bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of N‐glycolylneuraminic‐acid‐specific lectin from Scylla serrata
Authors: MERCY P. D. , RAVINDRANATH Mepur H.
Published: March 2005

A sialic‐acid‐binding lectin with specificity for N‐glycolylneuraminic acid (NeuGc) was purified from the hemolymph of the marine crab Scylla serrata by affinity chromatography using thyroglobulin‐coupled agarose. The binding specificity of Scylla lectin distinguishes it from other known sialic‐acid‐specific lectins found in Limulus polyphemus and Limax flavus, which show a broader range of specificity for sialic acids. The molecular mass of the purified lectin is about 55 kDa. Under reducing conditions (SDS/PAGE), it resolved into two subunits of 30 kDa and 25 kDa. NeuGc inhibited hemagglutination activity of the purified lectin at a concentration as low as 0.6 mM, whereas N‐acetylneuraminic acid (NeuAc) even at a concentration of 100 mM, failed to inhibit hemagglutination. This finding was supported by potent inhibition of hemagglutination by bovine and porcine thyroglobulins, which contain a NeuGcα2‐6Gal as terminal component of oligosaccharide residues. Neither glycoproteins (glycophorin NN; porcine submaxillary mucin), which contain NeuAcα2‐3Gal/GalNAc and NeuAcα2‐6GalNAc, nor human acid glycoprotein, which contains NeuAcα2‐3/α2‐6 Gal, or colominic acid, a sialopolymer with NeuAcα2‐8NeuAc, inhibited the lectin activity. The specificity of the lectin for NeuGc appears to account for the fact that it agglutinates rabbit and mice erythrocytes, but not human A, O, AB, rat or chicken erythrocytes, which contain NeuAc. The inability of the lectin to agglutinate erythrocytes (horse) that prominently express NeuGc could be due to O‐acetylation of NeuGc. In support of this, bovine submaxillary mucin, which contains O‐acetylated NeuGc inhibited the hemagglutination of the lectin better after removal of O‐acetyl groups by base treatment. The unique specificity of Scylla lectin is of diagnostic potential for human cancer tissues expressing NeuGc, since NeuGc is not found in normal human tissues.
Cholesterol 7α‐hydroxylase is up‐regulated by the competitive inhibitor 7‐oxocholesterol in rat liver
Authors: BREUER Olof , SUDJANA‐SUGIAMAN Elly , EGGERTSEN Gösta , CHIANG John Y. L. , BJÖRKHEM Ingemar
Published: March 2005

Rats of the Sprague‐Dawley strain were infused intravenously with a fat emulsion (Intralipid, trademark of Kabi Pharmacia, Uppsala, Sweden) containing 7‐oxocholesterol. This resulted in an increased cholesterol 7α‐hydroxylase activity in liver microsomes as compared to controls and was accompanied by increased levels of cholesterol 7α‐hydroxylase mRNA and microsomal cholesterol 7α‐hydroxylase protein. Rats were also fed a cholestyramine‐supplemented diet and infused with 7‐oxocholesterol. These animals excreted about half as much bile acids in faeces as cholestyraminefed controls.

Addition of 7‐oxocholesterol to liver microsomes from normal rats in amounts corresponding to those present in microsomes from 7‐oxocholesterol‐treated rats inhibited the cholesterol 7α‐hydroxylase activity by about 75%.

Cholesterol induced a type‐I binding spectrum when added to a purified bacterial‐expressed cholesterol 7α‐hydroxylase (P‐450c72–24). 7‐Oxocholesterol competitively inhibited the cholesterol binding spectrum, while 7β‐hydroxycholesterol did not interfere with binding of cholesterol to the enzyme.

It is concluded that treatment with the competitive inhibitor 7‐oxocholesterol leads to a reduced bile acid biosynthesis and, as a consequence of reduced bile acid inhibition, a compensatory increase in cholesterol 7α‐hydroxylase synthesis. The high enzyme activity measured in microsomal preparations from 7‐oxocholesterol‐treated rats may be due to a continuous conversion of 7‐oxocholesterol into less inhibitory metabolites, e.g. 7β‐hydroxycholesterol. The latter compound was found in high concentrations in liver microsomes from rats treated with 7‐oxocholesterol.

The physiological importance of these results is discussed in relation to the previous findings that 7‐oxocholesterol is accumulated in liver after cholesterol feeding and that 7‐oxocholesterol is formed from cholesterol during lipid peroxidation.
Human α and β parvalbumins
Authors: FÖHR Ursula G. , WEBER Barbara R. , MÜNTENER Markus , STAUDENMANN Werner , HUGHES Graham J. , FRUTIGER Séverine , BANVILLE Denis , SCHÄFER Beat W. , HEIZMANN Claus W.
Published: March 2005

α and β parvalbumins are Ca2+‐binding proteins of the EF‐hand type. We determined the protein sequence of human brain α parvalbumin by mass spectrometry and cloned human β parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. β parvalbumin differs in 54 positions from α parvalbumin and lacks the C‐terminal amino acid 109. From MS analyses of α and β parvalbumins we conclude that parvalbumins generally lack posttranslational modifications.

α and β parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase‐chain‐reaction techniques. Whereas α parvalbumin was found in a number of adult human tissues, β parvalbumin was restricted to preterm placenta. The pattern of α parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, α parvalbumin was found in extrafusal and intrafusal skeletal‐muscle fibres whereas, in man, α parvalbumin was restricted to the muscle spindles. Different functions for α and β parvalbumins are discussed.
Interaction of myeloperoxidase with peroxynitrite
Authors: FLORIS René , PIERSMA Sander R. , YANG Guang , JONES Peter , WEVER Ron
Published: March 2005

Polymorphonuclear neutrophils generate both nitric oxide and superoxide and these molecules can combine to form peroxynitrite. Neutrophils also contain myeloperoxidase which reacts with peroxynitrous acid (HOONO). On mixing myeloperoxidase with HOONO compound II was formed. Compound I could not be detected as an intermediate. The apparent second‐order rate constant of formation of compound II was strongly pH‐dependent (2.5×105 M−1· s−1 at pH 8.9 and 6.2×106 M−1· s−1 at pH 7.2). The pKa of this effect is 6.9 and it was concluded that the enzyme reacts with the protonated form of the peroxide, that is peroxynitrous acid, with a pH‐independent second‐order rate constant of 2.0×107 M−1· s−1 at 12°C. The interaction of HOONO with lactoperoxidase was studied for comparison. As was observed for myeloperoxidase, compound I could not be detected as an intermediate. The apparent second‐order rate constant of compound II formation is pH‐dependent and is 3.3×105 M−1· s−1 at pH 7.4 and 8.4×104 M−1· s−1 at pH 9.0. In contrast, horseradish peroxidase reacts with HOONO to form compound I, which is subsequently followed by the formation of compound II. The second‐order rate constant for the formation of compound I is 3.2×106 M−1· s−1 and is pH‐dependent, the pKa for this effect is 6.8. Catalase (up to 3 μM) does not affect the rate of decomposition of peroxynitrite and no compound I formation is observed. Since nitrite may be present in the peroxynitrite preparation and to discriminate between the reaction of the enzyme with nitrite or peroxynitrite, the effect of nitrite on myeloperoxidase was studied. The dissociation constant for the myeloperoxidase‐nitrite complex is pH‐dependent and has values of 580 μM at pH 6.0 and 55 mM at pH 8.5.
Minimal 16S rRNA binding site and role of conserved nucleotides in Escherichia coli ribosomal protein S8 recognition
Authors: MOUGEL Marylène , ALLMANG Christine , EYERMANN Flore , CACHIA Claire , EHRESMANN Bernard , EHRESMANN Chantal
Published: March 2005

Escherichia coli ribosomal protein S8 was previously shown to bind a 16S rRNA fragment (nucleotides 584–756) with the same affinity as the complete 16S rRNA, and to shield an irregular helical region (region C) [Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert‐Bezançon, Ebel, J. P., Ehresmann, B. & Ehresmann, C. (1987). J. Mol. Biol. 198, 91–107]. Region C was postulated to display characteristic features: three bulged adenines (A595, A640 and A642), a non‐canonical U598–U641 pair surrounded by two G · C pairs. In order to delineate the minimal RNA binding site, deletions were introduced by site‐directed mutagenesis and short RNA fragments were synthesized. Their ability to bind S8 was assayed by filter binding. Our results show that the RNA binding site can be restricted to a short helical stem (588–605/633–651) containing region C. The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition. Single and double mutations were introduced by site‐directed mutagenesis in fragment 584–756, and their effect on S8 binding was measured. It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642. U598 is also crucial and the highly conserved G597 · C643 pair cannot be inverted. These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation. The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.
Protein O‐mannosylation in Candida albicans
Authors: WESTON Anthony , NASSAU Pamela M. , HENLY Charlotte , MARRIOTT Michael S.
Published: March 2005

A protein‐O‐d‐mannosyltransferase (PMT) assay was optimised using a microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV. [14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac‐YNPTSV‐NH2 was resistent to proteolysis and was apparently a better acceptor for O‐mannosylation. This peptide had a Km value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (Km 2.2 mM) which was further improved by blocking both ends: Ac‐YATAV‐NH2 (Km 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin‐YATAV‐NH2 had a Km of 0.075 mM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac‐ATA‐NH2, whilst no incorporation was observed with Ac‐GTG‐NH2.
Structures of the glycopeptidolipid antigens of two animal pathogens: Mycobacterium senegalense and Mycobacterium porcinum
Authors: LÓPEZ MARÍN Luz María , LANÉELLE Marie‐Antoinette , PROMÉ Danielle , DAFFÉ Mamadou
Published: March 2005

The structures of the major glycolipid antigens of two animal pathogens Mycobacterium senegalense and Mycobacterium porcinum were elucidated by a combination of fast‐atom bombardment mass spectrometry, nuclear magnetic resonance spectroscopy, chemical analyses and radiolabeling experiments. Five glycoconjugates belonging to the class of C‐mycoside glycopeptidolipids were characterized in each species. They shared with those recently described in M. peregrinum the same unusual distribution of the disaccharides on the alaninol end of the molecules. Both species showed the presence of the novel sulfated glycopeptidolipid. In addition, some acetylated forms of the glycolipids were also present in the species examined. Identical seroreactivities were observed between the glycolipid antigens extracted from M. senegalense, M. porcinum and M. peregrinum and an antiserum raised against the whole lipid antigens of M. peregrinum. These data reinforce the close taxonomic relationships between the three mycobacterial species and demonstrate the antigenicity of the new variants of mycobacterial glycopeptidolipids.
The anatomy of a kinase and the control of phosphate transfer
Authors: JOÃO Heidi C. , WILLIAMS Robert J. P.
Published: March 2005

Table of contents

Basic features of phosphoglycerate kinase (PGK), The sequences and crystal structures of PGK NMR studies of the structure of the protein PGK, Thermal analysis of yeast PGK, Additional data from NMR studies

Substrate binding, Nucleotide binding site: MgATP and MgADP, The function of magnesium, The triose substrate binding site, NMR analysis of substrate binding: summary, General anion‐binding site

The conformation change, Kinetic studies of the reaction, Site‐specific mutagenesis studies

Effects of mutations on protein structure in the absence of substrates, Mutations in the interdo‐main region, Mutations in the basic patch

Effect of Gri3P binding on mutant proteins, Mutations in the interdomain region, Mutations in the basic patch

Effects of mutations on kinetics of PGK, Mutations in the interdomain region, Mutations in the basic patch

Conclusions from experiments with mutants

Kinetic effects of low sulphate concentration, Interdomain region, Basic patch region

The so‐called hinge of proteins, Features of PGK

Common features with other NTP‐utilising systems, The nucleotide‐binding site, The catalytic loop, Domain closure

Comments on mechanical devices in multi‐protein enzymes
Sweet potato β‐amylase
Authors: TODA Hiroko , NITTA Yasunori , ASANAMI Shogo , KIM Jun Pyong , SAKIYAMA Fumio
Published: March 2005

The complete amino acid sequence of a subunit of sweet potato β‐amylase, a homotetramer, was established by sequence analysis of peptides obtained by digestions with Achromobacter protease I and Staphylococcus aureus V8 protease and by cyanogen bromide cleavage of the S‐carboxyme‐thylated subunit. The subunit of the enzyme is a single polypeptide consisting of 498 amino acid residues. It showed 50–60% identity in the amino acid sequence with those of β‐amylases from soybean and barley, while it about 25% with those of three bacterial β‐amylases deduced from the cDNA sequences.

Sweet potato β‐amylase was completely inactivated with 2,3‐epoxypropyl α‐d‐[U‐14C]glucopyranoside. Sequence analysis of the inactivated enzyme revealed that Glu187 was specifically esterified by the affinity labeling with the above reagent, proposing that Glu187 is a potent candidate involved directly in the catalysis with this plant β‐amylase.
Prosomatostatin processing in Neuro2A cells
Authors: BRAKCH Noureddine , BOILEAU Guy , SIMONETTI Mario , NAULT Christiane , JOSEPH‐BRAVO Patricia , RHOLAM Mohamed , COHEN Paul
Published: March 2005

Proline residues located near the processing sites of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin 28 and somatostatin 14 [Gomez, S., Boileau, G., Zollinger, L., Nault, C., Rholam, M. & Cohen, P. (1989) EMBO J. 8, 2911–2916]. In this study, site‐directed and regional mutagenesis of the human prosomatostatin cDNA coupled with analysis by circular‐dichroism and Fourier‐transform‐infrared spectroscopies of the native and mutated peptide sequences were used to elucidate the role of proline in proteolytic processing. Glycine was substituted for proline a position –5 and the β‐turn‐promoting sequence Pro‐Arg‐Glu‐Arg, located near the somatostatin‐14 cleavage site and predicted to form a β‐turn structure, was replaced by Ser‐Ser‐Asn‐Arg or Tyr‐Lys‐Gly‐Arg, which have been shown by X‐ray diffraction to form β turns in other proteins. Analysis of the prosomatostatin‐derived peptides produced by expression of the mutated cDNA species in Neuro2A cells indicated that while Pro‐5 → Ala abolished cleavage at the dibasic site, the formation of mutants [Gly−5] prosomatostatin, [Ser−5, Ser−4, Arg−3] prosomatostatin and [Tyr−5, Lys−4, Gly−3] prosomatostatin did not affect cleavage at the dibasic site but produced modifications in both the relative proportions of the generated hormones and in precursor processing efficiency. Moreover, spectroscopical analysis showed that whereas these substitutions did not modify the presence of a β turn structure in the corresponding peptide sequences, replacement of Pro‐5 Ala resulted in a dramatic increase in α‐helix accompanied by the significant decrease of other structures including β turn.

The data support the hypothesis that the proline residue near the processing site for somatostatin‐14 production is an important structural feature for conferring on the cleavage domain the adequate conformation for accessibility to processing enzymes and permitting production of equivalent amounts of both hormones.
Basic features of class‐I alcohol dehydrogenase: variable and constant segments coordinated by inter‐class and intra‐class variability
Authors: PERSSON Bengt , BERGMAN Tomas , KEUNG Wing Ming , WALDENSTRÖM Ulrika , HOLMQUIST Barton , VALLEE Bert L. , JÖRNVALL Hans
Published: March 2005

The enzymatic and structural properties of alligator liver alcohol dehydrogenase have been determined. Aliphatic and alicyclic alcohols serve as substrates for this first reptilian form of the enzyme characterized, with Km values decreasing rapidly from methanol to hexanol, as for the human class I enzymes, and a Km of 1.2 mM for ethanol at pH 9.9. The N‐terminus of the 374‐residue protein chain is acetyl‐blocked.

The enzyme is related in descending order to class I > III > V > II of the structurally characterized mammalian alcohol dehydrogenases. This observation is compatible with the presence of a I/III ancestral line. Differences of the enzyme classes exceed those of the species, suggesting an early origin of the classes. Within its enzyme class, the reptilian protein is most closely related to the avian from (82% residue identities), and is closer to the human than to the amphibian form (76%, versus 69%, respectively). This establishes class I alcohol dehydrogenase as an enzyme having fairly constant rate of change during much of vertebrate evolution, ∼10% residue differences/100 million years of separation between pairs compared.

Residues interacting with the substrate and coenzyme are largely conserved. In the alligator enzyme, there are only four replacements in the substrate pocket compared with the human class I γ subunit, and those are not known to have functional roles. These properties account for the kinetic parameters, and suggest distinct metabolic functions for the class I enzyme in vertebrates.

Comparisons of the enzymes of the different vertebrate lines reveal that segment patterns are characteristic features of the class I enzymes. Three segments are ‘variable’, while two are ‘constant’, and both these types of segment are identical with those of the classes. There is extensive variability in close proximity to the active site of the enzyme and this appears to constitute a fundamental property of class I liver alcohol dehydrogenases in general.
The proteolytic susceptibility of specific sites in myosin light chains is modulated by the filament conformation
Authors: ROULET Agnès , BURGAT Jean‐Marc , CARDINAUD Robert
Published: March 2005

The proteolytic susceptibilities of specific sites in the LC1 and LC2 N‐termini were modulated by ionic strength in myosin (a species able to form filaments) but not in S1. (a) In the presence of Ca2+ or Mg2+, the proteolytic susceptibility (apparent initial reaction rate) showed a sharp discontinuity at a critical ionic concentration similar for LC1′, LC2′ and LC2” cleavages. (b) The susceptibility of LC1′ and LC2” was higher at low ionic concentration in the more compact structure of the filament than in the dissociated form at high ionic concentration. (c) The ionic concentration effect was no longer observed with species unable to form filaments. (d) This effect occurred at a critical ionic concentration markedly different from the critical concentration at which the monomer—filament equilibrium was found.

These observations lead to the following conclusions. (a) The ionic concentration effect is an attribute of the filament structure. (b) In the filament the faster cleavage at sites (LC1′ and LC2”) near the LC1 and LC2 N‐termini are due to an extended configuration of the N‐terminal segment binding to a site in the filament structure. (c) The slower rate of formation of LC2′ in the filament indicates that the N‐terminal segment of LC2 binds more tightly to the structure than that of LC1. (d) The critical ionic concentration is not that of the filament—monomer equilibrium but corresponds to the order—disorder transition of the heads in the filament. These results suggest that the N‐termini of the light chains (here in striated muscles) play a role in a secondary regulatory mechanism. The analysis of these regions may contribute to our understanding of the altered activity and regulation seen in such diseases as idiopathic dilated cardiomyopathy [Margossian, S. S., White, H. D., Caulfield, J. B., Norton, P., Taylor, S. & Slayter, H. S. (1992) Circulation 85, 1720–1733].
Intestinal arginine metabolism during development
Authors: BLACHIER François , M'RABET‐TOUIL Hamida , POSHO Leta , DARCY‐VRILLON Béatrice , DUÉE Pierre‐Henri
Published: March 2005

The capacity for l‐arginine metabolism was studied in villus enterocytes isolated from pigs at birth, after 2–8 days suckling and after weaning. Immediately after birth, enterocytes were able to convert 1 mM l‐citrulline, 2mM l‐glutamine or 1 mM l‐ornithine to l‐arginine. In 2–8‐day‐old animals, the net production of l‐arginine from l‐citrulline (2.00±0.45 nmol · 106 cells−1· 30 min−1), or from l‐ornithine (0.29±0.06 nmol · 106 cells−1· 30 min−1) was similar to the values obtained at birth. Furthermore, 40% of l‐arginine synthetized de novo from l‐citrulline were released into the incubation medium. In 2–8‐day‐old animals, the production of l‐arginine from l‐glutamine represented only 5% of the production at birth (the latter being 0.73±0.15 nmol · 106 cells−1± 30 min−1). In enterocytes isolated from post‐weaned pigs, no significant production of l‐arginine from either l‐glutamine or l‐ornithine was detected. In contrast, although the l‐arginine production from l‐citrulline was very low in post‐weaned animals, it was significantly enhanced in the presence of l‐glutamine, representing 23% of the production measured in suckling animals. The capacity of enterocytes to cleave l‐arginine to l‐ornithine and urea was very limited at birth, but was increased more than threefold in 2‐day‐old animals. This was concomitant with a marked increase in arginase activity. In post‐weaned animals, the flux through arginase in intact enterocytes, and the arginase activity were both threefold higher than in 2–8‐day‐old animals. It is concluded that enterocytes isolated from neonatal pigs exhibit the capacity for a net production of l‐arginine since the metabolism of this amino acid is oriented to anabolism rather than catabolism. The results are discussed in relation to l‐arginine metabolism in the neonatal liver.
Role of Tyr201 and Tyr385 in substrate activation by p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: ESCHRICH Klaus , van der BOLT Frank J. T. , de KOK Arie , van BERKEL Willem J. H.
Published: March 2005

The crystal structure of the enzyme‐substrate complex of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4‐hydroxybenzoate interacts with the side chain of Tyr201, which is in close contact with the side chain of Tyr385. The role of this hydrogen bonding network in substrate activation was studied by kinetic and spectral analysis of Tyr→Phe mutant enzymes.

The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr201→Phe and Tyr385→Phe) with the physiological substrate are comparable with those of the corresponding mutant proteins of p‐hydroxybenzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Ballou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341–17349]. Enzyme Tyr201→Phe has a high Km for NADPH and produces only 5% of 3,4‐dihydroxybenzoate/catalytic cycle. Uniike the wild‐type enzyme, the Tyr201→Phe mutant does not stabilize the phenolate form of 4‐hydroxybenzoate. With enzyme Tyr385→Phe, flavin reduction is rate‐limiting and the turnover rate is only 2% of wild type. Despite rather efficient hydroxylation, and deviating from the description of the corresponding P. aeruginosa enzyme, mutant Tyr385→Phe prefers the binding of the phenolic form of 4‐hydroxybenzoate.

Studies with substrate analogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4‐fluorobenzoate differentially stimulates the stabilization of the 4a‐hydroperoxyflavin intermediate. Unlike wild type, both Tyr mutants produce 3,4,5‐trihydroxybenzoate from 3,4‐dihydroxybenzoate. The affinity of enzyme Tyr201→Phe for the dianionic substrate 2,3,5,6‐tetrafluoro‐4‐hydroxybenzoate is very low, probably because of repulsion of the substrate phenolate in a more nonpolar microenvironment.

In contrast to data reported for p‐hydroxybenzoate hydroxylase from P. aeruginosa, binding of the inhibitor 4‐hydroxycinnamate to wild‐type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including secondary binding of the inhibitor. Enzyme Tyr201→Phe does not stabilize the phenolate form of the inhibitor. In enzyme Tyr385→Phe, the phenolic pKa of bound 4‐hydroxycinnamate is increased with respect to wild type. It is proposed that Tyr385→Phe is involved in substrate activation by facilitating the deprotonation of Tyr201.
Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH‐tolerant enzyme
Authors: LESUISSE Emmanuel , SCHANCK Karin , COLSON Charles
Published: March 2005

The extracellular lipase of Bacillus subtilis 168 was purified from the growth medium of an overproducing strain by ammonium sulfate precipitation followed by phenyl‐Sepharose and hy‐droxyapatite column chromatography. The purified lipase had a strong tendency to aggregate. It exhibited a molecular mass of 19000 Da by SDS/PAGE and a pI of 9.9 by chromatofocusing. The enzyme showed maximum stability at pH 12 and maximum activity at pH 10. The lipase was active toward p‐nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups. Using trioleyl glycerol as substrate, the enzyme preferantially cleaved the 1(3)‐position ester bond. No interfacial activation effect was observed with triacetyl glycerol as substrate.
Effect of ionizing radiation and topoisomerase II inhibitors on DNA synthesis in mammalian cells
Authors: LALLEV Attanas , ANACHKOVA Boyka , RUSSEV George
Published: March 2005

We have used a novel, quantitative approach to study the effect of γ‐radiation and topoisomerase‐II inhibitors on the initiation of DNA synthesis in eukaryotic cells. We found out that mild γ‐irradiation caused an almost immediate decrease in the rate of initiation of genomic DNA replication and stimulated DNA repair. This held true for two different cell lines, Ehrlich ascites tumor cells and Friend transformed erythroid cells, although the effect of γ‐radiation on Friend cells was more pronounced. At the same time, the synthesis of mitochondrial DNA was not affected by the irradiation. The effect of topoisomerase‐II inhibitors on DNA initiation closely paralleled that of γ irradiation, but did not stimulate repair. The fact that γ‐radiation and topoisomerase‐II inhibitors, two types of agents that differ so profoundly, have practically the same effect on DNA synthesis speaks strongly in favour of the idea that eykaryotic cells have a general mechanism for coping with any disturbances in DNA integrity and chromatin structure. This mechanism is probably similar to the SOS‐mechanism of prokaryotic cells and includes, as an early step, a slowdown of the initiation of replicative DNA synthesis.
VAT‐1 from Torpedo electric organ forms a high‐molecular‐mass protein complex within the synaptic vesicle membrane
Authors: LINIAL Michal
Published: March 2005

VAT‐1 is an abundant 41‐kDa protein from Torpedo cholinergic synaptic vesicles. Most of VAT‐1 immunoreactivity (70%) is localized to the synaptic vesicle membrane while the rest (30%) copurifies with larger membranous fragments. VAT‐1 forms a high‐molecular‐mass complex within the synaptic vesicle membrane. The Stokes radius of the VAT‐1 complex is 4.85 nm and the sedimentation coefficient is 8.0 × 10−13 S. Using these values, the calculated apparent mass of the VAT‐1 complex is 176 kDa and the friction coefficient is consistent with that for a globular protein. Electrophoresis of solubilized synaptic vesicle proteins following cross‐linking resulted in a 40‐kDa ladder which was detected by VAT‐1 antibodies. This is in accord with VAT‐1 protein complex being composed primarily of VAT‐1 subunits. The hydrodynamic characteristics of the VAT‐1 protein complex suggest that it is composed of three or four VAT‐1 subunits. Synaptophysin, an abundant component of Torpedo synaptic vesicle membranes, which has a similar apparent size as VAT‐1, is not part of the VAT‐1 protein complex. Interactions between the subunits within the protein complex do not depend on disulfide bonds or on lowering the ionic strength. However, partial dissociation of VAT‐1 subunits from the complex occurs by chelating calcium ions.
The role of the proton‐pumping and alternative respiratory chain NADH:ubiquinone oxidoreductases in overflow catabolism of Aspergillus niger
Authors: PRÖMPER Claudia , SCHNEIDER Regina , WEISS Hanns
Published: March 2005

Mitochondria of fungi contain two respiratory chain enzymes concerned with the oxidation of matrix NADH. These are the proton‐pumping NADH:ubiquinone oxidoreductase, also called complex I, which has a high affinity for NADH, and a non‐proton‐pumping NADH:ubiquinone oxidoreductase, called alternative NADH dehydrogenase, which has a low affinity for NADH. The role of these two enzymes in normal and overflow catabolism has been studied in Aspergillus niger. Three strains were investigated, the wild‐type 732, the mutant nuo51 that was generated from the wild‐type by disrupting the gene of the (51‐kDa) NADH‐binding subunit of complex I and the citric acid over‐producing strain B60 that looses complex I concomitantly with the onset of the over‐production. Under standard growth conditions, respiratory energy transduction in the mutant nuo51 was decreased by 40% compared to the parental wild‐type and the strain B60. Respiratory electron transfer in the mutant nuo51, however, meets standard catabolic requirements. The intracellular levels of citric acid cycle intermediates in the mutant nuo51 were the same as in the other two strains. Under growth conditions which lead to uncontrolled catabolic flux through glycolysis, a dramatic catabolic overflow occurred in the mutant nuo51. Intracellular levels of citric acid cycle intermediates increased to 20‐fold normal levels. The strain B60, likewise lacking complex I under these conditions, excretes large amounts of citrate to moderate the intracellular catabolic overflow.
ADP‐stimulated fibrinogen binding is necessary for some of the inositol phospholipid changes found in ADP‐stimulated platelets
Authors: VICKERS John D.
Published: March 2005

ADP‐stimulation of washed human platelets suspended in Tyrode/albumin solution containing Ca2+ (2mM) and fibrinogen (0.4 mg/ml) causes extensive, reversible aggregation without appreciable secretion of granule contents. Under these conditions ADP (10 μM) stimulation decreased the amounts of phosphatidylinositol 4,5‐bisphosphate (PtdInsP2) and phosphatidylinositol 4‐phosphate (PtdInsP) at 10 s. Omitting fibrinogen from the suspending medium or blocking fibrinogen binding to the platelets using Arg‐Gly‐Asp‐Ser (RGDS, 0.23 mM) inhibited these decreases in PtdInsP2 and PtdInsP. In contrast, ADP‐induced decreases in PtdInsP2 and increases in PtdInsP at 60 s compared to 10 s were not affected by RGDS or the absence of fibrinogen. In platelets prelabelled with [3H]glycerol and [32P]phosphate, changes in labelling of the inositol phospholipids paralleled the changes in amount. The ADP‐induced changes in phosphatidic acid (PtdOH) at 10 s were unaffected by RGDS; this finding supported previous reports that phospholipase C was not the cause of the early decreases in PtdInsP2 and PtdInsP. These results indicate that the early decreases in PtdInsP2 and PtdInsP at 10 s are dependent on fibrinogen binding to the platelets and occur after fibrinogen binding which is activated by ADP stimulation. It is proposed that the fibrinogen‐dependent changes in PtdInsP2 and PtdInsP may have a feedback role augmenting platelet aggregation or other responses of platelets that might occur after fibrinogen binding, possibly due to effects on actin polymerisation.
Rabbit and rat liver nuclei both contain proteins which bind to the regions controlling apolipoprotein A‐I gene expression
Authors: REISHER Samuel R. , FISHER Edward A. , FEINSTEIN Sheldon I.
Published: March 2005

We have tested the 5′ flanking region of the apolipoprotein A‐I (apo A‐I) gene, which controls its expression in hepatic cells, for the ability to bind protein factors from rat and rabbit liver nuclei. We found that nuclear extracts from each species contain proteins which bind to three sites in the region which have been shown to be important for control of apo A‐I gene transcription. These results contrast with a previous report [Dai, P. H., Lan, S. S. F., Ding, X. H. & Chao, Y. S. (1990) Eur. J. Biochem. 190, 305–310] that no rabbit liver nuclear protein could be detected which binds to the rat apo A‐I upstream region and that this lack of binding could explain the failure of the rabbit liver to express the apo A‐I gene. We have also shown that the low levels of apo A‐I mRNA in the rabbit liver correlate with decreased transcription. Our data suggest that the lack of apo A‐I gene expression in liver is a result of transcriptional control but cannot be due to simple lack of protein binding to this region of DNA.
Mechanisms of activation of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase
Authors: COLLEN Desiré , VAN HOEF Berthe , SCHLOTT Bernhard , HARTMANN Manfred , GÜHRS Karl‐Heinz , LIJNEN H. Roger
Published: March 2005

The molecular basis of the marked interspecies variability in the response of plasma fibrinolytic systems to activation by streptokinase (SK) or recombinant staphylokinase (STAR) was studied using highly purified plasminogens and α2‐antiplasmins from five representative species (man, baboon, rabbit, dog and cow). Human plasminogen reacted rapidly and stoichiometrically with both SK and STAR to yield potent plasminogen activators (catalytic efficiencies, kcatKm, of 1.0 μM−1· s−1 and 0.3 μM−1· s−1, respectively). The complex with SK was insensitive to α2‐antiplasmin, which, however, rapidly inhibited the complex with STAR (second‐order rate constant, k1,app of 8 × 106 M−1· s−1). In a system composed of a 0.06‐ml 125I‐fibrin‐labeled plasma clot submerged in 0.30 ml plasma, both SK and STAR had potent fibrinolytic properties, causing 50% clot lysis in 2 h (EC50), with 120nM and 13 nM, respectively. Clot lysis with SK was non‐fibrin specific (residual fibrinogen <10%), whereas lysis with STAR was highly fibrin specific (residual fibrinogen 76%). Canine plasminogen reacted avidly with SK, but SK was rapidly degraded; it reacted rapidly and quantitatively with STAR to form a potent plasminogen‐activating complex (kcat/Km of 0.4 μM−1 s−1) which was sensitive to neutralization by α2‐antiplasmin (k1,app of 6 × 105 M−1· s−1). In a canine plasma milieu, SK was relatively potent (EC50 200 nM) and fibrin specific, whereas STAR was very potent (EC50 1.3 nM) but poorly fibrin specific. Baboon and rabbit plasminogen did not form stable stoichiometric complexes with SK, but reacted stoichiometrically and quantitatively with STAR. The complexes with STAR, however, had low catalytic efficiencies for the activation of their autologous plasminogens (kcat/Km 0.02 μM−1· s−1) and reacted more slowly with α2‐antiplasmin (k1,app 5–10 × 105 M−1· s−1). Bovine plasminogen was virtually unreactive towards both SK and STAR as well as to their complexes with human plasminogen, as monitored by measurement of the initial activation rates. The resistance to fibrinogen degradation with STAR observed in the human system could be transferred to the canine system by reconstituting canine plasma, depleted of plasminogen and α2‐antiplasmin, with the human proteins. Conversely, the sensitivity to fibrinogen degradation of the canine system could be transferred to the human system by reconstituting depleted plasma with canine plasminogen and α2‐antiplasmin.

It is concluded that the variability in the response of mammalian plasma fibrinolytic systems to activation with SK or STAR is determined mainly by the extent of complex formation of these compounds with plasminogen, by the catalytic efficiencies of the complexes for the activation of autologous plasminogen and by the rate of inhibition of these complexes by α2‐antiplasmin.
Large‐scale preparation of fully deuterated cell components
Authors: VANATALU Kalju , PAALME Toomas , VILU Raivo , BURKHARDT Nils , JÜNEMANN Ralf , MAY Roland , RÜHL Martin , WADZACK Jörg , NIERHAUS Knud H.
Published: March 2005

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log‐phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, μ≤ 0.35 h−1, for acetate/succinate μ≤ 0.1 h−1) which avoids the accumulation of the substrate or of by‐products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source.

The cells were first grown in fed‐batch to high cell densities (above 50 g wet cells/1) under conditions of substrate limitations. A steady‐flow fermentation followed keeping the growth rate at about μ of 0.1 h−1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.
Gene regulation in rodent hepatocytes during development, differentiation and disease
Authors: XANTHOPOULOS Kleanthis G. , MIRKOVITCH Jovan
Published: March 2005

The expression of genes in the liver is mostly controlled at the transcriptional level and depends on the regulatory interactions between cis‐acting sequences and trans‐acting molecules. Proximal promoters and distant enhancers in combination with a number of hepatocyte‐enriched DNA‐binding proteins and general transcription factors interact specifically with these elements and control the expression of liver‐specific genes. Hepatocyte‐enriched regulatory proteins have been isolated from liver nuclear extracts, characterized, and their corresponding genes have been cloned. These include the hepatocyte nuclear factors 1, 3, 4 (HNF‐1, 3, 4), some members of the CAAAT/enhancer binding protein (C/EBP) family, and D site binding protein (DBP). These factors belong to larger families and are able to form heterodimers, perhaps with the exception of the HNF‐3 family, with other members of the same family. Interestingly, the majority of the genes encoding such proteins are themselves regulated at the transcriptional level, although both transcriptional and post‐transcriptional events modulate their expression during development, hepatoeyte differentiation and disease, suggesting that a transcriptional cascade may play a critical role in mammalian liver development and differentiation.
Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis
Authors: MICHALON Pascal , COUTURIER Roger , BENDER Klaus , HECKER Hermann , MARION Christian
Published: March 2005

The sensitive electric‐birefringence method was used to reveal structural differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orientation of the nucleosomal chains and the presence of extended DNA were analysed from the sign and amplitude of the steady‐state birefringence, and the conformational properties (overall dimensions and flexibility) were studied in relation to the orientational relaxation times.

In contrast to the higher eukaryotes, the birefringence of T. brucei brucei is negative and of low amplitude, corresponding to that of H1‐depleted rat liver nucleosomes. Furthermore, the relaxation times are very small, about 10 μs. If salt is added, the birefringence as well as the relaxation time decreases dramatically, indicating that condensation affects T. brucei brucei chromatin although it behaves like nucleosome filaments, with less stable DNA‐protein interaction than for the higher eukaryotes. However, this condensation does not induce the formation of regular higher‐order structure. This complies with the hypothesis that typical histone H1 is absent from T. brucei brucei chromatin and that a protein or protein domain fulfils the role of histone H1.

The accessibility and structural role of histone‐like proteins in T. brucei brucei chromatin were also investigated using limited proteolysis with enzymes covalently bound to nylon spheres. The analysis of protein products obtained after digestion with immobilized trypsin and subtilsin shows that proteins a and d, which are classified as H3 and H4 histones, respectively, are the first to be attacked.

The changes in chromatin conformation indicate that chromatin undergoes a structural transition, leading to decondensation, as indicated by incrases in negative birefringence and relaxation time, and to a change in its orientation mechanism, indicated by the appearance of a permanent moment.

This result is very interesting since, in rat liver, H4 was very resistant and was the last histone to be attacked, suggesting internal location and its inovolvement in nucleosome stabilization rather than higher‐order condensation.

Therefore, in T. brucei brucei chromatin, the charcteristic properties a and d (their composition and interaction with DNA), as well as their external location on the nucleosome surface, suggest that if these proteins play a role similar to that played by H3 and H4 in higher eukaryotes, probably through their N‐terminal regions and interaction either with DNA or protein domains, the mechanisms involved in chromatin compaction are quite different. Since there is no binding with H1, it may be hypothesized that a (non‐histone) protein, or core protein domains, may play a part in chromatin compaction and gene expression regulation comparable to that of H1 in higher eukaryotes.
Role of the B1 short arm in laminin self‐assembly
Authors: SCHITTNY Johannes Constantin , SCHITTNY Christiane Martha
Published: March 2005

Laminin self‐assembles into a basement membrane polymer through specific low‐affinity interactions. Recently, it was shown that the terminal short‐arm domain (domains VI and V) of the B1 chain (fragment E4) possesses one of the laminin self‐interaction sites [Schittny, J. C. & Yurchenco, P. D. (1990) J. Cell Biol. 110, 825–832], but that the binding partner(s) of this domain is unknown. Using affinity retardation chromatography we now investigate the domain(s) fragment E4 binds to. The elution of E4 was clearly retarded on immobilized laminin and fragment E1′ (three‐chain short‐arm complex excluding the distal part of the B1 chain), but not on immobilized E4 in calcium containing buffer and at 37°C. Under the same conditions, E1′ strongly interacts with immobilized E4. In addition, E1′ is able to non‐covalently cross‐link soluble E4 to immobilized E4. No further interaction of laminin and E4 with additinal fragments (P1′, A, B2 and B1 chain short‐arm complex without B1‐domains VI–IV and without globules; E8, distal long arm and G1–3; E3, long‐arm G subdomains 4 and 5) could be demonstrated. These data are interpreted as evidence that (a) the primary laminin‐laminin bonds are formed between the short arms of laminin, that (b) the terminal B1 short‐arm domain (E4) can interact with the short arm(s) of the A and/or B2 chain(s) (domain E1′), but does not self‐interact, and that (c) due to at least three self‐binding sites, laminin polymerization behaves co‐operatively.
C‐terminal phosphorylation of the serum‐response factor
Authors: JANKNECHT Ralf , ERNST Wolfram H. , HOUTHAEVE Tony , NORDHEIM Alfred
Published: March 2005

The serum‐response factor (SRF) is essential for the induction and repression of the protooncogene c‐fos. Phosphorylation of SRF has been implicated to be involved in these processes and five phosphorylation sites have already been mapped within the N‐terminal region. Here we show that in vivo additional phosphorylation of SRF does occur. This modification is located primarily within amino acids 206–289, which probably contain more than one phosphorylation site. Microsequencing allowed the identification of one phosphorylation site at Ser253, which is a potential target of casein kinase II. Mutational analysis revealed that, in contrast to N‐terminal phosphorylation, Ser253 phosphorylation does not affect DNA‐binding properties. In addition, phosphorylation at Ser253 does not seem to change transactivation activity of SRF but rather influences its contribution to transcriptional repression. Thus, C‐terminal phosphorylation of SRF may modulate c‐fos basal repression.
Probing the membrane topology of Candida tropicalis cytochrome P450
Authors: SANGLARD Dominique , SENGSTAG Christian , SEGHEZZI Wolfgang
Published: March 2005

The membrane topology of two alkane‐inducible cytochromes P450 from the yeast Candida tropicalis, alk1 and alk2, was tested by construction of fusion proteins with part of invertase and histidinol dehydrogenase (invHIS4C) and expression in a Saccharomyces cerevisiae his4 mutant. Depending on the localization of invHIS4C on the endoplasmic reticulum (ER) cytoplasmic or luminal side, the enzyme converts histidinol to histidine and allows the his4 yeast strain to grow on histidinol‐supplemented medium. The N‐terminal segments of alk1 and alk2 were fused to invHIS4C at three different locations that follow the first alk1 and alk2 transmembrane domains or a second putative transmembrane domain of alk1. The combination of this in vivo assay with subcellular immunoprecipitations of the expressed fusion proteins allowed us to establish that both P450s contain only one transmembrane domain with their N‐terminus located in the ER lumen. Deletions performed in these fusion proteins removing the first transmembrane domain of alk1 (TM) resulted in a less efficient targeting to the ER membrane but did not prevent their insertion in these membranes. Furthermore deletion of a negatively charged peptide preceding the first alk1 transmembrane domain (L) in an invHIS4C protein fused after this domain caused the N‐terminal to have a positive net charge and to be oriented in the cytoplasm thus translocating the remaining protein into the ER lumen. The presence of the second hydrophobic segment, however, prevented the complete translocation of this fusion protein into the ER lumen. This study describes the first assessment of P450 membrane topology using an in vivo technique.