bluuebunny/crossref_metadata_2025 huggingface dataset:
Subunit Structure and Interactions of the Phloem Proteins of Cucurbita maxima (Pumpkin)
Authors: READ Steve M. , NORTHCOTE Don H.
Published: March 2005

The two major proteins from the phloem exudate of Cucurbita maxima (pumpkin), PP1 and PP2, were stable in the absence of reducing agents after modification of their accessible cysteine residues with iodoacetamide. This permitted their purification without precautions to prevent oxidation.

PP2, a lectin specific for oligomers of N‐acetyl‐D‐glucosamine, was shown by sedimentation‐equilibrium ultracentrifugation to be a dimer of Mr, of 48000. Neither dithiothreitol nor tri‐(N‐acetyl‐D‐glucosamine) altered this value. The constituent polypeptides were linked by two buried disulphide bridges. PP2 behaved aberrantly on gel‐filtration on both Sephadex and Bio‐Gel unless tri‐(N‐acetyl‐D‐glucosamine) was added to the elution buffer; the Mr, was then measured as 46000. Other proteins which bind oligomers of N‐acetyl‐D‐glucosamine are also retarded on gel‐filtration.

Soluble phloem filaments were prepared by collection of exudate into deaerated buffer containing iodoacetamide but no reducing agent. Oxidative gellation of the filaments was prevented by rapid modification of their many accessible cysteine residues, and is assumed to have maintained the degree of polymerisation found in vivo. Those disulphide bridges which were present allowed the incorporation of approximatically 60% of the PP1 and 80% of the PP2 into polymeric material. It is concluded that PP1 and PP2 are both structural proteins present in the filaments observable in vivo.

PP2 had an elongated binding‐site for oligomers of N‐acetyl‐D‐glucosamine. It is suggested that this lectin immobilises bacteria and fungi to the cross‐linked filaments which seal wounded phloem sieve‐tubes, and thus maintains sterility.
Relationship between Oligosaccharide‐Lipid Synthesis and Protein Synthesis in Mouse LM Cells
Authors: GRANT Stephen R. , LENNARZ William J.
Published: March 2005

Previous studies from several laboratories have reported that inhibition of protein synthesis results in a concomitant reduction in synthesis of oligosaccharide‐diphosphoryldolichol. We have investigated this phenomenon in LM cells grown in defined culture medium. The results of this study indicate that incubation of LM cells with cycloheximide at a concentration sufficient to totally arrest polypeptide synthesis, results in a rapid reduction in the synthesis of [3H]mannose‐labeled or [3H]glucosamine‐labeled oligosaccharide‐lipid within 2 min. Cycloheximide treatment had only a slight inhibitory effect on synthesis of GDP‐Man. Furthermore, during the first 5 min after the addition of cycloheximide to LM cells, when [3H]mannose incorporation into oligosaccharide‐lipid was maximally reduced, the specific activity of the GDP‐Man pool was identical to that observed in control cells. Labeling in vivo of the lipid‐linked saccharide precursors of oligosaccharide‐lipid revealed that cycloheximide had virtually no effect on the synthesis of Man‐P‐Dol, GlcNAc‐PP‐Dol, GlcNAc2‐PP‐Dol, and β‐Man‐(GlcNAc)2‐PP‐Dol (Dol = dolichol). In agreement with this finding, results of experiments in vitro using microsomes prepared from LM cells indicate that cycloheximide did not directly inhibit the enzymes responsible for the synthesis of these lipid‐linked saccharide precursors.

Supplementation of mouse LM cells by preincubation for 1 h with dolichyl phosphate (5 μg/ml) resulted in a 300% stimulation of oligosaccharide‐lipid synthesis when compared to non‐supplemented cells. However, dolichyl phosphate supplementation of cycloheximide‐treated cells failed to restore oligosaccharide‐lipid synthesis to the level observed in control cells. In control cells pre‐labeled oligosaccharide‐lipid turned over rapidly and, as expected, cycloheximide addition to the chase medium significantly retarded this turnover process. However, preincubation of cells with exogenous dolichyl phosphate had little or no effect on the turnover of oligosaccharide‐lipid in control or cycloheximide‐treated cells. These findings argue against dolichyl phosphate deficiency as the primary cause of reduced oligosaccharide‐lipid synthesis when protein synthesis is blocked. The implications of these results, and an alternative hypothesis to explain the effect of inhibition of protein synthesis on oligosaccharide‐lipid synthesis based on elevation of intracellular GTP levels, are discussed.
Ethane Release during Metabolism of Aldehydes and Monoamines in Perfused Rat Liver
Authors: MÜLLER Armin , SIES Helmut
Published: March 2005

Infusion of aldehyde such as acetaldehyde, propionaldehyde or benzaldehyde to perfused rat liver leads to an increase in hepatic ethane production. Half‐maximal effect was obtained with about 20 μM acetaldehyde, a concentration range found in plasma during ethanol metabolism. Compounds which metabolically generate aldehydes such as monoamines (benzylamine, phenylethylamine) as substrates for monoamine oxidase or ethanol as substrate for alcohol dehydrogenase [A. Müller and H. Sies (1982) Biochem. J. 206, 153–156] are also able to elicit ethane release. Results obtained with inhibitors of hepatic aldehyde metabolism (pargyline or cyanamide) or of monamine oxidase (pargyline or tranylcypromine) suggest that metabolism of the aldehydes is required for ethane production. Radical scavenging by the addition of the flavonoid, cyanidanol, or by pretreatment with vitamin E (α‐tocopherol) abolished ethane release, in agreement with lipid peroxidation as a source of alkane production during aldehyde metabolism.
Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum‐free medium
Authors: BROAD Thomas E. , HAM Richard G.
Published: March 2005

Fibroblasts from ovine skin, and from the perirenal and subcutaneous adipose tissues of sheep were grown at clonal densities in medium MCDB 202 supplemented with 1 μg/ml bovine insulin, 1 μM dexamethasone, 100 ng/ml fibroblast growth factor and 20 μg/ml of the lipid preparation described by Bettger, W. J., Boyce, S. T., Walthall, B. J. and Ham, R. G. [(1981) Proc. Natl Acad. Sci, USA, 78, 5588–5592].

When maintained as a confluent monolayer in this medium, the fibroblasts from the adipose tissues spontaneously underwent an adipose differentiation. This was accelerated by substituting medium F12 for medium MCDB 202, and by raising the CO2 tension from 2% to 7.5% in air over the cultures. The differentiation was inhibited by deleting FGF from the growth medium, or by coating the culture surface with fibronectin or poly‐D‐lysine. Differentiation also failed to occur when the defined supplements were replaced with fetal bovine serum.

The synthesis of triacylglycerol by the cells, as seen by the increased specific activity of [14C]acetate incorporated into this lipid class, was accompanied by an increase in the specific activity of glycerol‐3‐phosphate dehydrogenase.
The location of redox‐sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli
Authors: POOLMAN Bert , KONINGS Wil N. , ROBILLARD George T.
Published: March 2005

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane.

Treatment of right‐side‐out membrane vesicles from E. coli ML 308–225 with the membrane‐impermeable oxidant ferricyanide resulted in inhibition of L‐proline uptake without having significant effect on the magnitude of the ΔμH+. Subsequent addition of reducing agents restored proline transport activity. The membrane‐impermeable SH‐reagent glutathione hexane maleimide inhibited proline transport in right‐side‐out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide.

Electron transfer in the respiratory chain of right‐side‐out vesicles led to the generation of a ΔμH+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4‐(2‐arsonophenyl)azo‐3‐hydroxy‐2, 7‐naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group.

Treatment of insideout membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside‐out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a ΔμH+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.
Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides
Authors: TRAYER Hylary R. , TRAYER Ian P.
Published: March 2005

The unique fast‐reacting cysteine residue (SH1) of myosin subfragment 1 (S1), prepared by chymotryptic digestion, and cysteine 373 of actin have been labelled selectively with the fluorescent probes, N‐(bromoacetyl)‐N′‐(1‐sulpho‐5‐naphthyl)ethylenediamine (1, 5‐BrAEDANS) and 5‐(iodoacetamido)fluorescein (5‐IAF), whose spectral properties render them a particularly effective donor‐acceptor pair in fluorescence energy transfer studies. The transfer efficiency of 40–45% represented a spatial separation of the chromophores of about 5 nm, which is in reasonable agreement with the value of 6 nm reported earlier for similarly labelled S1, prepared by papain digestion, and actin [Takashi, R. (1979) Biochemistry, 18, 5164–5169]. This transfer efficiency did not change when the doubly‐labelled binary complex was formed: (1) with acto‐S1(A1) or acto‐S1(A2) at 10–200 mM KC1, pH 7–8 and different buffer conditions; (2) with either S1 isoenzyme and regulated actin (i. e. actin with tropomyosin and troponin) both in the presence and absence of Ca2+ or when the donor and acceptor attachment sites were reversed. Analysis of donor and acceptor polarized fluorescence showed that the chromophores are not randomly orientated (i.e. χ2≠ 2/3), but they do have some motion relative to either protein. From a knowledge of the limiting values for χ2, the intersite distance for donor and acceptor chromophores was calculated to be in the range 3.9–6.7 nm.

Addition of MgATP to the doubly‐labelled acto‐S1 complex eliminated energy transfer but this was recovered when ATP hydrolysis was completed. By utilizing the known binding constants between S1, actin and either MgADP or MgAdoPP[NH]P (magnesium adenosine 5′‐[βγ‐imido]triphosphate) [Konrad, M. and Goody, K. (1982) Eur. J. Biochem. 128, 547–555; Greene, L. E. and Eisenberg, E. (1980) J. Biol. Chem. 255, 543–548], the concentrations of all species present at equilibrium were determined. Experimental conditions were chosen to maximise the amount of ternary acto‐S1‐nucleotide complex (∼ 50%) and minimise the amount of binary complex (≤ 2%). The spatial separation of the chromophore interaction sites in the ternary complex was found to be the same with both nucleotides and indistinguishable from that found with the binary complex. A similar strategy was employed to compare the conformations of the binary and ternary complexes by 1H‐NMR spectro‐scopy. In these experiments about 90% of the S1 was in the form of the ternary complex. There was no noticeable change in the acto‐S1 spectra upon addition of either MgAdo PP[NH]P or MgADP. These observations support the conclusion that there is no large change in structure in the ‘rigor’ binary acto‐S1 complex when it binds either ADP or Ado PP[NH]P.
Heterogeneity of brush‐border‐membrane vesicles from rat small intestine prepared by a precipitation method using Mg/EGTA
Authors: STIGER Bruno , MURER Heini
Published: March 2005

Brush border membrane vesicles from rat small intestine were isolated by a Mg/EGTA precipittion method. Further fractionation either by free flow electrophoresis or by sucrose density gradient centrifugation leads to subfractions which differ with respect to enzyme enrichment factors, transport properties for D‐glucose and protein pattern analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A relative enrichment of (Na++ K+)‐ATPase is found in one fraction, whereas in another fraction maltase, aminopeptidase M and alkaline phosphatase are relatively enriched. The fractions show different properties of D‐glucose transport under tracer exchange conditions and a different inhibition of D‐glucose transport by phlorizin and phloretin. These results indicate that the vesicles obtained from rat small intestine by this cation precipitation method are not homogeneous. The inhomogeneity cannot be due to a crosscontamination by membranes other than from the cell envelopment, as none of the fractions show a significant enrichment of succinate–cytochrome c oxidoreductase, KCN‐resistant NADH oxidoreductase or glucosaminidase. The inhomogeneity might be due either to a crosscontamination by basal‐lateral membranes or to membranes derived from epithelial cells not yet fully differentiated.
Distribution of enzymes of fatty acid and ketone body metabolism in periportal and perivenous rat‐liver tissue
Authors: KATZ Norbert R. , FISCHER Wilhelm , GIFFHORN Susanne
Published: March 2005

The zonal distribution of cytosolic acetyl‐CoA carboxylase, mitochondrial 3‐hydroxyacyl‐CoA dehydrogenase and 3‐hydroxybutyrate dehydrogenase was studied in microdissected liver tissue.

In fed male and female rats the activity of the lipogenic enzyme acetyl‐CoA carboxylase was 1.6‐times higher in the perivenous than in the periportal zone of the liver acinus.

3‐Hydroxybutyrate dehydrogenase, which is involved in the formation of the ketone body, 3‐hydroxybutyrate, exhibited a similar distribution pattern with a 1.5–1.8‐times higher activity in the perivenous than in the periportal zone.

In contrast, the activity of 3‐hydroxyacyl‐CoA dehydrogenase, the third enzyme in ß‐oxidation, was about equally distributed between the periportal and the perivenous zone of the liver acinus.

The results indicate a predominance of lipogenesis in the perivenous, mainly glycolytic zone of the liver acinus. Furthermore, these data support the hypothesis that ß‐oxidation supplies energy for basic and anabolic processes like gluconeogenesis in the periportal zone, while it provides acetyl‐CoA for ketogenesis besides energy for basic needs in the perivenous zone.
Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus Presence of two forms of H2A in the sipunculid chromatin
Authors: KMIECIK Daniel , COUPPEZ Maurice , BELAICHE Denise , SAUTIERE Pierre
Published: March 2005

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with α‐chymotrypsin and from structural studies of tryptic peptides of the protein.

By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non‐conservative.

Most of the evolutionary changes are mainly observed in the basic amino‐terminal and carboxy‐terminal regions of the molecule, which are the primary DNA‐binding sites. Few conservative point changes are observed in the central region (residues 18–118) which interacts strongly with histone H2B to form the dimer H2A‐H2B. 60% of the H2A molecules were found phosphorylated on the amino‐terminal residue, N‐acetyl‐serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 ± 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.
Nucleotide sequence of cloned cDNA coding for honeybee prepromelittin
Authors: VLASAK Reinhard , UNGER‐ULLMANN Claudia , KREIL Günther , FRISCHAUF Anna‐Maria
Published: March 2005

Total mRNA from venom glands of young queen bees was transcribed into cDNA and cloned into the PstI site of plasmid pBR322. The nucleotide sequence of two clones with inserts containing genetic information for prepromelittin is presented. The longer insert encompasses 374 base pairs including 52 nucleotides before the initiation codon and a 3′ non‐coding region of 112 base pairs. The 70 amino acids of prepromelittin represent the total coding capacity of the mRNA from which this insert is derived. Southern blot analysis with this cloned cDNA has shown that it hybridizes with a single EcoRI fragment of honeybee DNA which contains about 3000 base pairs.
The role of protein L16 and its fragments in the peptidyltransferase activity of 50‐S ribosomal subunits
Authors: MAIMETS Tovio , USTAV Mart , VILLEMS Richard
Published: March 2005

Two large polypeptide fragments of ribosomal protein L16 were obtained by limited hydrolysis with trypsin and chymotrypsin. The chymotryptic fragment, lacking nine N‐terminal amino acid residues, is fully active in the restoration of the peptidyltransferase activity of the LiCl‐stripped 50‐S ribosomal subunits, whereas the tryptic fragment, lacking an additional six residues, is inactive. We also show that under the optimized ionic conditions protein L16 is not needed for the peptidyltransferase activity.
A 1H‐NMR study of isolated domains from human plasminogen Structural homology between kringles 1 and 4
Authors: LLINAS Miguel , DE MARCO Antonio , HOCHSCHWENDER Susan M. , LAURSEN Richard A.
Published: March 2005

Kringles 1 and 4 from human plasminogen are polypeptide domains of Mr∼ 10000 each of which can be isolated by proteolysis of the zymogen. They have been studied by 1H‐NMR spectroscopy at 300 MHz and 600 MHz. The spectra, characteristic of globular structures, show striking analogies that point to a close conformational relatedness among the two kringles, consistent with their high degree of amino acid conservancy and homology. The interaction of both kringles with p‐benzylaminesulfonic acid (BASA), an antifibrinolytic drug that binds to a lysinebinding site, results in better resolved, narrower lines for both spectra. Aromatic and methyl‐region spectra of BASA complexes of kringles 1 and 4 were compared and the latter was studied by two‐dimensional NMR spectroscopy. Analysis of the CH3 multiplets in terms of their resonance patterns, and the amino acid compositions and sequences of the two kringles, leads to the identification of most signals and to some assignments. In particular, a doublet at‐1 ppm, exhibited by both kringles and also found in reported proton spectra of homologous bovine prothrombin fragments, has been assigned to Leu46, a residue that is conserved in all of the kringles studied to date by 1H‐NMR. Since this resonance is somewhat more sensitive to BASA than other methyl signals, it is likely that Leu46 is proximal to the lysine‐binding site. Nuclear Overhauser experiments reveal that Leu46 is surrounded by a cluster of closely interacting hydrophobic and aromatic side chains. Kringle 4 was also compared with a derivative chemically modified at Trp72 with dimethyl(2‐hydroxy‐5‐nitrobenzyl)sulfonium bromide. As judged from the proton spectra, the modified kringle 4 retains globularity and is perturbed mainly in the aromatic region, in analogy to that which is observed for the unmodified kringle upon BASA binding. Furthermore, although previous studies have indicated no retention of the modified kringle by lysine‐Sepharose, the NMR studies point to a definite interaction between BASA and the kringle derivative. The spectroscopic data also suggest that the His31 imidazole is not significantly affected by the ligand and that the lysine‐binding site is structured mostly by hydrophobic side chains, including Trp72 in the case of kringle 4, and probably Tyr72 in kringle 1.
Topography of the total protein population from cultured cells upon fractionation by chemical extractions
Authors: LENSTRA Johannes A. , BLOEMENDAL Hans
Published: March 2005

Chemical extractions are proposed as a major tool for a fractionation of cellular proteins. As a model system, proteins from cultured hamster lens cells have been divided by independent extractions into seven subcellular fractions, corresponding to water‐soluble proteins and the proteins from membranes, microfilaments (and other deoxycholate‐soluble proteins), intermediate filaments, microtubules, polysomes and nuclei respectively. The latter two fractions have been subfractionated yielding ribosomal proteins, the elongation and initiation factors of the protein‐synthesis machinery, chromatin proteins and non‐chromatin proteins.

The protein compositions of the fractions have been analyzed by one‐dimensional and two‐dimensional gel electrophoresis. This resulted in an almost complete topography of the proteins detected on two‐dimensional gels of total‐cell lysates.

Comparison of two‐dimensional patterns of proteins from the total‐cell lysate and proteins from hamster erythrocytes or from liver, muscle or brain tissue showed that the different cell types have only few proteins in common. Two proteins are common to all of these cell types, namely actin and a 68‐kDa protein. The latter protein was, like actin, vimentin and the tubulin subunits, also present in most cell fractions. Evidence is presented that this protein is identical to a 68‐kDa heat‐shock protein.
Ribosomal subunits and ribosomal proteins of Tetrahymena thermophila
Authors: PÉTRIDOU Barbara , CUNY Marguerite , GUÉRIN Marie‐France , HAYES Francoise
Published: March 2005

Proteolytic degradation of ribosomal proteins occurs during the preparation of subunits of the cytoplasmic ribosomes of the protozoa Tetrahymena thermophila and the isolated subunits are inactive. Addition of 5 mM iodoacetamide to cell suspensions before extraction inhibits proteolytic activity and permits isolation of active subunits. The protein complements of these subunits have been characterized in two different two‐dimensional electrophoretic systems, and their molecular weights have been determined.
Isolation and purification of poly(ADP‐ribose) glycohydrolase from pig thymus
Authors: TAVASSOLI Manoochehr , TAVASSOLI Mahvash H. , SHALL Sydney
Published: March 2005

Poly(ADP‐ribose) glycohydrolase has been purified about 12 300‐fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 μmol min−1 mg protein−1. The molecular weight was estimated to be 59000 by gel filtration through Sephadex G‐100 in a non‐denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weigth, 61 500 and 67 500. The Km value for poly(ADP‐ribose) is estimated to be 1.8 μM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP‐ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP‐ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single‐stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double‐stranded DNA was not inhibitory.
Dimeric glutamyl‐tRNA synthetases from wheat Kinetic properties and functional structures
Authors: THOMES Jean Claude , RATINAUD Marie Hélène , JULIEN Raymond
Published: March 2005

The Michaelis constants in the tRNA aminoacylation reaction have been studied for the three dimeric glutamyl‐tRNA synthetases C, P and E. The values were found to be: for tRNA, 0.20 μM, 0.20 μM, 0.20 μM and 0.44 μM; for glutamic acid, 10 μM, 83 μM and 83 μM; for MgATP, 0.46 mM, 0.38 mM and 0.26 mM. MgATP concentrations higher than 2 mM induce pronounced inhibition.

The presence of the cognate tRNA is required for [32P]PPi‐ATP isotopic exchange. In the absence of tRNA no hyperbolic saturation of the enzymes by glutamic acid occurs in our experimental conditions.

Analysis of the enzymic activity as a function of enzyme concentration leads to the conclusion that the active forms are dimers which are in equilibrium with inactive monomers. The values of the dissociation constants Kd were found to be 43 nM, 53 nM and 87 nM for glutamyl‐tRNA synthetases C, P and E respectively.
Carbohydrate composition and electrophoretic properties of tomato polygalacturonase isoenzymes
Authors: MOSHREFI Massoud , LUH Bor S.
Published: March 2005

Two polygalacturonase isoenzymes, PG I and PG II, were extracted from Murrieta tomato and purified by gel exclusion and ion‐exchange chromatography. The kinetic constants and activation energies of the purified isoenzymes have been determined. Polygalacturonase I has two polypeptide chains (Mr= 47500 and 41400) whereas polygalacturonase II is a single polypeptide (Mr= 47500) as shown by electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate. Both isoenzymes are glycoproteins. Through gas liquid chromatography, polygalacturonase II was shown to contain 4.6% neutral hexoses and 1.5% amino sugars. There are eight d‐mannose, two l‐fucose, two d‐xylose and three N‐acetylglucosamine residues per mole of PG II. The carbohydrate portion of PG II was shown to be attached to the protein part through an N‐acetylglucosaminylasparaginyl bond.
Detergent solubilisation of the rabbit neutrophil receptor for chemotactic formyl peptides
Authors: BALDWIN Jocelyn M. , BENNETT Jonathan P. , GOMPERTS Bastien D.
Published: March 2005

Digitonin was found to be the only detergent (out of 24 tested) capable of solubilising the chemotactic formyl peptide receptor from rabbit neutrophil membranes in a form which retained its [3H]fMet‐Leu‐Phe binding activity. The solubilised material retained many of the characteristics of the membrane‐bound receptor. [3H]fMet‐Leu‐Phe binding to the digitonin extract was measured at 4°C using an equilibrium dialysis assay. Binding was saturable and of high affinity (Kd= 3.5±0.7 nM). The potencies of a series of synthetic peptides as inhibitors of [3H]fMet‐Leu‐Phe binding to the soluble receptor showed the same rank order as for inhibition of the membrane‐bound receptor. In addition, binding to both preparations was sulphydryl dependent showing a parallel inhibition by p‐chloromercuribenzene sulphonate which could be partially reversed by subsequent incubation with dithiothreitol.
Nucleic acid sequence of the region of the genome encoding capsid protein VP1 of neurovirulent and attenuated type 3 polioviruses
Authors: STANWAY Glyn , CANN Alan J. , HAUPTMANN Rudi , MOUNTFORD Roger C. , CLARKE Lindsay D. , REEVE Peter , MINOR Philip D. , SCHILD Geoffrey C. , ALMOND Jeffrey W.
Published: March 2005

Three closeiy related strains of poliovirus type 3 have been used to study the molecular basis of attenuation in the currently used Sabin vaccine of this serotype. Plaque‐purified derivatives of these strains possess closely similar serological and biochemical properties yet differ markedly in neurovirulence for monkeys. Molecular cloning via an RNA cDNA method has facilitated comparative nucleotide sequencing. Initial efforts have concentrated on the region of the genome encoding VP1. Only minor structural differences between neurovirulent and attenuated type 3 strains were detected, in contrast to the major differences observed between the vaccine strains of poliovirus type 1 and its virulent precursor P1/Mahoney. These observations suggest that the molecular basis of attenuation of type 3 Sabin vaccine virus does not involve the VP1 polypeptide and, therefore, that mutations conferring the attenuated phenotype probably lie elsewhere in the genome.
Dogfish insulin
Authors: BAJAJ Mona , BLUNDELL Thomas L. , PITTS James E. , WOOD Stephen P. , TATNELL Michele A. , FALKMER Sture , EMDIN Stefan O. , GOWAN Linda K. , CROW Hal , SCHWABE Christian , WOLLMER Axel , STRASSBURGER Wolfgang
Published: March 2005

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid‐ethanol extraction and salt precipitation. The amino acid sequences of the performic‐acid‐oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co‐ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln→His, B21Glu→Pro, B22Arg→Lys, B25Phe→Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half‐maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin.

The use of interactive computer graphics model‐building predicts that the dogfish insulin can attain a threedimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model‐building studies.
Non‐reactivity of the selenoenzyme glutathione peroxidase with enzymatically hydroperoxidized phospholipids
Authors: GROSSMANN Adelbert , WENDEL Albrecht
Published: March 2005

Selenium‐containing glutathione peroxidase (EC 1.11.1.9) was purified 6000‐fold from bovine red blood cells to apparent homogeneity. Lipoxygenase (EC 1.13.11.12) was enriched 20‐fold from soybean acetone powder. Linoleic acid was peroxidized with lipoxygenase and then used as a substrate in the glutathione peroxidase reaction. Analogous experiments were conducted with synthetic 1,2‐dilinoleoyl‐l‐α‐glycerophosphocholine and with natural bovine heart cardiolipin. The peroxidized phospholipids were reactive with glutathione peroxidase only after enzymatic attack by phospholipase A2 (EC 3.1.1.4). This result implies that the membrane‐protective function of glutathione peroxidase includes preceeding phospholipase action and excludes a direct interaction of this enzyme with membrane‐bound lipid hydroperoxides.
β‐Agarases I and II from Pseudomonas atlantica. Purifications and some properties
Authors: MORRICE Lora M. , McLEAN Maltland W. , WILLIAMSON Frank B. , LONG William F.
Published: March 2005

The agarose‐degrading system of Pseudomonas atlantica has been re‐examined. In addition to the previously reported extracellular endo‐β‐agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333–335] a second, membrane‐bound endo‐enzyme activity, β‐agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6‐anhydro‐α‐l‐galactopyranosyl‐(1→3)‐d‐galactose] and also to degrade partially 6‐O‐methylated agarose to neoagarobiose and 61‐O‐methyl‐neoagarobiose. Novel assays were devised for β‐agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of β‐agarase I and II.

β‐Agarase I was purified 670‐fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G‐100. The enzyme was resolved from the small amount of extracellular β‐agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32 000. Activity was observed against agar over the pH range 3.0–9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30°C but only for up to 2 h at 40°C.

β‐Agarase II was partially purified (5‐fold) from the soluble fraction of disrupted cells by chromatogm raphy on Sephadex G‐100, hydroxyapatite and DEAE‐Sepharose CL‐6B. This preparation was free of β‐agarase I and disaccharidase. β‐Agarase II was stimulated by NaCl, optimally in the range 0.10–0.20 mol dm−3 (2.4‐fold the activity at 0.010 mol dm−3 NaCl). Alkali earth metal (0.002 mol dm−3 CaCl2 or 0.005 mol dm−3 MgCl2) gave 1.2‐fold the normal activity. Optimum activity was over pH 6.5–7.5.
Detection of conformational changes in chloroplast coupling factor 1 by 8‐anilino‐1‐naphthalene‐sulphonate fluorescence changes
Authors: PICK Uri , FINEL Moshe
Published: March 2005

Chloroplast coupling factor 1 (CF1) contains a high‐affinity binding site for 8‐anilino‐1‐napthalene sulphonate (ANS, Kd= 5–6 μM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue‐shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1.

Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 μM to 22 μM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n=1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals.

The affinity of latent CF1 for ADP (Kd=12 μM) is considerably higher than for ATP (Kd=95 μM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 μM. Mg increases the apparent affinity for ATP (Kd=28 μM) but not for ADP or Pi.

Binding of ATP to the tight‐sites does not inhibit the ADP or Pi‐induced fluorescence quenching but decreases the affinity for ADP (Kd=34 μM) and for inorganic phosphate (Kd=320 μM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites.

Activation of a Mg‐specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis.

ATPase activation also influences the ANS responses to Ca and Mg. Ca‐ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg‐ATPase activation specifically increases the Mg‐induced fluorescence enhancement.

The fluorescence of CF1‐bound ANS is enhanced by Dio‐9 and quenched by phloridzin, quercetin, Nbf‐Cl and FITC. Nbf‐Cl and FITC completely inhibit the ADP‐induced fluorescence quenching whereas Dio‐9 inhibits the Mg‐induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site.

ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.
Study of the Hansenula anomala yeast flavocytochrome‐b2–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c
Authors: THOMAS Marie‐Antoinette , FAVAUDON Vincent , POCHON François
Published: March 2005

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn‐cytochrome c appears to be in the form of a stable though non‐covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal‐free porphyrin‐cytochrome c, the precursor of Zn‐cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate‐reduced state showed a simple binding equilibrium (Kd=0.1 μM at I=0.03 M, 10°C) with a stoichiometry of one Zn‐cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn‐porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.
Study of the Hansenula anomala yeast flavocytochrome‐b2‐cytochrome‐c complex
Authors: THOMAS Marie‐Antoinette , GERVAIS Michel , FAVAUDON Vincent , VALAT Pierre
Published: March 2005

The reversible association of the Zn2+ ‐substituted Hansenula anomala cytochrome c dimer (Thomas et al., preceding paper in this issue) to flavocytochrome b2 in oxidized or lactate‐reduced state has been investigated by fluorimetry. The same method has been used for the determination of Zn‐cytochrome c complexing to defined proteolytic fragments of flavocytochrome b2, either heme‐b2‐containing monomers or a flavin‐linked tetramer. All these fragments but the isolated cytochrome b2 core showed binding stoichiometries, Kd values and ionic strength dependences quite similar to those found for native flavocytochrome b2. These data allowed localization of the single high‐affinity binding site of cytochrome c on a particular globule in the dehydrogenase domain of the flavocytochrome b2 protomers. Quenching of the Zn‐porphyrin c fluorescence in the various complexes occurred with only minor changes of the fluorescence lifetime and did not show any direct relationship to the presence or the redox state of the heme b2 group.
On the formation of an oxygen‐tolerant three‐component nitrogenase complex from Azotobacter vinelandii
Authors: SCHERINGS Gerard , HAAKER Huub , WASSINK Hans , VEEGER Cees
Published: March 2005

Conditions are defined in which the oxygen‐labile nitrogenase components from Azotobacter vinelandii can be protected against oxygen inactivation by the so‐called Fe/S protein II. It is demonstrated that oxygen protection can be achieved by complex formation of the three proteins. Complex formation was studied by gel chromatography. Only when the three proteins are in the oxidized state and MgCl2 is present, can an oxygen‐tolerant complex be isolated. Quantitative SDS/polyacrylamide gel electrophoresis of such complexes, yielded an average ratio of nitrogenase component 2/nitrogenase component 1 (Av2/Av1) of 2.4 ± 0.5. Protection by Fe/S protein II was correlated with the amount of [2 Fe‐2 S] clusters present in the protein and not by the amount of protein. Measurements of the amount of iron and sulfide of Fe/S protein II showed that the iron and sulfide content of the protein was variable. The maximum values found indicate that Fe/S protein II contains two [2Fe‐2S] clusters per dimer of 26 kDa. Full protection by Fe/S protein II was obtained with a ratio of Fe/S protein II/Av1 of 1.1 ± 0.2; the Fe/S protein II containing two [2Fe‐2S] clusters per dimer of 26 kDa. When Fe/S protein II contains less [2 Fe‐2S] clusters, more protein is necessary to obtain full protection. The three‐component nitrogenase complex is also oxygen stable in the presence of MgATP or MgADP.

Analysis in the ultracentrifuge showed that the major fraction of the reconstituted complex has a sedimentation coefficient centered around 34S. A small fraction (<30%) sediments with values centered around 111 S. This suggests an average mass for the oxygen‐stable nitrogenase complex of 1.5 MDa. Taking into account the determined stoichiometry of the individual proteins, the molecular composition of the oxygen‐stable nitrogenase complex is presumably 4 molecules of Av1, 8–12 molecules of Av2 and 4–6 molecules of Fe/S protein II containing two [2Fe‐2S] clusters per dimer of 26 kDa.
Synthetic peptides reproducing the site phosphorylated by cAMP‐dependent protein kinase in protein phosphatase inhibitor‐1
Authors: CHESSA Gavino , BORIN Gianfranco , MARCHIORI Fernando , MEGGIO Flavio , BRUNATI Anna Maria , PINNA Lorenzo A.
Published: March 2005

The hexapeptide (Arg)2‐Pro‐Thr‐Pro‐Ala (T1) and octapeptide (Arg)2‐Pro‐Thr‐Pro‐Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor‐1, a physiological target of cAMP‐dependent protein kinase, and five related peptides were synthesized by the method in solution.

The phosphorylation rates of such peptides by cAMP‐dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)2‐Ala‐Ser‐Val‐Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C‐terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing Km and depressing V. On the other hand the N‐terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the Km, without affecting the V. The extension of the hexapeptide T 1 on its C‐terminal side to give the derivative (Arg) 2‐Pro‐Thr‐Pro‐Ala‐Thr‐Val‐Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced.

Therefore the local structural features of the phosphorylatable site of inhibitor‐1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.
Molecular properties of the apamin‐binding component of the Ca2+‐dependent K+ channel Radiation‐inactivation, affinity labelling and solubilization
Authors: SCHMID‐ANTOMARCHI Heidy , HUGUES Michel , NORMAN Robert , ELLORY Clive , BORSOTTO Marc , LAZDUNSKI Michel
Published: March 2005

Radiation‐inactivation was used to assess the functional size of the apamin‐binding component of the Ca2+‐dependent K+ channel. The amount of specific binding of 125I‐apamin to receptors in synaptic membranes of rat cortex decayed exponentially with increasing doses of ionizing radiation and target size analysis was consistent with a relative molecular mass of 250000 ± 20000 for the 125I‐apamin receptor. Analysis on sodium dodecyl sulfate gels following covalent cross‐linking of 125I‐apamin to its receptor in a synaptosomal membrane preparation from rat cortex revealed a single labelled polypeptide chain ofMr= 33000 ± 2000 in the presence of protease inhibitors. Our results suggest that the Ca2+‐dependent K+ channel from rat cortex is an oligomeric structure of Mr= 250000 ± 20000 containing an apamin‐binding subunit of Mr= 33000 ± 2000. The apamin‐binding component of the Ca2+‐dependent K+ channel from rat synaptosomes was solubilized using detergents such as sodium cholate or 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane sulfonate. Phospholipids did not increase the stability of the apamin‐binding component during the solubilization. Binding of apamin to its solubilized receptor is reversible and saturable. The dissociation constant of the apamin‐receptor complex is 40–150 pM. the rates constants of association and dissociation being 3.2 × 106 M−1 s−1 and 1.4 × 10−4 s−1 respectively. These binding characteristics are similar to those found for the membrane‐bound apamin receptor.
A solution equivalent of the 2Zn→4Zn transformation of insulin in the crystal
Authors: RENSCHEIDT Heribert , STRASSBURGER Wolfgang , GLATTER Uwe , WOLLMER Axel , DODSON Guy G. , MERCOLA Daniel A.
Published: March 2005

Circular dichroic spectroscopy clearly reveals a solvent‐induced conformational change of insulin in the presence of zinc ions. The spectral change corresponds to an increase in helix content. The transition observed in solution is an equivalent of the 2Zn → 4Zn insulin transformation in the crystal. This is inferred from a series of observations. (1) The spectral effects are compatible with the refolding of the B‐chain N‐terminus into a helix known from crystal studies. (2) The spectral effects are induced by the very same conditions which are known to induce the 2Zn → 4Zn insulin transformation in the crystal (i.e. threshold concentrations of NaCl, KSCN, NaI, for example). (3) They fail to be induced by the same conditions that fail to induce the crystal transformation (e.g. Ni2+ instead of Zn2+).

It is concluded that the potential to undergo the transition resides in the hexamer since neither insulin dimers nor monomeric des‐pentapeptideB26–30‐insulin respond detectably to high halide concentration. Secondly the ability of zinc ions to accommodate tetrahedral coordination allows the transition which is not permitted by other divalent metal ions. Thirdly the transition is independent of the off‐axial tetrahedral zinc coordination sites since it occurs in [AlaB5]insulin which lacks the B5 histidine necessary for their formation. A symmetrically rearranged hexamer thus appears possible with two tetrahedrally coordinated zinc ions on the threefold axis; this is consistent with the observation that in native insulin two zinc ions per hexamer are sufficient to produce the full spectral effect. The amount of additional helix derived from the circular dichroic spectral change, however, cannot settle whether the transition comprises only three or all six of the subunits to yield a symmetrical hexamer. Finally the transformation in solution evidently still occurs in an intramolecularly A1‐B29‐cross‐linked insulin in spite of the partially reduced flexibility.
Labeling of phospholipids in vesicles and human erythrocytes by photoactivable fatty acid derivatives
Authors: BERKHOUT Theo A. , van AMERONGEN Aard , WIRTZ Karel W. A.
Published: March 2005

The photoactivable glycolipid probes, 2‐(4‐azido‐2‐nitrophenoxy)palmitoyl[1‐I4C]glucosamine (compound A) and 12‐(4‐azido‐2‐nitrophenoxy)stearoyl[1‐14C]glucosamine (compound B) were synthesized essentially as described before [Iwata, K. K. et al. (1978) Prog. Clin. Biol. Res. 22, 579–589]. These probes were used to label phospholipid vesicles and erythrocyte membranes. A chromatographic method was developed to quantify the individual probe‐phospholipid adducts involving both phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. For both membranes as well as for both probes a phospholipid labeling pattern was obtained which appeared to reflect the relative content of fatty acid double bonds in each phospholipid class. The distinct labeling of phosphatidylserine in intact erythrocytes strongly suggested that the probes spontaneously and rapidly redistributed between the two halves of the membrane bilayer. In addition, both probes yielded an extensive labeling of the membrane proteins. Analysis by dodecylsulfate‐polyacrylamide gel electrophoresis and autoradiography has indicated that the protein labeling pattern was different, depending on whether the ‘shallow’ probe (compound A) or ‘depth’ probe (compound B) were used.