bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in membrane‐associated proteins during sporulation in Bacillus subtilis
Authors: RHAESE Hans J. , REICHENBACH Angelika , STAMMINGER Gudrun
Published: March 2005

Membrane proteins from vegetative and sporulating cells of Bacillus subtilis were separated by the two‐dimensional gel electrophoresis system using isoelectric focusing and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (O'Farrell technique). Membrane proteins were isolated according to published procedures.

The gels were stained with Coomassie blue. Three different concentrations of proteins were analyzed to detect even minor constituents. Over two hundred different membrane proteins were identified in vegetative cells by their isoelectric point (pI) and molecular weight (Mr). Analysis of membrane proteins from cells harvested during and at the end of logarithmic growth (A600∼ 0.8; T0) and every hour thereafter until T4 showed that in the wild‐type strain 55 proteins are degraded mostly at the beginning or sporulation. Many others (76 proteins) are newly synthesized during sporulation. About 16 proteins are synthesized at times during sporulation but again degraded within 1 h or less. Others (uncertain proteins, 65) are degraded and resynthesized again. This observation is in agreement with experiments previously published by Andreoli et al. [Andreoli, A. J., Kao, M., Chui, R., Cabrera, J., and Wong, S. K. S (1981) in Sporulation and Germination (Levinson, H. S., Sonenshein, A. L., and Tipper, D. J., eds) pp. 168–173, American Society for Microbiology, Washington] using Bacillus cereus.

Experiments with the early blocked asporogenous mutant JH 649 (spoof) showed that few proteins (40%) are degraded and even fewer (30%) are newly synthesized between A600∼ 0.8 and T4.

Protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, o‐phenanthroline) have no effect on the protein patterns. The experiments presented here show that proteins involved in differentiation in B. subtilis can be identified by the two‐dimensional gel electrophoresis system and with the aid of asporogenous mutants.

In order to assure that no cytoplasmic proteins are contaminating the membrane preparations, several cytoplasmic enzyme activities have been measured. Their concentration was found to be always below 0.005% of total protein, which is below the level of detection by Coomassie blue staining.
Inhibition of cell proliferation by interferons
Authors: GEWERT Dirk R. , MOORE Graham , TILLERAY Vivienne J. , CLEMENS Michael J.
Published: March 2005

Treatment of Daudi cells with human lymphoblastoid interferons for up to 5 days progressively inhibits cell proliferation. For the first 3 days cells continue to grow but with prolonged doubling times; subsequently, net proliferation ceases and is accompanied by a loss of cell viability. We have investigated the changes in labelling of DNA with radioactive precursors which occur during the first phase of the response to interferon treatment. We have shown previously [Gewert et al. (1981) Eur. J. Biochem. 116, 487–492] that inhibition of incorporation of [3H]thymidine into DNA can be accounted for by impairment of thymidine transport and thymidine kinase activity. In spite of this inhibition, the total intracellular dTTP pool is larger in interferon‐treated than in control cells. Because of these changes it has been necessary to use other methods to determine whether interferon treatment inhibits the overall rate of DNA synthesis. The results of experiments employing (a) moderately high thymidine concentrations or (b) incorporation of radioactivity from deoxynucleoside triphosphates into DNA in detergent‐lysed or permeabilised cell systems indicate that there is in fact relatively little inhibition of the overall rate of DNA synthesis in cells exposed to up to 100 units/ml of interferons for at least 48 h. Furthermore, a similar proportion of cells incorporate [3H]thymidine in control and interferon‐treated cultures and there is only a small decrease in the number of cells in S phase after interferon treatment, as revealed by fluorescence‐activated cell sorting. These results indicate that cell proliferation may be regulated in this system by a mechanism in which there is a loss of coordination between the initiation of DNA synthesis and the subsequent events required for cell division.
Isolation and characterization of a mutant from Saccharomyces cerevisiae lacking fructose 1,6‐bisphosphatase
Authors: GANCEDO Carlos , DELGADO Marco A.
Published: March 2005

Mutants lacking fructose 1,6‐bisphosphatase activity have been isolated from Saccharomyces cerevisiae and genetically purified. Mutants were unable to grow on gluconeogenic carbon sources. Revertants that grew on glycerol have regained the fructose 1,6‐bisphosphatase activity, showing that there is no bypass in yeast for this enzymatic step. No significant differences were found in the growth of the mutants and the parental strains in other carbon sources. Other mutants lacking fructose 1,6‐bisphosphatase activity but pleiotropically affected in the derepression of several enzymes sensitive to catabolite repression were also isolated. All the mutants isolated were of nuclear origin and defined three complementation groups.
The structure of the B subunit of calcineurin
Authors: AITKEN Alastair , KLEE Claude B. , COHEN Philip
Published: March 2005

The complete primary structure of the B subunit of calcineurin (protein phosphatase 2B) has been determined by automated sequence analysis. The protein consists of a single polypeptide chain of 168 residues, relative molecular mass 19200. The structure shows 35% identity with the sequence of calmodulin and 29% with troponin C. Homology is mainly confined to the regions of the four putative Ca2+‐binding loops. The results demonstrate that the B subunit is a new member of this family of Ca2+‐binding proteins. The N‐terminal glycine residue is blocked with the C14‐saturated fatty acid myristic acid and the first four residues are very similar to those of the catalytic subunit of cyclic‐AMP‐dependent protein kinase which also contains a myristoyl blocking group.
Differentiation of receptor sites for [3H]nitrendipine in chick hearts and physiological relation to the slow Ca2+ channel and to excitation‐contraction coupling
Authors: RENAUD Jean‐François , KAZAZOGLOU Théodossia , SCHMID Annie , ROMEY Georges , LAZDUNSKI Michel
Published: March 2005

The properties of interaction of the Ca2+ channel antagonist [3H]nitrendipine have been investigated in chick hearts at various stages of in ovo and post‐natal development and in cultured cells. The dissociation constant of the [3H]nitrendipine‐receptor complex is between 0.4 nM and 0.5 nM for intact ventricle and cultured cells. [3H]Nitrendipine binding is antagonized by nitrendipine analogs. The order of efficacy of the different dihydropyridine molecules is nitrendipine < nimodipine < nifedipine < nisoldipine with Kd values ranging from 0.5 to 4 nM. Inhibition of [3H]nitrendipine binding by other antiarrhythmic molecules like amiodarone, F13004 and bepridil was observed. Half‐maximum inhibitions (K0.5) were found for verapamil and D600 at concentrations between 0.23 and 0.26 μM.

The potency of organic Ca2+ blockers to depress by 50% the maximum amplitude of spontaneous beating of heart cells is closely related to K0.5 values obtained from [3H]nitrendipine binding experiments.

Electrophysiological results indicate that the slow channel is insensitive to nitrendipine at the younger stage of development (3‐day‐old) whereas, in adult like cells, nitrendipine (50 nM) abolished both slow action potential due to the slow Ca2+ channel and contraction. The maximum binding capacity for [3H]nitrendipine is found to increase during development of the embryonic heart from 40 fmol/mg protein at day 3 to 100 fmol/mg protein at day 14, to stay relatively stable until day 18. Then the number of sites increases rapidly to reach a second plateau at 210 fmol/mg protein on day 4 after hatching. Treatment with 6‐hydroxydopamine results in 35% increase in [3H]nitrendipine binding, whereas reserpine treatment is without effect. Developmental properties of nitrendipine‐sensitive Ca2+ channels have been compared with those of tetrodotoxin‐sensitive Na+ channels and muscarinic receptors. These results indicate that nitrendipine receptors exist at the early stage of development (3‐day‐old‐hearts) but that they do not correspond to functional slow Ca2+ channels, that in ovo development corresponds both to an increase of the number of [3H]nitrendipine receptors and to the transformation of silent Ca2+ channels into functional Ca2+ channels, and that there is a regulation of the level nitrendipine‐sensitive Ca2+ channels by innervation.
The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody
Authors: ROUSSELET Annie , FAURE Grazyna , BOULAIN Jean‐Claude , MÉNEZ André
Published: March 2005

Five derivatives of Naja nigricollis toxin α, spin‐labeled on a single amino group, were prepared. The toxin derivatives were purified to homogeneity by ion‐exchange and high‐pressure liquid chromatographies. The modified amino groups are localized at residue 1 and lysines 15, 27, 47 and 51. Competition data show that incorporation of spin label at residues 27 or 47 reduces the affinity of the toxin for the nicotinic acetylcholine receptor (AcChR), while incorporation at residues 1 or 15 diminishes toxin affinity for a monoclonal toxin‐specific immunoglobulin (M1). Classical and/or saturation transfer electron spin resonance (ESR) analysis was carried out on each derivative, either in the free state or bound to AcChR or M1. The data obtained give the following indications.

In the free state, the nitroxides incorporated at residues 1, 15, 47 and 51 have their own rapid motion, while that at residue 27 has no residual mobility and reflects the toxin rotation.

Binding of AcChR to the toxin reduces the motion of the nitroxide bound to Lys47. Binding of M1 to the toxin immobilizes the two nitroxides fixed on residues 1 and 15. ESR spectra show that Lys27‐bound nitroxide remains immobilized upon binding of either AcChR or M1.

The change in nitroxide immobilization observed upon AcChR or M1 binding correlates well with the variation of nitroxide accessibility to a water‐soluble paramagnetic N2+i ion.

Binding of the labeled Lys47 toxin derivative to AcChR yields a complex ESR signal, disclosing the existence of a physical difference between the two toxin binding sites on AcChR.

All the data indicate that AcChR and M1 bind at two topographically distinct sites on the toxin surface.
A temperature‐jump study of the electron transfer reactions in Hansenula anomala flavocytochrome b2
Authors: TEGONI Mariella , SILVESTRINI Maria Chiara , LABEYRIE Françoise , BRUNORI Maurizio
Published: March 2005

Temperature‐jump experiments on flavocytochrome b2 were carried out at different levels of heme reduction at pH 7.0 and 6.0, and as a function of pyruvate concentration.

The relaxation, corresponding to an increase in the concentration of reduced heme, is in no case a simple process. At pH 7.0 the mean reciprocal relaxation time is 1/τ*= 190 s−1, independent of enzyme concentration, wavelength of observation and percentage of heme reduction. Flavin semiquinone has been identified as the major electron donor to the heme in this process. At the same pH the presence of pyruvate in the millimolar concentration range increases the relaxation rate and affects its amplitude. The latter effect could be accounted for by a change in redox equilibria between heme and flavin upon pyruvate binding.

At pH 6.0 the relaxation pattern depends more clearly on the level of heme reduction. A rapid process (τ−1= 2500 s−1), predominant at high percentages of reduced heme, has been assigned to the reduction of heme by flavin hydroquinone, while the slower process (τ−1= 350 s−1), essentially the only one present at or below 50% of heme reduction, has been ascribed to the reduction of heme by flavin semiquinone. These results are discussed in relation to the catalytic mechanism of the enzyme.
Purification and properties of a cAMP‐independent nuclear protein kinase from Dictyostelium discoideum
Authors: RENART Margarita F. , SASTRE Leandro , SEBASTIÁN Jesús
Published: March 2005

A cyclic‐AMP‐independent nuclear protein kinase has been purified from Dictyostelium discoideum amoebae. The purification procedure involves chromatography of DEAE‐Sephadex, phosphocellulose and heparin‐Sepharose. The purified enzyme phosphorylates threonine and serine of acidic proteins as casein and phosvitin. Phosphorylation of casein is stimulated by spermine. The kinase requires Mg2+ and can utilize both ATP and GTP as phosphoryl donors. Heparin is a potent inhibitor of the enzyme, being the protein kinase activity fully inhibited at concentrations of 0.5 μg/ml. One polypeptide of molecular mass 38 kDa was the major protein band present in the purified kinase preparation as estimated by NaDodSO4 denaturing polyacrylamide gel electrophoresis. This band belongs to the protein kinase because it is the only one that is observed associated with the protein kinase activity when the enzyme preparation is centrifuged in glycerol gradients. The 38‐kDa polypeptide is also the major product of autophosphorylation of the enzyme preparation. The enzymatic properties allow to classify the enzyme as a type‐II casein kinase. However, its structural properties are different from the mammalian type‐II casein kinases and make the D. discoideum enzyme more similar to the plants type‐II casein kinases.
Calmodulin content and activity of Ca2+‐ATPase and phospholipase A2 in rat Kupffer cells
Authors: BIRMELIN Manfred , MARME Dieter , FERBER Ernst , DECKER Karl
Published: March 2005

A protein resembling calmodulin was isolated from non‐parenchymal and parenchymal cells of rat liver by affinity chromatography. The biological activity of the purified protein was assessed by the bovine brain cAMP phosphodiesterase assay. A highly sensitive radioimmunoassay as well as the cAMP phosphodiesterase method were employed to determine the calmodulin content of crude extracts from monolayer cultures of rat Kupffer cells and hepatocytes.

An ATP‐dependent, calmodulin‐enhanced calcium transport was demonstrated in a membrane fraction of the non‐parenchymal cells. Phospholipase A2 activity specific for 2‐arachidonoyl phosphatide and with a pH optimum of 8.1 was measured in homogenized Kupffer cells; it was stimulated by agents previously shown to enhance prostaglandin synthesis in Kupffer cells, e.g. zymosan particles and lipopolysaccharide isolated from Salmonella minnesota. The increase in activity was completely prevented by pretreatment with or simultaneous addition of R 24571, a known calmodulin antagonist. However, if this inhibitor or calmodulin was added to the cell‐free extract phospholipase A2 activity was not influenced. Phospholipase A1 activity could be detected at pH 5 only, showing a slight decrease in the homogenate of stimulated macrophages. Acyltransferase activity was high but independent of treatment of the Kupffer cells.
Blockade of the galactose‐binding sites of ricin by its linkage to antibody
Authors: THORPE Philip E. , ROSS Walter C. J. , BROWN Alex N. F. , MYERS Christopher D. , CUMBER Alan J. , FOXWELL Brian M. J. , FORRESTER J. Tony
Published: March 2005

A method is described for preparing specific cytotoxic agents by linking intact ricin to antibodies in a manner that produces obstruction of the galactose‐binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non‐specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose‐binding capacity were separated by affinity chromatography on Sepharose (a β‐galactosyl matrix) and asialofetuin‐Sepharose.

Fluorescence‐activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti‐Thy1.1‐ricin conjugate that passed through a Sepharose column had markedly diminished capacity to bind non‐specifically to Thy1.2‐expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin‐Sepharose column displayed no detectable non‐specific binding. Both fractions of conjugate were potent cytotoxic agents for Thy1.1‐expressing AKR‐A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50% at a concentration of 2–5 pM. Comparable inhibition of EL4 cells was only achieved with 3000–7500‐fold greater concentrations of conjugate. By contrast, the fraction of anti‐Thy1.1‐ricin that retained Sepharose‐binding capacity showed marked non‐specific binding and toxicity to EL4 cells.

A conjugate with diminished galactose‐binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T‐lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen‐expressing rat T‐leukaemia cells. This finding is of particular importance because isolated ricin A‐chain disulphide‐linked to W3/25 antibody is not cytotoxic. The property of the B‐chain in intact ricin conjugates that facilitates delivery of the A‐chain to the cytosol thus appears to be independent of galactose recognition.

It is concluded that the ‘blocked’ ricin conjugates combine the advantages of high potency, which is often lacking in antibody‐A‐chain conjugates, with high specificity, which previously was lacking in intact ricin conjugates.
Nucleotide‐mediated interactions of eukaryotic elongation factor EF‐2 with ribosomes
Authors: NYGÅRD Odd , NILSSON Lars
Published: March 2005

Eukaryotic elongation factor EF‐2 isolated from rat liver microsomal salt‐wash showed two types of nucleotide‐dependent interactions with reconstituted empty 80S ribosomes when analyzed by gradient centrifugation. Stable EF‐2 ribosome complexes were only formed with GTP analogues with reduced or no ability to serve as substrates for the EF‐2 and ribosome‐dependent GTPase. GTP‐stimulated complex formation was only demonstrable after glutaraldehyde fixation, unless a GTP‐regenerating system was included throughout the gradient centrifugation. GDP and to a lesser extent GMP and guanosine also stimulated the less stable type of complex formation as demonstrable after fixation. Under the same conditions some complex formation was also observed with ATP and its analogue adenosine 5′‐[β, γ‐methylene]triphosphate, although with less efficiency than with the corresponding guanosine nucleotides.

The results in combination with available data indicate that EF‐2 has two binding states with different affinities on the 80S ribosome: a high‐affinity pre‐translocation state specific for EF‐2 · GTP and a low‐affinity post‐translocation state, in which EF‐2 · GDP is bound to the ribosome in a less stable and specific complex.
Isolation of the contact regions of the neighboring mammalian ribosomal proteins L6 and L29
Authors: NIKA Heinz , HULTIN Tore
Published: March 2005

Proteins L6 and L29 in the 60‐S subparticle of mammalian ribosomes interact in situ under gentle conditions by intermolecular disulfide bond formation. For identifying the contact regions of the two proteins the disulfide complex was isolated from whole ribosomes by preparative polyacrylamide gel electrophoresis and subjected to cyanogen bromide cleavage. For effective cleavage non‐oxidizing conditions had to be maintained throughout the preparation. A simple and generally applicable method of high‐efficiency gel pre‐electrophoresis with anionic and cationic thiols was developed. Under these conditions reversibly cross‐linked CNBr fragments of L6 and L29 could be isolated in high yield (Mr approx. 13000 and 7000, respectively). After [14C]carboxymethylation of the reduced disulfide links smaller contact sequences were obtained by pepsin digestion and characterized by two‐dimensional peptide mapping. These smaller contact peptides were contained within the corresponding CNBr fragments. Both contact peptides were hydrophilic and relatively basic.
Purification, characterization, and kinetic mechanism of S‐adenosyl‐l‐methionine: vitexin 2″‐O‐rhamnoside 7‐O‐methyltransferase of Avena sativa L
Authors: KNOGGE Wolfgang , WEISSENBÖCK Gottfried
Published: March 2005

An O‐methyltransferase catalyzing the transfer of the methyl group of S‐adenosyl‐l‐methionine to the A‐ring 7‐hydroxyl group of vitexin 2′‐O‐rhamnoside has been isolated from oat primary leaves and purified 180‐fold by protein fractionation with (NH4)2SO4 and chromatography on DEAE‐cellulose and S‐adenosyl‐l‐homocysteine‐sepharose. Km values for S‐adenosyl‐l‐methionine and the flavonoid substrate were 1.6 μM and 15 μM, respectively. The lack of methyl transfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7‐O‐methylvitexin 2″‐O‐rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono‐iso Theorell‐Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.
Coordinated two‐disk nucleation, growth and properties, of virus‐like particles assembled from tobacco‐mosaic‐virus capsid protein with poly(A) or oligo(A) of different length
Authors: SCHÖN Astrid , MUNDRY Karl‐W.
Published: March 2005

Assembly of nucleoprotein rods from tobacco mosaic virus (TMV) coat protein and poly(A) depends on the presence of 20S disks in a manner very similar to nucleation and growth of virions in reconstitution with TMV RNA. Products assembled with (A)∼ 5000 appear to have the same buoyant density in CsCl, the same nucleotide/protein ratio and the same nuclease stability, as reconstituted and native TMV. Their rate of formation is very similar to the rate of reconstitution with TMV RNA when high‐molecular‐mass (A)∼ 5000 is used, but becomes a function of chain length particularly with (A)≤185. The composition of assembly products can be described sufficiently with the relation between number of capsid polypeptide monomers/particle, np, to the number of nucleotide residues/chain, nnt, of np= 1/3 (nnt+ 50) with two important restrictions: (1) particles of less than four turns of helically arranged capsid subunits are unstable, and (2) particles with about 150 or less nucleotides per chain deviate in structure from mature virus and virus‐like (= longer) assembly products. This is indicated by changes in both buoyant density in CsCl and optical properties, while ‘dislocation’ of the disk to the helical arrangement of capsid subunits (‘helicalization’) and nuclease stability already become established with chains as short as (A)∼ 58 ± 20. Consequently, we suggest that assembly proceeds through three distinct phases: (1) nucleation (resulting in helicalization) by interaction of nucleic acid with the first disk; (2) stabilization of the primary (unstable!) nucleation complex by addition of a second disk and formation of a four‐turn virus‐like and stable nucleoprotein helix, which is then fit for (3) elongation by addition of further disks. The question of what makes the TMV protein disk select specifically TMV RNA during virion assembly is discussed in some detail.
Isoforms of an endogenous lectin in rabbit bone marrow
Authors: FITZGERALD John E. , CATT James W. , HARRISON F. Lynne
Published: March 2005

A lectin which may mediate inter‐erythroblast associations during red blood cell development in rabbit bone marrow has previously been purified and characterised. We have now detected different forms of this lectin in purified preparations and crude tissue extracts, by isoelectric focusing in agarose gels followed by rocket immunoelectrophoresis and by indirect antibody staining of focused proteins blotted onto nitrocellulose paper. These minor antigens are probably isoforms of the bone marrow lectin previously characterised.
Propionyl‐coenzyme A carboxylase of Mycobacterium smegmatis
Authors: HAASE F. Carl , BEEGEN Helga , ALLEN S. H. George
Published: March 2005

Propionyl‐CoA carboxylase has been purified to homogeneity and examined in the electron microscope. The native carboxylase presents a profile with a large central subunit to which smaller subunits are attached. The central subunit has two prominent profiles, one circular (100 Å) with a central hole and the other rectangular (70 × 100 Å). The six polypeptides of this subunit appear to be arranged in a cylindrical structure. Six spherical (50 Å) biotin‐containing peripheral subunits are attached in sets of three to the two opposite circular faces of the central subunit. A model of the 18‐S carboxylase is presented.
Kinetics for the inhibition of acetylcholinesterase from the electric eel by some organophosphates and carbamates
Authors: FORSBERG Åke , PUU Gertrud
Published: March 2005

The inhibition kinetics for some organophosphates (paraoxon, diisopropylfluorophosphate, sarin, VX, soman and soman isomers) and carbamates (physostigmine, neostigmine, pyridostigmine and carbaryl) in the reaction with acetylcholinesterase from electric eel have been studied. Dissociation constants and rate constants for the irreversible step were determined. The great differences in inhibitory power of the organophosphates were almost entirely due to differences in affinity. A possible correlation between affinity and bonding rate is discussed.
Synthesis and properties of (4‐13C)NAD+
Authors: OBERFRANK Margit , HULL William E. , RETEY János
Published: March 2005

Starting from (13C)formic acid, acetone and cyanoacetamide samples of (4‐13C)nicotinic acid and (4‐13C)‐nicotinamide were synthesised in an overall and additive yield of 11%.1H‐NMR and mass spectroscopy showed 90% enrichment of 13C in the expected position. NADase‐catalysed exchange between thionicotinamide–adenine dinucleotide and (4‐13C)nicotinamide furnished (4‐13C)NAD+ which was purified, characterized and quantified by 1H‐NMR and 13C‐NMR spectroscopy and by enzymic assay. The 13C‐NMR signal of (4‐13C)β‐NAD+ (146.09 ppm) was broadened and shifted (147.83 ppm) upon binding to yeast alcohol dehydrogenase.
Bacteriorhodopsins with chromophores modified at the β‐ionone site
Authors: MURADIN‐SZWEYKOWSKA Maria , PARDOEN Johannes A. , DOBBELSTEIN Dick , van AMSTERDAM  , LUGTENBURG Johan
Published: March 2005

The binding to bacterioopsin of the all‐trans isomers of retinal analogues lacking the six‐membered ring and differing in length of the conjugated chain, as well as the light‐driven action of the proton pump of the resulting bacteriorhodopsin analogues, were studied. The ‘opsin shifts’ in these modified bacteriorhodopsins are all around 2700 cm−1 and do not depend on the number of double bonds in the chromophore. These experimental results suggest that the 4800 cm−1‘opsin shift’ in unmodified bacteriorhodopsin consists of a contribution of about 2700 cm−1 due to the interaction of the protonated Schiff‐base with the counterion. The extra 2100 cm−1 shift in bacteriorhodopsin is due to the specific interaction of the cyclohexene ring and the protein. Only the bacteriorhodopsin analogue with the same number of conjugated double bonds in the chromophore as bacteriorhodopsin itself shows light‐driven proton pump action.
Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N‐glycosylated
Authors: ORLEAN Peter , SCHWAIGER Hubert , APPELTAUER Ute , HASELBECK Anton , TANNER Widmar
Published: March 2005

α Factor specifically inhibits the synthesis of N‐glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in α cells or in a/α diploids. a Factor has the same effect of α cells. The synthesis of O‐glycosylated proteins is not inhibited. Although the mating pheromones act like a ‘physiological tunicamycin’, the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N‐glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in α‐factor‐treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by α factor; but only in the former case did carbohydrate‐free protein accumulate in the cells.

α Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N‐glycosylated proteins are required for the G1/S‐phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature‐sensitive N‐glycosylation mutant is shifted to its restrictive temperature.
Mechanism of inhibition of polypeptide chain initiation in heat‐shocked Ehrlich ascites tumour cells
Authors: PANNIERS Richard , HENSHAW Edgar C.
Published: March 2005

The rate of polypeptide synthesis is inhibited by 80% in Ehrlich cells incubated at 43°C compared to those at 37°C. The regulatory site of translation resides at polypeptide chain initiation. Polypeptide synthesis does not recover at the higher temperature; however, the inhibition is reversed by returning the cells to 37°C. Neither new RNA synthesis or protein synthesis is required for recovery at 37°C, eliminating degradation of mRNA and irreversible denaturation of a protein essential for polypeptide chain initiation. The concentration of 40‐S initiation complexes was found to be reduced markedly in heat‐shocked cells compared to controls. This was confirmed in the cell‐free protein‐synthesizing systems prepared from heat‐shocked and control cells. Reversible alteration in the activity of components affecting eIF2 function is, therefore, a likely mechanism of regulation in heat‐shocked Ehrlich cells. In extracts from heat‐shocked cells, Met‐tRNA synthetase activity was unaltered compared to control extracts.
Assay of ATP synthesis using single giant mitochondria
Authors: CAMPO Maria Luisa , BOWMAN Charles L. , TEDESCHI Henry
Published: March 2005

Two assays of the capacity of single giant mitochondria or mitoplasts to phosphorylate ATP from Pi and ADP have been developed. One depends on the placement of a single mitochondrion next to a glycerinated myofibril, by micromanipulation. With the appropriate controls, contraction of the myofibril serves as an indication of ATP synthesis. The other assay similarly requires the isolation of one single mitochondrion but the assay of ATP synthesized uses the luciferin‐luciferase reaction with a conventional photometric system. With the latter, we found that either impalement with microelectrodes or electrophoretic microinjection of two dyes, Lucifer Yellow CH or pyranine, into the inner space have no effect on the phosphorylative capacity of mitochondria or mitoplasts. The electric potential across the mitochondrial membrane was monitored during the assay and found to be small and generally positive inside.
Alterations of oxidative phosphorylation reactions in mitochondria isolated from hypothyroid rat liver
Authors: EZAWA Ikuko , YAMAMOTO Michiko , KIMURA Satoshi , OGATA Etsuro
Published: March 2005

Several parameters of oxidative phosphorylation reaction were studied in vitro in isolated liver mitochondria from hypothyroid rats.

When succinate was employed as a respiratory substrate for mitochondria incubated in a mannitol/sucrose/phosphate buffer, and measurements were performed during initial additions of ADP, the magnitude of state 3 and state 4 respiration was not different between mitochondria from the hypothyroid and those from the control rats.

During the course of repetitive additions of ADP and consequently of sequential transitions from state 4 to state 3 and back to state 4, mitochondria from hypothyroid animals showed a gradual decline in the rate of both state 3 and state 4 respiration whereas those from normal animals did not. The total succinate dehydrogenase activity was not different between the two types of mitochondria, and the decline in state 3 and state 4 respiration was not accompanied by any change in the apparent Km for ADP or in the corrected ADP/O ratio [Biochem. Biophys. Res. Commun. (1973) 53, 988].

The amount of oxygen consumed during the state‐4→ 3→4 transition was lower in the hypothyroid than in the control mitochondria.

These alterations were reversed if the hypothyroid animals were injected with thyroxine intraperitoneally (2μg/100g body weight) for 3 weeks before isolation of mitochondria.

These results indicate that the fall of respiratory activity in hypothyroidism may result from the decrease not only of respiratory activities of state 3 and state 4, but also of the energy spent in the process of the state‐4→3→4 transition, while the coupling efficiency per se remains normal. These properties become manifest when mitochondria respond to pulses of ADP load, a situation likely to occur in situ.
Aldehyde dehydrogenase from human liver
Authors: HEMPEL John , von BAHR‐LINDSTRÖM Hedvig , JÖRNVALL Hans
Published: March 2005

Analysis of CNBr fragments and other peptides from human liver cytoplasmic aldehyde dehydrogenase enabled determination of the complete primary structure of this protein. The monomer has an acylated amino terminus and is composed of 500 amino acid residues, including 11 cysteine residues. No evidence of any microheterogeneity was obtained, supporting the concept that the enzyme is a homotetramer. The disulfiram‐sensitive thiol in the protein, earlier identified through its reaction with iodoacetamide, is contributed by a cysteine residue at position 302, while the cysteine which in horse liver mitochondrial aldehyde dehydrogenase is reactive with coenzyme analogs appears to correspond to either Cys‐455 or Cys‐463. Analysis of glycine distribution and prediction of secondary structures to localize βαβ regions typical for coenzyme‐binding are not fully unambiguous, but suggest a short region around position 245 as a likely segment for this function. In this region, sequence similarities to parts of a bacterial aspartate‐β‐semialdehyde dehydrogenase and a mammalian alcohol dehydrogenase were noted. Otherwise, no extensive similarities were detected in comparisons with characterized mammalian enzymes of similar activity or subunit size as aldehyde dehydrogenase (glyceraldehyde‐3‐phosphate dehydrogenase and glutamate dehydrogenase, respectively).
Structural analysis of the Ss sialoglycoprotein specific for Henshaw blood group from human erythrocyte membranes
Authors: DAHR Wolfgang , KORDOWICZ Maria , JUDD W. John , MOULDS John , BEYREUTHER Konrad , KRÜGER Jürgen
Published: March 2005

The N‐terminal structures of the MN and Ss erythrocyte membrane sialoglycoproteins (glycophorins A, B) from two Henshaw (He) blood‐group heterozygotes were determined by manual sequencing of tryptic glycopeptides and various secondary fragments. No structural alteration of the MN glycoprotein could be detected. The He‐specific portion of the Ss glycoprotein was found to exhibit the N‐terminal sequence Trp‐Ser+‐Thr+‐Ser+Gly‐(+= glycosylation). Thus it differs at three positions from its normal counterpart which possesses ‘N’ activity and exhibits the N‐terminal structure Leu‐Ser+‐Thr+‐Thr+‐Glu‐. Analysis of the Ss glycoprotein from 15 He‐negative erythrocyte samples did not reveal any of the three He‐specific structural alterations. The presence of a glycine residue at the fifth position of the blood‐group‐M‐active MN glycoprotein as well as in the He‐specific Ss glycoprotein provides an explanation for the occurence of antisera (anti‐Me) reacting with the M and He antigens.
Polymorphism of α‐actinin from human blood platelets Homodimeric and heterodimeric forms
Authors: CACHE Yannick , LANDON Françoise , OLOMUCKI Anna
Published: March 2005

In purified solutions of α‐actinin from human blood platelets, three polypeptides a, b and c, of approximately 100 kDa, were observed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were identified as α‐actinin subunits on the basis of their cross‐reactivity with antibodies against skeletal muscle α‐actinin and their interaction with F‐actin. After electrophoresis in the absence of sodium dodecyl sulfate, six forms were observed: aa, ab, bb, ac, bc, cc. The similarity between the one‐dimensional peptide maps of a and b and the absence of b in the presence of calcium‐dependent protease inhibitors indicated that b is probably derived from the a subunit. The c subunit differs from the a subunit. The results provide evidence that there are actually only two platelet α‐actinin subunits a and c which give rise to three isoforms: two homodimers aa and cc, and one heterodimer ac.
Photoaffinity labeling of the glucagon receptor with a new glucagon analog
Authors: WRIGHT David E. , HORUK Richard , RODBELL Martin
Published: March 2005

The preparation, purification and characterization of N‐4‐azidophenylamidinoglucagon are described. This photoreactive peptide was found to be 50% as potent as native glucagon in competing with 125I‐labeled glucagon for binding to glucagon receptors on rat liver plasma membranes. Similarly, the analog was 50% as potent as native glucagon in its ability to stimulate adenylate cyclase. The photoreactive glucagon analog was radioiodinated to high specific activity with iodine‐125 and was used to label rat liver plasma membrane proteins. Analysis of labeled membrane proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed covalent incorporation predominantly into a protein of relative molecular mass, Mr, of 50000–60000. Occasionally a protein of Mr 170000–180000 was also labeled. Irradiation of membranes in the presence of unlabeled glucagon or GTP selectively inhibited the labeling of the 50000–60000‐Mr protein(s). As a result of these studies we suggest that the sodium‐dodecyl‐sulfate‐dissociated glucagon receptor is a 50000–60000‐Mr protein.
Biosynthesis and processing of phytohemagglutinin in developing bean cotyledons
Authors: VITALE Alessandro , CERIOTTI Aldo , BOLLINI Roberto , CHRISPEELS Maarten J.
Published: March 2005

Phytohemagglutinin (PHA) is a family of tetrameric isolectins which accumulate in the protein bodies of developing Phaseolus vulgaris cotyledons. Each tetramer contains erythroagglutinating (E) or lymphocyte‐mitogenic (L) subunits, or a combination of both. The subunits have Mr around 33000, E being slightly larger than L. Phytohemagglutinin is a glycoprotein, and its carbohydrate moiety contains N‐acetylglucosamine, mannose, fucose and xylose, indicating that this protein has complex oligosaccharide sidechains. Several steps in the biosynthesis and in the cotranslational and post‐translational processing of the glycopolypeptides of PHA have been identified.

The polypeptides of PHA are synthesized by polysomes attached to the endoplasmic reticulum.

The glycosylation of the polypeptides is a cotranslational process, in which each PHA polypeptide usually acquires two oligosaccharide sidechains.

The oligosaccharides of PHA isolated from the endoplasmic reticulum are susceptible to digestion with α‐mannosidase and endo‐β‐AT‐acetylglucosaminidase H indicating that they are of the high‐mannose type.

In the presence of tunicamycin two unglycosylated polypeptides of PHA are synthesized, indicating that the differences in Mr between the E and L subunits of PHA are not due to differences in glycosylation alone.

Transport of PHA to the protein bodies is mediated by the Golgi apparatus where at least part of the oligosaccharide chains of PHA are modified [Chrispeels, M. J. (1983) Planta (Berl.) 157, 454–461, and 158, 140–151]. The modified oligosaccharide chains of PHA are then gradually trimmed to a smaller size when the protein is already in the protein bodies. This processing results in an increase in the mobility of the PHA subunits in denaturing polyacrylamide gels.
Isolation of short interspersed repetitive DNA sequences present in the regions of initiation of mammalian DNA replication
Authors: ANACHKOVA Boyka , TODOROVA Maria , VASSILEV Lyubomir , RUSSEV George
Published: March 2005

Nascent DNA chains containing the putative replication origins were isolated from cells of human embryonic lung fibroblasts, Hela, Ehrlich ascites tumour and Guerin ascites tumour as described earlier [Russev, G., and Vassilev, L. (1982) J. Mol Biol. 161, 77–87]. It was demonstrated that the synthesis of these nascent chains correlated with the ability of cells to initiate semiconservative DNA replication. Reassociation and electrophoretic analysis showed that the nascent chains from all four cell lines contained middle repetitive DNA in the form of short interspersed sequences. Mouse repetitive sequences were isolated and hybridized to Escherichia coli, chicken call and rat DNA and to homologous hnRNA. The kinetics of hybridization indicated that the repetitive sequences found in the vicinity of the replication origins were order‐specific and were not heavily transcribed.

Reassociation experiments, in which homologous DNA isolated from nuclei digested with micrococcal nuclease to different extents was used as a driver, showed that these repetitive sequences were organized into nucleosomes like the bulk of the chromatin.
Replication of M13mp7 single‐stranded DNA in vitro by the 9‐S DNA polymerase α from calf thymus
Authors: GROSSE Frank , KRAUSS Gerhard
Published: March 2005

The replication of M13 single‐stranded DNA by the 9S DNA polymerase α from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short Oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single‐stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase a contains a potent DNA primase activity which enables it to initiate replication on a single‐stranded template in the presence of the four NTPs. However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA.
A nuclear‐Overhauser‐enhancement study of the solution structure of a double‐stranded DNA undecamer comprising a portion of the specific target site for the cyclic‐AMP‐receptor protein in the gal operon
Authors: CLORE G. Marius , GRONENBORN Angela M.
Published: March 2005

A 500‐MHz 1H‐NMR study on a double‐stranded non‐self‐complementary DNA undecamer comprising a portion of the specific target site for the cyclic AMP receptor protein in the gal operon is presented. Using pre‐steady‐state nuclear Overhauser effect (NOE) measurements, all exchangeable imino, non‐exchangeable base, methyl, and H1′, H2′ and H2″ sugar proton resonances are assigned in a sequential manner. In addition, some of the H3′ sugar proton resonances are also assigned and some of the exchangeable amino proton resonances identified. The relative magnitudes of the intranucleotide and internucleotide NOEs are indicative of a right‐handed B‐type conformation for the duplex undecamer in solution.
Influence of fatty acids on energy metabolism
Authors: SCHOLZ Roland , SCHWABE Ursula , SOBOLL Sibylle
Published: March 2005

Changes in metabolic rates (oxygen consumption, ketogenesis, 14CO2 production from labelled fatty acids, glycolysis) following the addition of octanoate or oleate were studied in isolated livers from fed and starved rats perfused with Krebs‐Henseleit bicarbonate buffer in a non‐recirculating system. The following results were obtained.

The infusion of fatty acids Caused a large increase in the rate of oxygen consumption. The effect was greater with octanoate than with oleate and was half‐maximal with fatty acid concentrations (free plus albumin bound) around 0.1 mM.

The effects of oleate were only partially suppressed when the perfusate contained albumin concentrations near the physiological range.

When fatty acids were oxidized at high rates, the glycolytic rate was diminished by 50%.

The increase in oxygen consumption could not be explained fully by the increased ATP demand for fatty acid metabolism or by a compensation for the diminished extramitochondrial ATP generation.

In the presence of phenylalkyl oxirane carboxylic acid, an inhibitor of the transport of long‐chain acyl‐CoA derivates into the mitochondria, ketogenesis and 14CO2 production from labelled oleate were strongly inhibited, whereas the increase in oxygen consumption was only slightly affected.

In the presence of antimycin A, the increase in oxygen consumption due to fatty acids was totally abolished.

Following pretreatment of rats with ciprofibrate (induction of enzymes for peroxisomal β‐oxidation of long‐chain fatty acids), ketogenesis (but not 14CO2 production) from oleate was enhanced threefold. The increase in oxygen consumption, however, was not affected.

In conclusion, the increase in hepatic oxygen consumption due to addition of fatty acids reflects a mitochondrial process; it is, in part, independent of the ATP demand of the cell. An uncoupling‐like effect of fatty acids on the respiratory chain and its possible physiological significance in ketogenesis are discussed.
Influence of fatty acids on energy metabolism
Authors: SOBOLL Sibylle , GRUNDEL Sonja , SCHWABE Ursula , SCHOLZ Roland
Published: March 2005

The uncoupling‐like effect of fatty acids [Scholz, R., Schwabe, U., and Soboll, S. (1984) Eur. J. Biochem. 141, 223–230] was further substantiated in experiments with perfused rat livers by two ways: firstly the kinetics of changes in metabolic rates (oxygen consumption, ketogenesis, fatty acid oxidation) were analysed; secondly subcellular contents of adenine nucleotides and pH gradients across the mitochondrial membrane were determined following fractionation of freeze‐fixed and dried tissues in non‐aqueous solvents. The following results were obtained.

The relaxation kinetics of the increase in oxygen consumption following fatty acid infusion revealed two components, a rapid one with a half‐time around 10s and a slow one with a half‐time of more than 100s.

The rapid component was similar to the kinetics of fatty acid oxidation (ketogenesis and 14CO2 production from labelled fatty acids) whereas the half‐time of the slow component was in the range of half‐times observed with the increase in oxygen consumption following addition of carbonylcyanide p‐trifluoromethoxyphenylhydrazone.

In the presence of fatty acids, the cytosolic ATP concentrations and ATP/ADP ratios decreased, whereas the corresponding parameters for the mitochondrial space were either increased (oleate) or decreased (octanoate). The effects of oleate were dependent on the albumin concentrations in the perfusate. The normally large difference between cytosolic and mitochondrial ATP/ADP ratios became smaller. Similar observations were obtained with uncoupling agents.

The pH gradient across the mitochondrial membrane as calculated from the subcellular distribution of 5,5 di‐methyl[2‐14C]oxazolidine‐2,4‐dione was inversed following the addition of both carbonylcyanide p‐trifluoromethoxyphenylhydrazone and fatty acids, i.e. the mitochondrial matrix became more acidic than the cytosol. The pH gradient was not affected when oleate was added in the presence of high albumin concentrations.

The data support the hypothesis that the increase in hepatic oxygen consumption due to octanoate or oleate is, in part, caused by a mechanism similar to uncoupling of oxidative phosphorylation. This mechanism seems not to be an artifact of isolated systems; it may be of physiological importance for processes in which reducing equivalents are removed independently of the ATP demand of the hepatocyte.