bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of subunit b of the Fo complex from Escherichia coli ATP synthase by a hydrophobic maleimide and its effects on Fo functions
Authors: SCHNEIDER Erwin , ALTENDORF Karlheinz
Published: March 2005

Purified F0 from Escherichia coli ATP synthase was labelled with N‐(7‐dimethylamino‐4‐methyl‐coumarinyl)‐maleimide (DACM), a hydrophobic reagent which forms a stable, strongly fluorescent adduct with SH groups. Sodium dodecyl sulfate gel electrophoresis clearly demonstrated that subunit b was exclusively labelled, most likely at Cys‐21, the only cysteine residue in E. coli F0. The amount of two molecules of DACM bound per F0, which was calculated from the absorption spectrum at 380 nm, is in full agreement with the postulated stoichiometry of two copies of subunit b/F0 complex. Thus the label provides a useful tool for simply detecting subunit b in protein chemical studies.

DACM‐labelled F0 was incorporated into liposomes and assayed for H+ translocating activity and its capacity to bind purified F1. Whereas the initial rate of H+ uptake was inhibited about 40% the reconstitution of a dicyclohexylcarbodiimide‐sensitive F1 F0 ATPase activity was completely unaffected. In a second set of experiments we reconstituted an F0 complex from DACM‐labelled purified subunit b and an ac complex. In contrast to the results obtained with intact, DACM‐labelled F0, both H+ translocating activity and F1 binding capacity were greatly reduced. Our data indicate that cysteine‐21, probably together with other amino acids, is involved in maintaining a proper interaction of the hydrophobic N‐terminal region of subunit b with the ac complex. This interplay seems to be a prerequisite for at least the in vitro assembly of a functional F0 complex.
Characterization of extracellular matrix‐associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture
Authors: WANG Zu‐Wu , IRIMURA Tatsuro , NAKAJIMA Motowo , BELLONI Paula N. , NICOLSON Garth L.
Published: March 2005

A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix‐associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per‐cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix‐associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix‐associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High‐speed gel permeation chromatography profiles on Fractogel TSK HW‐55(S) of matrix‐associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav= 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan‐sulfate‐specific endo‐β‐glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav= 0.43). Size analysis and anion‐exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N‐sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix‐associated sulfated glycosaminoglycans.
Schistosoma mansoni surface glycoproteins
Authors: PAYARES Gilberto , SIMPSON Andrew J. G.
Published: March 2005

Surface glycoproteins from newly transformed schistosomula of Schistosoma mansoni have been identified by surface radioiodination and lectin‐affinity chromatography. From the glycoconjugates bound by the three lectins used, concanavalin A, peanut agglutinin and fucose‐binding protein, only in the concanavalin‐A‐bound fractions were glycoproteins identified. Changes in concanavalin‐A‐binding glycoproteins were detected after transformation and early maturation of the schistosomula.

Some glycoproteins disappeared (Mr 38000, 29000 and 25000), some appeared independently of host molecules (Mr 19000), others only appeared after culture in human serum (Mr 45000).

Two major glycoproteins of Mr 32000 and 16000 were detected on all stages examined. Within the total set of surface glycoproteins identified on 3‐h schistosomula only the strong Mr‐38000–32000 complex was found to be antigenic. Thus many major low‐molecular‐mass surface glycoproteins of the parasite are not recognised as antigens by immune animals. The separation of only the Mr‐38000–32000 antigens by concanavalin A affinity chromatography indicates the feasibility of using this method in conjunction with immunoaffinity columns to purity these molecules.
Synthesis, transport and processing of cathepsin C in Morris hepatoma 7777 cells and rat hepatocytes
Authors: MAINFERME Francis , WATTIAUX Robert , von FIGURA Kurt
Published: March 2005

The synthesis, transport and processing of cathepsin C was studied in Morris hepatoma 7777 cells by metabolic labelling, immunoprecipitation and characterization of labelled polypeptides by gel electrophoresis and fluorography. The largest detectable precursor of cathepsin C was a polypeptide of Mr= 92 500. Even 3 min after synthesis this precursor was accompanied by four polypeptides with Mr values ranging from 63 000 to 54 000, indicating cleavage of the precursors within the endoplasmic reticulum. The early forms of cathepsin C were associated with low‐buoyant‐density organelles containing the markers of endoplasmic reticulum and Golgi complex. About 30% of these early forms were secreted within 3 h after synthesis. The remaining 70% were transferred into dense lysosomes and processed between 2 and 3 h after synthesis to a mixture of the least five major and nine minor polypeptides with Mr values ranging from 73 000 to 12 000. These forms remained stable for at least 3 days. In freshly isolated hepatocytes cathepsin C was processed to forms closely related to those found in the hepatoma cells. Cathepsin C was synthesized in Morris hepatoma 7777 cells as a glycoprotein with mannose‐6‐phosphate residues that mediated mannose‐6‐phosphate‐specific receptor‐dependent uptake in human skin fibroblasts. In contrast to hepatocytes, synthesis of mannose‐6‐phosphate receptors in Morris hepatoma 7777 cells was below the limit of detection. The hepatoma cells did not express at the cell surface these or other receptors mediating endocytosis of lysosomal enzymes. Further, processing and transport of newly synthesized cathepsin C was largely resistant to NH4Cl. Apparently, cathepsin C is transferred in Morris hepatoma 7777 cells by a mechanism independent of mannose‐6‐phosphate‐specific receptors.
Properties of two isoforms of human blood platelet α‐actinin
Authors: LANDON Francoise , GACHE Yannick , TOUITOU Hélène , OLOMUCKI Anna
Published: March 2005

The structural and functional properties of the aa (2 × 97 kDa) and cc (2 × 94 kDa) isoforms of platelet α‐actinin have been compared. Structural differences between aa and cc are revealed by their peptide maps, obtained from limited proteolysis, and by their immunological cross‐reactivity. Both isoforms stimulate the Mg ATPase activity of actomyosin, bind to F‐actin (high‐speed sedimentation) and cross‐link or gel actin filaments (low‐speed sedimentation and viscometry), in a calcium‐dependent manner. The study of the interaction with F‐actin indicates that the binding of 1 molecule of aa or ccα‐actinin/9‐11 actin monomers is sufficient to produce maximal gelation in the presence of EGTA. CaCl2 at 0.1 mM strongly inhibits the binding of aa to F‐actin and weakly that of cc, while it inhibits similarly the cross‐linking of either aa or cc. The cross‐linking efficiency of cc is 9, 7, 1.7 and 1.3 times higher than that of aa at 4, 20, 30 and 37°C, respectively. The bb form (2 × 96 kDa), which is a proteolytic product of aa [Y. Gache et al. (1984) Biochem. Biophys. Res. Commun. 124, 877‐881], behaves roughly as aa, but the calcium sensitivity of its binding to F‐actin is intermediate between that of aa and cc. These results suggest that the effect of Ca2+ concentration on the binding of platelet α‐actinin to F‐actin may be partly dissociated from the effect on the cross‐linking.
Heterogeneity in the metabolism of the arachidonoyl molecular species of glycerophospholipids of rabbit alveolar macrophages
Authors: NAKAGAWA Yasuhito , KURIHARA Koichi , SUGIURA Takayuki , WAKU Keizo
Published: March 2005

The relative incorporation of [3H]arachidonic acid (20:4) into individual molecular species containing 20:4 at the 2 position (18:1 ‐ 20:4, 16:0 ‐ 20:4 and 18:0 ‐ 20:4 species) of diacyl and ether‐linked glycerophosphocholine, glycerophosphoethanolamine and glycerophosphoinositol of rabbit alveolar macrophages has been measured by reversed‐phase high‐performance liquid chromatography (HPLC).

The rate of incorporation of [3H]20:4 into the molecular species of glycerophospholipids was greatly influenced by their structures. The reversed‐phase HPLC analysis allowed elucidation of the influence of structural differences, such as the nature of the polar head group, the fatty chain at the 1 position and the chemical form of the bond of the fatty chain attached at the 1 position on the uptake of [3H]20:4 by comparison of the specific radioactivities of arachidonoyl molecular species having the same structures, except that one of the three kinds of moiety was different. The specific radioactivities of the molecular species containing choline head groups were significantly higher than those containing ethanolamine and inositol moieties. The specific radioactivities of diacyl molecular species were considerably higher than those of ether‐linked molecular species. The nature of the fatty chain attached at the 1 position also influenced the uptake of [3H]20:4 into glycerophospholipids. The arachidonoyl molecular species containing 18:1 at the 1 position were preferentially labelled with [3H]20:4 as compared to the corresponding 16:0 ‐ 20:4 and 18:0 ‐ 20:4 species either of diacyl or ether‐linked glycerophospholipids.

The present results suggest that the acyltransferase involved in the incorporation of 20:4 into glycerophospholipids has selectivity for the structures of glycerophospholipids and the order of selectivity of this enzyme for the arachidonoyl molecular species, deduced in the present experiments, was as follows: choline head group > ethanolamine and inositol groups, acyl bond > ether and vinyl ether bonds, 18:1 fatty chain > 16:0 and 18:0 fatty chains at the 1 position. Comparison of the metabolic activities of all major arachidonoyl molecular species of glycerophospholipids having a single structure is reported here for the first time.
The primary structure of ribosomal protein L2 from Bacillus stearothermophilus
Authors: KIMURA Makolo , KIMURA Junko , WATANABE Keiichi
Published: March 2005

The complete amino acid sequence of ribosomal protein L2 from the moderate thermophile Bacillus stearothermophilus has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with Staphylococcus aureus protease, trypsin and chymotrypsin, as well as by chemical cleavage with o‐iodosobenzoic acid. The protein contains 275 amino acid residues and has a calculated molecular mass of 30201 Da. Comparison of this sequence with sequences of the corresponding proteins from Escherichia coli and from spinach and tobacco chloroplasts reveals that 60% of the residues of protein L2 from B. stearothermophilus are identical to those of the protein from E. coli and 45% are identical to those found in the two chloroplast proteins. There are extended regions of totally conserved sequence at positions 54–58 (GGGHK), 81–86 (EYDPNR), and 224–230 (MNPVDHP) in all four proteins.
Structure of bacillomycin F, a new peptidolipid antibiotic of the iturin group
Authors: PEYPOUX Francoise , MARION Dominique , MAGET‐DANA Régine , PTAK Marius , DAS Bhupesh C. , MICHEL Georges
Published: March 2005

The structure of a new antibiotic of the iturin group, bacillomycin F, has been demonstrated. It is a mixture of homologous peptidolipids, essentially C51H80N12O14 and C52N82N12O14. The lipid moiety consists of minor isoC15, anteisoC15β‐amino acids and major isoC16, isoC17 and anteisoC17β‐amino acids. The peptide sequence was determined by studying the peptides obtained after mild HCl hydrolysis and by Edman degradation of bacillomycin F treated with N‐bromosuccinimide. The sequence was confirmed by two‐dimensional NMR spectrometry and fast‐atom‐bombardment mass spectrometry gave the molecular masses of the homologous compounds. Bacillomycin F is a cyclic peptidolipid; its complete structure is given in the paper.
Antitermination is required for readthrough transcription of the maize rbcL gene by a bacteriophage promoter in Escherichia coli
Authors: GATENBY Anthony A. , CUELLAR Richard E.
Published: March 2005

Sequences upstream of the 5′ end of the rbcL gene in maize chloroplast fragment Bam 9, have a polar effect on the expression of rbcL from an external upstream bacteriophage PL promoter in Escherichia coli. This polarity can be suppressed by the bacteriophage transcription antitermination protein N or Q. The requirement for transcription antitermination is abolished if DNA upstream of rbcL is removed by a deletion. We have also investigated the ability of RNA polymerase initiating transcription at PL in the presence of N to transcribe through the normal rbcL transcription terminator and into sequences beyond. RNA polymerase initiating at PL can traverse rbcL and its 5′ and 3′‐flanking regions in the presence of N.
Assignment of resonances in the 1H NMR spectrum of human lysozyme
Authors: BOYD Jonathan , DOBSON Christopher M. , REDFIELD Christina
Published: March 2005

Assignments in the 1H NMR spectrum for more than 120 resonances arising from 38 of the 130 amino acid residues of human lysozyme are presented. Assignments have been achieved using a combination of one and two‐dimensional NMR techniques. Two‐dimensional double‐quantum correlated spectroscopy and relayed coherence transfer spectroscopy were found to be particularly useful for the identification of spin systems in the aromatic and methyl regions of the spectrum. These spin systems were assigned to specific residues in human lysozyme with reference to the X‐ray crystal structure using one‐dimensional nuclear Overhauser enhancement (NOE) data and a computer‐based search procedure. Unique assignments were found for resonances of 27 amino acid residues even when a distance constraint on NOE effects of 0.7 nm was used in the search procedure; for the remaining residues closer constraints or additional information were required. The assignments include all but one of the resonances in the aromatic region of the spectrum and all the methyl group resonances in the region upfield of 0.6 ppm. The assignments presented here provide a basis for a comparison of the NMR spectra of human lysozyme and the more widely studied hen lysozyme.
Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6‐bisphosphate
Authors: VAN SCHAFTINGEN Emile , OPPERDOES Fred. R. , HERS Henri‐Géry
Published: March 2005

The activity of pyruvate kinase present in a crude extract of the bloodstream form of Trypanosoma brucei was greatly increased by fructose 2,6‐bisphosphate, which converted the saturation curve for phosphoenolpyruvate from a sigmoid into a hyperbola with no change in V. Phosphate and arsenate had an effect opposite to that of fructose 2,6‐bisphosphate and the apparent Ka for fructose 2,6‐bisphosphate was shifted from 75 nM to 1.5 μM by the presence of 5 mM phosphate. Fructose 1,6‐bisphosphate had effects similar to those of fructose 2,6‐bisphosphate but at approximately 4000‐fold higher concentrations. Pyruvate kinases of Crithidia luciliae and of Leishmania major, two trypanosomatids which are like T. brucei in containing glycosomes, were also stimulated by fructose 2,6‐bisphosphate and inhibited by phosphate.
Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation
Authors: ANTRANIKIAN Garabed , HERZBERG Christina , GOTTSCHALK Gerhard
Published: March 2005

Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate. In contrast to citrate lyase ligase from C. sporosphaeroides and Streptococcus lactis, the enzyme from C. sphenoides was under stringent regulatory control. The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase. These enzymes were purified from in vivo32P‐labeled C. sphenoides cells, which were grown on low‐phosphate medium containing 40 mM citrate and 1 mCi [32P]orthophosphate. During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled. Growth experiments with 14C‐labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide. Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated. Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation.
Synthesis, intracellular processing and secretion of thrombospondin in human endothelial cells
Authors: VISCHER Peter , BEECK Hannelore , VOSS Bruno
Published: March 2005

The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse‐chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from ‘high‐mannose’ to ‘complex’ structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N‐glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa.

Pulse‐chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio‐Gel P‐4 and concanavalin‐A–Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition.
Purification and characterization of a protein from chicken gizzard, which inhibits actin polymerization
Authors: SCHRÖUER Elke , WEGNER Albrecht
Published: March 2005

An actin‐polymerization‐inhibiting protein, that occurs in crude preparations of vinculin from chicken gizzard, has been purified by DEAE‐cellulose and carboxymethyl ion‐exchange chromatography. According to sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis and to gel filtration the polymerization‐inhibiting protein is heterogeneous and the molecular mass ranges from 20 kDa to 80 kDa. After treatment with acid the polymerization‐inhibiting activity was found to migrate on a SDS/polyacrylamide gel as a single band of molecular mass about 32 kDa. The mechanism of the action of the polymerization‐inhibiting protein on actin assembly was investigated by the effect on the kinetics of actin polymerization. The polymerization‐inhibiting protein blocks elongation of actin filaments at substoichiometric ratios but does not nucleate actin filaments. The equilibrium constant for binding of the polymerization‐inhibiting protein to the barbed end of an actin filament was estimated to be 2 × 106 M−1 in 100 mM KCl and 2 mM MgCl2, and 35 × 106 M−1 in 2 mM MgCl2.
Specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of the polypeptide cardiac stimulant anthopleurin‐A
Authors: GOOLEY Paul R. , NORTON Raymond S.
Published: March 2005

The specific assignment of resonances in the 300‐MHz 1H nuclear magnetic resonance (NMR) spectrum of anthopleurin‐A, a polypeptide cardiac stimulant from the sea anemone Anthopleura xanthogrammica, is described. Assignments have been made using two‐dimensional NMR techniques, in particular the method of sequential assignments, where through‐bond and through‐space connectivities to the peptide backbone NH resonances are used to identify the spin systems of residues adjacent in the amino acid sequence. Complete assignments have been made of the resonances from 33 residues out of a total of 49, and partial assignment of a further 3. The resonances from several of the remaining residues have been identified but not yet specifically assigned. A complicating factor in making these assignments is the conformational heterogeneity exhibited by anthopleurin‐ A in solution. The resonances from a number of amino acid residues in the minor conformer have also been assigned. These assignments contribute towards identification of the origin of this heterogeneity, and permit some preliminary conclusions to be drawn regarding the secondary structure of the polypeptide.
d‐Cysteine desulfhydrase of Escherichia coli
Authors: NAGASAWA Toru , ISHII Takafumi , KUMAGAI Hidehiko , YAMADA Hideaki
Published: March 2005

d‐Cysteine‐specific desulfhydrase is found in some intestinal bacteria. Escherichia coliW 3110 ΔtrpED 102/F′ΔtrpED 102 was found to have the highest enzyme activity. The enzyme was purified from E. coli W3110 ΔtrpED102/F′ΔtrpED 102 in six steps. After the last step the enzyme appeared to be homogeneous by the criteria of polyacrylamide gel electrophoresis, analytical ultracentrifugation and double diffusion in agarose. The enzyme has a molecular mass of about 67000 Da and consists of two subunits identical in molecular mass. The enzyme exhibits absorption maxima at 278 nm and 418nm, which are independent of pH (6.5–10.5), and contains 2 mol pyridoxal phosphate/mol enzyme. The holoenzyme is resolved to the apoenzyme by incubation with phenylhydrazine, and reconstituted on the addition of pyridoxal phosphate. d‐Cysteine desulfhydrase also catalyzes the β‐replacement reaction of the chlorine of 3‐chloro‐d‐alanine with thioglycolic acid to yield S‐carboxymethyl‐d‐cysteine. Its catalytic and immunological properties are compared with those of 3‐chloro‐d‐alanine dehydrochlorinase.
The pH dependence of the electron self‐exchange rate of azurin from Pseudomonas aeruginosa as studied by 1H‐NMR
Authors: GROENEVELD Cornelis M. , CANTERS Gerard W.
Published: March 2005

The electron self‐exchange rate of azurin from Pseudomonas aeruginosa has been measured by proton NMR as a function of temperature under various conditions of pH, buffer and ionic strength. The rate does not appear very sensitive to variations in the latter parameters. Qualitative thermodynamic compensation is observed for the entropy and enthalpy of activation, with a compensation temperature of 309 ± 7 K, and an average exchange rate of 1.3×106 M−1 s−1. The observed high entropy of activation contributes significantly to the high exchange rate. The data are analyzed by considering an encounter complex in which two azurin molecules associate along their hydrophobic patches. Copper‐to‐copper electron transfer over a distance of 1.5 nm in the complex is facilitated by the favourable disposition of the His‐117 ligands.
Structural and functional studies of hemoglobin Poissy a2β256 (D7) Gly → Arg and 86 (F2) Ala → Pro
Authors: LACOMBE Claire , CRAESCU Constantin T. , BLOUQUIT Yves , KISTER Jean , POYART Claude , DELANOE‐GARIN José , AROUS Nicole , BARDAKDJIAN Josiane , RIOU Jean , ROSA Jean , SCHAEFFER Corinne , GALACTEROS Frédéric
Published: March 2005

Hemoglobin Poissy α2β256 (D7) Gly → Arg and 86 (F2) Ala Pro, is a new variant of the β chain with two substitutions within the second exon of the corresponding gene. The electrophoretic mobilities are identical to those of Hb Hamadan α2β256 (D7) Gly → Arg as is the fingerprint of the tryptic hydrolysate of the two abnormal β chains. The second substitution β86 Ala → Pro was detected by high‐pressure liquid chromatography.

Hb Poissy has a threefold increase in oxygen affinity with low Hill coefficient and diminished Bohr effect, which are restored to normal upon addition of 2,3‐bisphosphoglycerate. Since the functional properties of Hb Hamadan (β56 Gly → Arg) have been described as normal, the abnormal function of Hb Poissy may be attributed to the β86 (F2) Ala → Pro substitution.

Hb Poissy exhibits a mild instability and a greater reactivity of the thiol groups of the β93 (F9) Cys residues in the deoxy form than does Hb A. The oxidation rate of Hb Poissy is biphasic indicating a large inequivalence between the α and β hemes. Thereafter NMR studies demonstrated that the β86 Ala Pro substitution produces a displacement of the F helix closer to the heme plane and a large increase in the dynamic fluctuations of the tertiary structure on the proximal side of the β hemes. These results lead to the conclusion that the β86 Ala Pro susbstitution produces a destabilization of the F helix extending downwards to the FG corner and altering both the β hemes and the α1β2 contacts.
Enzymatic basis for the calcium‐induced decrease of membrane protein methyl esterification in intact erythrocytes
Authors: GALLETTI Patrizia , INGROSSO Diego , IARDINO Patrizia , MANNA Caterina , PONTONI Gabriele , ZAPPIA Vincenzo
Published: March 2005

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes.

When the cells were incubated with l‐[methyl‐3H]methionine, 40 μM CaCl2 and 10 μM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 μM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition.

In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed.

l‐Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations.

Conversely in the Ca2+‐loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%.

When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187‐treated erythrocytes is significantly lower than the control, up to 5 μM ATP. This result suggests that in the Ca2+‐loaded erythrocytes the ATP intracellular concentration is significantly lowered.

The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading.

The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels.

The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet‐dependent reactions as well as in post‐translational protein methylation related to the ageing process is also discussed.
Laurate binding to human serum albumin
Authors: PEDERSEN Anders Overgaard , HUST Birthe , ANDERSEN Signe , NIELSEN Frede , BRODERSEN Rolf
Published: March 2005

Multiple binding of laurate (n‐dodecanoate) to human serum albumin was studied by a kinetic dialysis method. With this method the concentration of unbound ligand can be determined by measuring the rate of exchange of radioactive label across a dialysis membrane under conditions of equilibirium.

Determination of the rate constant of exchange, in the absence of albumin, revealed that laurate does not change its state of aggregation over a concentration range from 0.1 μM to 500 μM, indicating the prevalence of a monomer.

Binding of laurate to albumin, at pH 7.5, 37°C, was investigated through 220 determinations of binding equilibria in the range of 0–10 mol laurate/mol albumin, corresponding to a concentration range of unbound laurate from 1 nM to 0.1 mM.

Binding data were analyzed in terms of stepwise binding. Considering the stochastic errors of the experimental data, we generated a variety of possible (equal goodness of fit) solutions to the stoichiometric binding equation. This analysis resulted in reasonably well‐defined values for the first two step constants, with an indication of negative interaction. The variation of the higher step constants excluded any mechanistic conclusions, although the binding isotherms, defined by these varying sets of binding constants, fitted the experimental data well within a chosen probability limit.
Leukotriene C4 metabolism by hepatoma cells deficient in the uptake of cysteinyl leukotrienes
Authors: WECKBECKER Gisbert , KEPPLER Dietrich O. R.
Published: March 2005

Uptake and metabolism of the cysteinyl leukotrienes C4 and E4 (LTC4 and LTE4) were studied in AS‐30D hepatoma cell suspensions and compared with rat hepatocytes. The hepatoma cells were deficient in the uptake of [3H]LTC4 and [3H]LTE4 but took up, in control experiments, l‐[14C]glutamine and [14C]adenosine in a time‐dependent manner. By contrast, isolated hepatocyte suspensions incubated under the same conditions took up [3H]LTC4 and [3H]LTE4 as well as l‐[14C]glutamine and [14C]adenosine.

The hepatoma cells deficient in the uptake of cysteinyl leukotrienes metabolized extracellular [3H]LTC4 to [3H]LTD4 and to [3H]LTE4. Addition of acivicin, an inhibitor of γ‐glutamyltransferase, largely prevented metabolism of [3H]LTC4 by the hepatoma cells. Sonication of the cells did not enhance the formation of [3H]LTD4 and [3H]LTE4 from [3H]LTC4.

We conclude that ectoenzymes of AS‐30D hepatoma cells catalyze the conversion of LTC4 to LTE4 via LTD4. As compared to hepatocytes, these neoplastic cells have lost the uptake system for cysteinyl leukotrienes and may serve in studies on leukotriene metabolism by cell‐surface enzymes.
Structural, immunological and kinetic comparisons of NADP‐dependent malate dehydrogenases from spinach (C3) and corn (C4) chloroplasts
Authors: FERTE Nathalie , JACQUOT Jean‐Pierre , MEUNIER Jean‐Claude
Published: March 2005

This paper compares structural, immunological and kinetic properties of corn (C4) and spinach (C3) NADP‐malate dehydrogenases. These chloroplastic enzymes are regulated in vivo by thiol– disulfide interchange. Both in their oxidized (inactive) and reduced (active) states these enzymes have a dimeric structure with molecular masses for the subunit ranging from 28 kDa to 38 kDa according to the procedure used for the determination. These enzymes are thus structurally related. The use of specific antibodies showed that they are also immunologically related although not identical. Finally both enzymes showed close kinetic properties with comparable Kcat and Km. Since C4 plants have approximately ten times more NADP‐malate dehydrogenase activity than C3 plants, these data suggest that the differences in activities are probably related to the enzyme content of each plant type.
Reaction of cimetidine with Fenton reagent
Authors: ZBAIDA Shmuel , KARIV Rami , FISCHER Paula , SILMAN‐GREENSPAN Judith , TASHMA Zeev
Published: March 2005

Cimetidine sulfoxide, N‐desmethylcimetidine, N‐desmethylcimetidine sulfoxide, cimetidine guanylurea, and the 5‐hydroxymethylimidazole derivative of cimetidine sulfoxide were isolated from the reaction of cimetidine with Fenton reagent. The product distribution from the reaction of cimetidine with Fenton reagent clearly simulates the metabolism of cimetidine by the mixed‐function oxidase.
Molecular cloning, full‐length sequence and preliminary characterization of a 56‐kDa protein induced by human interferons
Authors: WATHELET Marc , MOUTSCHEN Sabine , DEFILIPPI Paola , CRAVADOR Alfredo , COLLET Marc , HUEZ Georges , CONTENT Jean
Published: March 2005

Among the various proteins which are induced when human cells are treated with interferon, a predominant protein of unknown function, with molecular mass 56 kDa, has been observed. With the aim of exploring the molecular basis of the regulation of this protein and of its mRNA, in order to understand its biological function and its possible contribution to the various antiviral and non‐antiviral actions exerted by interferons, we cloned a full‐length cDNA copy of the 1.8 – 1.9 × 103‐base 56‐kDa‐protein mRNA and determined its sequence. The cDNA contains 1662 nucleotides derived from the 56‐kDa‐protein mRNA, including a poly(A) tail of 20 residues. Primer extension experiments indicate that the 5′ end of this cDNA clone is probably located only 13 nucleotides downstream of the actual cap site of the 56‐kDa‐protein mRNA. It consists of a 64‐nucleotide‐long 5′‐non‐coding segment, a coding segment of 1434 nucleotides terminated by a TAG triplet and a 141‐nucleotide 3′‐non‐coding segment. The encoded protein of 478 amino acid residues has a molecular mass of 55335 Da and a single potential site for N‐glycosylation. The protein contains an excess of basic amino acids and most of them are localized in the carboxy‐terminal half of the molecule. A single [35S]methoinine‐labeled 56‐kDa protein was obtained using an SP64 construction to allow the cell‐free transcription and translation of the cloned cDNA. Microinjection of this labeled protein in Xenopus oocytes indicates that the 56‐kDa protein is cytoplasmic.
Proteins from the prokaryotic nucleoid
Authors: LOSSO M. Adele , PAWLIK Roman T. , CANONACO Maria A. , GUALERZI Claudio O.
Published: March 2005

Escherichia coli DNA‐binding proteins NS1, NS2 and NS (NS1 + NS2) react with the protein‐protein bifunctinol cross‐linking reagents dimethylsuberimidate and dimethyladipimidate to yield oligomers up to hexamers. The former reagent, with the longer arm, is more efficient than the other shorter one. Both one‐ and two‐dimensional gel electrophorses show that the cross‐linked trimers are homogeneous, while the dimers appear heterogeneous, suggesting that at least two types of dimers but geometrically equivalent trimers are formed. In the presence of DNA, the cross‐linking reaction with either reagent yields fewer dimers and more of the larger products.

The yield of cross‐linked products of various sizes was determined for NS1, NS2 and NS as a function of the protein concentration (0.03–3000 μM), From the results obtained in these experiments, we derived a model of quaternary structure in which dimers and tetramers are predominant in very dilute solutions of the proteins. Above a critical concentration (10–50 μM), interactions among tetramers become increasingly important, yielding octamers and perhaps larger products. Our data do not support a recently proposed model in which the DNA is packaged around a protein disc consisting of 8–10 NS dimers.
Structural features of the DNA template required for transcription in vitro by yeast RNA polymerase B (II)
Authors: ARCANGIOLI Benoit , LESCURE Bernard
Published: March 2005

Yeast RNA polymerase II initiates in vitro transcription at two sites located within the vector DNA and the cloned promoter, on a recombinant plasmid DNA containing the yeast iso1 cytochrome c promoter. Both initiation sites are found within a DNA fragment hypersensitive to osmium tetroxide modification. Using a series of yeast iso1 cytochrome c promoter deletions, we have characterized an upstream DNA sequence required for optimal transcription from this site and shown in this case a correlation between osmium sensitivity and the capacity of RNA polymerase to initiate. However, perturbation of the double helix is not sufficient to generate a transcription initiation site. Insertion of 28 alternating AT residues at the EcoRV site of pBR322 generates an site hypersensitive to osmium tetroxide modification, that does not serve as a transcription start site.
Immunological and biosynthetic studies on the mammalian 2‐oxoglutarate dehydrogenase multienzyme complex
Authors: HUNTER Anne , LINDSAY John Gordon
Published: March 2005

High‐titre, monospecific, polyclonal antisera have been raised against purified mitochondrial 2‐oxoglutarate dehydrogenase complex (OGDC) from ox heart and two of its three constituent enzymes, 2‐oxoglutarate dehydrogenase (E1) and lipoyl succinyltransferase (E2). These specific antisera have been employed to monitor molecular events in the biosynthesis, import and maturation of this multimeric assembly. Lipoamide dehydrogenase (E3) elicits a poor antibody response in comparison to the other polypeptides of the complex.

In cultured pig kidney cells (PK‐15), incubated with [35S]methionine in the presence of uncouplers of oxidative phosphorylation, appearance of stable higher‐Mr forms of the individual enzymes can be detected by specific immunoprecipitation and fluorographic analysis. In the case of 2‐oxoglutarate dehydrogenase, E1, the initial cytoplasmic translation product has a subunit Mr value of 1500–3000 greater than in the mature enzyme while the precursor of the lipoyl succinyltransferase, E2, contains an additional sequence of Mr 6000–8000. Competition studies have revealed the immunological similarity of the precursor molecules to the native subunits.

On removal of uncouplers, processing of accumulated precursors is rapidly initiated and is complete within 40 min. Interestingly, antiserum to native 2‐oxoglutarate dehydrogenase complex fails to recognise E2 precursor molecules (pre‐E2), which can be immunoprecipitated, however, by antibodies raised against the denatured E2 subunit. It is concluded that pre‐E2 is conformationally dissimilar to native E2, which exists normally as a highly ordered, multimolecular aggregate in the native complex.
The complete amino acid sequence of adenylate kinase from baker's yeast
Authors: TOMASSELLI Alfredo G. , MAST Edeltraud , JANES Wolfgang , SCHILTZ Emile
Published: March 2005

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP ⇄ MgADP + ADP) from baker's yeast has been determined.

Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were sequenced with either a solid‐phase sequencer or a gas‐phase sequencer.

Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by endoproteinase Lys‐C and cyanogen bromide cleavages.

The N‐terminus is blocked by an acetyl group as shown by proton magnetic resonance.

Carboxypeptidase A digestion of the whole protein showed that the C‐terminal sequence is ‐Lys‐Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2‐iodosobenzoic acid cleavages.

The enzyme is a monomer of 220 amino acids with Mr 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putative active‐site region of the enzyme. After position 111, however, there is an insertion of 32 residues in the yeast species, similar to the adenylate kinase and the GTP:AMP phosphotransferase from beef heart mitochondria.
Purification and characterization of calcitonin receptors in rat kidney membranes by covalent cross‐linking techniques
Authors: BOUIZAR Zhor , FOUCHEREAU‐PERON Martine , TABOULET Jacqueline , MOUKHTAR Mohsen S. , MILHAUD Gérard
Published: March 2005

We have characterized the binding parameters of renal receptors (Scatchard analysis revealed the presence of two binding sites: site I, Ka1= 1.29×109 M−1, number of binding sites = 9.9 × 106/μg protein; site II, Ka2= 0.93 × 108M−1, number of binding sites = 4.27×108/μg protein) and studied the effect of solubilization. The high‐affinity sites are preserved during affinity chromatography and the process results in a 6080‐fold purification of those sites. The lower‐affinity sites are also preserved but the overall purification factor is about 40% lower than that obtained using molecular sieving. The purification of the renal calcitonin receptor by molecular sieving (Sephacryl S‐200) is accompanied by total loss of the high‐affinity site; however, the low‐affinity site is enriched over 1642‐fold. Binding parameters were obtained for the purified fractions. Synthetic salmon calcitonin was also bound to renal membranes using the bifunctional reagent disuccinimidyl suberate and photo affinity cross‐linking using hydroxysuccinimidyl azidobenzonate reagent. Cross‐linked receptor eluted in the same volume as solubilized membranes specifically binding salmon calcitonin (S‐200 chromatography).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified fractions showed several protein bands with apparent molecular masses ranging from 18000 Da to 100000 Da in the presence or absence of a reducing agent (2‐mercaptoethanol). Autoradiography of polyacrylamide gels of cross‐linked calcitonin receptor showed only three protein bands specifically binding salmon calcitonin. Their molecular masses were 70000 Da, 40 000 Da and 33 000 Da respectively. The 40 000‐Da molecule represents a major band (47% total binding species). This suggests that these three proteins are the principal components of the calcitonin receptor and that S‐S bonds are not involved in the assembly of the receptor subunits.
Amino‐acid‐transfer reactions in isolated nuclei of Ehrlich ascites tumor cells
Authors: ROTHBARTH Karsten , WERNER Dieter
Published: March 2005

Nuclear enzymatic activities incorporating amino acids into acid‐insoluble material were investigated with respect to their differentiation from protein biosynthesis, reaction optima, requisites and localization. The product of the reaction was analyzed with respect to its localization and nature.

(1)The nuclear activities are not inhibited by a number of inhibitors for protein biosynthesis. (2) The reaction optima found are similar to those of other residual nuclear syntheses including the stringent dependence on ATP. (3) All naturally occurring amino acids are utilized with different efficiencies. Their incorporation is neither cooperative nor competitive which points to individual incorporation mechanisms. Aminoacylation of tRNA may be involved because the incorporation is RNase‐sensitive and aminoacylation of tRNA can be shown under the reaction conditions. (4) The enzymatic activities are exclusively nuclear. Significant activity with unchanged characteristics is released by sonication. (5) 70% of the radiolabel incorporated is exported across the nuclear envelope during the incubation. The residual 30% of the radiolabel is distributed without enrichment in any nuclear subfraction. (6) The products are exclusively of polypeptide nature. Since distinct nuclear proteins (e.g. histones) which are definitely preformed in the cytoplasm by protein biosynthesis become radiolabelled by the incorporation of radiolabelled amino acids, it is evident that the incorporation takes place at preformed polypeptides. This is unequivocally proven by the incorporation of radiolabelled amino acids into exogenous proteins by means of the solubilized nuclear activities.

The results indicate that the nuclear activity under investigation reflects a nuclear modification system for polypeptides which may be of similar importance as other post‐translational modification systems.
Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells
Authors: RICARD Jacques , NOAT Georges
Published: March 2005

A theory is proposed to explain the physical bases of the ionic control of the activity of an enzyme system, located on a plant cell surface, and probably involved in cell‐wall synthesis and extension. The model, which is based on various previously published experimental results, involves several assumptions: a cell‐wall pectin methyl esterase de‐esterifies pectins and thus creates the fixed negative charges of the cell wall; various enzymes incorporate uncharged carbohydrates in cell‐wall material; cell‐wall extension implies the sliding of cellulose microfibrils; the enzymes responsible for carbohydrate incorporation are activated by protons in the pH range 4–8 and have very similar pH dependencies: the cell‐wall pectin methyl esterase is inhibited by protons in the same pH range.

The mathematical derivation of this model, written in the form of a hypercycle, indicates that it is equivalent to a set of two antagonistic enzyme reactions: an enzyme reaction conditioned by pectin methyl esterase which results in the increase of fixed charge density of the cell wall; a number of ‘growth enzymes’, which produce extension and building up of the cell wall and therefore a decrease of charge density. The mathematical study of this model shows it may display a very high co‐operativity of its response to slight changes of pH. This co‐operativity means that the cell wall charge density may dramatically increase or decrease, within a very narrow pH range. The steep response of this system appears to be the direct consequence of different pH sensitivities of pectin methyl esterase and of the other cell‐wall enzymes involved in cell growth. Calcium, which tightly binds to the cell wall, may diminish or even suppress this abrupt charge transition.

This model suggests a novel theory of the ionic control of cell‐wall expansion. The very basis of this theory is the existence of an electrostatic potential difference, ΔΨ, between the inside and the outside of the cell wall. When this ΔΨ value is large, the local proton concentration is high. Therefore the enzymes involved in cell wall extension and building up are active, but pectin methyl esterase is not. Therefore, the cell wall extends and the charge density decreases. The ΔΨ value then declines, as well as the local proton concentration. Under these conditions, the pectin methyl esterase becomes activated, whereas the ‘growth enzymes’ are not. This activation of pectin methyl esterase restores the initial, or an even higher, electrostatic potential difference, which in turn results in a decrease of local pH.

In the following papers the basic ideas of this model are subjected to experimental control.
New phase supports for liquid‐liquid partition chromatography of biopolymers in aqueous poly(ethyleneglycol)‐dextran systems
Authors: MÜLLER Werner
Published: March 2005

New phase supports for liquid‐liquid partition chromatography, using aqueous poly(ethyleneglycol)‐dextran systems have been developed by grafting linear polyacrylamide chains on to premanufactured chromatographic supports carrying primary or secondary aliphatic hydroxyl functions on their surface. Columns prepared from such supports have a higher binding capacity for the dextran‐rich stationary phase and much higher performances than columns prepared from cellulose, the previously used phase support for this system. Test separations of DNA restriction fragments, ranging from 11 base pairs to 3829 base pairs, document a high resolution for DNA fragments larger than 200 base pairs.