bluuebunny/crossref_metadata_2025 huggingface dataset:
A Raman spectroscopic study on the interaction of an ion‐channel protein with a phospholipid in a model membrane system (gramicidin A/l‐α‐lysophosphatidylcholine)
Authors: ASLANIAN Dimitrina , NÉGRERIE Michel , CHAMBERT Régis
Published: March 2005

The interaction of the ion channel polypeptide gramicidin A with the L‐α‐lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1:9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700–1700 cm−1 and 2800–3100 cm−1 at 10°C.

The Raman spectrum of L‐α‐lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all‐trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains.

Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan‐stacking interactions suggested by some authors exsist, they are very weak ones.
The effect of 1‐cyclohexyl‐3‐(2‐morpholinoethyl)carbodiimide on the rat liver microsomal glucose‐6‐phosphatase system
Authors: MURATALIEV Marat B. , VULFSON Evgeni N.
Published: March 2005

The effect of 1‐cyclohexyl‐3‐(2‐morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose‐6‐phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose‐6‐phosphatase, pyrophosphate: glucose and carbamoyl‐phosphate:glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose‐6‐phosphate, pyrophosphate, and carbamoyl‐phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that (a) CMC inactivates the microsomal glucose‐6‐phosphate translocase, (b) the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; (c) pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose‐6‐phosphate translocase; (d) pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo.
Partial characterization of the human β‐myosin heavy‐chain gene which is expressed in heart and skeletal muscle
Authors: LICHTER Peter , UMEDA Patrick K. , LEVIN James E. , VOSBERG Hans‐Peter
Published: March 2005

A human myosin heavy‐chain gene, cloned in γCharon 4A phage (and as a clone designated γgMHC‐1), was shown to code for a cardiac myosin heavy chain of the β‐type. The 5′ end of the 14200‐base‐pair genomic DNA clone is located in the head region of the myosin chain. The 3′ end was shown to extent to the COOH terminus and includes the 3′‐nontranslated sequence of the corresponding mRNA. The identification of γgMHC‐1 as coding for a cardiac β‐myosin heavy chain was achieved by heteroduplex mapping using genomic cardiac myosin heavy‐chain DNA of rabbit as a probe and, furthermore, by DNA sequence analysis of three selected subregions of the cloned DNA including the 3′‐nontranslated sequence. It was demonstrated by the S1 nuclease protection technique that the β‐myosin heavy‐chain gene is transcribed in human heart muscle. In addition, we have found by the same technique that it is also expressed in human skeletal muscle.
Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa
Authors: SCHOMBURG Ulrich , GROSSE Frank
Published: March 2005

DNA topoisomerase II was purified from calf thymus nuclei by a simple and fast four‐step procedure: selective ammonium sulfate precipitation, chromatography on blue‐Sepharose and hydroxyapatite, followed by ultracentrifugation on a glycerol gradient. Starting from 300 g thymus glands, this procedure yields 0.7 mg of homogeneous topoisomerase II. The final product is free of any nucleolytic, proteolytic or topoisomerase I activity. Dodecylsulfate/polyacrylamide gel electrophoresis reveals two bands with apparent molecular masses of 175 and 150 kDa. Analytical gel filtration and sedimentation on isokinetic sucrose gradients were used to determine the Stokes' radius as 6.4 nm and the sedimentation coefficient as 9.5 S, indicating a dimeric structure for the native enzyme. The purified topoisomerase II is strictly dependent on ATP or dATP, the Km values of which were 0.14 mM and 0.5 mM, respectively. Mg2+ is an essential cofactor for the reaction at concentrations between 0.5–8 mM, with an optimum at 4 mM. Mg2+ can be substituted by Mn2+ at concentrations between 0.2–0.4 mM. Both the relaxation and the catenation reaction exhibit a salt optimum at 130 mM NaCl. At concentrations below 30 mM and above 200 mM, the enzyme is inactive. The pH is optimal between 8 and 9.5 using Tris buffers.
Lactate dehydrogenase and glyceraldehyde‐phosphate dehydrogenase are single‐stranded DNA‐binding proteins that affect the DNA‐polymerase‐α–primase complex
Authors: GROSSE Frank , NASHEUER Heinz‐Peter , SCHOLTISSEK Stephan , SCHOMBURG Ulrich
Published: March 2005

Affinity chromatography on double‐stranded (ds) and single‐stranded (ss) DNA‐cellulose columns was employed to find analogs of the Escherichia coli and T4 single‐stranded DNA binding proteins (SSB proteins) in calf thymus. The interaction of several purified SSB proteins with the pure DNA‐polymerase‐α‐primase complex on DNA synthesis on activated DNA and on primase‐initiated M13 DNA served as a criterion for a possible involvement of one of these proteins in the process of DNA replication. Two SSB proteins were purified to essential homogeneity. These most abundant proteins exhibited apparent relative molecular masses of 35000 (SSB‐35) and 37000 (SSB‐37) for the protomers and 140000 and 80000 for the native enzymes. Both proteins resisted elution with 0.5 mg/ml dextran sulfate and were eluted from the ssDNA‐cellulose with 0.2 M and 1 M NaCl, respectively. SSB‐35 stimulated the DNA‐polymerase‐α–primase complex from the same organism up to fivefold over a broad range of DNA covering. By contrast, SSB‐37 inhibited the primase‐initiated replication of M13 DNA. Like most eukaryotic SSB proteins, these proteins showed a 300‐fold preference for binding to ssDNA over dsDNA in a nitrocellulose filter binding assay, as well as strong binding to several DNA and RNA homopolymers. Furthermore, we provide evidence for a cooperative mode of binding for SSB‐37. Although SSB‐35 and SSB‐37 behave as typically eukaryotic SSB proteins in all assays employed, we tested these SSB proteins for dehydrogenase activities as well. SSB‐35 was found to be identical with lactate dehydrogenase and SSB‐37 was identical with a dimeric form of glyceraldehyde‐3‐phosphate dehydrogenase. These results imply that further studies are mandatory in order to prove the authenticity of eukaryotic SSB proteins.
Characterization of dehydropeptidase I in the rat lung
Authors: HIROTA Takashi , NISHIKAWA Yuko , TANAKA Minoru , IGARASHI Takashi , KITAGAWA Haruo
Published: March 2005

The activity of dehydropeptidase I in rat tissues decreases in the order of lung > kidney > liver‐spleen > other tissues, while aminopeptidase activity is high in the kidney, and lower in the lung than in other tissues. Dehydropeptidase I was solubilized from the membrane fraction of rat lung by treatment with papain and purified by DEAE‐cellulose column chromatography, affinity chromatography on concanavalin‐A‐Sepharose and high‐performance liquid chromatography gel filtration. The purified preparation was found to be homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The relative molecular mass was estimated to be 150000 by gel filtration, comprising a homodimer of two 80000‐Mr subunits. The enzyme activity was inhibited by cilastatin, o‐phenanthroline and ATP. This enzyme catalyzed the hydrolysis of S(substituent)‐l‐cysteinyl‐glycine adducts such as l‐cystinyl‐bis(glycine) and N‐ethylmaleimide‐S‐l‐cysteinyl‐glycine, as well as the conversion of leukotriene D4 to E4. Furthermore it catalyzed a hydrolytic splitting of l‐Leu‐l‐Leu, but not S‐benzyl‐l‐cysteine p‐nitroanilide, which is a good substrate for aminopeptidase. Our enzyme preparation was immunologically identical to the rat renal dehydropeptidase I. The physiological significance of the pulmonary dehydropeptidase I on the metabolism of glutathione and its adducts is discussed.
Specificity of human glucosylceramide β‐glucosidase towards synthetic glucosylsphingolipids inserted into liposomes
Authors: SARMIENTOS Francesco , SCHWARZMANN Günter , SANDHOFF Konrad
Published: March 2005

The behaviour of highly purified glucosylceramide β‐glucosidase (glucosylceramidase, EC 3.2.1.45) from human placenta [Furbish, F. S., Blair, H. E., Shiloach, J., Pentchev, P. G. & Brady, R. B. (1977) Proc. Natl Acad. Sci. USA 74, 3560–3563] was investigated in the absence of detergents with structurally modified glucosyl‐ceramides inserted into unilamellar liposomes. The reaction between the water‐soluble enzyme and the liposomal substrates was significantly dependent on the structure of the lipophilic aglycon moiety of glycolipids: glucosyl‐N‐acetyl‐sphingosines (d‐erythro and l‐threo) were better substrates than the corresponding glucosylceramides. The l‐threo derivatives were poorer substrates with higher apparent Km values than the corresponding d‐erythro derivatives. For glucosyl‐3‐keto‐ceramide and glucosyl‐dihydro‐ceramide (d‐erythro), higher Km values were found than for glucosylceramide. Sphingosine, glucosylsphingosine and glucosyl‐N‐acetyl‐sphingosine were the most effective inhibitors of the hydrolysis of glucosylceramide. d‐erythro‐Ceramide and d‐galactosyl‐N‐acetyl‐d‐erythro‐sphingosine inhibited the hydrolyis of amphiphilic glucosylceramide but not that of water‐soluble 4‐methyl‐umbelliferyl‐β‐glucoside, suggesting a hydrophobic binding site of the enzyme for the aglycon moiety of its membrane‐bound substrate.

Dilution experiments suggested that at least a fraction of the enzyme associates with the liposomes and degrades the lipid substrate even in the absence of activator proteins.

Acidic phospholipids incorporated into liposomes caused a powerful stimulation (30–40‐fold) of the glucosylceramide β‐glucosidase, whereas acidic sphingolipids (sulphatide, gangliosides GM1 and GD1a) incorporated into liposomes stimulated this enzyme only moderately (3–10‐fold).
Conformational changes induced by domain assembly within the β2 subunit of Escherichia coli tryptophan synthase analysed with monoclonal antibodies
Authors: FRIGUET Bertrand , DJAVADI‐OHANIANCE Lisa , GOLDBERG Michel E.
Published: March 2005

The effects of domain assembly on the conformation of the F1 (N‐terminal) and F2 (C‐terminal) domains of the β2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were analysed using six monoclonal antibodies which recognize six different epitopes of the native β2 subunit (five carried by the F1 domain and one carried by the F2 domain). For this purpose, the affinity constant of each monoclonal antibody for the isolated domains F1 or F2, the associated domains in the trypsin‐nicked apo‐β2 and in the native apo‐β2 subunits were determined, both with the intact immunoglobulin and the Fab fragment. It was found that the association of the F1 and F2 domains within β2 is accompanied by structural changes of the two domains, as detected by variations of their affinity constants for the monoclonal antibodies.
Characterization of two carnosine‐degrading enzymes from rat brain
Authors: KUNZE Norbert , KLEINKAUF Horst , BAUER Karl
Published: March 2005

From rat brain extracts, two carnosine‐degrading enzymes have been identified and partially purified by ion‐exchange chromatography, hydrophobic interaction chromatography on phenyl‐Sepharose CL‐4B and gel filtration. These enzymes, exhibit distinct differences in their chemical characteristics and substrate specificities.

One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine‐degrading enzyme hydrolyzes βAla‐Arg considerably faster than carnosine and, therefore, has been tentatively designated βAla‐Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km= 25 mM) and βAla‐Arg (Km= 2 mM). Homocarnosine and γ‐aminobutyryl‐arginine are not degraded by βAla‐Arg hydrolase.

Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carno‐sinase is also not inhibited by bestatin but βAla‐Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50= 50 nM). While carnosinase is strongly inhibited by thiol‐reducing agents such as dithioerythritol and 2‐mercaptoethanol, βAla‐Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal‐chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, βAla‐Arg hydrolase requires Mn2+ ions for full enzymatic activity.

Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.
Primary structure of a low‐molecular‐mass N‐linked oligosaccharide from hemocyanin of Lymnaea stagnalis
Authors: VAN KUIK J. Albert , SIJBESMA Rint P. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , WOOD Edward J.
Published: March 2005

Hemocyanin from the freshwater snail Lymnaea stagnalis is a high‐molecular‐mass copper‐containing oxygen‐transport protein, which occurs freely dissolved in the hemolymph. It is a glycoprotein contaning fucose, xylose, 3‐O‐methylmannose, 3‐O‐methylgalactose, mannose, galactose, N‐acetylgalactosamine and N‐acetylglucosamine residues as sugar constituents. The N‐glycosidic carbohydrate chains of this glycoprotein were released by hydrazinolysis of a pronase digest and subsequently fractionated as oligosaccharide‐alditols on Bio‐Gel P‐4 followed by Lichrosorb‐NH2. Investigation with 500‐MHz 1H‐NMR spectroscopy, in conjunction with sugar and methylation analysis revealed the lowest‐molecular‐mass glycan chain to have the structure:
Glycosphingolipid biosynthesis in early chick embryos
Authors: FELDING‐HABERMANN Brunhilde , JENNEMANN Richard , SCHMITT Jürgen , WIEGANDT Herbert
Published: March 2005

Early chick embryos at the age of 22 h (neurula stage) and 48 h (20–25 somite stage) were explanted from eggs and cultured in vitro in the presence of radioactive sugar precursors. Metabolically labelled glycosphingolipids were isolated. Amongst these, neutral and acidic components, the latter including sulfatide and gangliosides, were identified. Cleavage by exoglycosidases, as well as immunostaining with antibodies on thin‐layer plates, showed that at both embryonic stages glycosphingolipids were synthesized that belong to the globo series (globoside, Forssman glycolipid), the lacto series (lactoneotetraosylceramide, nLc4Cer, and two nLc4Cer‐based gangliosides, a monosialo and a disialo species), and the ganglio series (ganglioside Gtet1a and higher sialylated derivatives).
Serine hydroxymethyltransferase
Authors: SCHIRCH Verne , SCHIRCH Douglas , MARTINI Filippo , BOSSA Francesco
Published: March 2005

Homogeneous preparations of cytosolic serine hydroxymethyltransferase from rabbit liver were incubated with several different proteases. Chymotrypsin rapidly cleaves a tetradecapeptide from the NH2‐terminal end of the enzyme with the enzyme retaining full catalytic activity. Trypsin digestion results in the release of several small peptides from the NH2‐terminal end of the enzyme. The remaining core protein is reduced in molecular mass by about 3500 Da. With L‐serine as substrate the core protein has 1.5 times the activity of the native enzyme. The difference in activity is due to a change in Vmax since the Km values for L‐serine and tetrahydrofolate are unchanged. When allothreonine is used as the substrate the activity of the trypsin‐treated enzyme is unchanged. Ks values for glycine and several folate compounds are also unchanged for the trypsin‐treated enzyme. The relative distribution of three glycine‐enzyme complexes shows only small differences between the native and trypsin‐digested enzyme. Thermal denaturation studies show that the trypsin‐digested enzyme has a thermal transition three degrees lower than the native enzyme but the same enthalpy of denaturation. These results suggest that the 25–30 amino acid residues from the NH2‐terminal end of the enzyme are not important in determining the catalytic activity and structural stability of the purified enzyme. Several other proteases had no observable effect on the activity and size of the enzyme. All of the proteases tested inactivated the apoenzyme and digested it into small fragments. The loss of enzyme activity in frozen liver is probably the result of the enzyme slowly being converted to the apoenzyme form, which is susceptible to protease degradation.
Overproduction and high‐yield purification of phospholipase A from the outer membrane of Escherichia coli K‐12
Authors: DE GEUS Pieter , RIEGMAN Nico H. , HORREVOETS Anton J. G. , VERHEIJ Hubertus M. , DE HAAS Gerard H.
Published: March 2005

The cloned gene for the outer‐membrane‐bound phospholipase A from Escherichia coli was placed under control of the strong PL resulted in a 250‐fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild‐type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12‐Sulfobetaine and anion‐exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29‐kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.
Accessibility of F0 subunits from Escherichia coli ATP synthase
Authors: DECKERS‐HEBESTREIT Gabriele , ALTENDORF Karlheinz
Published: March 2005

1. Antisera have been raised against denatured and non‐denatured subunits a, b and c of the F0 complex of the ATP synthase from Escherichia coli. The subunit specificity of the antibodies has been established with immunoblot analysis or enzyme‐linked immunosorbent assay (ELISA).

2. In inside‐out oriented membrane vesicles the binding avidities of both sets of antisera, against denatured and non‐denatured subunits of F0, were similar in the presence as well as in the absence of the F1 part. F1‐depleted everted membrane vesicles always produced an efficient binding of the different antisera. In the presence of F1 no antibody recognition could be observed with the anti‐a antisera, while anti‐b and anti‐c antisera showed strong binding. However, a higher membrane protein concentration was necessary for the same antibody binding as in F1‐stripped vesicles.

3. In membrane vesicles with right‐side‐out orientation the recognition of the three F0 subunits was dependent on the antisera set used. Antisera raised against denatured subunits showed no binding to the membrane vesicles, only in case of anti‐(dedecylsulfate‐denatured b) antiserum could a slight affinity be detected. An antigen‐antibody recognition with all three F0 subunits occurred when the antisera against non‐denatured subunits were incubated with membrane vesicles of right‐side‐out orientation. The membrane protein concentration which was necessary to produce a significant binding was 10–100‐fold higher compared to that of F1‐depleted everted membrane vesicles.
Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm
Authors: STENGEL Dominique , HENRY Dominique , TOMOVA Svetlana , BORSODI Anna , HANOUNE Jacques
Published: March 2005

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by α‐chymotrypsin. The purification (26000‐fold from the particulate fraction or 125 000‐fold from the whole‐sperm proteins) was achieved by conventional procedures (DEAE‐Trisacryl, Ultrogel AcA 34, DEAE‐Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5–10% and a final specific activity of 19 γmol cyclic AMP formed mg protein−1 min−1 at 30°C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half‐lives of 27 min, 50 min and 160 min were obtained at 30°C, 20°C and 4°C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4°C. The purified enzyme exhibited a Km value similar to that of the native enzyme (Km= 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non‐competitive manner. This indicates that (a) adenosine acts on the catalytic component itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a ‘P‐site’ specificity. The purified adenylate cyclase, which has an apparent molecular mass of 38 kDa on a high‐performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10818–10826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.
Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat‐shock
Authors: PIPER Peter W. , CURRAN Brendan , DAVIES Mark W. , LOCKHEART Alan , REID Georg
Published: March 2005

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat‐shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25°C to 38°C transient increases of 6–7‐fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein‐synthesis inhibitor cycloheximide, but was not observed in cells bearing the rnal.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat‐shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half‐life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress.

Synthesis of PGK enzyme in glucose‐grown cultures is efficient after mild (25°C to 38°C) or severe (25°C to 42°C) heat‐shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat‐shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high‐copy‐number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat‐shock and translated efficiently at supra‐optimal temperatures.
Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems
Authors: NAKANISHI Kazuhiro , KIMURA Yukitaka , MATSUNO Ryuichi
Published: March 2005

We studied kinetics and the equilibrium relationship for the thermolysin‐catalyzed synthesis of N‐(benzyloxy‐carbonyl)‐l‐aspartyl‐l‐phenylalanine methyl ester (Z‐Asp‐PheOMe) from N‐(benzyloxycarbonyl)‐l‐aspartic acid (Z‐Asp) and l‐phenylalanine methyl ester (PheOMe) in an aqueous‐organic biphasic system. This is a model reaction giving a condensation product with dissociating groups. The kinetics for the synthesis of Z‐Asp‐PheOMe in aqueous solution saturated with ethyl acetate was expressed by a rate equation for the rapid‐equilibrium random bireactant mechanism, and the reverse hydrolysis reaction was zero‐order with respect to Z‐Asp‐PheOMe concentration. The courses of synthesis of Z‐Asp‐PheOMe in the biphasic system were well explained, by the rate equations obtained for the aqueous solution and by the partition of substrate and condensation product between the both phases. The rate of synthesis in the biphasic system was much lower than in aqueous solution due to the unfavorable partition of PheOMe in the aqueous phase.

The equation for the equilibrium yield of Z‐Asp‐PheOMe in the biphasic system was derived assuming that only the non‐ionized forms of the substrate and condensation product exist in the organic phase. It was found theoretically and experimentally that the yield of Z‐Asp‐PheOMe is maximum at the aqueous‐phase pH of around 5, lower than for synthesis in aqueous solution. The effect of the organic solvent on the rate and equilibrium for the synthesis of Z‐Asp‐PheOMe could be explained by the variation in the partition coefficient.

The effect of the partitioning of substrate on the aqueous‐phase pH change was also shown.
Characterization of the elongation factors from calf brain
Authors: CRECHET Jean‐Bernard , PARMEGGIANI Andrea
Published: March 2005

The properties of EF‐1α from calf brain have been investigated and compared with those of EF‐Tu. EF‐1α binds GDP and GTP in a 1:1 stoichiometry, showing the same affinity for both nucleotides (K′d= 2–4 μM). EF‐1β strongly enhances the dissociation rate of the EF‐1α· GDP complex and to a lesser extent of the EF‐1α· GTP complex. Aminoacyl‐tRNA (aa‐tRNA) stabilizes EF‐1α· GTP much less efficiently than the EF‐Tu · GTP complex. Unlike EF‐Tu, EF‐1α sustains the binding of aa‐tRNA to the ribosome also in the presence of GDP or in the absence of any nucleotide, though to a lesser degree than with GTP. Kirromycin enhances the dissociation rate of both EF‐1α· GTP and EF‐1α· GDP but especially that of the latter. This effect results in an increase of the exchange rate of the EF‐1α‐bound nucleotide with free nucleotides. Although in this regard the effect of kirromycin mimicks that of EF‐1β, the antibiotic is incapable of increasing the EF‐1α· GDP/GTP exchange rate when aa‐tRNA and ribosomes are present. Therefore, unlike EF‐1β, kirromycin cannot enhance the rate of poly(Phe) synthesis. On the other hand, the failure of kirromycin to induce a GTP‐like conformation of EF‐1α· GDP, as in the case of EF‐Tu · GDP, explains its inability to inhibit peptide bond formation in the eukaryotic system.
Characterization of the elongation factors from calf brain
Authors: CRECHET Jean‐Bernard , PARMEGGIANI Andrea
Published: March 2005

The GTPase activity of purified EF‐1α from calf brain has been studied under various experimental conditions and compared with that of EF‐Tu. EF‐1α displays a much higher GTPase turnover than EF‐Tu in the absence of aminoacyl‐tRNA (aa‐tRNA) and ribosomes (intrinsic GTPase activity); this is due to the higher exchange rate between bound GDP and free GTP. Also the intrinsic GTPase of EF‐1α is enhanced by increasing the concentration of monovalent cations, K+ being more effective than NH+4. Differently from EF‐Tu, aa‐tRNA is much more active than ribosomes in stimulating the EF‐1α GTPase activity. However, ribosomes strongly reinforce the aa‐tRNA effect. In the absence of aa‐tRNA the rate‐limiting step of the GTPase turnover appears to be the hydrolysis of GTP, whereas in its presence the GDP/GTP exchange reaction becomes rate‐limiting, since addition of EF‐1β enhances turnover GTPase activity. Kirromycin moderately inhibits the intrinsic GTPase of EF‐1α; this effect turns into stimulation when aa‐tRNA is present. Addition of ribosomes abolishes any kirromycin effect. The inability of kirromycin to affect the EF‐1α/guanine‐nucleotide interaction in the presence of ribosomes shows that, differently from EF‐Tu, the EF‐1α· GDP/GTP exchange reaction takes place on the ribosome.
Translational control of ADP‐ribosylation in eucaryotic cells
Authors: LEDFORD Barry E. , JACOBS Donna F.
Published: March 2005

Starvation of the mouse hepatoma cell line Hepa for an essential amino acid (Trp, His, Leu, Ile or Phe) stimulated the incorporation of [3H]adenosine as ADP‐ribose monomer into an 80000‐Mr protein, P80. Two‐dimensional electrophoresis of Hepa proteins showed that P80 was the only protein labeled under starvation conditions. Time course experiments showed that the ADP‐ribosylation of P80 was a consequence rather than the cause of reduced translational activity. Cycloheximide treatment and incubation at reduced temperatures also reduced the rate of protein synthesis and stimulated the ADP‐ribosylation of P80. Starvation‐dependent ADP‐ribosylation of P80 was shown to occur in three other cell lines (Chang, Neuro‐2a, and chick comb fibroblasts).
Quantification of ATP‐producing and consuming processes of Ehrlich ascites tumour cells
Authors: MÜLLER Marianne , SIEMS Werner , BUTTGEREIT Frank , DUMDEY Renate , RAPOPORT Samuel Mitja
Published: March 2005

ATP production of Ehrlich ascites tumour cells was estimated on the basis of their coupled respiration and lactate formation. ATP‐consuming processes were assessed from the effects of selective inhibitors of RNA synthesis, protein synthesis and proteolysis, Na+/K+‐ATPase and Ca2+‐ATPase on respiration. The extent of protein synthesis and proteolysis were also determined directly. From these values and those of the inhibition of respiration by selective inhibitors, a P/O ratio of 2.2 was calculated. About 75% of the total ATP consumption could be assigned to specific processes. The major ATP‐consuming processes of tumour cells in an amino‐acid‐enriched medium, in which they are in an approximate steady state, are protein synthesis with about 30% of total ATP consumption, and Na+/K+‐ATPase with about 20%, while RNA synthesis, ATP‐dependent proteolysis and Ca2+‐ATPase contribut about 10% each. In an amino‐acid‐free glucose medium, protein synthesis is reduced to a third, with a corresponding decrease of respiration, whereas the rate of the other ATP‐consuming processes is unchanged.
Cooperative DNA binding by lambda integration protein – a key component of specificity
Authors: MINTER Stephen J. , CLORE G. Marius , GRONENBORN Angela M. , DAVIES R. Wayne
Published: March 2005

Quantitative analysis of nitrocellulose filter binding data by the method of Clore, Gronenborn and Davies [(1982) J. Mol. Biol. 155, 447–466] has been used to show that lambda integration protein (Int) exhibits cooperativity in binding to specific recognition sites within the attachment site region (λattP) of bacteriophage lambda DNA. Optimal values of the equilibrium constant obtained were 3.0(±1.0) × 1010 M−1 for the P′ site using a model of three sites with equal affinity and 1.9(±0.4) × 1010 M−1 for the P1 site on a two‐site model. The value of the cooperativity parameter α is 172±10666 in all cases. The occurrence of a consensus recognition sequence is necessary but not sufficient for strong binding; cooperative interaction between Int molecules binding to adjacent members of an array of binding sites is also essential. The occurrence of binding site arrays distinguishes λattP very clearly from other DNA sequences containing single recognition sites by chance.
Purification, subunit structure and pharmacological effects on cardiac and smooth muscle cells of a polypeptide toxin isolated from the marine snail Conus tessulatus
Authors: SCHWEITZ Hugues , RENAUD Jean‐François , RANDIMBIVOLOLONA Nicole , PRÉAU Catherine , SCHMID Annie , ROMEY Georges , RAKOTOVAO Lala , LAZDUNSKI Michel
Published: March 2005

The most active component in smooth muscle contraction, isolated from the whole venom of the marine snail Conus tessulatus, has a molecular mass of about 55 kDa. The toxin protein, tessulatus toxin, appeared to be constituted by two distinct polypeptide bands of 26 kDa and 29 kDa. The pure toxin caused a marked contraction of both guinea‐pig ileum and rabbit aorta at nanomolar concentrations. Tessulatus‐toxin‐induced contraction was indirectly prevented by classical inhibitors of the voltage‐dependent Ca2+ channel.

Tessulatus toxin caused a large increase in the initial rate of 45Ca2+ uptake by cardiac cells. This uptake was insensitive to Ca2+ channel blockers at concentrations 100–1000 fold higher than those known to block voltage‐dependent Ca2+ channels in these cells. Voltage clamp experiments have confirmed that tessulatus toxin was not directly active on the Ca2+ current.

Tessulatus‐toxin‐stimulated 45Ca2+ influx was inhibited by dichlorobenzamil and suppressed when Na+ was substituted by Li+, indicating that the toxin acted via activation of the Na+/Ca2+ exchange system in cardiac cells. Activation by tessulatus toxin of the Na+/Ca2+ exchange system occurred via a toxin‐stimulated Na+ entry into cardiac cells and was observed in the same range of toxin concentration which produced 45Ca2+ entry. The Na+ entry system that was activated by tessulatus toxin was insensitive to classic inhibitors of known Na+ entry systems in cardiac cells. Possible mechanisms by which tessulatus toxin induced Na+ entry into cardiac cells and contractions in smooth muscles are discussed.

Tessulatus toxin is cytotoxic when used at high concentrations.
Isolation and characterization of a yeast mutant defective in cholinephosphotransferase
Authors: HOSAKA Kohei , YAMASHITA Satoshi
Published: March 2005

A yeast mutant defective in cholinephosphotransferase (cpt) was isolated as a revertant from a choline‐sensitive mutant, which exhibited lowered phosphatidylinositol synthesis. A block at the cholinephosphotransferase step in the mutant was indicated by the enzyme defect and the accumulation of CDP‐choline in the cells with a decrease in phosphatidylcholine synthesis. The defect was due to a single recessive mutation in a nuclear gene. The residual activity in the mutant showed an increased apparent Km for CDP‐choline and an altered sensitivity to Tween 20. Thus the structural gene may be affected in the mutant. The occurrence of an intact ethanolaminephosphotransferase in the mutant indicates the distinctness of the genes encoding cholinephosphotransferase and ethanolaminephosphotransferase in yeast. The present selection method was also effective for isolating mutants defective in the other steps of the CDP‐choline pathway and choline transport.
Conformational changes in the oligonucleotide duples d(GCGTTGCG). d (CGCAACGC) induced by formation of a cis‐syn thymine dimer
Authors: KEMMINK Johan , BOELENS Rolf , KONING Thea M. G. , KAPTEIN Robert , van der MAREL Gijs A. , van BOOM Jacques H.
Published: March 2005

In order to obtain insight into the repair mechanism of DNA containing thymine photo‐dimer, the conformation of the duplex d(GCGGCG). d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two‐dimensional nuclear Overhauser enhancement (2D NOE) spectrosopy and two‐dimensional homonuclear Hartmann‐Hahn spectroscopy have been applied to assign all the non‐exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis‐syn cyclobutane‐type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B‐DNA structure, the main conformational change occurring at the site of the thymine dimer.
Comparison of enterobacterial common antigen from different species by serological techniques
Authors: KUHN Hella‐Monika , BASU Sumanta , MAYER Hubert
Published: March 2005

Enterobacterial common antigen (ECA) was isolated from a number of selected species (including Salmonella montevideo, Shigella sonnei and Plesiomonas shigelloides) using the extraction method described by Männel and Mayer [Eur. J. Biochem. 86, 361–370 (1978)]. ECA of all these species behaved identically in enzyme‐linked immunosorption assay (ELISA) and in its inhibition using monoclonal anti‐ECA antibodies. Immunoblotting showed a ladder‐like pattern of at least 20 bands for all preparations tested. ECA modified at its lipid moiety (e.g. by phospholipases A2 and D or by mild acid hydrolysis) lost its coating capacity leaving, however, the serological reactivity as detected by inhibition assays intact. In contrast, reduction of the carboxylic groups of 2‐acetamido‐2‐deoxy‐D‐mannopyranosyluronic acid destroyed the serological reactivity. Deacylated ECA was also not detectable in immunoblotting. Chemical reacylation restored the reactivity of deacylated ECA in ELISA and in immunoblot and thus proved the essential function of fatty acids for the physicochemical properties of the molecule. 2‐Acetamido‐2‐deoxy‐D‐glucopyranose was identified as the reducing end of the ECA sugar chain after splitting off the lipid moiety by phospholipase D.
Disulfide‐bonded heparan sulfate proteoglycans associated with the surfaces of cultured bovine vascular endothelial cells
Authors: HISS Donovan , SCOTT‐BURDEN Timothy , GEVERS Wieland
Published: March 2005

A predominant species of heparan sulfate proteoglycan that consisted of at least two subunits linked by disulfide bonding was isolated from cell layers of normal (‘cobblestone’) bovine vascular endothelial cells in culture. Treatment of the parent molecules with dithiothreitol caused their complete cleavage and permitted the subsequent separation of the larger and smaller subunits on Sepharose CL‐4B columns. Removal of dithiothreitol by dialysis resulted in the reformation of large disulfide‐bonded molecules but such recombination of the subunits was prevented by prior reductive alkylation using iodoacetamide. Buoyant density gradient analysis as well as gel chromatography on Sepharose CL‐6B columns, following alkaline borohydride and nitrous acid treatment of individual carbohydrate‐rich subunits, showed that the latter consisted of core proteins associated solely with heparan sulfate glycosaminoglycans. The sizes of the latter were estimated by chromatographic techniques to be approximately 50 kDa in the case of the larger and 114 kDa for the smaller subunit. This is the first description of disulfide‐bonded proteoheparan sulfates in endothelial cells.
Phosphorus‐31 nuclear‐magnetic‐resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver
Authors: DESMOULIN Franck , COZZONE Patrick J. , CANIONI Paul
Published: March 2005

A quantitative analysis of the phosphorus‐31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30‐mm sample cell.

The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well‐oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25±0.05 and free magnesium concentration 0.5 mM.

Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn‐glycerol 3‐phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occured during ethanol oxidation. No significant pH variation is observed during ethanol infusion.

In perfused liver ofrats subjected to 48‐h fasts, the concentrations of cytosolic phosphorylated metabolities are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%.

During a 4‐min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min−1 (100 g body weight)−1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02±0.05 at the end of the 4‐min hypoxic period.
Role of cytosolic free calcium and phospholipase C in leukotrien‐B4‐stimulated secretion in human neutrophils
Authors: LEW P. Daniel , MONOD Antoinette , WALDVOGEL Francis A. , POZZAN Tullio
Published: March 2005

Various leukotriene analogues were tested for their capacity to raise the cytosolic free calcium concentration, [Ca2+]i, and to stimulate exocytosis in human neutrophils. Their order of potency for both parameters was LTB4 > the stereochemical isomer of LTB4, (5S, 12S)‐LTB4 ≫ the sulphidopeptides LTD4, LTC4. The correlation between [Ca2+]i and secretion indicates that an increase of [Ca2+]i above a threshold level of about 300 nM is necessary for stimulating secretion with LTB4. This threshold is about an order of magnitude higher than that required for the chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe).

The increase in [Ca2+]i elicited by LTB4 was unaffected by increasing cellular cAMP, while secretion was completely inhibited. These results indicate, that similar to fMet‐Leu‐Phe, leukotrienes generate other signals in addition to [Ca2+]i elevations. Contrary to previous claims, leukotrienes stimulate polyphosphoinositide hydrolysis, as indicated by the increase in [3H]inositol trisphosphate, InsP3, observed upon stimulation of myo‐[3H]inositol‐labelled neutrophils with LTB4 or (5S‐12S)‐LTB4. The two InsP3 isomers [Ins(1,4,5)P3 and Ins(1,3,4P3] were separated by high‐pressure liquid chromatographed and, as reported for other cell types, the formation of Ins(1,4,5)P3 precedes that of Ins(1,3,4)P3. Maximal stimulatory doses of LTB4 or (5S, 12S)‐LTB4 produce about 50% the amount of InsP3 generated by equimolar concentrations of fMet‐Leu‐Phe.

The present observation suggest that, though the transmembrane signalling systems activated by LTB4 and fMet‐Leu‐Phe are the same, the different efficacy of these two agonists at stimulating neutrophil functions is due, at least in part, to a different degree of activation of phospholipase C.
The pH dependence of the active‐site serine DD‐peptidase of Streptomyces R61
Authors: VARETTO Louis , FRÈRE Jean‐Marie , NGUYEN‐DISTÊHE Martine , GHUYSEN Jean‐Marie , HOUSSIER Claude
Published: March 2005

Titration of the active‐site serine DD‐peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac2‐L‐Lys‐D‐Ala‐D‐Ala and enzyme inactivation by the β‐lactam compounds benzylpenicillin, N‐acetylampicillin and ampicillin relies on the acidic form of an enzyme's group of pK∼ 9.5. It is proposed that protonation of a lysine ɛ‐amino group facilitates initial binding by charge pairing with the free carboxylate of the substrate and the β‐lactam molecules. Lowering the pH from 7 to 5 has no effect on the second‐order rate constant of enzyme acylation by benzylpenicillin and N‐acetylampicillin but results in a decreased rate constant of acylation by ampicillin and Ac2‐L‐Lys‐D‐Ala‐D‐Ala. Protonation of the side‐chain amino group of ampicillin and a decreased efficacy of the initial binding of the peptide to the enzyme seem to be responsible for the observed effects. Whatever the molecule bound to the enzyme, there is no sign for the active involvement of an enzyme's histidine residue of pK 6.5 – 7.0 in the hydrolysis pathway.
Purification and characterization of rat liver glutaminase
Authors: HEINI Hans G. , GEBHARDT Rolf , BRECHT Andreas , MECKE Dieter
Published: March 2005

Phosphate‐dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400‐fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein.

For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o‐phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion‐exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half‐life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h.

The relative molecular mass of the protein (Mr170 500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate‐dependent glutaminase present in kidney.