US have been determined by measuring the changes in tyrosine fluorescence upon refolding. Under conditions where the energy of stabilization of the native protein is small (in marginally native conditions) the fluorescence‐detected kinetics of folding coincide with the ones measured by other probes. However, when refolding is carried out under conditions where the native protein is highly stabilized (in strongly native conditions), the fluorescence changes become progressively slower than folding monitored by tyrosine absorbance or by 2′CMP binding. Under these conditions the fluorescence‐detected kinetics of folding show properties of proline isomerization reactions. In addition they match proline isomerization measured during refolding of Us by a two‐step assay for non‐native proline isomers.

The following conclusions are drawn. (1) There is a kinetic intermediate between US and the native enzyme in the folding pathway of RNase A. This has been shown by the divergent progress curves monitored by different probes. Although the intermediate contains non‐native proline isomers, it is folded and hence sensitive to agents that affect the stability of native proteins. (2) Fluorescence‐detected folding closely follows proline isomerization during folding. Refolding of US is not blocked by incorrect prolinc isomers, but US is able to fold to a structural intermediate before proline isomerization occurs. (3) The quantum yield of the fluorescence of native RNase A depends on the precise spatial location of the tyrosine side chains with respect to their hydrogen bond acceptors and to the disulfide bonds. Therefore the native fluorescence is not regained until all essential proline peptide bonds are in the right configuration.
A Native‐Like Intermediate on the Ribonuclease A Folding Pathway
Authors: SCHMID Franz X. , BLASCHEK Heidi
Published: July 2010

A folded intermediate IN has been detected in the slow refolding reaction of ribonuclease A, US→N, where US is an unfolded species which contains non‐native isomers of X‐Pro peptide bonds and N is the native enzyme. IN has been characterized previously as a structured folding intermediate: it shows a tyrosine absorbance like native ribonuclease, but it still contains at least one non‐native proline peptide bond and its tyrosine fluorescence differs from the native enzyme.

Here we have used three probes to characterize further the structure of IN in comparison to native ribonuclease A. (1) The accessibility of the tyrosine hydroxyl groups to ionization has been investigated in IN. (2) The binding of the specific inhibitor cytidine 2′‐phosphate (2′CMP) to IN was measured. (3) The regain of enzymatic activity during folding was monitored to decide whether IN is an enzymatically active intermediate.

The results indicate that IN possesses a compact folded structure which shields part of the tyrosine residues from the aqueous environment like native ribonuclease A. The active‐site region of IN has folded to the native conformation. IN binds 2′CMP as well as native ribonuclease A does and it shows enzymatic activity similar to the native enzyme.

We conclude that unfolded molecules, which have only proline‐93 in the non‐native trans configuration, can refold to the native‐like intermediate IN before proline isomerization takes place. Proline‐93 is located far away from the active‐site region in an external bend and therefore does not block folding. It isomerizes in the folded molecule to the native cis configuration.
360‐MHz Nuclear Magnetic Resonance and Laser Photochemically Induced Dynamic Nuclear Polarization Studies of Bile Salt Interaction with Porcine Colipase A
Authors: COZZONE Patrick J. , CANIONI Paul , SARDA Louis , KAPTEIN Robert
Published: July 2010

Porcine pancreatic colipase possesses a particular three‐dimensional surface domain containing six out of the seven aromatic residues of the molecule in a highly hydrophobic environment [P. Canioni and P. Cozzone (1979) Biochimie (Paris) 61, 343–354; J. Wieloch, B. Borgström, K. E. Falk and S. Forsen (1979) Biochemistry, 18, 1622–1628]. The domain corresponds to the 49–57 and 77–86 β‐sheet fragments brought into spatial proximity by protein folding [P. Canioni, P. Cozzone and L. Sarda (1980) Biochim. Biophys. Acta, 621, 29–42]. The identification of this specific domain as being the lipid binding site on colipase is proposed on the basis of the NMR and photochemically induced dynamic nuclear polarization (photo‐CIDNP) studies of the complexes of colipase with organized bile salt micelles.

The binding of taurodeoxycholate micelles specifically perturbs the proton NMR ring resonances of Tyr‐I and Tyr‐II (Tyr‐56 and Tyr‐57) and His‐II (His‐86) together with several aliphatic resonances, reflecting the involvement of the hydrophobic aromatic domain in micelle fixation. In the presence of colipase, specific shifts and broadening of the methyl groups at position 18 and 21 of the sterane ring of taurodeoxycholate are observed and suggest that the hydrophobic side of the bile salt is primarily involved. The strong photo‐CIDNP effects of Tyr‐I and Tyr‐II in the presence of lumiflavin dye, which have been described on free colipase [P. Canioni, P. Cozzone and R. Kaptein (1980) FFBS Lett. 111, 219–222] are totally suppressed in the presence of taurodeoxycholate or chenocholamine micelles, indicating that due to the protection by the bile salt, the aromatic surface residues are no longer accessible. The pH dependence of the NMR perturbations and photo‐CIDNP effects observed in the colipase‐micelle complexes confirms that (1) the hydrophobic aromatic domain is directly involved and (2) the driving force of the primary micelle binding is essentially hydrophobic.

A general model for micelle binding to colipase is proposed and involves a two‐step mechanism. Initially, the lipid hydrophobic binding site participates in the building of the bound taurodeoxycholate aggregate to form a mixed micelle through hydrophobic surface interactions (stoichiometric complex). At higher bile salt concentrations, polar forces might account for further growth of the micellar structure bound on colipase.
Heterogeneity of the Glycans O‐Glycosidically Linked to the Hinge Region of Secretory Immunoglobulins from Human Milk
Authors: PIERCE‐CRETEL Annick , PAMBLANCO Mercedes , STRECKER Gérard , MONTREUIL Jean , SPIK Geneviève
Published: July 2010

Pure secretory immunoglobulin A was isolated from human milk by fractionation in gradients of pH and (NH4)2SO4 concentration followed by gel filtration. The hinge region containing all the O‐glycosidically linked oligosaccharides was isolated en bloc after trypsin and pepsin hydrolysis and separated by gel filtration. The mixture of O‐glycosidically linked oligosaccharides contained N‐acetylneuraminic acid (NeuAC), fucose (Fuc), galactose (Gal). N‐acetylglucosamine (GlcNAc) and N‐acetylgalactosamine (GalNAc) in the molar ratio of 0.2:0.5:2.5:2:1 respectively. After β‐elimination several oligosaccharides were separated by a combination of ion‐exchange chromatography and gel‐filtration chromatography. The complete structure of four of these oligosaccharides was determined by methanolysis, methylation and mass spectrometry.

The structure of the four oligosaccharides which arc linked to serine or threonine residues of the hinge region are as follows: β‐Gal‐(1→3)‐GalNAc‐ol; α‐HeuAc‐(2→3)‐β‐Gal‐(1→3)‐GalNAc‐ol; β‐Gal‐(1→3)‐[β‐GlcNAc‐(1→6)]‐GalNAc‐ol; β‐Gal‐(1→3)‐[β‐Gal‐(1→4)‐β‐GlcNAc‐(1→6)]‐GalNAc‐ol.

These oligosaccharides are more complex and heterogenous than the oligosaccharides linked to serine residues of the hinge region from myeloma seram immunoglobulin A1.
Specificity of Twelve Lectins Towards Oligosaccharides and Glycopeptides Related to N‐Glycosylproteins
Authors: DEBRAY Henri , DECOUT Dominique , STRECKER Gérard , SPIK Geneviève , MONTREUIL Jean
Published: July 2010

Glycopeptides and oligosaccharides of either the N‐acetyllactosaminic or the oligomannosidic type derived from glycoproteins containing the N‐glycosylamine linkage were used to define the specificity of different lectins (concanavalin A, Lens culinaris agglutinin, Vicia faba agglutinin, Pisum sativum agglutinin, Ricinus communis agglutinins, soybean agglutinin, wheat germ agglutinin, Solanum tuberosum agglutinin, Datura stramonium agglutinin, Lotus tetragonolobus agglutinin, Ulex europeus agglutinin) by studying the inhibition of human red blood cell agglutination by these structures. The results obtained show that lectins considered ‘identical’ in terms of monosaccharide specificity, possess the ability to recognize fine differences in more complex structures. In fact, different lectins are able to recognize different saccharidic sequences on the same glycan structure. As these sequences are likely to be common to numerous glycoproteins, including cell membrane glycoproteins, the results obtained with lectins in the study of cell surface carbohydrates have to be very carefully interpreted.

Moreover, our results confirm previous data on the spatial configuration of the glycan moiety of glycoproteins deduced from the construction of molecular models; the fact that oligosaccharides bearing an α‐NeuAc‐(2→6)‐Gal unit are more powerful inhibitors than oligosaccharides bearing an α‐NeuAc‐(2→3)‐Gal unit could be related to the high rotational freedom of α‐2, 6 linkage; the observation that glycoasparagines, glycopeptides and glycoproteins possess a higher affinity for lectins than the related oligosaccharides could be explained by the fact that the glycan‐amino acid linkage leads to structures more rigid than those of the oligosaccharides themselves.
Isolation and Characterization of α‐Amylase Messenger RNA from Bank Vole Parotid Glands
Authors: VÜST Jens , THOMSEN Karl K. , LUND Torbert
Published: July 2010

Bank vole saliva contains two glycogen‐precipitable proteins, both of which show affinity for the α‐amylase inhibitor cycloheptaamylose. One of these proteins, amylase, has a molecular weight of 55000, judged from dodecylsulphate/acrylamide gel electrophoresis. The other has an apparent molecular weight of 59000 and has no amylase activity. We report here that tryptic peptide maps as well as amino‐acid composition analyses indicate extensive homology between the two proteins. We have also isolated total poly(A)‐containing mRNA from amylase‐rich bank vole parotid glands. These mRNAs were translated in the presence of [35S]methionine in an mRNA‐dependent cell‐free translation system from rabbit reticulocyte lysate. The radioactive translation products were examined by dodecylsulphate/polyacrylamide gel electrophoresis. Two major translation products with apparent molecular weights of approximately 56500 and 60500, respectively, were further characterized by tryptic peptide analyses. Our data indicate that the 56500‐Mr product is the biosyntetic precursor of amylase, whereas the 60500‐Mr translation product is a precursor of the 59000‐Mr amylase‐like protein. Both precursors appear to contain extra peptide material, presumably as amino‐terminal ‘pre’ or ‘signal’ peptides, in analogy with that found for other precursors of secretory proteins. Thus, amylase and the 59000‐Mr protein, although very similar, are translated from two separate mRNAs. These two messengers sediment in a sucrose gradient at about 17‐S, corresponding to lengths of about 1800 nucleotides.
An α‐Amanitin‐Resistant DNA‐Dependent RNA Polymerise II from the Fungus Aspergillus nidulans
Authors: STUNNENBERG Hendrik G. , WENNEKES Lambertus M. J. , SPIERINGS Titus , van den BROEK Hendrikus W. J.
Published: July 2010

An α‐amanitin‐resistant DNA‐dependent RNA polymerise II has been purified from the lower eukaryote Aspergillus nidulans to apparent homogeneity by extraction of the enzyme at low salt concentration, polymin P (polyethylene imine) fractionation, binding to ion‐exchangers and density gradient centrifugation. By this procedure 0.4 mg of RNA polymerise II can be purified over 6000‐fold from 500 g (wet weight) of starting material with a yield of 25% and a specific activity of 550 units/mg.

The subunit composition has been resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by two‐dimensional gel electrophoresis using a non‐denaturing gel in the first dimension and a dodecylsulphate slab gel in the second dimension. The putative subunits have molecular weights of 170000, 150000, 33000, 27000, 24000, 19000, 18000 and 16000. Only one form of RNA polymerise II could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerise II are clearly different from RNA polymerise I from A. nidulans but throughout comparable with other class II enzymes. It differs from all other class II enzymes by its insensitivity towards the toxin α‐amanitin, even at concentrations up to 400 μg/ml, and appears to be unable to bind O‐[14C]methyl‐γ‐amanitin at a concentration of 10 μg/ml of the toxin.

We conclude that the purified RNA polymerise from A. nidulans is a real, but exceptional, type of the class II RNA polymerises.
The Organization of Cholesterol Esters in Membranes of Mycoplasma capricolum
Authors: MELCHIOR Donald L. , ROTTEM Shlomo
Published: July 2010

The organization of cholesterol esters in Mycoplasma capricolum membranes was studied by differential scanning calorimetry. Cells grown in the presence of horse serum incorporated large amounts of cholesterol esters into their membranes. The cholesterolester‐containing membranes after incubation at low temperature showed an endotherm characteristic of a cholesterol ester crystalline→isotropic liquid transition that was identical in membranes both before and after thermal protein denaturation. This transition was not observed in membranes of cells grown in medium in which the horse serum was replaced by bovine albumin, fatty acids and unesterified cholesterol unless cholesterol esters were added to the growth medium. In membrane preparations obtained both from cells grown in horse serum and from cells grown with bovine albumin plus cholesterol and fatty acids, the free cholesterol content was sufficient to eliminate the bilayer order/disorder transition observed in isolated membrane phospholipids. Our studies indicate that the majority of cholesterol esters in M. capricolum membranes is not present in attached serum lipoprotein particles, nor is intimately associated with membrane protein, but exists as relatively large cholesterol ester droplets or pockets tightly associated with the membrane. The cholesterol esters in these pockets appear relatively pure, although the presence of small amounts of other membrane components is likely.
The Effect of the Chemical Nature of the Side Chains of the Amatoxins in the Inhibition of Eukaryotic RNA Polymerase B
Authors: WIELAND Theodor , GÖTZENDÖRFER Christa , VAISIUS Alain C. , ZANOTTI Giancarlo
Published: July 2010

The inhibition constants (Ki) of DNA‐dependent RNA polymerase B (or II) from calf thymus were measured for eight synthetically obtained bicyclic amanitin‐like thioethers, two R‐sulfoxides, and two S‐sulfoxides. These Ki values were compared with those of α‐amanitin, its 6′‐O‐methylether Ia (an R‐sulfoxide), the S‐sulfoxide, the sulfone, the S‐deoxo derivative (Id) of IA, and several previously described amatoxins. The necessity of a β‐methyl side chain in position 3 and a hydroxy group in proline‐2 was confirmed. Additionally, the presence of an isoleucine side chain in position 6 and the absence of a side chain in position 5 was recognized as important for binding to the enzyme. In the three sulfoxide samples examined, the R‐diastereomer was found to be a stronger inhibitor than the S‐form. The contribution of every structural element to biological activity has been discussed.
Murein Biosynthesis and O‐Acetylation of N‐Acetylmuramic Acid during the Cell‐Division Cycle of Proteus mirabilis
Authors: GMEINER Jobst , KROLL Hein‐Peter
Published: July 2010

The murein of Proteus mirabilis is unique with respect to the O‐acetylation of part of its N‐acetylmuramic acid residues. Working with synchronized cells and providing radioactive N‐acetylglucosamine with the medium, the murein was pulse‐labelled in vivo for 10‐min periods at different times of the cell cycle. After isolation of murein and enzymatic cleavage with endo‐N, O‐diacetylmuramidase from Chalaropsis, the distribution of radioactivity between uncrosslinked murein subunits (monomers) and the peptide‐crossliitked dimers was analyzed. The following results were obtained.

Murein synthesized at different times during the cell cycle did not show significant variation regarding the degree of crosslinkage and the degree of O‐acetylation. However, the degree of O‐acetylation of pulse‐labelled murein was found to be about sevenfold lower than that of murein from continuously labelled asynchronous cultures.

A pulse‐chase experiment revealed that only non‐O‐acetylated murein subunits were incorporated into the growing murein sacculus, part of which became O‐acetylated subsequently. Since a certain amount of newly incorporated subunits was lost during the chase, which reappeared in the sacculus about one generation later, limited murein turnover during the cell cycle became evident.

The degree of crosslinkage of both the O‐acetylated as well as the non‐O‐acetylated newly synthesized murein increased during subsequent growth.

The labelled peptide‐crosslinked dimers were cleaved by an endopeptidase isolated from P. mirabilis. The distribution of radioactivity between the resulting non‐O‐acetylated and O‐acetylated monomers indicated that most of the mono‐O‐acetylated dimer fraction carried radioactivity exclusively in the non‐O‐acetylated subunit which was not O‐acetylated during subsequent murein biosynthesis.

Since only a small portion of the labelled mono‐O‐acetylated dimer fraction carried radioactivity in both subunits, and only at specific times of murein biosynthesis, it was concluded that thin portion of the labelled mono‐O‐acetylated dimer fraction represented an intermediate in the O‐acetylation process.

From these results a defined sequence of steps of murein biosynthesis is proposed.
Purification and Characterization of DNA‐Dependent ATP Phosphohydrolases from KB Cells
Authors: DE JONG Peter J. , VAN DER VLIET Peter C. , JANSZ Hendrik S. , Jan TOMMASSEN P. M.
Published: July 2010

DNA‐dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single‐stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA‐dependent ATPases into three activities designated ATPase I. II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140000. ATPase I was further purified by gradient elution on ATP‐agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gele electrophoresis revealed only one broad polypeptide band co‐sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66000, 68000, 70000 and 71000. Comparison of the native molecule weight (140000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme.

The purified enzyme has an apparent Km of 0. 5 mM for ATP producing ADP + Pi. A maximum activity of 2100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent canons (Mg2+ > Ca2+ > Mn2+ > Co2+). A broad pH optimum (pH 6–8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.
Kinetic Studies with Rat‐Brain Succinic‐Semialdehyde Dehydrogenase
Authors: RIVETT A. Jennifer , TIPTON Keith F.
Published: July 2010

A simple procedure is described that gives an approximately 100‐fold purification of rat brain succinic‐semialdehyde dehydrogenase with a high yield. The enzyme exhibits a relatively low Km value for succinic semialdehyde (2. 5 μM) and is inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD+ (Ki= 150 μM), p‐Hydroxybenzaldehyde was shown to give competitive inhibition with respect to succinic semialdehyde and uncompetitive inhibition with respect to NAD+. Initial rate studies in the presence of a fixed concentration of this inhibitor allowed a more accurate estimation of the kinetic parameters for the uninhibited reaction. The results of these studies, together with analysis of the dead‐end inhibition by AMP and the effects of NAD+ and 3‐acetylpyridine‐adenine dinucleotide as alternative acceptors in the reaction, were consistent with the enzyme‐catalysed reaction obeying a compulsory‐order mechanism in which NAD+ was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it.
Studies on the Mechanism of Pyrophosphate‐Mediated Uptake of Iron from Transferrin by Isolated Rat‐Liver Mitochondria
Authors: KONOPKA Krystyna , ROMSLO Inge
Published: July 2010

Respiring rat liver mitochondria accumulate iron released from transferrin by pyrophosphate. The amount of iron accumulated is 1–1. 5 nmol mg protein−1 h−1, or approximately 60% of the amount of iron mobilized from transferrin.

The uptake declines if respiration is inhibited, substrate is deleted, or the experiments are run under anaerobic conditions. Substrate depletion and respiratory inhibitorsare less inhibitory under anaerobic conditions.

More than 80% of the amount of iron accumulated by aerobic, actively respiring mitochondria can be chelated by bath ophenanthroline sulphonate, and with deuteroporphyrin included, up to 30% of the amount of iron accumulated is recovered as deuteroheme. Iron accumulated by respiration‐inhibited mitochondria under aerobic conditions is not available for heme synthesis.

With time the uptake of iron increases eightfold relative to the uptake of pyrophosphate.

The results are compatible with a model in which ferric iron is mobilized from transferrin by pyrophosphate, ferric iron pyrophosphate is bound to the mitochondria, iron is reduced, dissociates from pyrophosphate and is taken up by the mitochondria. Ferrous iron thus formed is available for heme synthesis.
The Role of Penicillin‐Binding Proteins in the Action of Cephalosporins against Escherichia coli and Salmonella typhimurium
Authors: CHASE Howard A. , FULLER Christopher , REYNOLDS Peter E.
Published: July 2010

The binding of two radioactively‐labelled cephalosporins, LY97962 and LY121998, to the penicillin‐binding proteins of Escherichia coli and Salmonella typhimurium has been studied in detail. The concentrations of the cephalosporins that resulted in 50% saturation of the penicillin‐binding proteins were measured in growing cells as well as in preparations of disrupted cell envelopes. This technique has allowed the crypticity of an organism towards a particular β‐lactam to be measured directly, i.e. the extent to which a penicillin‐binding protein is accessible in intact cells. The cephalosporin LY97962 appeared to have no difficulty in penetrating the outer layers of the cell whereas access of cephalosporin LY121998 to the penicillin‐binding proteins appeared to be somewhat restricted.

At concentrations below the minimum growth inhibitory concentration the two cephalosporins inhibited cell division and caused the cells to grow as long filaments. When growing cells were incubated with the radioactive β‐lactam antibiotics at these concentrations, the β‐lactams bound almost exclusively to penicillin‐binding protein 3. The degree of binding of the cephalosporins to protein 1 A in cells growing in the presence of the minimum growth inhibitory concentrations of antibiotics varied with the duration of the incubation. After exposure for 5 min, very little binding to protein 1 A was apparent, whereas substantial binding was observed after 60–180 min. It was only at the end of this period of growth in the presence of the cephalosporins that lysis of the cells became apparent. This provided evidence that the binding of β‐lactams to protein 1 A of gramnegative organisms is an important step in the bactericidal action of certain cephalosporins. Protein 1 B was excluded as a target for the lethal action of these cephalosporins because binding to this protein was observed only at concentrations greater than those needed to inhibit growth of the cells.

Microscopical examination of the growth of S. typhimurium in the presence of cefotaxime at sub‐lytic concentrations showed that cells grew as filaments for at least six generations without lysis. Observations of the division of the filament into individual cells after removal of the antibiotic showed that the entire length of the filament was viable. It is concluded that inhibition of cell division as a result of the binding of β‐lactams to protein 3 of gram‐negative organisms is not in itself a lethal event for the cells, but requires concomitant binding of antibiotic to protein 1 A before lysis is initiated.
Structure and Configuration of Fructose 2,6‐Bisphosphate by 31P and 13C Nuclear Magnetic Resonance
Authors: HESBAIN‐FRISQUE Anne‐Marie , VAN SCHAFTINGEN Emile , HERS Henri‐Géry
Published: July 2010

31P and 13C magnetic resonance spectra of fructose 2,6‐bisphosphate, prepared as‐described by Van Schaftingen and Hers (preceding paper)‐ and of commercial fructose 1,6‐bisphosphate were obtained. Analysis of these spectra has confirmed the structure of fructose 2,6‐bisphosphate; this ester was present as a single anomeric form to which the β configuration could be assigned.
The Pattern of Protein Synthesis Induced by Heat Shock of HeLa Cells
Authors: SLATER Adrian , CATO Andrew C. B. , SILLAR Gillian M. , KIOUSSIS Joanna , BURDON Roy H.
Published: July 2010

While incubation of HeLa cells for 5–10 min at 45°C does not affect subsequent cellular growth and DNA synthesis at 37°C, there is an increased synthesis of specific proteins in the molecular weight region of 100000, 72000–74000 and 37000. The synthesis of these proteins reaches a maximum 2 h after the heat shock and while the proteins themselves are stable, their synthesis declines to normal levels 4 h afterwards. The increased synthesis is blocked by actinomycin D but the addition of cycloheximide or NaF during the ‘induction’ period is without effect.
Regeneration of Rhodopsin and Bacteriorhodopsin
Authors: TOWNER Paul , GAERTNER Wolfgang , WALCKHOFF Baerbel , OESTERHELT Dieter , HOPF Henning
Published: July 2010

The rate of regeneration of rhodopsin, from 11‐cis‐retinal and opsin, and bacteriorhodopsin from all‐trans‐retinal and bacterio‐opsin, in the presence or absence of compounds whose structures partially resemble retinal were measured. Some of these compounds severely slowed down the regeneration process, but did not influence the extent of regeneration. In the case of compounds with a carbonyl functional group they were not joined to the active site of the apo‐protein via a Schiff's base linkage since after treatment with NaBH4 an active apo‐protein remained. The most effective inhibitors of rhodopsin regeneration were molecules whose structure could be superimposed on 9‐cis or 11‐cis retinal up to carbon atom 11. These C13 and C15 molecules were not distinguished between aldehyde, ketone or alcohol functional groups.

The regeneration of bacteriorhodopsin was not inhibited by retinal analogues with short side chains. The most effective inhibitors were the all‐trans C17‐aldehyde (β‐ionylideneacetaldehyde) or C18‐ketone (β‐ionylidenepent‐3‐ene‐2‐one) which, compared to retinal, lack two or three carbon atoms from the end of the polyene chain. The inhibition was very dependent upon the presence of the all‐trans isomer and required aldehyde or ketone as functional group; nitriles and alcohols were less effective. However, similarly to retinol, the all‐trans C17 and C18 alcohols underwent a bathochromic shift and showed fine‐structured spectra when mixed with bacterio‐opsin.
Amino‐Acid Sequence of the 20000‐Molecular‐Weight Light Chain of Chicken Gizzard‐Muscle Myosin
Authors: MAITA Tetsuo , CHEN Jiann‐I , MATSUDA Genji
Published: July 2010

The light chain fraction was separated from chicken gizzard muscle myosin. After S‐carboxymethylation or performic acid oxidation, two light chain components (20000‐M, and 17000‐Mr chains) were isolated by chromatography on a column of DEAE‐cellulose in the presence of 4 M urea. Tryptic peptides of the S‐carboxymethylated 20000‐Mr chain were isolated, and their sequences were determined. The alignment of these tryptic peptides in the chain was deduced from the amino acid compositions and from the partial sequences of peptic peptides of the oxidized protein. The established sequence consists of 171 amino acids and its calculated molecular weight is 19692. Comparing the sequence with those of L‐2 chains from chicken and rabbit skeletal muscle myosins, 81 and 78 amino acid substitutions were recognized, respectively, including insertions and/or deletions.
Immunological Relatedness of Subunits of RNA Polymerase II from Insects and Mammals
Authors: KRÄMER Angela , BAUTZ Ekkehard K. F.
Published: July 2010

A procedure is described which allows rapid purification of RNA polymerase II from either embryos of Drosophila melanogaster or larvae of Chironomus thummi. The polypeptide compositions of the enzymes were compared in denaturing gels. Antisera were raised against the native enzymes and were tested for binding activity to separated subunits of each enzyme. Sheep antibodies to Drosophila RNA polymerase were found to react with most polypeptides of the insect RNA polymerases but only with the high‐molecular‐weight subunits of calf thymus RNA polymerase II. These were also found to cross‐react with a monoclonal antibody directed against the large subunits of the Drosophila enzyme. An antiserum to Chironomus RNA polymerase II reacted with the three largest subunits of this enzyme and subunits 0.1 and 2 of Drosophila RNA polymerase II. The purified IgG fractions of the polyspecific antisera inhibited the activity in vitro of the insect RNA polymerases to different degrees.
Fast Stages of Photoelectric Processes in Biological Membranes
Authors: DRACHEV Lei A. , KAULEN Andrey D. , KHITRINA Lyubov' V. , SKULACHEV Vladimir P.
Published: July 2010

Bacteriorhodopsin‐containing fragments of Halobacterium halobium membrane (bacteriorhodopsin sheets) were incorporated into a lecithin‐impregnated collodion film, and fast stages of flash‐induced electrogenesis were measured by two electrodes separated by this film.

It is found that a single turnover of bacteriorhodopsin results in an electrogenic response composed of three main stages of the following τ: the first < 200 ns, the second 15–70 μs and the third 10 ms. The second and third phases are of the same direction as an electric response to continuous illumination, whereas the first one is oppositely directed. The lts and ms stages were shown to correlate, in the first approximation, with formation and decomposition of the bacteriorhodopsin intermediate absorbing at 412 nm, respectively. Both the second and third phases of the photoelectric response are sums of at least two exponents.

The third stage is specifically inhibition by La3+ ions which are also shown to decrease the rate of regeneration of the original bacteriorhodopsin absorbing at 570 nm from the intermediate absorbing at 412 nm.

Acidification of the medium induces parallel inhibition of the second and third phases and of formation of the intermediate absorbing at 412 nm as if protonation of a group with pK= 3.6 were responsible for this inhibition. The first (opposite) phase survives acidification. It even increases at pH lower than 1.5. At such a low pH, one can show a good correlation of decays of photopotential and of a bacteriorhodopsin bathointermediate. The decays are biphasic (τ1= 200 μs and τ2= 2 ms), formation of both the photopotential and the bathointermediate being faster than 200 ns.

At higher pH, when a three‐phase photoelectric response is revealed, decay of the formed electric potential difference gives the average τ value of about 1 s. It can be accelerated by compounds that increase ionic conductance of biomembranes.

At pH below 4, fluoride is found to completely inhibit the second and third phases, so that only the first phase is observed.

The results are discussed in terms of a scheme postulating that the first electrogenic phase is a result of translocation of the protonated Schiff base inside the membrane due to a light‐induced conformation change in retinal or protein. The second and third phases are explained by H+ transfer from the Schiff base to the outer membrane surface and from inner (cytoplasmic) surface of membrane to the Schiff base, respectively.