bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Homologous Cytochromes b5 in a Single Cell
Authors: LEDERER Florence , GHRIR Rachid , GUIARD Bernard , CORTRIAL Sylvie , ITO Akio
Published: March 2005

The amino acid sequence of the heme‐binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of autotmatic and manual degradation of fragments generated bvy trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high‐pressure liquid chromatography.

The sequence fo microsomal cytochrome b5 is identical with the one published by Ozols and Heinemannn after completion of this study [Biochim. Biophys. Acta (1982) 704, 163–173.] The sequesnce of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight microsomal sequences published thus far. The non‐conservatie substitutions are located at the surface of the know three‐dimensional structure of the microsomal cytochrome b5 except of for the substitution of histidine‐15 by arginine.

This paper brings the final proof that two iso‐cytchromes b5 exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed.
Regulation of Photosynthetic Carbon Metabolism
Authors: WOODROW Ian E. , MURPHY Denis J. , WALKER David A.
Published: March 2005

The activation and Steady‐state kinetics of wheat chloroplast sedoheptulose‐1,7‐bisphosphatase at several cencentrations of inorganic phosphatge are examined. Inorganic phosphate competitively inhibits substrate binding to both the active and inactive forms of the enzyme and reduces the rate of enzyme activation. Modulation of the apparent Km of sedoheptulose‐1,7‐bisphosphatase and fructose‐1,6‐bisphosphatase for their substrates by inorganic phosphate is discussed in terms of the control of intermediate pool sizes in the reductive pentose phosphate pathway and of the flux of fixed carbon towards starch synthesis or export from the chloroplast.
Proteins of the 30‐S Subunit of Escherichia coli Ribosomes which Interact Directly with Natural mRNA
Authors: BROUDE Natalia E. , KUSSOVA Klaudia S. , MECVEDEVA Natalia I. , BUDOWSKY Edward I.
Published: March 2005

Ultraviolet irradiation (254 nm) of the complexes of MS2 phage RNA (mRNA) and the 30‐S aubunits of Escherichia coli ribosomes prepared at 0°C and 37°C in the presence and absence of intiation factor 3 (IF‐3) causes cross‐linking of mRNA with proteins S3, S4, S5, S7, S9, S18 and IF‐3. Hence, these proteins interact directly with mRNA within the complex 30‐S‐subunit. mRNA.

Additin of IF‐3 results in an increase o f the rate of complex formation and decrease of its dissociation rate. The addition of IF‐3 changes the relative amounts of cross‐linked proteins (mainly S3, S4 and S18).

Decreasing the temperature from 37°C to 0°C not only decelerates the complex formation rate but also changes the relative amount of cross‐linkes proteins, indicating the influence of the conditins on the structure of the complex.
Structure of Horseradish Perosidase Compound I
Authors: ADEDIRAN Suara A. , DUNFORD H. Brian
Published: March 2005

The kinetics of the reaction between horseradish peroxidase and p‐nitroperbenzoic acid to form compound I have been studied at 25°C in phosphate buffer pH 7.2 and ionic strength of 0.11 M by transient‐state and steady‐state methods. The second‐order rate constant for compound I formation for compound I formation ovtained by stopped‐flow measurements at 403 nm is (3.7 ± 0.2) × 107 M1 s1. for the disappearance of p‐nitroperbenzoic acid and appearance of p‐nitrobbenzoic acid using steady‐state kinetics measured at 265 nm the rate constant is (3.0 ± 0.6) × 107 M1 s1. The results provide and idependent confirmation that one and only one oxygen atom is incorporated from the oxidizing substrate into compound I.
Membrane Topology of ATP Synthase from Bovine Heart Mitochondira and Escherichia coli
Authors: MONTECUCCO Cesare , DABBENI‐SALA Federica , FRIEDL Peter , GALANTE Yves M.
Published: March 2005

The polypeptides exposed to lipids in the mebranous F0 sector of the mitochondrial and Escherichia coli ATP synthases were labelled with radioactive photoreactive lipids. Highly resolving gel electrophoretic conditions were used in order to separate all the eighteen components forming the bovine heart mitochondrial enzyme. The hydrophobic labelling was performed on fully active and inhibitor‐sensitive ATP synthases. In the mitochondrial enzyme prepared according to Serrano et al. (1976) [J. Biol. Chem. 251, 2453–2461] seven polypeptides of Mr 30500; 11500; 10500; 10000; 9500; 8500 and 4500 were labelled. The major amount of radioactivity was associated with the 30500‐Mr component, which is thought to be the adenine nucleotide carrier. In the preparation of Galante et al., (1979) which almost completely lacks this component [J. Biol. Chem. 254, 12372–12378] nine polypeptides of Mr 25000; 21000; 11500; 10500; 10000; 9500; 9200; 8500 and 4500 were labelled. In the ATPase synthase from E. coli the major amount of labelling was associated with subunit b and only a minor portion with subunit c.
Purification and Characterization of a β‐N‐Acetylglucosaminidase from Octopus vulgaris
Authors: CECCARINI Costante , D'ANIELLO Antimo , CACACE Marcello G. , ATKINSON Paul H.
Published: March 2005

We have purified a β‐N‐acetylglucosaminidase from the hepatopancreas of the octopus which we have called βI. The enzyme was homogeneous as judged by Sephadex column chromatography, isoelectric focusing, non‐denaturing gel electrophoresis at two different pH and with sodium dodecyl sulphate/polacrylamide gel electrophoresis. The native portein has an apparent molecular weight of 120000 and we can conclude that it is a tetramer made up of two α and two β subunits with apparent Mr of 27000 and 34000, respectively. Using NMR spectroscopy we have examined the specificity of β and have established that the enzyme hydrolyses the β1,4 linkage of N‐acetylglucosamine but at only a specific site of the substrates used, two glycopeptides isolated from linkage of N‐acetylglucosamine but at only a specfic site of the substrates used, two glycopeptides isolated from ovalbumin. To our knowledge this is the first known exoglycosidase which has both linkage and site specificty.
Molecular Interactions in Intermediate‐Sized Filaments Revealed by Chemical Cross‐Linking
Authors: QUINLAN Roy A. , FRANKE Werner W.
Published: March 2005

Certain glia cells, notably astrocytes and tumor cxells derived therefrom, express simultaneously two types of proteins of intermediate‐sized filaments, vimentin and glia filament protein (GFP). We have used an established human glioma (astrocytoma) cell culture line (U 333 CG/343 MG) in which both proteins are seen in partly overlapping fibrillar structures by immunofluorescene microscopy to examine the possible existence of heteropolymer filaments of these two proteins by using reversible oxidative cross‐linking facilitated by the 1,10‐phenanthroline‐cupric ion complex. Dimeric cross‐link products are characterized by one‐dimensional and two‐dimensional gel electrophoresis under non‐reduing and reducing conditions as well as by peptide mapping. The relatively large prportions of heterodimers of vimentin and GFP obtained in cytoskeletal filaments cross‐linked in this way, demonstrate the frequency of heteropolymer filaments in this cell as well as the frequency of face‐to‐face ‘pairs’ of GEP and vimentin in such filaments. Together with our related observations on heteropolymer filaments between vimentin and desmin in some smooth muscle cells [Ouinlan. R. A. and Franke, W. W. (1982) Proc. Natl Acad. Sci. USA, 79, 3452–3456]. we discuss this as evidence for common principles of molecular arrangments of vimentin, GFP and desmin, at least in the cysteine‐containing surface domains. The results are also discussed in relation to cytoskeletal changes during glial differentiation.
Amino‐Acid Sequence of Feather Keratin from Fowl
Authors: ARAI Kunio Murayama , TAKAHASHI Rieko , YOKOTE Yoshiko , AKAHANE Kiso
Published: March 2005

The amino acid sequence of a single polypeptide chain, B‐4, from fowl feather barbs has been determined. The B‐4 chain was found to consist of 96 amino acid residues and t o have a molecular weight of 10206 in the S‐carboxymethylated form. The kN terminus of this protein was an N‐acetylsrine residue. The B‐4 protein contained seven S‐carboxymethylcysteine residues, six of which are located in the N‐terminal region (residues 1–26), and other one in C terminus. The central region of teh peptide chain was rich in hydrophobic residues. There were homolohous amino acids at 66 positions in the sequences of the sequences of the feather keratins of fowl, emu and silver gull. The variation (substitution, deletion and insertion) in sequence was found to be localized in both terminal sections of the polypeptide chain.

The B‐4 protein structure was predicted to contain β‐Sheet structure is mostly located in the central region (residues 22–70). On the othere hand, both terminal regions are almost devoid of secondary structure.
Lipid Domains in Plasma Membrances from Rat Liver
Authors: SCHROEDER Friedhelm
Published: March 2005

The existence of fluid and solid lipid domains in isolated rat‐liver plasma membranes was evaluated using the fluorescent fatty acids trans‐parinaric and cis‐parinaric acid as probe molecules for solid and fluid membrane areas, respectively. The fluorescence probe 1,6‐diphenyl‐1,3,5‐hexatriene indicated that a phase transition was present in the liver plasma membrane between 18°C and 30°C. At intermediate temperatures, cis‐parinaric acid, which partitioned approximately equally into fluid and solid lipid areas, detected two lipid domains: the mole fractions of fluid and solid lipid domains at 24°C were 0.32 and 0.68 while the mole fractions of cis‐parinaric acid in each domain were 0.34 and 0.66, respectively. The dissociation constant, aqueous to membrane lipid partition coefficient, and bound to free ratio for trans‐parinaric acid were 7.0 ± 0.7 μM, 4.0 ± 0.6 × 106, and 83:17, respectively. The affinity of the membrane for cis‐parinaric acid was twofold lower than for trans‐parinaric acid. The trans‐parinaric acid partitioned preferentially into solid lipid. Kps/f= 3.30, while the cis‐parinaric acid partitioned equally between fluid and solid phases Kps/f= 0.92. Thus, the data demonstrate the coexistence of fluid and solid domains in rat liver plasma membranes.
Characterisation of Phosphoenolpyruvate Transport across the Erythrocyte Membrane
Authors: HAMASAKI Naotaka , MATSUYAMA Hiroyuki , HIROTA‐CHIGITA Chiyo
Published: March 2005

Phosphoenolpyruvate was found to be transported across the erythrocyte membrane by a carrier‐mediated transport system. The transport of phosphoenolpyruvate was competitively inhibited by inorganic phosphate (Ki= 24mM) and pyridoxal 5‐phosphate (Ki= 0.2 mM), whereas the transport was noncompetitively inhibited by L(+)‐lactate (Ki= 37 mM). Specific inhibitors for the inorganic anion transport system such as 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid and 4,4′‐dinitrostilbene‐2,2′‐disulfonic acid strongly inhibited the phosphoenolpyruvate transport. The transport was irreversibly inhibited by treating erythrocytes with pyridoxal 5‐phosphate and NaBH4. Transport activities of Phosphenolpyruvate and inorganic phosphate in the treated cells were similary inhibited by pyridoxal 5‐phosphate depending on its concetrations. 4,4′‐Dinitrostilbene‐2,2′‐disulfonic acid protected both transport activities against the pyridoxal 5‐phosphate/NaBH4 treatment.

The major integral membrane protein, band 3, was preferentially labelled by treating erythrocytes with pyridoxal 5‐phosphate and NaB[3H]H4. the radioactive incorporation into band 3 was confirmed by two‐dimensional gel electrophoresis combining isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the second dimension.

Taken together, these results suggest that band 3 mediates the transport of phosphoenolpyruvate as well as inorganic phosphate.
Photoaffinity Leabeling of Chloroplastic and Cytoplasimic Leycly‐rRNA Synthetases of Euglena gracilis, by the γ‐(p‐Azidoanilidae) of ATP
Authors: KRAUSPE Rainald , LAVRIK Olga I.
Published: March 2005

The photoreactive‐δ(p‐azidoanilidate) analog of ATP, AzAn ATP, was used to affinity‐label the chlorophlastic an cytoplasmic leucyl‐tRNA synthetases of Euglena gracilis. The analog is abvle to replacxe the substrate ATP is in the tRNA leucylation reaction catalyzed by both enzymes. In the presence of ATP, it is competitive inhibitor against ATP as well a sleucine for the two isoenzymes, as is also shown for the photoiactive‐ δ‐anilidate analog of ATP, AnATP, which does not serve as subtrate in the enzyme reaction.

During ultraviolet irradiaiton, the enzymes are irrevesibly inactivated by AzAnATP in a concenytration‐dependent and time‐dependent manner indicative of phtoaffinity labelling. Both ATP and leucine, but not tRNA, protect the enzymes against ultraviolet‐induced inactivation by AzAn ATP. Comparative kinetic characterization of the inactivation process differences in the active centers of two intracelluar isoenzymes.
Purification and Characterization of a Secreted Protease from Tetrahymena pyriformis
Authors: BANNO Yoshio , YANO Koh , NOZAWA Yoshinori
Published: March 2005

A simple major protease, sevreted into the medium during growth of Tetrahymana pyriformis strain W, has been purified about 4000‐fold by (NH4)2SO4 precipitation, ion‐excange chromatogrphy, gel filtrtion and affinity chromatography on organomercurial‐Sepharose. Thje purified protease was homogeneous as judged by polyacrylamide gel electrophoresis and was a monomeric protein with a molecular weight of 22000–23000. Amino acid alalysis showed that the enzyme was rich in acidic amino acids. In addition, the purified Tetrahymena proteaseconsists of mutiple forms with isoelectric point between pH 5.3 and 6.3. Optimum activity of the purified enzyme was in the pH range 6.5–8.0 with α‐N‐benzoyl‐Dl‐arginie‐P‐nitroanilide and with azocasein, while it was in the lower pH range (4.5–5.5) for denatuired hemoglobins. The purified enzyme was inhibited by componds effective against thiol proteases. Leupeptin and chymostatin were potent inhibitors but pepstatin was without effect. This enzyme is similar to cathepsin B and appears to be a major proteolytic enzyme in Tetrahymena.
Site‐Directed Fluorogenic Modification of Bacteriophodopsin by 7‐Chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole
Authors: ALLEGRINI Peter Roland , SIGRIST Hans , SCHALLER Johann , ZAHLER Peter
Published: March 2005

Site‐directed covalent mnodification of bacteriorhodopsin is achieved by reacting the hydrophobic probe 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole (NBd‐CI) at neutral pH with purple membranes. The basteriorhodosin fluorescence thus produced is specific for a nucleophgilic group. the spectral properties of NBd‐ modified bsterorhodopsin indicate covalent interaction of the probe with thge nuclephilic ɛ‐amino group of a luysine reside. Modification fo tyrosine can be exclude. As demonstrated by polypeptide fragmentation and subsequent sequence analysis, NBD binding is confined to lysine 41 within the primary structure of bacteriorhodopsin. Collisional fluorescence quendching with iodide demonstrrates that, In NBd‐treated purplke membranes, the covalently bound label is not accessible in the aqueous phase. A hjysdrophobic location for the introduced fluorophor is thereby implid.
Subcellular Distribution in Xenopus laevis Oocytes of a Microinjected Poly(A)‐Binding Protein from Artemia salina Gastrulae
Authors: DE HERDT Erik , MARBAIX Gérard , TENCER René , SLEGERS Herman
Published: March 2005

The poly(A)‐binding protein P38 of non‐polysomal mRNP from Artemia salina gastrulae was labelled by reductive methylation and microinjected into he cytoplasm of Xenopus laevis oocytes. The labelled protein has a half‐life of approximately 20 h and accumulated in the nucleus to a final concentration 3.15 timees higher than that reached by free diffusion. This fact suggests that P38, a cytoplasmic poly(A)‐binding protein, might also play some role in the nucleus of the cell.
Identification of Monkey Lung Procathepsin d‐II as a Pepsinogen‐C‐Like Acid Protease Zymogen
Authors: MORIYAMA Akihiko , KAGEYAMA Takashi , TAKAHASHI Kenji
Published: March 2005

Procathepsin d‐II (Mr=37500) was purifieed from Japanese monkey lung at pH 7.0, and was shown to be converted to the active form, cathepsin d‐II (Mr=33000) via an intermediate (Mr=35500) upon treatment at pH 3.0 and 14°C. Procathepsin d‐II was shown to be the inactive precursor of cathepsin d‐II based on the following results; the former was inactive toward heat‐denaturated casein at pH 5.4 whereas the latter was active; the former was not inactivated by diazoacetyl‐Dl‐norleucine methyl ester in the presence of Cu2+ ion at pH 6.0 whereas the latter was inactivattede rapidly under the same conditions; and the former had no affinity to pepstein‐Sepharose between pH5 and 7 whereas the latter was adsorbed to it.

With a rabbit antiserum against procathepsin d‐II, cathepsin d‐II, pepsinogen C and pepsinC of Japanese monkey were each found to give a single precipitin line which fused completely with each other on agarose plate. On the other hand, cathepsin d‐I purified from the monkey lung, and pepsinogens A (I, II, III‐1, III‐2 and III‐3) obtained from the monkey gastric mucosa failed to precipitate with the antiserum. With the antiserum against the monkey pepsinogen C, the same results were obtained. Further, procathepsin d‐II and pepsinogen C were shown to have the same amino‐terminal amino acid sequence, Ala‐Val‐Val‐Lys‐Val‐Pro‐Leu‐Lys‐Lys‐Phe‐Lys‐. All these results indicate a strong similarly of procathepsin d‐II abd cathepsin d‐II to pepsinogen C and pepsin C, respectively.
Inactivation‐Reactivation Process and Repression of Permease Formation Regulate Several Ammonia‐Sensitive Permeases in the Yeast Saccharomyces cerevisiae
Authors: GRENSON Marcelle
Published: March 2005

Two distinct regulatory mechanisms are responsible for the absence of general amino‐acid permease activity in cells of the wild‐type strain Σ 1278b of Saccharomyces cerevisiae grown in the presence of ammonium ions.

One is a reversible inactivation process which progressively develops upon addition of ammonium ions to a proline‐grown culture, and completely suppresses the permease activity within one hour. This inactivation process is absent in mutants altered at the MUT2, MUT4, or PGR genetic loci.

In these mutants, a repression of the formation of active permease may clearly be observed in the presence of ammonium ions. This second regulatory mechanism is absent in mutants affected at the GDHCR locus, which might code for a repressor molecule. It is also relieved in the presence of a glnts mutation (which makes the glutamine synthetase thermosensitive) suggesting glutamine as an effector.

Two other ammonia‐sensitive permeases, namely the proline permease and the ureidosuccinic‐acid permease, seem to be subject to the same double regulation.

Mutations affecting the structural gene of the anabolic NADP‐linked glutamate dehydrogenase (gdhA) seem to completely prevent repression of the general amino‐acid permease, while they partially suppress its inactivation in the presence of ammonium ions.
Translational Barrier in Central Region of Encephalomyocarditis Virus Genome
Authors: SVITKIN Yuri V. , AGOL Vadim I.
Published: March 2005

A fractionated cell‐free system from Krebs‐2 cells was prepared which contained ribosomes and a high‐speed supernatant. When this system was programmed with encephalomyocarditis virus RNA, the synthesis of a precursor of capsid proteins, polypeptide preA, proceeded at a rate not very different from that observed in unfractionated extracts, whereas the synthesis of more distally encoded proteins, in particular polypeptide F, was greatly retarded, if not abolished. A protein was purified from the cytoplasmic extracts of Krebs‐2 cells which greatly enhanced production of polypeptide F as well as other noncapsid proteins in the fractionated system. By several criteria, this protein was identified as eukaryotic elongation factor 2 (eEF‐2). By using the ADP‐ribosylation assay, it was found that the fractionated system contained about 15% of the amount of eEF‐2 present in the unfractionated extracts.

The results suggest that changes in the eEF‐2 content may affect the elongation rate differently at different regions of the RNA template.
The Pyruvate Dehydrogenase Complex of Escherichia coli K12
Authors: STEPHENS Paul E. , DARLISON Mark G. , LEWIS Hilary M. , GUEST John, R.
Published: March 2005

The nucleotide sequence of a 3780‐base pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain‐termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post‐infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino‐terminal residue and carboxy‐terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1).

The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr= 27049) of unknown function.
Carboxylesterases in Rat and Human Sera and Their Relationship to Serum Aryl Acylamidases and Cholinesterases
Authors: TSUJITA Takahiro , OKUDA Hiromichi
Published: March 2005

The three enzyme activites, carboxylesterase, aryl acylamidase and cholinesterase activities, have been found in rat and human sera. Rat serum carboxylesterase associated with serum aryl acylamidase activity, but not with serum cholinesterase activity, was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE‐cellulose, blue Sepharose and QAE‐Sephadex, and then electrophoresis. Evidence for the identity of the two enzymes, carboxylesterase and aryl acylamidase, was their co‐elution profiles and co‐purification in the different steps, including electrophoresis, with constant ratios of specific activities and percentage recoveries.

Human serum carboxylesterase associated with serum cholinesterase, purified earlier, was compared with the rat serum esterase. Human serum carboxylesterase and aryl acylamidase activities were inhibited by serotonin and neostigmine, whereas rat serum carboxylesterase and aryl acylamidase activities were not affected by these compounds. Tyramine activated human but not rat aryl acylamidase. Rat and human serum esterase activities were both strongly inhibited by the diisopropylfluorophosphate. Both esterases catalyzed the hydrolysis of short‐chain triacylglycerols, such as tributyrin, and medium‐chain monoacylglycerols, such as monocaprin, but not the hydrolysis of long‐chain triacylglycerols.
Fusion of Sulfatide‐Containing Vesicles of Phosphatidylcholine
Authors: CESTARO Benvenuto , CERVATO Giovanna , BARENGHI Livia , PISTOLESI Elvira , PIZZINI Giuliano
Published: March 2005

Positively charged albumin is described as a ‘useful tool’ to induce both aggregation and fusion of phosphatidylcholine vesicles containing sulfatide. Techniques that include light‐scattering, Sepharose chromatography, centrifugation, electron microscopy, trapped volume determination and scanning calorimetry demonstrate that extensive fusion occurs during aggregation when sulfatide concentrations are above 4–5 mol%. The rate of fusion increases with time for 1–2 h, then reaches a plateau. Fusion occurs extensively above the transition temperature of the phospholipid and is strongly inhibited by increasing concentration of vesicle cholesterol. The significance of both membrane fluidity and sulfatide‐phospholipid organization in the fusion mechanism are discussed.
Characterization of the Trimeric, Self‐Recognizing Geodia cydonium Lectin I
Authors: MÜLLER Werner E. G. , CONRAD Jürgen , SCHRÖDER Christina , ZAHN Rudolf K. , KURELEC Branko , DREESBACH Karin , UHLENBRUCK Gerd
Published: March 2005

A d‐galactose‐specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high‐pressure liquid gel chromatography, was found to be 36500 ± 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of d‐galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal d‐galactose residues are bound by β1→6 and/or β1→4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.
Localization of Ellipticine Derivatives Interacting with Membranes
Authors: TERCE François , TOCANNE Jean‐François , LANEELLE Gilbert
Published: March 2005

The interaction with membranes of three anti‐cancer drugs of the ellipticine family was studied by fluo‐ rescence quenching of membrane probes. The fluorescence of three probes, located at different levels in mem‐ branes, was quenched by addition of two types of ellipticine derivatives, one amphiphilic drug (9‐methoxy‐ ellipticine) and two dipolar molecules (9‐aminoellipticine and 9‐hydroxyellipticine). By comparing the quenching curves obtained, the following can be proposed.

a) 9‐Methoxyellipticine can penetrate deeper in the lipid layers than 9‐aminoellipticine and 9‐hydroxy‐ ellipticine can.

b) The three drugs are able to penetrate at least between the first methylene groups of the acyl chains of lipids in liposomes.

c) In an isolated bacterial membrane, only 9‐methoxyellipticine can be located in the region of the first methylene groups of lipids, the two dipolar drugs being adsorbed on the membrane surface.

It was also shown that cholesterol hindered the penetration of 9‐methoxyellipticine in the bilayer of liposomes.
N‐Acetyimuramoyl‐l‐alkaline Amidase of Escherichia coli K12
Authors: PARQUET Claudine , FLOURET Bernard , LEDUC Mireille , HIROTA Yukinori , VAN HEIJENOORT Jean
Published: March 2005

Various experiments were carried out in an attempt to determine the possible physiological function of the N‐acetylmuramoyl‐l‐alanine amidase purified from Eschrrichia coli K12 on the basis of its activity on N‐acetyl‐muramoyl‐l‐alanyl‐d‐7‐glutamyl‐meso‐diaminopimelic acid [MurNAc‐lAla‐dGlu(msA2pm)]. A Km value of 0.04 mM was determined with this substrate. Specificity studies revealed that compounds with a MurNAc‐lAla linkage are the most probable substrates of this enzyme in vivo. Purified amidase had no effect on purified peptidoglycan and only low levels (1–2.5%) of cleaved MurNAc‐lAla linkages were detected in peptidoglycan isolated from normally growing cells. However, the action of the amidase in vivo on peptidoglycan was clearly detectable during autolysis. The amidase activity of cells treated by osmotic shock, ether or toluene, as well as that of mutants with altered outer membrane composition was investigated. Attempts to reveal a transfer reaction catalysed by amidase were unsuccessful. Furthermore, by its location and specificity, amidase was clearly not involved in the formation of UDP‐MurNAc. The possibility that it might be functioning in vivo as a hydrolase degradingexogeneous peptidoglycan fragments in the periplasma was substantiated by the fact that MurNAc itself and MurNAc‐peptides could sustain growth of E. coli as sole carbon and nitrogen sources. Finally, out of 200 therniosensitive mutants examined for altered amidase activity, only two strains had less than 50 % of the normal level of activity, whereas ten strains were found to possess more than 50%,. In fact, two of the overproducers encountered presented a 4–5‐fold increase in activity.
Detection and Regulation of the mRNA for the Inhibitor of Extracellular cAMP Phosphodiesterase of Dictyostellum discoideum
Authors: ROSSIER Claude , FRANKE Jak ob , MULLENS Isabel A. , KELLEY Kenneth J. , KESSIN Richard H.
Published: March 2005

The inhibitor of the cAMP phosphodiesterase of Dictyostelium discoideum is a cysteine‐rich glycoprotein, which binds to the enzyme and inactivates it. When the inhibitor is removed, enzymatic activity is restored. Following translation in vitro of RNA from developing cells and immunoprecipitation with anti‐inhibitor serum, newly synthesized inhibitor can be detected by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography. The inhibitor can be labeled using [35S]cysteine but not [35S]methionine, in agreement with the previously determined amino acid composition, and can be detected after cell‐free translation only if it has been previously acetylated. Purified native inhibitor blocks immunoprecipitation of the inhibotor polypeptide synthesized in vitro.

No inhibitor mRNA was detected in growing cells. Translatable mRNA was present 2 h after the beginning of starvation, reached a maximal level after 3 h, and decreased thereafter. Addition of 1 mM cAMP at the beginning of starvation delayed the appearance of translatable inhibitor mRNA. In the presence of 5 μM adenosine cyclic‐3′,5′‐phosphorothioate, a slowly hydrolyzed cAMP analogue, no translatable mRNA could be detected. Following removal of the analogue, the mRNA appeared within one hour and inhibitor was secreted after another hour.
Serine Proteinase Inhibitors from Vipera ammodytes Venom
Authors: RITONJA Anka , TURK Vito , GUBENŠEK Franc
Published: March 2005

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long‐nosed viper Vipera ammodytes ammodytes by ion‐exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki= 3.4 × 10−10 and 5.6 × 10‐10 M), while the third one primarily inhibits chymotrypsin (Ki= 4.3 × 10‐9 M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N‐terminal group in the former inhibitors is blocked; arginine is the N‐terminal amino acid in the latter. Besides trypsin and α‐chymotrysin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitzpancreatic trysin inhibitor family of inhibitors.
Conformational Changes of Apigenin 7‐O‐(6‐O‐malonylglucoside), a Vacuolar Pigment from Parsley, with Solvent Composition and Proton Concentration
Authors: MATERN Ulrich , HELLER Werner , HIMMELSPACH Karl
Published: March 2005

Malonylated apigenin 7‐O‐glucoside was prepared from malonyl‐CoA and apigenin 7‐O‐glucoside using a malonyltransferase from parsley cell cultures. The enzyme product, as well as the flavonoid substrate, was analyzed by high‐resolution nuclear magnetic resonance spectroscopy, confirming that malonic acid had been attached to the primary hydroxyl of the glucose moiety of apigenin 7‐O‐glucoside. During these studies in several solvents, it became apparent that, in particular, the chemical shift of H‐6” A and the coupling constants J5,“6”A and J5,“6”B were dependent on solvent composition and proton concentration. Similar changes of sugar proton resonance frequencies were also observed with methyl 6‐O‐malonyl‐β‐d‐glucopyranoside, but not with apigenin 7‐O‐[6‐O‐(4‐coumaroyl)glucoside]. The change of coupling constants is ascribed to a conformational modification of the sugar portion in the malonylglucosides.

Malonylated flavonoid glycosides are exclusively located within the vacuoles of parsley cells. We propose that acylation of flavonoid glycosides which are synthesized in the cytoplasm facilitates transport of the substrates into the vacuole. The conformational modification which is induced by changes in proton concentration may provide a mechanism to trap flavonoids in situ.
Biosynthesis of the Major Glycoprotein Associated with Zymogen‐Granule Membranes in the Pancreas
Authors: HAVINGA Johan R. , STROUS Ger J. A. M. , POORT Cees
Published: March 2005

The biosynthesis of GP‐2, the major glycoprotein associated with zymogen‐granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse‐chase experiments, using [35S]methionine, were performed and the processing of GP‐2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

GP‐2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr= 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr= 78000–80000). Only the precursor form of GP‐2 is sensitive to digestion with the glycosidase endo‐β‐N‐acetylglucosaminidase H, indicating that the observed conversion reflects the processing of ‘high‐mannose’ oligosaccharides into complex type oligosaccharides.

Acinar cells cultured in the presence of increasing concentrations of the N‐glycosylation inhibitor tunicamycin synthesize 5–6 distinct precursor GP‐2 species with apparent molecular weights decreasing from 73000–61000. We conclude that GP‐2 contains five or six N‐linked carbohydrate chains.

From cell fractionation studies it was established that the precursor GP‐2 is present in a microsomal fraction with high density (> 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP‐2 is localized in low density microsomes (< 1.130 g/ml) probably Golgi vesicles. The GP‐2 in zymogen granule membranes is also in the mature form.
Revision of Enzyme Nomenclature
Authors: Dixon (Chairman) H. B. F. , Cornish‐Bowden (Secretary) A.
Published: March 2005

A new edition of Enzyme Nomenclature is being prepared. During preparation it has become clear that some of the references in the present (1978) edition and its four supplements [Eur. J. Biochem. 104, 1–4 (1980); 116, 423–435 (1981); 125, 1–13 (1982); 131, 461–472 (1983)] are incorrect. Unfortunately, it is impracticable to check all 4000 references to ensure that each does indeed refer to the enzyme indicated. Nonetheless, most of the errors are probably known to biochemists interested in the particular enzymes in whose listing the errors occur, and it would also be relatively easy for individual biochemists to check whether the entries for the enzymes of which they have specialist knowledge are correct. II you know a any errors, please write to Professor Edwin C. Webb. Vice‐Chancellor, Macquarie University, North Ryde, New South Wales, Australia 2113.

Although we are primarily concerned with actual errors, we would like to repeat the standing invitation for suggestions of new entries and improvements for old ones, including the citation of more appropriate references. We should be most grateful for all such help.
The Pyruvate Dehydrogenase Complex of Escherichia coli K12
Authors: STEPHENS Paul E. , DARLISON Mark G. , LEWIS Hilary M. , GUEST John R.
Published: March 2005

The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined using the dideoxy chain‐termination method. The aceF gene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho‐independent terminator. The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (aceE) of the pyruvate dehydrogenase component (E1) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon.

The amino terminus, carboxy‐terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein. The predicted molecular weight (Mr= 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78 000–89 000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration. The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains: the lipoyl domain and the subunit binding and catalytic domain. The lipoyl domain corresponds to the amino‐terminal segment of the protein. It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues. The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy‐terminal segment.
Kinetic Studies of Aminoglycoside Acetyltransferase and Phosphotransferase from Staphylococcus aureus RPAL
Authors: MARTEL Annie , MASSON Maryse , MOREAU Nicole , Le GOFFIC François
Published: March 2005

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6′‐N‐aminoglycoside acetyltransferase and a 2”‐O‐aminoglycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography.

The kinetic mechanisms of each activity have been determinated from studies of initial velocities, as well as product and dead‐end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed.

However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.
Improved Preparation of Lipoteichoic Acids
Authors: FISCHER Werner , KOCH H. Uwe , HAAS Renate
Published: March 2005

A procedure is described for measuring the extraction of lipoteichoic acids from gram‐positive bacteria in absolute terms. Virtually complete extraction was achieved from various bacteria by hot phenol/water if the cells were disrupted. Extraction of whole and delipidated cells and of the membrane fraction gave considerably lower yields.

Most of the nucleic acids co‐extracted from disrupted cells was removed by treatment with nucleases. Nuclease‐resistant nucleic acid, protein, polysaccharide, and teichoic acid were separated from lipoteichoic acid by anion‐exchange chromatography on DEAE‐Sephacel or hydrophobic interaction chromatography on octyl‐Sepharose. Purified preparations were essentially free of polymeric contaminants, retained their alanine ester substitution, and were in the sodium salt form.

Hydrophobic interaction chromatography also made it possible to recognize contamination of lipoteichoic acid with its deacylated and Iyso‐form, and to discriminate molecular species containing two and three, or two and four acyl groups.
Characterization of Benextramine as an Irreversible α‐Adrenergic Blocker and as a Blocker of Potassium‐Activated Calcium Channels
Authors: PLOTEK Yael , ATLAS Daphne
Published: March 2005

An irreversible α‐adrenergic blocker, benextramine [N,N′‐bis(o‐methoxybenzylamine‐n‐hexyl)‐cysteamine] was used as a probe to study the possible interrelationship between α‐adrenoceptors and the K+‐activated Ca2+‐channels.

Benextramine, a tetraamine disulfide, acts irreversibly both on the α1‐adrenoceptor (t1/2= 3 min) and the α2‐adrenoceptors. These studies were carried out on rat brain synaptosomes, [3H]prazosin and [3H]clonidine binding. Benextramine blocked Ca2+ influx in rat brain synaptosomes under both depolarizing (75 mM KCl) and normal conditions (5 mM KCl). Its action at the channel is reversible with IC50= 10 ± 5 μM of the net Ca2+ influex. This makes benextramine a most potent Ca2+ blocker compared to verapamil or nicardipine (IC50= 200 μM and 170 μ, respectively).

Pretreatment of rat brain slices with benextramine gave a synaptosomal preparation which was devoid of either α1‐adrenergic or α2‐adrenergic binding capacity due to the irreversible binding of benextramine, but with an undistrubed Ca2+ influx. Thus, these results suggest that the α‐adrenoceptors and the Ca2+‐channels are independent of each other, and that full occupancy of the α‐receptors does not affect the net calcium flux.
The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes
Authors: ANDUS Tilo , GROSS Volker , TRAN‐THI Thuy‐Anh , SCHREIBER Gerhard , NAGASHIMA Mariko , HEINRICH Peter C.
Published: March 2005

The biosynthesis and secretion of α2‐macroglobulin, transferrin, α1‐acid glycoprotein and α1‐proteinase inhibitor were studied in rat hepatocyte primary cultures. After labelling with [35S]methionine, two forms, which can be separated electrophoretically differing by molecular weight, were found for each of the four glycoproteins. The following molecular weights were estimated for the intracellular precursors and the secreted forms: α2‐macroglobulin, 176 000 and 182 000; transferrin, 84 000 and 86 000; α1‐acid glycoprotein, 39 000 and 43 000–60 000; α1‐proteinase inhibitor, 49 000 and 54 000.

Carbohydrate moieties could be removed from intracellular forms by treatment with endoglucosaminidase H indicating that their oligosaccharide chains were of the high‐mannose type. The extracellular forms were sensitive to sialidase. They incorporated [3H]galactose and [3H]fucose showing that their oligosaccharide chains were of the complex type.

Pulse‐chase experiments revealed a precursor‐product relationship for the high‐mannose and the complex type glycoproteins. In the hepatocyte medium newly synthesized albumin was detected after 30 min and newly synthesized glycoproteins after 60 min.

Unglycosylated α2‐macroglobulin (162 000), transferrin (79 000), α1‐acid glycoprotein (23 000), and α‐proteinase inhibitor (41 000) were found in the cells as well as in the medium, when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked reduction of the secretion of α2‐macroglobulin, α1‐acid glycoprotein and α1‐proteinase inhibitor, whereas the secretion of transferrin was less affected.
A Simple Model for Studies on the Regulation of Cholesterol Synthesis Using Freshly Isolated Hepatocytes
Authors: CIGHETTI Giuliana , GALLI Giovanni , KIENLE Marzia GALLI
Published: March 2005

Rat hepatocytes isolated by the procedure described here showed 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity in the range of that reported for rat liver at the maximum of the circadian cycle, even if they were taken from rats at the time of the minimum.

The enzyme was present in cells as both its active dephosphorylated (20 ± 8%) and the inactive phosphorylated forms. The enzyme activity and the ratio between the two forms were unaltered during 3 h of cell incubation.

25‐Hydroxycholesterol (50 μM) induced about 50% inhibition of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity during 1 h incubation but the relative amount of the two forms was not modified by the sterol.

Cells isolated by the described procedure may therefore be a useful tool in studies on the regulation of cholesterol neogensis, both through the synthesis of the enzyme, which can be shown by measuring the activity after complete dephosphorylation of the enzyme, and via the rapid reversible shift of the inactive to the active form, resulting from the ratio between the two enzyme forms. The latter mechanism for the modulation of cholesterol synthesis cannot be tested in cell cultures because full activation of the enzyme occurs during hepatocyte plating.
Polymorphism of α‐Actinin
Authors: KOBAYASHI Ryoji , ITOH Hideaki , TASHIMA Yohtalou
Published: March 2005

Heterogeneity of α‐actinins from rabbit skeletal muscles was studied. Polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate has made it possible to distinguish two closely related α‐actinins from rabbit fast, white muscle. One isoprotein (designated type I α‐actinin) appears to be preferentially located in the psoas muscle, while the other (designated type II α‐actinin) appears to be perferentially located in the longissimus dorsi muscle. Electrophoretic analyses have further shown that the two isoproteins are present as mixtures in most rabbit white, fast‐twitch muscles. A standard polyacrylamide gel–sodium dodecyl sulfate/polyacrylamide gel sequential electrophoretic procedure was developed to resolve the different α‐actinins dimers and to determine their subunit compositions. By this technique, both type I and type II α‐actinins appeared to be homodimers. No heterodimeric species of α‐actinin were detected. α‐Actinin of red, slow‐twitch muscles was similar to type II α‐actinin of fast, white muscle on one‐dimensional and two dimensional gels. However, slow, red muscle α‐actinin was significantly different from fast, white muscle α‐actinins in terms of one‐dimensional peptide mapping and immunological cross‐reactivity.
The Precursor of Rat Liver Mitochondrial Glutamate Dehydrogenase has Enzymatic Activity
Authors: FELIPO Vicente , MIRALLES Vicente , KNECHT Erwin , HERNANDEZ‐YAGO José , GRISOLIA Santiago
Published: March 2005

The cytosolic precursor for the mitochondrial glutamate dehydrogenase of rat liver was synthesized in a cell‐free reticulocyte lysate using messenger RNA from rat liver. To check whether this precursor had enzymatic activity, a highly sensitive fluorimetric method, which can measure picogram quantities of enzyme, was used together with competitive dissociation of the precursor from an immunoprecipitate with inactive glutamate dehydrogenase. Glutamate dehydrogenase activity, corresponding to that estimated from incorporation of [35S]‐methionine, was detected in the precursor. The significance of this finding is discussed.
Some Kinetic Properties of 4‐Hydroxyphenylpyruvate Dioxygenase from Pseudomonas sp. Strain P.J.874
Authors: RUNDGREN Marianne
Published: March 2005

Steady‐state kinetic properties of purified 4‐hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P.J. 874 were examined by a 14CO2 method at pH 7.5 and 377deg;C. The results conform to a mono‐iso‐ordered bi‐bi mechanism with binding of 4‐hydroxyphenylpyruvate before O2 and release of CO2 before homogentisate. A Theorell‐Chance mechanism can not be excluded.

The apparent DV and DV/Km ratios were about 1.1 and 1.2, respectively, for 4‐hydroxy[2,6‐2H2]phenylpyruvate when the initial O2 consumption was measured. The stoichiometry between the consumption of O2 and the formation of homogentisate and CO2 was 1:1:1: Loss of hydrogen at C2 or C6 of the ring of the substrate is thus not the rate‐limiting step during the reaction. Instead, this appears to be the conversion of an isomerized enzyme form.