bluuebunny/crossref_metadata_2025 huggingface dataset:
A pantothenate derivative is covalently bound to mitochondrial proteins in Neurospora crassa
Authors: LAKIN‐THOMAS Patricia L. , BRODY Stuart
Published: March 2005

The presence of protein‐bound pantothenate in Neurospora crassa was investigated by labelling a pantothenate auxotroph (pan‐2) with [14C]pantothenate and examining mycelial homogenates on dodecyl sulfate/polyacrylamide gels. Five peaks of radioactivity were found, with apparent molecular masses of 200, 140, 22, 19, and 9 kDa. The 200‐kDa peak was identified as fatty acid synthetase, based on its absence in a fatty acid synthetase mutant. The 22‐kDa and 19‐kDa peaks co‐purified with mitochondrial markers on sucrose gradients. When purified mitochondria were fractionated, the 19‐kDa protein was associated with the inner membrane and the 22‐kDa protein was enriched in the soluble mitochondrial fraction. The label was quantitatively recovered from the mitochondrial proteins as 4′‐phosphopantetheine after mild alkaline hydrolysis. Although the function of this post‐translational modification of mitochondrial proteins is not known, several possibilities are discussed: the 4′‐phosphopantetheine may act as a carrier group in an enzymatic reaction, or it may perform a regulatory function as part of an enzyme complex.
Regulation of the activity of eukaryotic peptide elongation factor 1 by autocatalytic phosphorylation
Authors: TUHÁČKOVÁ Zdena , ULLRICHOVÁ Jitka , HRADEC Jan
Published: March 2005

Highly purified peptide elongation factor 1 from rabbit reticulocytes liberates the terminal phosphate from [γ‐32P]GTP and incorporates it into its own protein. Approximately one phosphate residue becomes bound by one molecule of the factor. Only the eEF‐1α subunit of the factor (Mr 53000) becomes phosphorylated as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by autoradiography and by the incubation of [γ‐32P]GTP with individual subunits of the elongation factor separated by chromatofocusing in the presence of 5 M urea. The phosphorylation also takes place, though to a lesser extent, if the factor is incubated with Na2H32PO4, probably due to the presence of endogenous GTP bound in the molecule of the factor. The content of endogenous GTP in various factor preparations was 0.21–0.43 mol/mol factor. Phosphorylation of the peptide elongation factor is ribosome‐independent, acid‐labile and apparently autocatalytic since no other proteins are required for this reaction. Preincubation of the factor with GTP or with inorganic phosphate results in the phosphorylation of the factor and is followed by an enhanced binding of phenylalanyl‐tRNA to 80S ribosomes in the presence of poly(U). This is accompained by a dephosphorylation of the factor protein and thus the reversible autophosphorylation of the factor apparenly activates its binding site for aminoacyl‐tRNA. This is supported by the observation that sodium fluoride, which inhibits the dephosphorylation of the factor, blocks the factor‐catalyzed binding of aminoacyl‐tRNA to ribosomes. The incorporation of phosphate into factor protein also inhibits the formation of an eEF‐1 · GDP complex, which is inactive in protein synthesis. Thus GDP liberated by the GTPase activity of the factor cannot affect its binding site for aminoacyl‐tRNA. This may be the other reason for the enhanced activity of the phosphorylated factor. The autocatalytic GTP‐dependent phosphorylation of the peptide elongation factor 1 apparently modifies its function and may thus play a regulatory role in protein synthesis.
Expression in mammalian cells of the diaminopimelic acid decarboxylase of Escherichia coli permits cell growth in lysine‐free medium
Authors: JOUANNEAU Jacqueline , STRAGIER Patrick , BOUVIER Jean , PATTE Jean‐Claude , YANIV Moshe
Published: March 2005

The lysA gene of Escherichia coli encodes for a diaminopimelic acid decarboxylase (EC 4.1.1.20) which allows the conversion of diaminopimelic acid into lysine in bacteria. It has been cloned in an eukaryotic expression vector containing upstream the SV40 early promoting sequence, and downstream mouse α‐globin maturating sequences. The recombinant plasmid pSB99 (4800 base pairs) has been introduced into several mammalian cell lines by contransfection with a second selectable marker i.e. the polyoma‐transforming DNA. Selection for morphologically transformed rat cells which contained the intact lysA sequences, allowed the determination of the concentration of diaminopimelic acid in the lysine‐free medium that permitted cell growth. lysA‐expressing clones were directly selected in a medium containing 10 mM diaminopimelic acid, after transfection with pSB99 alone. Southern blot analysis on selected clones have shown that they contain up to 30–50 integrated copies of the plasmid in tandem arrangement. Finally, we demonstrated that lysA‐expressing clones incorporate a significant amount of radiolabelled [3H]diaminopimelic acid in acid‐insoluble material. The recombinant plasmid can serve as a selectable marker, in growth medium in which lysine was replaced by its direct bacterial precursor.
Time course of polynucleosome relaxation and ADP‐ribosylation
Authors: NIEDERGANG Claude P. , de MURCIA Gilbert , ITTEL Marie‐Elisabeth , POUYET Jean , MANDEL Paul
Published: March 2005

Isolated rat pancreatic polynucleosomes were poly(ADP‐ribosylated) with purified calf thymus poly(ADP‐ribose) polymerase. A time course study was performed using an NAD concentration of 200 μM and changes in nucleosomal structure were investigated by means of electron microscopy visualization and sedimentation velocity determinations. In parallel, analyses of histone H1 poly(ADP‐ribosylation) and determinations of DNA polymerase α activity on ADP‐ribosylated polynucleosomes were done at different time intervals.

A direct kinetic correlation between ADP‐ribose incorporation, polynucleosome relaxation amd histone H1 hyper‐ADP‐ribosylation was established. In addition, DNA polymerase α activity was highly stimulated on ADP‐ribosylated polynucleosomes as compared to control ones, suggesting increased accessibility of DNA to enzymatic action.

Because of the strong evidence implicating histone H1 in the maintenance of higher‐ordered chromatin structures, the present study may provide a−basis for the interpretation of the involvement of the histone H1 ADP‐ribosylation reaction in DNA rearrangements during DNA repair, replication or gene expression.
Isolation and characterization of the Fnr protein, the transcriptional regulator of anaerobic electron transport in Escherichia coli
Authors: UNDEN Gottfried , GUEST John R.
Published: March 2005

The Fnr protein, the transcriptional regulator of the expression of anaerobic respiratory functions in Escherichia coli, was purified to homogeneity from soluble extracts of a strain harbouring the fnr gene in an expression vector.

The identity of the isolated protein was confirmed by comparing its amino‐terminal sequence with that predicted from nucleotide sequence of the fnr gene. It appeared that eight amino‐terminal amino acids had been removed post‐translationally from the bulk of the isolated Fnr protein.

The molecular mass of the isolated protein (Mr 28000) was consistent with a monomeric state, but sedimentation coefficients for the cellular (4.1 S) and the isolated (2.9 S) Fnr protein suggest that it may exist as a dimer in the bacterial cells.

The Fnr protein bound DNA. However, the binding activity was not specific for the regulatory regions of relevant genes and it could not be stimulated by a variety of conditions or potential effectors.

Two of the four cysteine residues of the Fnr protein were alkylated by iodoacetic acid and this could have functional significance in rendering the protein redox‐sensitive.
Structure of the cytochrome c oxidase complex of rat liver
Authors: JARAUSCH Jochen , KADENBACH Bernhard
Published: March 2005

Isolated rat liver cytochrome c oxidase was cross‐linked with the cleavable reagents dimethyl‐3,3′‐dithiobispropionimidate (DTBP), 3,3′‐dithiobis(succinimidyl)propionate (DSP) and cupric di(1,10‐phenanthroline) (CuP). The cross‐linked products were separated by high‐resolving two‐dimensional dodecyl sulfate gel electrophoresis, which separates all thirteen polypeptides of the mammalian enzyme. With cupric di(1,10‐phenanthroline) seven polypeptides (I–III, Va, Vb, VIIb and VIII) were cross‐linked with each other and with itself, indicating the occurrence of free –SH groups in these polypeptides and a rearrangement of the native structure of the complex by cupric di(1,10‐phenanthroline). With dimethyl‐3,3′‐dithiobispropionimidate or 3,3′‐dithiobis(succinimidyl)propionate all nuclear‐coded polypeptides, with the exception of polypeptide VIIa, formed cross‐linked products with the three ‘catalytic’ polypeptides I–III, which are coded on mitochondrial DNA. Five additional cross‐linked pairs were found between nuclear coded polypeptides. The close arrangement of nuclear coded polypeptides with the catalytic polypeptides suggests a regulatory function of these polypeptides.
Structure of the cytochrome c oxidase complex of rat liver
Authors: JARAUSCH Jochen , KADENBACH Bernhard
Published: March 2005

The orientation of the thirteen polypeptides of rat‐liver cytochrome c oxidase in the inner mitochondrial membrane was studied by proteolytic digestion of mitoplasts and sonicated particles. After separation by sodium dodecylsulfate gel electrophoresis proteins were transferred on nitrocellulose, and individual polypeptides were identified by incubation with polypeptide‐specific antisera, followed by fluorescein‐isothiocyanate‐conjugated protein A.

The three catalytic polypeptides I–III and seven nuclear coded polypeptides (IV, Vb, VIa, VIc, VIIa, VIIb and VIII) were found accessible to proteases from the cytoplasmic phase. Polypeptides II, IV, Va, Vb and VIa were accessible from the matrix phase, indicating a transmembraneous orientation of polypeptides II, IV, Vb and VIa. Together with data on cross‐linking and on cytochrome‐c‐protected labeling of polypeptides, a model of the cytochrome c oxidase complex was developed. It is suggested that the cytochrome c binding site on polypeptide II is surrounded by several nuclear‐coded polypeptides, which may modulate the affinity of the enzyme towards cytochrome c.
Collagen‐stimulating factor from embryonic brain has ascorbate‐like activity and stimulates prolyl hydroxylation in cultured muscle cells
Authors: KALCHEIM Chaya , DUKSIN Dan , BACHAR Esther , VOGEL Zvi
Published: March 2005

Embryonic rat‐brain extract contains a collagen‐stimulating factor which enhances the production of collagen types I, III, IV and V by cultured rat muscle cells. Here we report on the partial characterization and possible mechanism of action of a low‐molecular‐mass fraction with ascorbate‐like activity isolated from embryonic rat brain extracts. This activity eluted very close to ascorbate when filtered through Bio‐Gel P‐2 and Sephadex G‐10. The peak of biological activity showed properties of a reducing agent. Both the biological and reducing activities were lost when the fraction was treated with the enzyme ascorbate oxidase. This factor enhanced in a time‐dependent manner, the secretion of procollagen, pulse‐labeled with [3H]proline. Incubation of the muscle cultures with the factor increased by 15‐fold the ratio of hydroxyproline to proline residues in secreted macromolecules over controls. A fourfold increase in the above ratio was obtained for the celluar proteins. Crude homogenates from control and factor‐stimulated cultures were tested for prolyl hydroxylase activity using [3H](Pro‐Gly‐Pro)n as a substrate. Cultures treated with the collagen‐stimulating factor showed a 5–50‐fold increase in prolyl hydroxylation activity compared to controls. No effect on prolyl hydroxylation was found when the factor was added in vitro to either control or stimulated enzyme preparations. Our results suggest that the collagen‐stimulating factor contains ascorbate‐like activity which promotes the secretion of collagenous proteins by increasing hydroxylation of proline residues in their polypeptide backbone.
Inhibition of the aminocylation of selected tRNA molecules by an estrogen‐regulated factor on uterine ribosomes.
Authors: WHELLY Sandra M. , BARKER Kenneth L.
Published: March 2005

Administration of estradiol to ovariectomized mature rats for 1 h induces a transient increase in the peptide elongation rate on uterine ribosomes. An inhibitor of the peptide elongation rate, which appears to be regulated by estrogen treatment in vivo, can be extracted from ribosomes of estrogen‐deprived rats. The extracted inhibitor or a native inhibitor‐ribosome complex affects the rate of the peptide elongation reaction in a uterine cell‐free protein synthesis system by inhibiting the ability of selected tRNAs in the assay to be charged with amino acids by their respective aminoacyl‐tRNA synthetases. The degree of inhibition of charging of the affected tRNAs ranges from 22% to 78%, the order of inhibition being Pro > Val > Arg > Try > Leu > Glu > Ile > Gly > His > Ser > Lys. Inhibition results from a specific dose‐dependent, and presumably reversible, effect of the inhibitor on tRNA, but not on the aminoacyl‐tRNA synthetase. The effect does not result from removal of A‐C‐C terminal nucleotides from the 3′ end of tRNA, but does inhibit the ability of selected tRNAs to bind to the aminoacyl‐tRNA synthetases. We propose that regulation of the peptide elongation rate on uterine ribosomes by estradiol occurs through the estradiol‐induced inactivation of a ribosome‐associated inhibitor, which causes a reversible alteration to selected tRNAs. The modified tRNAs are unable to bind to their respective aminoacyl‐tRNA synthetase to become charged with an amino acid thus causing the availability of selected aminoacyl‐tRNAs to become rate‐limiting in the sequential elongation of peptides.
Lipid requirement of the vanadate effect on the binding of calcium and ATP to the calcium transport ATPase of the sarcoplasmic reticulum
Authors: MEDDA Pankaj , HASSELBACH Wilhelm
Published: March 2005

Lipid deprivation of the sarcoplasmic reticulum calcium‐transport ATPase neither affects the enzyme's affinity for ATP nor that of calcium. In contrast, vanadate binding is almost completely abolished. Lipid substitution by oleic acid which at a ratio of 0.3 mg/mg protein completely reactivates the calcium‐dependent ATP hydrolysis restores vanadate binding. Concomitantly the mutual interactions between vanadate and calcium or ATP and ADP, respectively are restored. The vanadate‐induced disappearance of the enzyme's ATP binding sites as well as its high‐affinity binding sites for calcium follow the same time course. Conversely, the displacement of vanadate by calcium proceeds in parallel with the recovery of ADP binding. In lipid‐restituted preparations as well as in native membranes vanadate induces the disappearance of external high‐affinity and simultaneously the appearance of internal low‐affinity calcium binding sites.
Immunochemical measurement of histone H3 in non‐nucleosomal compartments of cultured mammalian cells
Authors: SENSHU Tatsuo , AKIYAMA Kyoichi , OHSAWA Takako , TAKAHASHI Kei
Published: March 2005

We measured histone H3 in the non‐nucleosomal compartment of cultured mammalian cells by enzyme‐linked immunoelectrotransfer blot assay of cytosolic proteins using affinity‐purified rabbit anti‐H3 IgG, and peroxidase‐linked second antibodies.

The cytosolic H3 level was estimated to be 0.5–1.0% of the nucleosomal H3 content in MH‐134SC cells (mean generation time 11 h) and 3–4% in HeLa cells (mean generation time 22 h). It showed characteristic changes under the inhibition of DNA and/or protein synthesis and during the cell cycle of HeLa cells. These indicate an inverse relationship between the cytosolic H3 level and the replicating activity of nuclear DNA. The possible implication of the non‐nucleosomal histones in the regulation of histone gene expression is discussed.
Human plasma actin‐depolymerizing factor
Authors: CHAPONNIER Christine , PATEBEX Patricia , GABBIANI Giulio
Published: March 2005

The plasma and serum of humans and various animal species exert an actin‐depolymerizing activity. Human actin‐depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE‐cellulose and blue‐Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin‐sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin‐A–Sepharose column. Incubation of ethanol‐fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90‐kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes.
Stimulation of peptidyltransferase reactions by a soluble protein
Authors: GANOZA Maria Clelia , ZAHID Nasir , BAXTER Ross MacPherson
Published: March 2005

The requirements for peptide‐bond synthesis and transesterification reactions of Escherichia coli 70S ribosomes, 50S native or reconstructed 50S subunits were examined using fMet‐tRNA as donor substrate and puromycin or α‐hydroxypuromycin as acceptors.

We report that the soluble protein EF‐P, purified to apparent homogeneity, stimulates the synthesis of N‐formylmethionylpuromycin or N‐formylmethionylhydroxypuromycin by 70S ribosomes or reassociated 30S and 50S subunits.

In the presence of EF‐P, 70S ribosomes are significantly more efficient than 50S particles in catalysing either peptide‐bond synthesis or transesterification.

The involvement of 50S subunit proteins in EF‐P‐stimulated peptide‐bond formation and transesterification was studied. 50S subunits were dissociated by 2.0 M LiCl into core particles and ‘split' proteins, several of which were purified to homogeneity. When added to 30S·A‐U‐G·f[35S]Met‐tRNA, 50S cores or 50S cores reconstituted with L6 or L11 promoted peptide‐bond synthesis or transesterification poorly. EF‐P stimulated peptide‐bond synthesis by both these types of core particles to approximately the same extent. On the other hand, EF‐P stimulated a low level of transesterification by cores reconstituted with L6 and L11. In contrast, core particles reconstituted with L16 exhibited both peptide‐bond‐forming and transesterification activities and EF‐P stimulated both reactions twentyfold and fortyfold respectively. Thus different proteins differentially stimulate the intrinsic or EF‐P‐stimulated peptide‐bond and transesterification reactions of the peptidyl transferase.

Ethoxyformylation of either 50S subunits or purified L16 used to reconstitute core particles, resulted in loss of peptide‐bond formation and transesterification. Similarly ethoxyformylation of EF‐P resulted in a 25–50% loss of its ability to stimulate both reactions. 30S subunits were resistant to treatment by this reagent. These results suggest the involvement of histidine residues in peptidyltransferase activities. The role of EF‐P in the catalytic mechanism of peptidyltransferase is discussed.
The NADP+‐binding site of ferredoxin‐NADP+ reductase
Authors: CIDARIA Dante , BIONDI Pier Antonio , ZANETTI Giuliana , RONCHI Severino
Published: March 2005

The flavoprotein ferredoxin‐NADP+ reductase is inactivated and loses its ability to bind NADP+ during covalent modification of a lysine by 5‐dimethylaminonaphthalene‐1‐sulfonyl chloride (dansyl chloride) [Zanetti, G. (1976) Biochim. Biophys. Acta 445, 14–24]. The substrate NADP+ gives almost complete protection against inactivation and modification. These observations are extended in this report by the characterization of an octapeptide containing the dansyl‐lysine which was isolated by high‐performance liquid chromatography from tryptic digests of protein modified with radiolabeled reagent. The amount of this peptide was severely reduced in protein modified in the presence of NADP+. The sequence of the dansyl‐peptide, only partially obtained by Edman degradation, was completed by analysis of the fragments resulting from thermolysin digestion of the purified tryptic dansyl‐peptide. Thus, the octapeptide containing the essential lysine residue has the following sequence: H2N‐Ser‐Val‐Ser‐Leu‐Cys‐Val‐Lys‐Arg‐COOH. A comparison with corresponding sequences of other known NADP+‐dependent dehydrogenases is attempted.
Characterization of human glucosylsphingosine glucosyl hydrolase and comparison with glucosylceramidase
Authors: VACCARO Anna Maria , MUSCILLO Michele , SUZUKI Kunihiko
Published: March 2005

Properties of glucosylsphingosine (gluco‐psychosine) glucosyl hydrolase were studied in detail in cultured human fibroblasts and placenta and were compared with those of glucosylceramidase. The two activities, that are deficient in tissues of Gaucher patients, showed minor but consistent differences. The pH optima were 4.8 for psychosine hydrolysis and 5.3 for glucosylceramide hydrolysis. In the presence of oleic acid, taurocholate activated glucosylceramidase more than 10‐fold, while it activated psychosine hydrolysis only by about 30%. Triton X‐100 was stimulatory for glucosylceramidase but was strongly inhibitory for psychosine hydrolysis. Phospholipids, that increase many times glucosylceramidase activity, were moderately inhibitory to enzymatic hydrolysis of psychosine. The psychosine hydrolase activity was slightly more heat‐stable than the glucosylceramidase activity. The Km values for the two substrates were similar; 1.7 × 10−5 M for psychosine and 2.7 × 10−5 M for glucosylceramide. The V for glucosylceramide was, however, 100‐times that for psychosine hydrolysis. Psychosine acted as a potent non‐competitive inhibitor (Ki= 1.8 × 10−5 M), while glucosylceramide was a weak inhibitor against psychosine hydrolysis. Within the limit of glucosylceramide solubility, psychosine hydrolysis could not be inhibited by more than 50%. Furthermore, the Dixon plot of glucosylceramide inhibition showed an anomalous slope. The ratio of the two activities was similar in fibroblasts, in the placenta mitochondria‐lysosomal fraction and in a partially purified placental preparation. These findings are best explained by the hypothesis that, although the two substrates are hydrolyzed by a single enzyme, they share an overlapping but not identical catalytic site while binding to hydrophobic sites unique for the respective substrates.
Purification of RNA polymerase and transcription‐termination factor Rho from Erwinia carotovora
Authors: NWANKWO Donald O. , GUTERMAN Sonia K.
Published: March 2005

Erwinia carotovora RNA polymerase consists of the holoenzyme structure σ2ββ′σ as found in Escherichia coli and other bacteria. E. carotovora RNA polymerase can synthesize RNA using λ, T7 of T4 DNA as templates; however, it is two times less active on these templates and is more temperature‐sensitive than the E. coli enzyme. The α subunit of the E., carotovora enzyme is lower in molecular mass than its E. coli counterpart. The σ factors from E. coli and E. carotovora are similar in size and in their ability to stimulate RNA synthesis by core enzyme on DNA templates such as T7 DNA. An additional protein of 115000 Da molecular mass, termed γ, is found associated with E. carotovora RNA polymerase. The γ protein is tightly associated with the polymerase subunits as it is not dissociated by gel filtration in buffer containing 0.5 M NaCl. It can be purified by passing the Agarose 1.5 m enzyme through coupled Bio‐Rex 70 and DEAE‐cellulose columns.

The γ‐protein, when present in excess over the σ subunit, inhibits holoenzyme activity on T7 DNA but not on poly[d(A‐T)] and may thus interfere with σ activity. The γ protein by itself cannot transcribe T7 DNA or poly[d(A‐T)], nor does it stimulate core enzyme activity on T7 DNA.

E. carotovora rho has a subunit molecular mass of 48000 Da and exhibits RNA‐dependent phosphohydrolysis of adenosine ribonucleoside triphosphate. E. coli and E. carotovora rho are indistinguishable immunologically, as total fusion of precipitin bands is observed. E. carotovora rho elutes from a phosphocellulose column at a salt concentration of about 0.21 M KCl, compared to that of 0.29 M KCl for E. coli rho. The poly(C)‐dependent ATPase activity of E. carotovora rho is more‐temperature sensitive and is six to ten times less active than that of E. coli rho. E. carotovora rho is capable of terminating RNA transcripts, as indicated by a decrease in RNA synthesis using λ or T7 DNA as template and E. carotovora or E. coli polymerase as the transcribing enzyme.
Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean
Authors: LORD J. Michael
Published: March 2005

The biosynthesis of the lectins and the other major storage proteins, the 11S globulins and the 2S albumins, which are found in protein bodies has been studied in developing castor bean endosperm cells. Newly synthesized proteins were radiolabelled by incubating intact endosperm tissue with [35S]methionine. The intracellular distribution of radiolabelled proteins was determined after fractionating endosperm homogenates by sucrose density gradient centrifugation. Pulse‐chase experiments revealed that all the major protein body components are initially segregated in precursor form into the lumen of the endoplasmic reticulum. The lectin precursors appeared as a group of 64000–68000‐Mr glycosylated polypeptides, the 11S globulins as a group of 46000–55000‐Mr polypeptides and the 2S albumins as a single 32500‐Mr polypeptide. These precursors were transferred from the endoplasmic reticulum to a population of transporting vesicles. The subsequent disappearance of the precursors from this vesicle fraction was accompanied by the accumulation of mature polypeptides in the protein body matrix (lectins and 2S albumins) or in the insoluble protein body crystalloid complexes (11S globulins). The castor bean proteins studied all exist as heterodimers in the protein bodies. After intracellular transport an endoproteolytic step is required to release each subunit of the heterodimer from the appropriate single polypeptide precursor.
Interaction of the pneumococcal amidase with lipoteichoic acid and choline
Authors: BRIESE Thomas , HAKENBECK Regine
Published: March 2005

The choline‐containing lipoteichoic acid (LTA, Forssman Antigen) of Streptococcus pneumoniae suppresses the activity of the pneumococcal autolysin, an N‐acetyl‐muramoy‐L‐alanine‐amidase (amidase) in aqueous solution [Höltje and Tomasz (1975) Proc. Natl Acad. Sci. USA 72, 1690‐1694]. The interaction between LTA and enzyme was used to establish a purification by affinity chromatography on LTA‐Sepharose. The amidase could be eluted from the column with choline only.This implies that binding of the enzyme to LTA is mediated via the choline residues of the LTA. Upon binding to the LTA‐Sepharose, the amidase converted from the applied E‐form (an inactive from of the amidase) to the active C‐form, a process which up to now was known to be mediated only by the pneumococcal choline‐containing wall teichoic acid.

Similar interactions between LTA and amidase seemed to occur in membrane fractions derived from cholinegrown cells: the membrane‐associated enzyme was present in the C‐form and could be detached completely with choline, suggesting that the amidase is bound to the membrane attached LTA rather than being a membrane protein itself. This was supported by the absence of amidase of amidase activity in membrane fractions derived from ethanolamine‐grown pneumococci, in which choline containing LTA is absent.

The LTA‐Sepharose‐associated amidase was not inhibited, but retained its activity. The enzyme was also not inhibited by lipase‐digested LTA. Both are conditions where the LTA is not present in micelles, unlike in aqueous solution. Therefore, mere binding to the LTA is probably not responsible for the inhibitory effect, but inhibition is a manifestation of an inaccessibility of the substrate for the amidase when bound to micellar LTA.

When the interactions between choline and amidase were investigated, it was found that high choline concentrations (2%) inhibited the enzyme completely. Even in vivo, 2% choline in the culture medium led to phenotypically amidase‐deficient pneumococci. Furthermore, in vitro, low choline concentrations (0.1%) suppressed the wallmediated conversion. On the other hand, with high choline concentrations (2%) conversion took place in the absence of cell walls. Depending on how the amidase has been converted, the apparent Mr of the resulting C‐amidase was different: the cell‐wall‐converted enzyme was of high Mr, Whereas the choline‐converted and the LTA‐Sepharose‐eluted enzyme showed an apparent low molecular mass known for the E‐form, when analyzed on sucrose gradiens. Prolonged dialysis of the low molecular mass C‐amidase resulted in ‘re‐conversion’ to the inactive E‐form, suggesting that the activity of the C‐form is dependent on enzyme‐attached choline molecules.

The interactions between choline and amidase led to an improved affinity chromatography system for the enzyme on choline‐Sepharose.
Purification and characterization of two aminopeptidases from guinea‐pig small‐intestinal mucosa
Authors: MORGAN John Gerard , DONLON John
Published: March 2005

Two electrophoretically distinct cytosolic peptide hydrolases from guinea‐pig small‐intestinal mucosa have been highly purified by a six‐step procedure comprising extraction from mucosal homogenate, ammonium sulphate fractionation, DEAE‐cellulose chromatography, chromatofocusing, calcium phosphate chromatography and Sephadex G‐100 gel filtration. They have similar apparent molecular masses as determined by gel filtration (Mr= 68 000) or by sodium dodecyl sulphate gel electrophoresis (Mr= 72 000). Both are aminopeptidases with optimum activity at pH 7.6. They are strongly inhibited by p‐hydroxymercuribenzoate, o‐phenanthroline and bestatin. Although both hydrolyse some dipeptides some dipeptides they have a distinctive kinetic preference for tripeptides composed of aromatic or non‐polar residues. Their affinities for some tripeptides are particularly high and also the hydrolysis of some substrates exhibits biphasic kinetics. These two aminotripeptidases are similar but they can be differentiated from each other and from a number of other aminopeptidases.
Reduced 6,6,8‐Trimethylpterins
Authors: RANDLES David , ARMAREGO Wilfred L. F.
Published: March 2005

The substrates of dihydropteridine reductase (EC 1.6.99.7), quinonoid 7,8‐dihydro(6H)pterins, are unstable and decompose in various ways. In attempting to prepare a more stable substrate, 6,6,8‐trimethyl‐5,6,7,8‐tetrahydro(3H)pterin was synthesised and the quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin derived from it is extremely stable with a half‐life in 0.1 M Tris/HCl (pH 7.6, 25°C) of 33 h.

Quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin is not a substrate for dihydropteridine reductase but it is reduced non‐enzymically by NADH at a significant rate and it is a weak inhibitor of the enzyme: I50 200 μM, pH 7.6, 25°C when using quinonoid 6‐methyl‐7,8‐dihydro(6H)pterin as substrate.

6,6,8‐Trimethyl‐5,6,7,8‐tetrahydropterin is a cofactor for phenylalanine hydroxylase (EC 1.14.16.1) with an apparent Km of 0.33 mM, but no cofactor activity could be detected with tyrosine hydroxylase (EC 1.14.16.2). Its phenylalanine hydroxylase activity, together with the enhanced stability of quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin, suggest that it may have potential for the treatment of variant forms of phenylketonuria.
Glycosphingolipides neutres des lignées lymphoblastoïdes de maladie de Fabry établies par transformation par virus Epstein‐Barr
Authors: SALVAYRE Robert , MARET Arlette , NEGRE Anne , DOUSTE‐BLAZY Louis
Published: March 2005

Neutral glycosphingolipids of Fabry's disease lymphoid cell lines established by Epstein‐Barr viral transformation

Human lymphoid cell lines established by Epstein‐Barr viral transformation of peripheral B‐lymphocytes from normal subjects and from Fabry patients, were investigated for their ability to biosynthesize neutral glycosphingolipids from [14C]galactose and [14C]glucose as precursors.

Galactose was taken up in the presence of high concentrations of glucose and selectively utilised by the cells in the synthesis of galactosphingolipids. The pattern of neutral glycosphingolipids labelled from [14C]galactose was slightly modified with time of labelling in either lymphoid cell line: the first labelled glycosphingolipid was lactosylceramide (LacCer) in the normal line and globotetraosylceramide (GbOse4Cer) in the Fabry line. After labelling for 96 h, a steady state was reached and the percentage of every type of labelled glycosphingolipid was stable in each cell line; however, differences in the neutral sphingolipid composition appeared between the various cell lines. When using radiolabelled glucose as precursor, the major part of the radioactivity was incorporated into neutral lipids and phospholipids; neutral sphingolipids were much less labelled than when using galactose.

Catabolism of endogeneous labelled glycosphingolipids (synthesized by the cells during the ‘pulse’) was studied after cultivating the cells without radiolabelled precursors (‘chase’). In the cells from normal subjects, all the neutral glycosphingolipids were slowly degraded (half‐life time around 15 – 25 days for LacCer and GbOse3Cer). In contrast, in a lymphoid line from a Fabry patient, no appreciable degradation of GbOse3Cer occurred during 30 days. This block in the catabolism of GbOse3Cer is in good agreement with the previously reported deficiency of α‐galactosidase A activity in this Fabry lymphoid cell line [Salvayre, R. et al. (1981) Biochim. Biophys. Acta 659, 445 – 456] and demonstrates that α‐galactosidase B does not hydrolyze GbOse3Cer in the living cell (in contrast to the situation in vitro).
Purification and properties of NADH/NADPH‐dependent p‐hydroxybenzoate hydroxylase from Corynebacterium cyclohexanicum
Authors: FUJII Takaaki , KANEDA Toshi
Published: March 2005

Crude soluble extracts of Corynebacterium cyclohexanicum, grown on cyclohexanecarboxylic acid, were found to contain 4‐hydroxybenzoate 3‐hydroxylase which functions with NADH as well as NADPH. The purified enzyme preparation was electrophoretically homogeneous and contained FAD as prosthetic group. The relative molecular mass of the enzyme was estimated to be about 47000 by native and denaturated acrylamide gel electrophoresis, indicating that it is monomeric. The enzyme was stable at 60°C for 10 min. The enzyme was highly specific for p‐hydroxybenzoate. The activity was inhibited by several aromatic analogues of p‐hydroxybenzoate such as p‐aminobenzoate, p‐fluorobenzoate, o‐hydroxybenzoate, m‐hydroxybenzoate, 2,4‐dihydroxygenzoate, and 2,5‐dihydroxybenzoate. The Km value for NADH was fairly constant, about 45 μM, in the pH range 7.0 – 8.4, whereas the Km value for NADPH increased from 63 μM to 170 μM as the pH rose from 7.0 to 8.4. V values in the same pH range, however, were approximately constant in both cases; about 30 μmol min−1 mg−1 for NADH, and 26 μmol min−1 mg−1 for NADPH, and 26 μmol min−1 mg−1 for NADPH. Mg2+ was required for full activity of the enzyme in low concentrations of phosphate buffer. The enzyme was inhibited by Cl− which was non‐competitive with respect to NADH, NADPH and p‐hydroxybenzoate.
Spermidine increases the accuracy of type II restriction endonucleases
Authors: PINGOUD Alfred
Published: March 2005

The non‐specific cleavage of DNA by type II restriction endonucleases (BamHI, BsuRI, EcoRI, EcoRV, HindIII, PstI and SalI) can be effectively suppressed by spermidine in millimolar concentrations, regardless of whether the non‐specific cleavage is induced by high concentrations of enzyme under optimal buffer conditions or by high pH, low ionic strength, organic solvents and Mn2+ ions. The increased specificity of restriction endonucleases in the presence of spermidine is due to an enhancement of the cleavage rate at the canonical site and a slowing down of the cleavage rate at related sites. It is argued that spermidine is essential for the high accuracy of restriction endonucleases in vivo.
Histone H5 promotes the association of condensed chromatin fragments to give pseudo‐higher‐order structures
Authors: THOMAS Jean O. , REES Christine , PEARSON Edwin C.
Published: March 2005

We describe two distinct situations in which chicken erythrocyte chromatin fragments associate in solution. The erythrocyte‐specific histone H5 is implicated since chromatins that do not contain H 5 do not show this behaviour.

Well‐defined oligomers of between ∼ 6 and ∼ 18 nucleosomes prepared at low ionic strength condense and associate when the ionic strength is raised to 75 mM, forming pseudo‐higher‐order structures. The associated forms, probably predominantly dimers, are stabilized by migration of about 10% of the H 5, and of the minor lysine‐rich histone H 1, from the non‐associated forms, probably reflecting the preference of H 5 for higher‐order structures observed previously [Thomas, J. O. and Rees, C. (1983) Eur. J. Biochem. 134, 109–115]. Since the final (H 1 + H 5) content of the aggregate at 75 mM is never higher than that of the fragment prepared at low ionic strength, migration is probably to a small proportion of sites that have inevitably become vacant due to handling losses at the higher (but not at low) ionic strength. H 5 thus maximizes its interactions in the condensed state of chromatin and even maintains the association of two or more fragments without continuity of the DNA. Aggregates of oligomers larger than about 18 nucleosomes may be too long to withstand hydrodynamic shear forces in the absence of such continuity.

During nuclease digestion of nuclear chromatin, H 5 and, to a lesser extent, H 1, are released from the ends of very short fragments and bind to larger oligomers of various sizes leading to heterogeneous aggregates that survive exposure to low ionic strength. These aggregates, in contrast to those described above, have up to 60% more H 5 and 20% more H 1 than chromatin prepared at low ionic strength. Whether the excess H 5 and H 1 bind non‐specifically or to a second low‐affinity binding site on each nucleosome is not known.

The associated forms described above (1) are well defined and potentially useful for structural studies, whereas the other aggregates (2) seem less likely to be directly relevant to the native structure of chromatin.
A complex from cultured Chinese hamster ovary cells containing nine aminoacyl‐tRNA synthetases
Authors: MIRANDE Marc , LE CORRE Daniel , WALLER Jean‐Pierre
Published: March 2005

The size distribution of the 20 aminoacyl‐tRNA synthetses from wild‐type Chinese hamster ovary (CHO) cells and from the mutant cell line tsH1, containing a temperature‐sensitive leucyl‐tRNA synthetase, was determined by gel filtration. Nine aminoacyl‐tRNA synthetases, specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine and proline, which coeluted as high‐Mr entities (Mr∼ 1.2 × 106), were further co‐purified to yield a multienzyme complex, the polypeptide composition of which was identical to that previously determined for the complex from rabbit liver. Immunoprecipitates obtained from crude extracts of wild‐type and tsH1 mutant cells, using specific antibodies directed to the lysyl‐tRNA or methionyl‐tRNA synthetase components of the complex, displayed the same polypeptide compositions as that of the purified complex, thereby establishing the heterotypic nature of this complex. Although the activity of leucyl‐tRNA synthetase from the mutant cells, grown at a permissive temperature, was low compared to that from the wild‐type, the polypeptide of Mr 129000, corresponding to this enzyme, was present in similar amounts and occurred exclusively as a component of the high‐Mr complex. Finally, we report that attempts to demonstrate phosphorylation of the components of the complex from cultured CHO, HeLa and C3 cells were unsuccessful.
Purification and characterization of two Cl−‐activated aminopeptidases hydrolysing basic termini from human skeletal muscle
Authors: MANTLE David , LAUFFART Brenda , McDERMOTT John R. , KIDD Alison M. , PENNINGTON Ronald J. T.
Published: March 2005

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion‐exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl− or Br− anions resulted in a 20‐fold or 10‐fold increasae in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05–5 mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 μM), p‐hydroxymercuriphenylsulphonic acid (0.1 μM), EDTA (10 mM), 1,10‐phenanthroline (100 μM), N‐ethylmaleimide (1 mM) and But‐Thr‐Phe‐Pro (0.5 mM). The molecular mass was 72000 Da (gel filtration). Only the arginyl and lysyl 7‐amino‐4‐methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10–250 μM) were estimated as follow: Arg‐Ame, K1m= 70 μM; KIIm= 270 μM; Lys‐Amc KIm= 280 μM, KIIm= 400 μM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2‐terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2‐terminal residues), but via cleavage of internal peptide bonds.

These results suggest that I and II may be isoenzymes of a Cl−‐requiring, thiol‐type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.
Intramolecular interactions, mesomerism and dynamics in actinomycin D studied by 15N NMR spectroscopy
Authors: JURETSCHKE Hans‐Paul , LAPIDOT Aviva
Published: March 2005

We present a detailed conformational study of 15N‐labelled actinomycin D in different organic solvents using 1H, 15N and two‐dimensional (2D) NMR techniques at 30.4 MHz and 50.6 MHz. The assignment of the threonine and valine 15N resonances to the individual residues on the α‐ or β‐lactone rings was achieved via heteronuclear shift‐correlated 2D NMR experiments.

The solvent perturbation studies allow an estimation of the solvent accessibility of the nitrogens and carbonyl groups. Evidence is presented that the pentapeptide rings of actinomycin D have different conformations in polar and in apolar solyents.

The chromophoric N10 is efficiently solvent‐protected, the solvent‐dependence of its 15N resonance resulting from solvent interactions at other positions of the molecule and from solvent‐dependent changes in the twisting of the chromophoric system. The chromophoric 2‐amino nitrogen is shown to exhibit a strong sp2 character due to the formation of a conjugated system with the carbonyl group at C1. Such a conjugation requires a nonplanar chromophoric ring system. Additionally, a hydrogen bond connecting the 2‐amino and the 1‐carbonyl group was detected. In some solvents, two resonances appear for the 2‐amino nitrogen implying the presence of the 2‐amino group in two different conformations.

The possible implications of the non‐planarity of the chromophore for the intercalation process and for the biological activity of the drug are discussed.
Effects of various amino acid replacements on the conformational stability of G‐actin
Authors: STRZELECKA‐GOLASZEWSKA Hanna , VENYAMINOV S. Yu. , ZMORZYǸSKI S. , MOSSAKOWSKA Malgorzata
Published: March 2005

Circular dichroic spectra of native, EDTA‐treated and heat‐denatured G‐actin from chicken gizzard smooth muscle are virtually the same as those of rabbit skeletal muscle actin. The rates of changes produced by EDTA or heat in the secondary structure are, however, higher in the case of gizzard actin. Similar differences were found in the rates of inactivation as measured by loss of polymerizability during incubation with EDTA or Dowex 50. The results are explicable in terms of local differences in the conformation at specific site(s) important for maintaining the native state of actin monomer. Involvement of the ATP binding site was shown by measuring the equilibrium constant for the binding of ATP to the two actins. Difference in the conformation of some additional site(s) is indicated by a higher rate constant of inactivation of nucleotide‐free actin observed for gizzard actin. No significant difference was found in the equilibrium constant for the binding of Ca2+ at the single high‐affinity site in gizzard and skeletal muscle actin. Comparison of inactivation kinetics of actin from chicken gizzard, rabbit skeletal, bovine aorta, and bovine cardiac muscle suggests that the amino acid replacements Val‐17 → Cys‐17 and/ or Thr‐89 → Ser‐89 have a destabilizing effect on the native conformation of G‐actin. The results indicate that deletion of the acidic residue at position 1 of the amino acid sequence has no effect on the conformation of the ATP binding site and the high‐affinity site for divalent cation as well.
Mechanism of action of gentamicin components
Authors: TANGY Frédéric , MOUKKADEM Maryam , VINDIMIAN Eric , CAPMAU Marie‐Louise , LE GOFFIC François
Published: March 2005

The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied. By means of equilibrium dialysis and of statistical interpretation of the data it was found that [3H]gentamicin C2 and 6′‐N‐[3H]methylgentamicin C1a interact with three classes of sites on tight‐coupled 70‐S species: (a) a first class concerning the tight and non‐cooperative interaction with one drug molecule (Kd= 0.6 μM), (b) a second class in which about five Gm molecules bind cooperatively (mean Kd= 10 μM), and (c) a third class of very high capacity in which up to 70 drug molecules may interact. The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from highaffinity sites towards lower‐affinity sites. The alteration of a ribosomal protein, L6, in a gentamicin‐resistant mutant of E. coli abolished the multiclass and the cooperative aspects of ribosomes–gentamicin interaction. The large ribosomal subunits from E. coli MRE 600 strain interact cooperatively with Gm, whereas 50‐S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity. The small subunits from both strains behave identically towards Gm. A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis.