bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific dephosphorylation by phosphatases 1 and 2A of a nuclear protein structurally and immunologically related to nucleolin
Authors: SCHNEIDER Helge R. , MIESKES Gottfried , ISSINGER Olaf‐Georg
Published: March 2005

A new nuclear substrate (N‐60) for phosphatase 1 and 2Ac has been described. In contrast to nucleolin (C23), to which it is structurally and immunologically related, N‐60 becomes dephosphorylated to 51% and 41% by phosphatases 1 and 2Ac, respectively, within 10 min.

Incubation up to 20 min led to a complete dephosphorylation of N‐60. The two other phosphatases tested (2B and 2C) did not dephosphorylate protein N‐60 to the same extent as phosphatases 1 and 2Ac.

In the case of nucleolin only 18% phosphate was released by all four phosphatases tested. The activity of both phosphatases, 1 and 2A, could be blocked by tumour promoter okadaic acid (100 nM) when N‐60 was used as a substrate. These results support the notion that the observed okadaic‐acid‐induced hyperphosphorylation of N‐60 in intact human fibroblasts may be caused by specific inhibition of phosphatases involved in the process of rDNA transcription.
The glycogen‐binding subunit of protein phosphatase‐1g from rabbit skeletal muscle
Authors: HUBBARD Michael J. , COHEN Philip
Published: March 2005

The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis.

A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation.

PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.
Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol‐specific phospholipase C and serum phospholipase D
Authors: TOUTANT Jean‐Pierre , ROBERTS William L. , MURRAY Nicole R. , ROSENBERRY Terrone L.
Published: March 2005

Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol‐specific phospholipase C (PtdIns‐specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns‐specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766–18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns‐specific PLC with the consequent release of hydrophilic AChE. Although serum anchor‐specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns‐specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.
Characterization of the lipid A component of genuine smooth‐form lipopolysaccharide
Authors: JIAO Binghua , FREUDENBERG Marina , GALANOS Chris
Published: March 2005

The smooth‐form lipopolysaccharide of Salmonella abortus equi had earlier been separated into three distinct fractions, a long‐chain fraction with an O chain containing 20–50 repeating units, a short‐chain fraction consisting of an R lipopolysaccharide and another with 1–6 repeating units, and an R fraction identical to the lipopolysaccharide synthesized by Ra, b‐mutant bacteria [Galanos et al. (1988) J. Chromatogr. 440, 397–404]. In this paper, the corresponding lipid A from each fraction was prepared by a newly elaborated procedure based on hydrolysis of the fractions in calcium acetate buffer (pH 3.5) followed by separation of the resulting free lipid A from the polysaccharide on a Sephadex G‐100 column. Chemical analysis revealed that lipid A of the R fraction contained the expected spectrum and amounts of fatty acids and it proved to be structurally identical to lipid A of previously studied Salmonella R mutants. In contrast, the lipid A of the long‐chain fraction contained only about 60% fatty acids compared to that of the R fraction. The lipid A of the short‐chain fraction also expressed a reduced substitution pattern of acyl residues.
Involvement of glutathione peroxidase activity in the stimulation of 5‐lipoxygenase activity by glutathione‐depleting agents in human polymorphonuclear leukocytes
Authors: HATZELMANN Armin , SCHATZ Monika , ULLRICH Volker
Published: March 2005

We recently demonstrated activation of 5‐lipoxygenase activity in human polymorphonuclear leukocytes (PMN) on preincubation of the cells with glutathione‐depleting agents, namely 1‐chloro‐2,4‐dinitrobenzene (Dnp‐Cl) and azodicarboxylic acid bis[dimethylamide] (diamide). In this paper we show that Dnp‐Cl, but not diamide, impairs the reduction of added organic peroxides in whole PMN. Also, since co‐incubation of fatty acid hydroperoxides with arachidonate caused activation of 5‐lipoxygenase, we propose that Dnp‐Cl increases the peroxide level in PMN which is required for the onset of lipoxygenase activity. This could be substantiated in PMN homogenates by a glutathione‐dependent depression of arachidonate 5‐lipoxygenation. At higher arachidonate concentrations and in the presence of Ca2+ the glutathione effect was not observed but additional glutathione peroxidase also blocked this maximally stimulated 5‐lipoxygenase. Together with other experiments, it became obvious that the formation of leukotrienes, but also of 15‐lipoxygenase products, requires a sharply defined threshold level of fatty acid hydroperoxides which are generated by the lipoxygenases and counteracted by glutathione‐dependent peroxidase(s). Dnp‐Cl influences this equilibrium by removing glutathione and thereby inhibiting glutathione‐dependent peroxidase activity. Form our data we conclude that it is the physiological function of the peroxidase activity in PMN to determine an efficiently regulated threshold level of hydroperoxide products, below which no activation of 5‐lipoxygenase or 15‐lipoxygenase can occur.
Differentiation of 3T3‐L1 pre‐adipocytes induced by inhibitors of poly(ADP‐ribose) polymerase and by related noninhibitory acids
Authors: JANSSEN Onno E. , HILZ Helmuth
Published: March 2005

To analyze a possible involvement of ADP‐ribosylation reactions in 3T3‐L1 pre‐adipocyte differentiation, ADP‐ribosyltransferase activities is permeabilized cells as well as endogenous amounts of protein‐bound mono‐and poly(ADP‐ribose) residues were determined. Also, in vivo labeling with [3H]adenosine of ADP‐ribose residues linked to high‐mobility‐group (HMG) proteins was performed. As an additional probe, the effects of ADP‐ribosylation inhibitors and non‐inhibitory analogs were studied. Basal and total poly(ADP‐ribose) polymerase activities markedly increased prior to the appearance of the differentiation marker glvcerol‐3‐phosphate dehydrogenase. Despite these apparent changes in activity, however, neither protein‐bound poly(ADP‐ribose) residue nor mono(ADP‐ribosyl) groups in histones, nor the NAD content, changed significantly under these conditions. Furthermore, although HMG protein‐associated [3H]ADP‐ribose was reduced in differentiating [3H]adenosine‐labeled cells, the data suggest altered precursor pool labeling rather than a specific decrease in ADP‐ribosylated HMG proteins. Non‐participation of ADP‐ribosylation reactions in 3T3‐L1 differentiation is further supported by experiments with inhibitors and non‐inhibitory analogs. Benzamide at 0.3–3 mM per se without effect on differentiation, was able to induce specific gene expression when combined with insulin (10−12–10−7 M). Similar effects were seen with benzoate as well as with nicotinamide, 3‐aminobenzamide and their corresponding acids. The data indicate that benzamide and analogs have profound effects on chromatin functions that are not mediated by ADP‐ribosylation reactions.
Dephosphorylation of the human T lymphocyte CD3 antigen
Authors: ALEXANDER Denis , GORIS Jozef , MARAIS Richard , ROTHBARD Jonathan , MERLEVEDE Wilfried , CRUMPTON Michael J.
Published: March 2005

Previous studies demonstrated that activation of T lymphocytes by phorbol ester or mitogenic lectin leads to phosphorylation of Ser 126 of the CD3 antigen γ chain, whereas treatment with ionomycin results in phosphorylation of both Ser 123 and 126 [Davies, A. A. et al. (1987) J. Biol. Chem. 262, 10918 – 10921]. In the present study, the dephosphorylation of Ser 123 and Ser 126 of the γ chain was investigated. Phorbol‐ester‐induced phosphorylation of the γ‐chain Ser 126 in vivo was reversed following removal of phorbol ester. Dephosphorylation of both Ser 123 and 126 was also observed in vitro using the microsome fraction of T lymphocytes. In order to identify the phosphatases acting at these two sites, the immunoprecipitated γ chain was used as substrate either following treatment with protein kinase C in vitro, in which case phosphorylation occurs mainly at Ser 123, or following in vivo phosphorylation of Ser 126. Purified oligomeric forms of the polycation‐stimulated phosphatases were more effective in dephosphorylating both phosphorylated forms of the γ chain compared with equivalent amounts of ATP, Mg2+ ‐dependent phosphatases or calcineurin. By using phosphopeptide analogues of the CD3 γ chain containing Ser 123 or Ser 126 as substrates (A3 and A6), it was shown that polycation‐stimulated phosphatases selectively dephosphorylated Ser 123 compared to Ser 126.

In order to determine which phosphatases dephosphorylate the γ chain in microsomes, A3 and A6 were used as substrates for characterising phosphatases in microsomes from human T leukaemia Jurkat 6 cells. Three phosphopeptide phosphatases (250—400 kDa) co‐eluted through five purification steps with three forms of polycation‐stimulated phosphorylase phosphatase. The partially purified A3/A6 phosphopeptide phosphatases were insensitive to Ca2+, calmodulin and inhibitor‐1, and dephosphorylated A3 preferentially compared with A6. A latent form of microsomal ATP, Mg2+ ‐dependent phosphorylase phosphatase was stimulated 10‐fold by trypsinisation, but did not dephosphorylate phosphopeptides A3 and A6. The results show that high‐Mr forms of polycation‐stimulated phosphatases are the only enzymes in human T leukaemia cell microsomes which dephosphorylate γ chain phosphopeptides.

The data point to an important role for polycation‐stimulated phosphatases in regulating the phosphorylation state, and so function(s), of the CD3 antigen.
The novel pituitary polypeptide 7B2 is a highly‐conserved protein coexpressed with proopiomelanocortin
Authors: MARTENS Gerard J. M. , BUSSEMAKERS Marion, J. G. , AYOUBI Torik A. Y. , JENKS Bruce G.
Published: March 2005

In the amphibian intermediate pituitary gland the biosynthetic activity for production of the precursor protein proopiomelanocortin (POMC) can be physiologically manipulated; POMC synthesis is high in animals adapted to a black background and low in white‐adapted animals. In order to study genes associated with POMC gene expression we applied a differential hybridization technique involving screening of a pituitary cDNA library with probes derived from RNA of inactive and physiologically activated intermediate pituitary cells of the amphibian Xenopus laevis. A differentially hybridizing Xenopus pituitary cDNA clone encoded the novel polypeptide 7B2. This Mr‐21 000 secretory granule‐associated protein of unknown function is shown to be highly conserved between Xenopus and human (83% amino acid sequence similarity). Conserved segments within the 7B2 structure included the N‐terminal portion, three pairs of basic amino acids which are potential recognition sites for proteolytic enzymes, and three regions sharing similarity with putative GTP‐binding domains. Levels of 7B2 mRNA were about 3% of POMC mRNA levels in Xenopus pituitary glands. In the intermediate pituitary the amount of both POMC and 7B2 mRNA was much higher in black‐adapted toads than in white‐adapted animals. These physiologically‐induced changes in POMC and 7B2 mRNA levels were not found in the anterior pituitary. We conclude that the POMC and 7B2 genes are coexpressed and that modulation of the activity of these genes is tissue‐specific.
Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis
Authors: NUTKINS Jennifer C. , WILLIAMS Dudley H.
Published: March 2005

The skin secretion of the frog Xenopus laevis has been fractionated by reverse‐phase HPLC and the most polar components studied by fast‐atom‐bombardment mass spectrometry (FAB/MS). Esterification of the hydrophilic peptides with methanol and ethanol was employed to improve the sensitivity of the technique. A number of small, highly acidic peptides have been identified, and alcoholysis of the peptide bonds within a number of these permitted their sequencing by FAB/MS. The sequences confirmed that they originate from acidic spacer regions found in the precursors to peptide hormones, such as caerulein, which have already been found in the secretion. In addition, acidic peptides derived from the spaces of the precursor to the antimicrobial peptides, PGS (or the magainins) have been isolated. The release of these from the preproprotein cannot be fully accounted for by documented processing mechanisms, suggesting that a novel type of cleavage site has been identified.
Kinetics and specificity of serine proteases in peptide synthesis catalyzed in organic solvents
Authors: GAERTNER Hubert , PUIGSERVER Antoine
Published: March 2005

Initial rates of peptide‐bond synthesis catalyzed by poly(ethylene glycol)‐modified chymotrypsin in benzene were determined using high‐performance liquid chromatography. Enzymatic synthesis of N‐benzoyl‐l‐tyrosyl‐l‐phenylalanine amide from N‐benzoyl‐l‐tyrosine ethyl ester and l‐phenylalanine amide was found to obey Michaelis‐Menten kinetics an to be consistent with a ping‐pong mechanism modified by a hydrolytic branch. The catalytic activity of modified chymotrypsin was dependent on both water concentration and type of organic solvent, the highest synthesis rate being obtained in toluene. Since the chymotrypsin specificity in the organic phase was actually altered, the enzyme's apparent kinetic parameters were determined for different substrates and compared to those obtained with other serine proteases in benzene. Both N‐benzoyl‐l‐tyrosine ethyl ester and N‐α‐benzoyl‐l‐lysine methyl ester were comparable acyl donors in benzene and the (kcat/Km)app value of modified chymotrypsin was only 10‐fold smaller than that obtained with poly(ethylene glycol)‐modified trypsin in the synthesis of N‐α‐benzoyl‐l‐lysyl‐l‐phenylalanine amide. The change in chymotrypsin specificity was also confirmed through the binding of trypsin inhibitors in benzene. The overall results suggest that hydrophobic bonding between the enzyme and its substrate should not be taken into account during catalysis in the organic phase. In general, if hydrophobic interactions are involved in the binding of substrates to the active site in aqueous media, the replacement of water by hydrophobic solvents will induce some change in enzyme specificity. Moreover, secondary residues of enzyme‐binding sites may also exert a significant influence on specificity since, as observed in this study, chymotrypsin exhibited high affinity for cationic substrates and cationic inhibitors as well in apolar solvents.
Immunoblotting analysis of protein‐protein crosslinks within the 50S ribosomal subunit of Escherichia coli
Authors: REDL Bernhard , WALLECZEK Jan , STÖFFLER‐MEILICKE Marina , STÖFFLER Georg
Published: March 2005

50S ribosomal subunits of Escherichia coli have been crosslinked with the bifunctional imidoester dimethyl‐suberimidate and the protein‐protein crosslinks have been analyzed by immunoblotting, using antisera specific for the individual ribosomal proteins of the large ribosomal subunit. Crosslinked protein pairs which occurred in yields higher than 5% have been unambiguously identified. Thus 13 crosslinks have been identified, namely L1‐L33, L5‐L7/12, L6‐L19, L7/12‐L10, L7/12‐L11, L9‐L28, L10‐L11, L13‐L20, L16‐L27, L17‐L32, L18‐L22, L19‐L25 and L27‐L33. These data, together with the results which we will be presenting elsewhere, contribute considerably to our knowledge of the protein topography of the 50S ribosomal proteins as determined by immunoelectron microscopy. We can now propose the approximate locations of ten proteins that have not previously been localized.
Positive and negative regulatory elements control the expression of the UGA4 gene coding for the inducible 4‐aminobutyric‐acid‐specific permease in Saccharomyces cerevisiae
Authors: VISSERS Stephan , ANDRE Bruno , MUYLDERMANS Francine , GRENSON Marcelle
Published: March 2005

In Saccharomyces cerevisiae, the pathway of 4‐aminobutyric acid catabolism, for use as a nitrogen source, involves a specific permease (encoded by the UGA4 gene) and two enzymes (encoded by the UGA1 and UGA2 genes, respectively). The synthesis of these proteins is induced by 4‐aminobutyrate. It also requires the product of the UGA3 gene. Here, we describe four additional regulatory mutations which provide evidence for the existence of both positive and negative regulatory elements which control the final expression of the UGA4 gene. Some of them simultaneously control the expression of the UGA1 and UGA2 genes. Three classes of mutant with a constitutive 4‐aminobutyrate‐specific permease have been isolated. (a) Recessive mutations in the UGA43 gene suggest that the product of the UGA43 gene behaves like a trans‐acting negative regulator of UGA4 gene expression. (b) The semi‐dominant mutation (uga11), closely linked to the UGA4 gene, might affect the receptor of the UGA43 gene product. In these two classes of mutant, only the permease is constitutive. (3) The uga81 mutation, closely linked to the UGA3 gene. makes the whole UGA regulon constitutive.

On the other hand, recessive mutations at the UGA3 gene locus lead to non‐inducibility of the UGA regulon. Hence the UGA35 gene product behaves like a second trans‐acting positive regulator in addition to UGA3.
Structure/activity relationships of scorpion α‐toxins
Authors: KHARRAT Riadh , DARBON Hervé , ROCHAT Hervé , GRANIER Claude
Published: March 2005

Chemical modifications of tyrosine and tryptophan residues of scorpion α‐neurotoxins II and III from Androctonus australis Hector were performed as well as modification of the two arginines and the α‐amino group of toxin I. The pharmacological potencies of each derivative were assessed in vivo by LD50 measurement and in vitro by competition experiments with 125I‐toxin for synaptosomal receptors. Arginine residues in positions 2 and 60 and the α‐amino group of Androctonus toxin I were derivatized by p‐hydroxyphenylglyoxal; the corresponding modified toxins exhibit low pharmacological potencies. Tryptophan 38 of toxin II and tryptophan 45 of toxin III were modified by nitrophenylsulfenyl chloride, leading respectively to a poorly and a fully active derivative. The tetranitromethane modification of tyrosine residues in positions 60, 5 and 14 of toxin III induced respectively 60%, 40% and 30% of loss of biological activity. Circular dichroic analysis indicated that for every derivative, except the nitrophenylsulfenyl derivative of Trp‐45 of AaH III, the conformation of the toxin was not altered by derivatization. Conformational integrity was also confirmed by full activity of the derivatives in radioimmunoassays. Taken together, the results suggest that aromatic residues belonging to the conserved hydrophobic surface, to the C‐terminal and to the loop region 37–44 are involved in the molecular mechanisms by which scorpion α‐toxins act. Charged residues in the N‐terminal and C‐terminal also contribute to the high efficacy of the binding process. It appears that all important residues are clustered on one face of the toxin, suggesting a multipoint interaction with the proteins of the sodium channel.
Purification and characterisation of a growth factor from porcine bone
Authors: MAYER Hubert , KUKOSCHKE Karl‐Gustav
Published: March 2005

A growth factor was extracted from porcine bone matrix by demineralisation and purified by heat and acid treatment, hydroxyapatite chromatography and gel filtration under dissociative conditions and reverse‐phase HPLC. Using the mitogenic response of osteoblast‐progenitor cells from embryonic chicken, a mitogenic activity was purified 3000‐fold. The mitogenic protein thus purified shows an apparent molecular mass of 13.5 kDa in both the nonreduced and reduced form on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The mitogenic activity is sensitive to proteinase K, dithiothreitol, and resistant to DNAse, RNase, heat (70°C) and pH (3–10). The factor stimulates the proliferation of osteoblast‐progenitor cells from embryonic chick at a concentration of 1 ng/ml. It is active on cells from skin, periosteum and sternum and has no or little activity on cells of the calvaria, intestine or kidney of embryonic chick or on mouse AKR‐2B/Balb c/3T3 cell line.
Studies on the active site of rat glutathione S‐transferase isoenzyme 4–4
Authors: van OMMEN Ben , PLOEMEN Jan H. T. M. , RUVEN Hendrik J. , VOS Ria M. E. , BOGAARDS Jan J. P. , van BERKEL Willem J. H. , van BLADEREN Peter J.
Published: March 2005

The active site of glutathione S‐transferase isoenzyme 4–4, purified from rat liver, was studied by chemical modification. Tetrachloro‐1,4‐benzoquinone, a compound previously shown to inactivate glutathione S‐transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro‐1,4‐benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue.

Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro‐1,4‐benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate.

The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro‐1,4‐benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue.

Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro‐1,4‐benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70–80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S‐transferase 4 subunit.
Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei
Authors: LACAVE Charlotte , LANEELLE Marie‐Antoinette , DAFFE Mamadou , MONTROZIER Henri , LANEELLE Gilbert
Published: March 2005

Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long‐chain 3‐hydroxyacids, branched at position 2), confirming previous proposals.

Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids: di‐unsatured mycolates, oxomycolates and wax‐ester mycolates (ester of dicarboxymycolic acid and 2‐icosanol or 2‐octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer‐Villiger oxidation step, as has been proposed on the basis of structural considerations.

By observing the labelling of the different mycolate pools in the cell, i.e. the organic‐solvent‐extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cell‐wall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall‐linked mycolates while wax‐ester mycolates appeared first as wall‐linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall‐linked arabino‐galactan.
Inactivation of the gene encoding the 14‐kDa subunit VII of yeast ubiquinol
Authors: SCHOPPINK Peter J. , BERDEN Jan A. , GRIVELL Leslie A.
Published: March 2005

The single nuclear gene encoding the 14‐kDa subunit VII of yeast ubiquinol:cytochrome c oxidoreductase has been inactivated by one‐step gene disruption, as verified by Southern blot analysis and immunoblotting. The resulting mutant has no ubiquinol:cytochrome c oxidoreductase activity and is respiratory‐deficient. Immunoblotting shows that cells lacking the 14‐kDa protein, also have lowered steady‐state levels of other subunits of complex III, the nuclear‐encoded 11‐kDa subunit VIII, the Rieske Fe‐S protein and the mitochondrially encoded cytochrome b can be detected spectrally. The steady‐state levels of the transcripts from genes encoding these proteins are not reduced, implying that the mutation exerts its pleiotropic effects at a posttranscriptional level. The residual amounts of subunits of complex III are recovered in the mutant mitochondria, suggesting that import is unaffected. The results strongly suggest that the 14‐kDa protein plays an essential role in the biosynthesis of the complex, most probably at the level of assembly. Field‐inversion gel electrophoresis was used to separate chromosomes of HR2 wild type and the (14‐kDa‐protein)° mutant, after which the gene encoding the 14‐kDa protein was located on chromosome IV by Southern blot analysis.
Assembly kinetics and identification of precursor proteins of complex I from Neurospora crassa
Authors: VIDEIRA Arnaldo , WERNER Sigurd
Published: March 2005

Complex I from Neurospora crassa was fractionated using chaotropic agents and various chromatographic techniques. Several subunits were isolated. Polyclonal antibodies directed against the holocomplex or individual subunits were raised in rabbits, and employed to analyse the composition and assembly of this respiratory chain enzyme in vivo.

N. crassa cells were pulse‐labelled with radioactive amino acids. The time course of incorporation of radioactivity into complex‐I polypeptides was studied by immunoprecipitation. The labelling kinetics of whole complex I was found to be similar to that of cytochrome oxidase, displaying a half‐maximal labelling time of 10 min. Newly synthesized individual polypeptide subunits (about 23 species) assembled into the holoenzyme at markedly different rates. Two mitochondrially synthesized proteins, a 29‐kDa polypeptide (the ND‐1 gene product) and a 12‐kDa polypeptide were the fastest components to appear in the enzyme. We estimate that the precursor pool sizes of all components range between 1–25% of the amounts present in the final complex.

Precursors of polypeptides of complex I were synthesized in an heterologous cell‐free system and immunoprecipitated with subunit specific antibodies. Six isolated precursors were compared with the corresponding mature proteins. It appears that four subunits (apparent molecular masses of 22, 25, 31 and 33 kDa) are initially synthesized as larger‐molecular‐mass precursors. Two subunits (apparent molecular masses of 12.5 and 14 kDa) are made with the same size as their mature forms.
Characterization of the tetraphenylboron‐induced calcium release from skeletal sarcoplasmic reticulum
Authors: SOLER Fernando , FERNANDEZ‐BELDA Francisco , GOMEZ‐FERNANDEZ Juan C.
Published: March 2005

Release of Ca2+ from skeletal sarcoplasmic reticulum vesicles was studied by the spectrophotometric stopped‐flow technique using tetraphenylboron as a releasing agent. The extent of Ca2+ release shows a sigmoidal response, with respect to the tetraphenylboron concentration, being dependent on Ca2+ preloading and Ca2+ ATPase activity, since these experiments were performed on actively loaded vesicles.The release process has a rapid component with an apparent rate constant of 6–8s−1, showing a linear relationship between the rapid rate of Ca2+ release and the Ca2+ content of the vesicles. The release is not mediated by the reversal of the Ca2+ pump. Since the amphipathic anion tetraphenylboron was unable to elicit a Ca2+‐release response when added to a preparation of sarcoplasmic reticulum phospholipid vesicles, it is suggested that there may be an interaction with some membrane protein(s) at the hydrophobic/hydrophilic interface leading to the opening of some specific Ca2+‐release pathway.
Repression of cytochrome P‐450c gene expression by cotransfection with adenovirus E1a DNA
Authors: SOGAWA Kazuhiro , HANDA Hiroshi , FUJISAWA‐SEHARA Atsuko , HIROMASA Takako , YAMANE Miyuki , FUJII‐KURIYAMA Yoshiaki
Published: March 2005

Gene expression of rat cytochrome P‐450c (P‐450c) depends upon inducible enhancers scattered in the 5′‐upstream region of the gene. We show that expression of the P‐450c gene is repressed by contransfection with adenovirus E1a DNA, regardless of the presence or absence of inducers, in a transient expression system of HeLa cells. Since cotransfection of either 13S or 12S E1a cDNA was effective in the repression, the region necessary for repression could be separated from that of transactivation of other adenovirus early genes. Moreover, we investigated the regions responsible for the inhibitory activity using in‐frame deletion mutants lacking internal or external portions of the E1a proteins. The sequence responsible for the repression was located in the aminoterminal half of the E1a proteins. The inducible expression of the chimeric plasmid containing a 24‐base‐pair enhancer sequence of the P‐450c gene placed in a heterologous promoter of SV40 was repressed by contransfection with E1a DNA, suggesting strongly that the inhibitory effect of the E1a proteins upon P‐450c gene expression was caused by blocking the enhancer activity.
Two out of the three kinds of subunits of inter‐α‐trypsin inhibitor are structurally related
Authors: GEBHARD Wolfgang , SCHREITMÜLLER Thomas , HOCHSTRASSER Karl , WACHTER Elmar
Published: March 2005

Inter‐α‐trypsin inhibitor is a 240‐kDa plasma‐protein complex of three different types of glycoproteins. Their stoichiometric relation in the complex is not yet known. One subunit results from proteolytic processing of a precursor protein composed of α1‐microglobulin and a double‐headed Kunitz‐type proteinase inhibitor protein. From this, only the inhibitor protein becomes part of the inter‐α‐trypsin inhibitor complex. Another subunit whose function is not yet understood is structurally unrelated to the first one as well as to other proteins of various data collections. Now we have obtained a cDNA clone coding for 837 amino acid residues of a precursor protein of the third subunit. Its primary structure is 40% identical to that of the completely known second‐subunit precursor. Peptide sequences obtained from isolated inter‐α‐trypsin inhibitor represent a distinct part of only about two thirds of the predicted polypeptide precursor, suggesting that its maturation is very similar to that of the second subunit. Therefore, we conclude that the deduced primary structure covers about 98% of the mature third subunit.
Complete structure of the gene for phosphoenolpyruvate carboxylase from maize
Authors: MATSUOKA Makoto , MINAMI Ei‐ichi
Published: March 2005

Phosphoenolpyruvate carboxylase is a key enzyme in photosynthesis in some plants that exploit the C4 photosynthetic pathway for the fixation of CO2. We cloned the gene for this enzyme from maize genomic libraries and analyzed its complete primary structure. The sequence of the cloned gene spans 6781 bp and consists of 10 exons and 9 introns. The site of initiation of transcription is located 84 nucleotides upstream from the first nucleotide of the initiation codon (position –84), as determined by the method of primer‐extension analysis. The analysis suggests that there is another initiation site located at position –81. The 5′‐flanking region of the gene lacks typical TATA and CCAAT elements in the anticipated regions, but there is a TATA‐similar sequence (TATTT) around the –30 regions as well as sequence homologous to the Sp‐1 protein‐binding site (CCGCCC). Six long, direct repeated sequences and a light‐responsive element are also present in the 5′‐flanking region. The results of Southern blot analysis indicated that the phosphoenolpyruvate carboxylase gene exists as a small multi‐gene family, but the enzyme that is expressed at high levels in green leaves and is involved in C4 photosynthesis is encoded by a single‐copy gene in the maize genome.
Gangliosides in normal human serum
Authors: SENN Hans‐Jürgen , ORTH Matthias , FITZKE Edith , WIELAND Heinrich , GEROK Wolfgang
Published: March 2005

The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid‐bound sialic acid was 10.5 ± 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1a, GM2, GT1b, MG‐3 (sialosyllactoneotetraosylceramide), GD1b and GQ1b. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.
Expression of yeast invertase in oocytes from Xenopus laevis
Authors: ROITSCH Thomas , LEHLE Ludwig
Published: March 2005

In an effort to understand factors that control glycosylation of proteins and processing of carbohydrate chains, invertase from Saccharomyces cerevisiae was expressed in a heterologous system. Microinjection of invertasespecific in vitro transcripts into oocytes from Xenopus laevis resulted in synthesis, glycosylation and secretion of enzymatically active invertase. It was found that although the number of carbohydrate chains acquired is the same as in yeast, the carbohydrate processing is different. This is consistent with the notion that the usage of a glycosylation site is determined by the protein part, whereas subsequent processing occurs in a host‐dependent manner. Both, high‐mannose and complex type glycans, most likely tri‐ and tetra‐antennary structures, were synthesized in oocytes. The data obtained suggests that in this system the core chains of yeast invertase remain high‐mannose type, whereas the more extensively processed polymannose chains are modified to complex oligosaccharides. In the presence of the glycosylation inhibitor, tunicamycin, and the glucosidase processing inhibitor, methyldeoxynojirimycin, secretion of invertase is significantly decreased whereas in the presence of the mannosidase inhibitor, deoxymannojirimycin, no influence of secretion is seen. This may suggest that glycosylation of invertase is important for early secretion events. Expression of invertase lacking the leader sequence results in loss of glycosylation and secretion in oocytes. This indicates that yeast signals for secretion are functional in this higher eukaryote.
Functional characterization of skeletal F‐actin labeled on the NH2‐terminal segment of residues 1–28
Authors: BERTRAND Raoul , CHAUSSEPIED Patrick , AUDEMARD Etienne , KASSAB Ridha
Published: March 2005

Rabbit skeletal α‐actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N‐(5‐sulfo‐1‐naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS‐peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1–28, most probably within the N‐terminal acidic region of residues 1–7. A minor labeling site was located on the segment that consists of residues 40–113. No label was incorporated into the COOH‐terminal moiety consisting of residues 113–375. The isolated EDANS—G‐actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS—F‐actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S‐1) and to tropomyosin. The complex formed between EDANS—F‐actin and S‐1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin—S‐1, suggesting that the EDANS‐substituted carboxyl site is also involved in the EDC crosslinking of native actin to S‐1. Moreover, the EDANS labeling of F‐actin resulted in a 20‐fold increase in the Km of the actin‐activated Mg2+. ATPase of S‐1. Thus, this labeling, while it did not much affect the rigor actin—S‐1 interaction, changes the actin binding to the S‐1—nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N‐terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N‐terminus of actin during its interaction with myosin and other actin‐binding proteins.
A novel role for aminoacyl‐tRNA synthetases in the regulation of polypeptide chain initiation
Authors: POLLARD Jeffrey W. , GALPINE Angela R. , CLEMENS Michael J.
Published: March 2005

Exposure of the temperature‐sensitive leucyl‐tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non‐permissive temperature of 39.5°C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF‐2 to participate in the formation of eIF‐2 · GTP · Met‐tRNAf ternary complexes and thus in the formation of 43S ribosomal pre‐initiation complexes. Associated with this decreased eIF‐2 activity is an increased phosphorylation of the eIF‐2α subunit. It has previously been shown in other systems that phosphorylation of eIF‐2α slows the rate of recycling of eIF‐2 · GDP to eIF‐2 · GTP catalysed by the guanine nucleotide exchange factor eIF‐2B. We show here that phosphorylation of eIF‐2α by the reticulocyte haem‐controlled repressor also inhibits eIF‐2B activity in cell‐free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF‐2α at the non‐permissive temperature in this system is consistent with impaired recycling of eIF‐2 in vivo.

Using a single‐step temperature revertant of tsH1 cells, TR‐3 (which has normal leucyl‐tRNA synthetase activity at 39.5°C), we demonstrate here that all inhibition of eIF‐2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF‐2 activity. In contrast, recharging tRNALeuin vivo in tsH1 cells at 39.5°C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF‐2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism.

Our data indicate a novel role for aminoacyl‐tRNA synthetases in the regulation of eIF‐2 function mediated through phosphorylation of the α subunit of this factor. However, in spite of the fact that cell‐free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF‐2α, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5°C.
The sequence‐specific assignment of the 1H‐NMR spectrum of an enzyme, horse‐muscle acylphosphatase
Authors: SAUDEK Vladimir , BOYD Jonathan , WILLIAMS Robert J. P. , STEFANI Massimo , RAMPONI Giampietro
Published: March 2005

A complete range of two‐dimensional NMR experiments was used for the assignment of the 1H‐NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long‐range patterns characteristics for secondary structure elements. This result had also provided the identification of the main‐chain α and amide proton resonances. Several of the completely assigned spin systems were then identified as being part of the secondary structure units which led, after analysis of the primary amino acid sequence, to unambiguous sequence‐specific assignments. The identification and assignment of the remaining side‐chain resonances was then completed and are reported here. These results provide a complete data base for the three‐dimensional structure determination of this enzyme in solution.
The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes
Authors: WAGNER Richard , APLEY Enno C. , GROSS Armin , FLÜGGE U. Ingo
Published: March 2005

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin‐5‐isothiocyanate(EITC)‐labeled l‐α‐dipalmitoylglycerophosphoethanolamine (Pam2GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator.

The temperature dependence of the anisotropy decay showed that above 25°C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (η1). Extrapolation to η1= 0 Pa · s yielded a correlation time of φ= 20 ± 5 ns, t= 30°C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr= 2.0 × 109 rad2· s−1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was η2∼ 12 mPa · s.

The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of η1= 1 mPa · s yielded a value of φr∼ 450 ns corresponding to Dr= 8.8 × 107 rad2· s−1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross‐sectional area of the protein part embedded in the membrane was calculated to be ∼ 9 nm2. This cross‐sectional area is large enough to include at most 14 membrane‐spanning helices.

Our result also indicated that at lipid/protein molar ratios > 1.5 × 104:1 aggregation occurred in the model membranes below 30°C. However, above 30°C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.
Functional analysis of the signal‐sequence processing site of yeast acid phosphatase
Authors: MONOD Michel , HAGUENAUER‐TSAPIS Rosine , RAUSEO‐KOENIG Isabelle , HINNEN Albert
Published: March 2005

A systematic study of the signal peptidase cleavage site of the main cell‐wall‐repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild‐type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core‐glycosylated protein accumulates in the cell.

Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (−7 to −4 relative to the processing site) thus preserving the important −3 to −1 region.