bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of vitamin B12 in anaerobic bacteria
Authors: MUNDER Michael , VOGT Joachim R. A. , VOGLER Bernhard , RENZ Paul
Published: March 2005

Experiments on the incorporation of erythrose and formate into the 5,6‐dimethylbenzimidazole moiety of vitamin B12 are described.

In one experiment, a 1:1 mixture of D‐[1‐13C]erythrose and D‐[1‐13C]threose was added to a Eubacterium limosum fermentation. The vitamin B12 formed was methylated at N3 of its 5,6‐dimethylbenzimidazole part and degraded to 1,5,6‐trimethylbenzimidazole. The 13C‐NMR spectrum of this compound exhibited a single prominent signal at 109.5 ppm due to 13C labeling in C7.

This shows that C1 of erythrose or threose was originally incorporated exclusively into C4 of the 5,6‐dimethylbenzimidazole moiety of vitamin B12.

In another experiment, sodium [13C]formate was added to a culture of E. limosum. The vitamin B12 isolated was transformed into 1,5,6‐trimethylbenzimidazole as before. The 13C‐NMR spectrum also showed one prominent signal at 142.8 ppm, evoked by 13C at C2.

These results demonstrate that erythrose is incorporated into the base part of vitamin B12 regiospecifically and that formate is the precursor of the C2.
Identification and molecular characterization of the calmodulin‐binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae
Authors: YE R. Ruby , BRETSCHER Anthony
Published: March 2005

A method has been developed for the rapid purification of yeast calmodulin in high yield. Using a 125I‐labeled calmodulin SDS/PAGE gel overlay procedure with either yeast or bovine calmodulin, we show that the bovine and yeast proteins recognize the same proteins in total yeast extracts. However, yeast calmodulin does not bind to many of the proteins in vertebrate cells identified using bovine calmodulin. A Δgt11 yeast genomic expression library was screened with yeast or bovine brain 125I‐calmodulin to identify sequences derived from calmodulin binding proteins. Twelve clones were recovered, all containing a common DNA insert; all bound calmodulin in a Ca2+‐dependent manner. The complete coding sequence was recovered and sequenced. The predicted protein sequence show > 50% identity to the A subunit of vertebrate protein phosphatase 2B. The gene was designated CMP1 and shown to reside on chromosome IV. Disruption or over‐expression of CMP1 have no obvious phenotype; yeast appears to contain one or more CMP1‐related genes. The protein product of the CMP1 gene is elevated by α‐factor treatment, suggesting an involvement of protein phosphatase 2B in the mating response.
Identification of proteins associating with poly(A)‐binding‐protein mRNA
Authors: BERGER Lloyd C. , BAG Jnanankur , Sells Bruce H.
Published: March 2005

Synthesis of poly(A)‐binding protein is regulated at the translational level. We have investigated the binding of proteins to this mRNA on the premise that the protein(s) of the mRNP complex may be involved in regulating the expression of the mRNA. We found the first 243 nucleotides of the 5′ untranslated region to contain sequences essential for RNP formation. A large, single‐stranded bulge structure encompassing stretches rich in adenine nucleotides and a potential stem‐loop domain appear to be the primary sites for protein binding. Removal of the 243‐nucleotide segment results in a drastic reduction in protein binding and a concomitant increase in translational efficiency in vitro. We suggest that proteins binding to this region, including poly(A)‐binding protein itself, may be essential for regulating translation of this mRNA.
Molecular characterization of six intermediate proteins in the processing of mouse protamine P2 precursor
Authors: CHAUVIÈRE Muriel , MARTINAGE Arlette , DEBARLE Michel , SAUTIÈRE Pierre , CHEVAILLIER Philippe
Published: March 2005

In mouse spermatozoa, DNA is compacted by two protamines mP1 and mP2. Protamine mP2 (63 residues) is synthesized in spermatid nuclei as a precursor pmP2 (106 residues) which is subsequently processed at the end of spermiogenesis [Yelick, P. C., Balhorn, R., Johnson, P. A., Corzett, M., Mazrimas, J. A., Kleene, K. C. & Hecht, N. B. (1987) Mol. Cell. Biol. 7, 2173–2179].

Six proteins, three of which were described earlier [Chauvière, M., Martinage, A., Debarle, M., Alimi, E., Sautière, P. & Chevaillier, Ph. (1991) C. R. Acad. Sci. 313, 107–112], have molecular and electrophoretic properties similar to those of pmP2. They were isolated from purified testis nuclei and characterized by amino acid composition, N‐terminal sequence and peptide mapping. From the amino acid compositions, it appears that all six proteins are rich in arginine, cysteine and histidine and are closely related to pmP2 and mP2. The N‐terminal sequence of each protein overlaps a distinct region of the N‐terminal part of pmP2. The C‐terminal part of protamine mP2 starting at arginine 15 is common to all proteins as assessed by amino acid compositions and peptide maps.

All these structural data demonstrate that the six isolated proteins are products of pmP2 precursor processing. The six intermediate proteins pmP2/5, pmP2/11, pmP2/16, pmP2/20, pmP2/26 and pmP2/32 which contain 102, 96, 91, 87, 81 and 75 residues, respectively, are generated from the pmP2 precursor after N‐terminal excision of 4, 10, 15, 19, 25 and 31 residues, respectively. The C‐terminal sequence of protamine mP2 is strictly identical to that of its precursor; therefore, no maturation occurs in this part of the molecule. At the present time, the proteolytic pathway involved in the amino‐terminal processing leading to the mature form of the protamine mP2 (63 residues) has not been elucidated. However, the different representation of six intermediates in the testis suggests that some stages of processing are faster than others or that some cleavage sites are preferred. The proteins described in this paper could result either from stepwise excision of N‐terminal residues or from non‐sequential cleavages.
Mössbauer study of the native, reduced and substrate‐reacted Desulfovibrio gigas aldehyde oxido‐reductase
Authors: BARATA Belarmino A. S. , LIANG Jin , MOURA Isabel , LEGALL Jean , MOURA José J. G. , HANH HUYNH Boi
Published: March 2005

The Desulfovibrio gigas aldehyde oxido‐reductase contains molybdenum and iron‐sulfur clusters. Mössbauer spectroscopy was used to characterize the iron‐sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde‐reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido‐reductase are organized as [2Fe‐2S] clusters. In the oxydized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (ΔEQ= 0.62 ± 0.02 mm/s and δ= 0.27 ± 0.01 mm/s) typical for the [2Fe‐2S]2+ state. Mössbauer spectra of the reduced clusters also show the characteristics of a [2Fe‐2S]1+ cluster and can be explained by a spin‐coupling model proposed for the [2Fe‐2S] cluster where a high‐spin ferrous ion (S= 2) is antiferromagnetically coupled to a high‐spin ferric ion (S= 5/2) to form a S= 1/2 system. Two ferrous sites with different ΔEQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe‐2S] clusters in the D. gigas enzyme. Taking this observation together with the re‐evaluated value of iron content (3.5 ± 0.1 Fe/molecule), it is concluded that, similar to other Mo‐hydroxylases, the D. gigas aldehyde oxido‐reductase also contains two spectroscopically distinguishable [2Fe‐2S] clusters.
Rat platelet phospholipase A2
Authors: RANSAC Stéphane , AARSMAN Anton J. , van den BOSCH Henk , GANCET Christian , de HAAS Gerard H. , VERGER Robert
Published: March 2005

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2‐dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2‐acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.
Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis
Authors: LAAN Harry , KOK Jan , HAANDRIKMAN Alfred J. , VENEMA Gerard , KONINGS Wil N.
Published: March 2005

Lactococcus lactis strains produce an extracellular subtilisin‐related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western‐blot analysis it was found that all monoclonal antibodies recognize all denatured proteinase components. The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule. In a L. lactis proteinase which was inactivated by the substitution Asp30 → Asn under native conditions, only one epitope could be detected. This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies.
Bilayer‐penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria
Authors: JORDI Wilco , HERGERSBERG Christoph , de KRUIJFF Ben
Published: March 2005

In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV‐DHFR, which exhibit mitochondrial import pathways with different characteristics. In‐vitro‐synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein [Rietveld, A. & de Kruijff, B. (1984) J. Biol. Chem. 259, 6704–6707; Dumont, M. E. & Richards, F. M. (1984) J. Biol. Chem. 259, 4147–4156]. Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in‐vitro‐synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria. In contrast, the purified and in‐vitro‐synthesized precursor fusion protein pCOX IV‐DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer. The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins. The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.
Multinuclear NMR spectroscopy of the cellular slime mold Polysphondylium pallidum
Authors: KLEIN Gérard , MARTIN Jean‐Baptiste , COTTER David A. , SATRE Michel
Published: March 2005

Polysphondylium pallidum microcysts and amoebae have been investigated by 31P‐ and natural‐abundance proton‐decoupled 13C‐NMR spectroscopy. Microcysts have been found to contain as prominent metabolites a phosphomonoester, inositol hexakisphosphate (1.4 mM), two phosphodiesters [glycerophosphocholine (5.5 mM) and glycerophosphoethanolamine (2.6 mM)], as well as nucleoside triphosphates (3 mM) and polyphosphates (>10 mM), the polyamines 1,3‐diaminopropane (3.5 mM), putrescine (16 mM) and spermidine (3 mM) and the sugar trehalose (31 mM). In vivo31P‐NMR has shown that the level of nucleoside triphosphates in microcysts was maintained metabolically and that the pH of their cytosol, deduced from the chemical shift of cytosolic Pi was 7.2. The absence of trehalose, glycerophosphocholine and glycerophosphoethanolamine in P. pallidum amoebae was the most remarkable difference from microcysts. Microcyst germination (excystment), induced by reduction of the ionic strength of the microcyst bathing medium, was monitored non‐invasively by 31P‐ and 13C‐NMR spectroscopy. The major modifications observed during excystment were the progressive disappearance of trehalose used as energy source, of glycerophosphocholine and glycerophosphoethanolamine used as membrane phospholipid precursors, and, finally, the appearance of NMR‐visible polyphosphates and of cellobiose. As a mirror situation, P. pallidum amoebae responded to a high‐ionic‐strength stress by production of trehalose, glycerophosphocholine, and glycerophosphoethanolamine, and induction of an encystment process.
Cecropia immunoresponsive factor, an insect immunoresponsive factor with DNA‐binding properties similar to nuclear‐factor KB
Authors: SUN Shao‐Cong , FAYE Ingrid
Published: March 2005

The immune genes in Hyalophora cecropia contain an upstream sequence that is homologous to the binding site of the mammalian nuclear‐factor KB (NF‐KB). These genes are strongly induced by bacteria, lipopolysaccharides and 4β‐phorbol 12‐myristate 13‐acetate. Induction of the immune genes involves the activation of a DNA‐binding protein complex that we have named Cecropia immunoresponsive factor (CIF). CIF specifically recognizes the KB‐like DNA sequences in the promoter regions of the Cecropia immune genes. The DNA binding activity of CIF correlates well with the transcriptional induction of the immune genes. Competition assays show that CIF has a DNA binding specificity similar to mammalian NF‐KB. The two factors also share other characteristics, including the pattern of induction and the migration on the native gel.
Characterization of ribulose‐1,5‐bisphosphate carboxylase/oxygenase carrying ribulose 1,5‐bisphosphate at its regulatory sites and the mechanism of interaction of this form of the enzyme with ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase activase
Authors: YOKOTA Akiho , TSUJIMOTO Noritaka
Published: March 2005

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5‐bisphosphate [Rbu(1,5)P2]. In the burst, Rbu(1,5)P2CO has its substrate‐binding sites occupied with Rbu(1,5)P2 for the initial few minutes, then both substrate‐binding and regulatory sites are occupied by Rbu(1,5)P2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5)P2 [A. Yokota (1991) J. Biochem. (Tokyo) 110, 246–252]. This study attempts the characterization of spinach Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the regulatory sites and the interaction of Rbu(1,5)P2CO activase with Rbu(1,5)P2CO purified with poly(ethylene glycol) 4000 without denaturation.

Binding of Rbu(1,5)P2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5)P2CO. The activation energy of Rbu(1,5)P2CO with Rbu(1,5)P2 at the regulatory sites was 40% larger than that without Rbu(1,5)P2 over 30°C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50°C. The optimum pH for the activity of Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the sites was 8.0–8.2, and increased by about pH 0.2 from that of Rbu(1,5)P2‐CO without Rbu(1,5)P2. The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5)P2CO.

The ATP‐hydrolyzing activity inherent to Rbu(1,5)P2CO activase was stimulated about twofold by 3–5 mM Rbu(1,5)P2. Rbu(1,5)P2CO in the inactive complex with Rbu(1,5)P2 experienced hysteresis and bound Rbu(1,5)P2 at the regulatory sites during activation in the presence of Rbu(1,5)P2CO activase. Evidence was obtained that Rbu(1,5)P2CO activase promoted the activation of Rbu(1,5)P2CO through binding to the large subunits of Rbu(1,5)P2CO.
Purification of a 29‐kDa hemocyte proteinase of Sarcophaga peregrina
Authors: KURATA Shoichiro , SAITO Hiromi , NATORI Shunji
Published: March 2005

Previously, we suggested the participation of a hemocyte proteinase in the dissociation of fat body of Sarcophaga peregrina (flesh fly) at metamorphosis. We have now purified this proteinase to near homogeneity from pupal hemocytes. It is a cysteine proteinase with a molecular mass of 29 kDa and has a unique substrate specificity hydrolyzing both Suc‐Leu‐Leu‐Val‐Tyr‐MCA and Z‐Phe‐Arg‐MCA (Suc, succinyl; MCA, methylcoumaryl‐7‐amide; Z, carbobenzoxy), which are substrates for chymotrypsin and cathepsin B, respectively. Partial similarity was found between the amino‐terminal sequence of this proteinase and that of catepsin B, including Pro, Glu and Arg residues conserved in the papain superfamily of enzymes.
On the interpretation of biochemical data by molecular dynamics computer simulation
Authors: van GUNSTEREN Wilfred F. , MARK Alan E.
Published: March 2005

The application of computer simulation to molecular systems of biochemical interest is reviewed. It is shown that computer simulation is a tool complementary to experimental methods, which can be used to access atomic details inaccessible to experimental probes. Examples are given in which computer simulation augments the experimental information by providing an atomic picture of high resolution with respect to space, energy or time.

The usefulness of a computer simulation largely depends on its quality. The most important factors that limit the accuracy of simulated results are discussed. The accuracy of different simulation studies can differ by orders of magnitude. The accuracy will depend on the type of biomolecular system and process studied. It will also depend on the choice of force field, the simulation set‐up and the protocol that is used. A list of quality‐determining factors is given, which may be useful when interpreting simulation studies appearing in the literature.
Mannitol‐specific enzyme II of the phosphoenolpyruvate‐dependent phosphotransferase system of Staphylococcus carnosus
Authors: FISCHER Roland , HENGSTENBERG Wolfgang
Published: March 2005

The enzyme IImannitol (EIImtl) of the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) catalyses the uptake and concomitant phosphorylation of mannitol by bacteria; it is specified by the gene mtlA. MtlA is located near the genes mtlF and mtlD in the staphylococcal genome, encoding the enzyme IIImtl and the mannitol‐1‐phosphate dehydrogenase, respectively.

We present the cloning of the whole operon by a novel complementation system which is generally suitable for cloning Gram‐positive PTS genes. The nucleotide sequence of a 2.5‐kbp subclone spanning mtlA has been determined. From the deduced amino acid sequence, it is predicted that the membrane‐protein EIImtl consists of 505 amino acid residues (54112Da). The protein has the expected hydropathy profile of an integral‐membrane protein. The NH2‐terminal part of the enzyme resides within the membrane, whereas the COOH‐terminus of the enzyme has the properties of a soluble protein. Comparison with the known amino acid sequence of EIImtl of Escherichia coli [Lee, C. A. & Saier, M. H. (1983) J. Biol. Chem. 258, 10 761–10 767] showed significant similarity. The motif containing the cysteine, which is the putative second phosphorylation site in EIImtl of E. coli [Pas, H. H. & Robillard, G. T. (1988) Biochemistry 27, 5835–5839], is well conserved in EIImtl of Staphylococcus carnosus. Chemical modification of the single active site cysteine residue by Ellman's reagent leads to total inactivation, which can be reversed by treatment with 2‐mercaptoethanol.
Tissue distribution and change in potato starch phosphorylase mRNA levels in wounded tissue and sprouting tubers
Authors: BHATT Anuj M. , KNOWLER John T.
Published: March 2005

Starch phosphorylase has been cloned from a λgt10 cDNA library of potato tuber mRNA. Selected recombinants have been used to demonstrate that phosphorylase mRNA is most abundant in tubers but is also detectable in stolon, root, stem and leaf tissue. The level of phosphorylase mRNA was greatly reduced in wounded stem and tuber tissue. The wounding‐induced decrease in phosphorylase mRNA levels is not reversed in the presence of sucrose or mannitol. Regional differences are described in the levels of phosphorylase and patatin mRNA in different parts of the tuber and in the shoot of sprouting potatoes.
Lysyl‐tRNA synthetase from yeast
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich
Published: March 2005

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occuring non‐cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl‐tRNA synthetases from yeast. Generally, both species of this class II aminoacyl‐tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90–1700, D values of the phosphorylated species in the range 40–770. The phosphorylated enzyme acts faster and less accurately.

In aminoacylation of tRNALys‐C‐C‐A(2′NH2) discrimination factors D1 vary over 30–980 for the native and over 8–300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof‐reading factors (II1) of 1.1–56 were calculated for the native enzyme, factors of 1.0–44 for the phosphorylated species. Post‐transfer proof‐reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys‐C‐C‐A. Pretransfer proof‐reading is the main correction step, posttransfer proof‐reading is less effective or negligible (II2∼ 1–8).

Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (ΔΔGI), were calculated from discrimination and proof‐reading factors. In contrast to class I synthetases, for lysyl‐tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three‐step process instead of the four‐step recognition observed for the class I synthetases. Plots of ΔΔGI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical ‘stopper model’ of these sites.
The primary structure and gene organization of human substance P and neuromedin K receptors
Authors: TAKAHASHI Kenzo , TANAKA Atsuo , HARA Masumi , NAKANISHI Shigetada
Published: March 2005

The gene organization and amino acid sequences of human substance P and neuromedin K receptors (SPR and NKR, respectively) are reported on the basis of molecular cloning and sequence determination of genomic DNA containing the respective receptor gene. The human SPR and NKR genes, unlike many other genes for G‐protein‐coupled receptors, (G protein, guanyl‐nucleotide‐binding‐regulatory protein), contain introns which interrupt the protein‐coding regions into 5 exons. The human SPR and NKR genes extend over 60 kb and 45 kb, respectively and are considerably larger than the human substance K receptor (SKR) gene consisting of 12 kb. All 4 introns, however, are located at equivalent positions of the three tachykinin receptor genes, suggesting that they evolved from a common ancestoral gene. Human SPR and NKR consist of 407 and 465 amino acid residues, respectively, each possessing structural features characteristic of the members of G‐protein‐coupled receptors. The human and rat receptors show a common tendency of distinctly segmented sequence conservation and divergence among the three receptors, and the observed sequence conservation and divergence would contribute to the emergence of similar but distinct properties of the three receptors. Furthermore, the amino acid sequences and the gene sizes are more closely related between SPR and NKR than between SKR and NKR, suggesting that the SPR gene evolved from the primordial NKR gene after a gene duplication to form the NKR and SKR genes.
Salt‐dependent and protein‐concentration‐dependent changes in the solution structure of the DNA‐binding histone‐like protein, HBsu, from Bacillus subtilis
Authors: WELFLE Heinz , MISSELWITZ Rolf , WELFLE Karin , GROCH Nicolas , HEINEMANN Udo
Published: March 2005

The solution structure of the histone‐like DNA‐binding protein, HBsu, from Bacillus subtilis in 2 mM sodium cacodylate, pH 7.5, is sensitive to the ionic strength of the buffer. This was shown by circular dichroism measurements at different concentrations of sodium chloride and potassium fluoride. The stability of HBsu is also influenced; at HBsu concentrations of about 0.1 mg · ml−1, melting temperatures of 32°C and 55°C were found in the absence of potassium fluoride and in the presence of 0.5 M potassium fluoride, respectively, exhibiting drastic ionic‐strength‐dependent differences in the temperature‐induced unfolding of HBsu.

Furthermore, at low ionic strength, circular dichroism spectra vary markedly depending on the HBsu concentration in the approximate range 0.2–3 mg · ml−1. Such protein‐concentration dependent differences in the spectra were not observed in the presence of 0.5 M potassium fluoride. Very similar circular dichroism spectra of HBsu and the histone‐like DNA‐binding protein of Bacillus stearothermophilus (HBst) at high ionic strength, indicate comparable structures of both proteins under these conditions. Estimation of the secondary structure content from the circular dichroism spectra yields data which are in satisfactory agreement with the values obtained from the crystal structure of HBst.

Transition temperatures of 45°C and 61°C were found in differential scanning calorimetric measurements performed with HBsu in potassium‐fluoride‐free buffer and in the presence of 0.5 M potassium fluoride, respectively. The thermodynamic data point to the melting of native HBsu dimers into two denatured monomers.
Target size analysis of prostaglandin endoperoxide synthase
Authors: RUF Hans Heinrich , SCHUHN Dietmar , DIETZ Reiner , NASTAINCZYK Wolfgang , NIELSEN Mogens
Published: March 2005

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono‐exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.
Protein phosphorylation and Mg2+influence light harvesting and electron transport in chloroplast thylakoid membrane material containing only the chlorophyll‐a/b‐binding light‐harvesting complex of photosystem II and photosystem I
Authors: HARRISON Michael A. , ALLEN John F.
Published: March 2005

A material containing only photosystem I (PSI) and the chlorophyll‐a/b‐binding light‐harvesting complex of PSII (LHC‐II) has been isolated from the chloroplast thylakoid membrane by solubilization with Triton X‐100. Fluorescence spectroscopy shows that, within the material, LHC‐II is coupled to PSI for excitation‐energy transfer and that this coupling is decreased by the presence of Mg2+, which also decreased PSI electron transport specifically at limiting light intensity. Inclusion of phosphorylated LHC‐II within the material did not alter its structure, but gave decreased energy transfer to PSI and inhibition of electron transport which was independent of light intensity, implying effects of phosphorylation on both light harvesting and directly on electron transport. Inclusion of Mg2+within the phosphorylated material gave decreased energy transfer, but slightly increased PSI electron transport. A cation‐induced direct promotion of PSI electron transport was also observed in isolated PSI particles. The PSI/LHC‐II material represents a model system for examining protein interactions during light‐state adaptations and the possibility that LHC‐II can contribute to the antenna of PSI in light state 2in vivois discussed.
Genetic analysis of epidermin biosynthetic genes and epidermin‐negative mutants of Staphylococcus epidermidis
Authors: AUGUSTIN Johannes , ROSENSTEIN Ralf , WIELAND Bernd , SCHNEIDER Uschi , SCHNELL Norbert , ENGELKE Germar , ENTIAN Karl‐Dieter , GÖTZ Friedrich
Published: March 2005

Epidermin is produced by Staphylococcus epidermidis Tü3298 which harbors the 54‐kb plasmid, pTü32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C, D, Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., Götz, F. & Entian, K.‐D. (1992) Eur. J. Biochem. 204, 57–68]. We isolated a number of stable mutants from strain Tü3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT181mcs and pCU1 led to their classification as epiA, epiB, epiC or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co‐transcribed from the epiA promoter. There is evidence that epiC and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S. carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D, and the DNA region comprising epiQ and P, host‐encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8‐kb DNA fragment of pTü32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of Gly10 by Glu.
Selective effect of poly(lysine) on the enhancement of the lyn tyrosine protein kinase activity
Authors: DONELLA‐DEANA Arianna , MARIN Oriano , BRUNATI Anna Maria , PINNA Lorenzo A.
Published: March 2005

A tyrosine protein kinase (TPK‐I), isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451–457] and recently identified as the product of the lyn oncogene [Brunati, A. M., Donella‐Deana, A., Ralph, S., Marchiori, F., Borin, G., Fischer, S. & Pinna, L. A. (1991) Biochim. Biophys. Acta 1901, 123–126], is stimulated by a variety of effectors, including poly(lysine), heparin and very high NaCl concentrations. The efficacy of these compounds is variably dependent on the nature of the phosphoacceptor peptide substrates. Here we show that poly(lysine), but neither NaCl nor heparin, specifically enhances the phosphorylation efficiency of lyn TPK for the peptide EDNEYTA (src peptide). It reproduces the main autophosphorylation site of pp60c‐src (Tyr416), which is entirely conserved in lyn TPK. The favourable effect of poly(lysine) is accounted for by both a dramatic drop of the Km value (70 μM versus 670 μM) and more than a threefold increase in Vmax. The effect is not so evident with a variety of different peptides, either unrelated to the src peptide (e.g. angiotensin II, AAYAA) or derived from the src peptide by single or multiple substitutions of the residues located on the N‐terminal side of tyrosine. In particular, the responsiveness to poly(lysine) is dramatically reduced whenever alanine is replaced for either asparagine at position – 2 or glutamic acid at position – 1, either in the src heptapeptide or in its shorter derivative, the pentapeptide NEYTA. In sharp contrast, the favourable effect of 2 M NaCl, which is invariably accounted for only by an increased Vmax, is especially evident with peptides like angiotensin II and AAYAA, whose phosphorylation is less sensitive to poly(lysine) stimulation. The phosphorylation of the src peptides are actually inhibited rather than stimulated by 2 M NaCl. Consistent with this, lyn TPK autophosphorylation is also dramatically stimulated by poly(lysine) while being inhibited by 2 M NaCl.

These data show that poly(lysine) deeply alters the selectivity of lyn TPK by conferring to it an enhanced activity and an especially high affinity toward peptides that reproduce the conserved autophosphorylation site of the TPK of the src family. It is suggested that endogenous compound, whose effect is mimicked by poly(lysine) in vitro, may play a relevant role in determining the specificity of lyn TPK in vivo and possibly of other TPK of the src family.
Inhibition of N‐acetylglucosamine kinase and N‐acetylmannosamine kinase by 3‐O‐methyl‐N‐acetyl‐D‐glucosamine in vitro
Authors: ZEITLER Reinhard , GIANNIS Athanassios , DANNESCHEWSKI Sabine , HENK Elfriede , HENK Thomas , BAUER Christian , REUTTER Werner , SANDHOFF Konrad
Published: March 2005

During the search for inhibitors of N‐acetylneuraminic acid biosynthesis, it was shown that 3‐O‐methyl‐N‐acetylglucosamine competitively inhibits the N‐acetylglucosamine kinase of rat liver in vitro with a Ki value of 17 μM. N‐Acetylmannosamine kinase is inhibited non‐competitively with a Ki value of 80 μM. In a human hepatoma cell line (HepG2), 3‐O‐methyl‐N‐acetyl‐D‐glucosamine (1 mM) inhibits the incorporation of 14C‐N‐acetylglucosamine and 14C‐N‐acetylmannosamine into cellular glycoproteins by 88% and 70%, respectively.
Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus
Authors: ARNONE M. I. , BIROLO L. , GIAMBERINI M. , CUBELLIS M. V. , NITTI G. , SANNIA G. , MARINO G.
Published: March 2005

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein.

Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez‐Carrion, M. (1984) J. Biol. Chem. 259, 723–728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323–326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (>60°C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25–75°C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line.

Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non‐linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non‐linear as well.
Conformation of MgATP bound to nucleotidyl and phosphoryl transfer enzymes 1H‐transferred NOE measurements on complexes of methionyl tRNA synthetase and pyruvate kinase
Authors: LANDY Steven B. , RAY Bruce D. , PLATEAU Pierre , LIPKOWITZ Kenny B. , RAO B. D. Nageswara
Published: March 2005

The conformations of MgATP bound to a nucleotidyl transfer enzyme, methionyl tRNA synthetase and a phosphoryl transfer enzyme, pyruvate kinase, were studied by transferred NOE (TRNOE) measurements in 1H NMR. The experiments were performed on D2O solutions at 276 MHz and 300 MHz, and 10°C in the presence of approximately a tenfold excess of substrate over the enzyme (sites). Selective inversion of chosen resonances was accomplished with an appropriately tailored DANTE sequence consisting of 100 phase‐alternating hard 1.8° pulses. NOE measurements were made in terms of difference spectra (with and without inversion) at 6–8 delay times ranging from 10–500 ms following the DANTE sequence. A full complement of ten NOE build‐up curves obtained for each enzyme complex was analyzed by using the complete relaxation‐matrix method (which includes all the non‐exchangeable protons in MgATP) suitably modified to include exchange between bound and free substrate. Molecular mechanics computations were used to examine the energetic implications of the NOE‐determined structure. The final structures obtained for MgATP bound to the two enzymes were very similar to each other, with a 3′‐endo sugar pucker and an anti conformation with a glycosidic torsional angle (O4‘–C1’–N9–C8) of 39°± 4°. Both enzymes contain multiple binding sites for MgATP and hence the structure obtained in each case represents an average due to chemical exchange. However, TRNOE experiments performed on a tryptic fragment of methionyl tRNA synthetase which has a single MgATP binding site, show that the same structure fits these measurements as well. This evidence, coupled with the striking similarity of the structures deduced, for the two enzyme complexes, and the reciprocal sixth‐power dependence of NOE on interproton distance, strongly suggests that the conformations at the individual binding sites of both the enzymes are virtually identical. This conclusion is in contrast with multiple conformations of MgATP bound to pyruvate kinase, proposed by Rosevear, P. R., Fox, T. L. & Mildvan, A. S. (1987) Biochemistry 26, 3487–3493.
Actin and neurofilament binding domain of brain spectrin β subunit
Authors: FRAPPIER Thierry , DERANCOURT Jean , PRADEL Louise‐Anne
Published: March 2005

Tryptic digestion of brain spectrin generates a number of fragments from α and β subunits; when these fragments are incubated with F‐actin or neurofilament light subunit, four of them with molecular masses below 30 kDa sediment with the cytoskeleton structures. A selective purification of these fragments by ammonium sulfate fractionation and butyl‐Sepharose chromatography has been achieved. Two fragments with molecular masses of 28 and 23 kDa bind to F‐actin. Native brain spectrin causes half‐maximal inhibition of the association at a concentration of 3 μM. Protein sequencing indicates that the actin‐binding domain is contained in the β subunit, in a stretch of amino acids at the N terminus from Ala43 (28‐kDa fragment) or from Met104 (23‐kDa fragment) and terminate probably at the C‐terminal Lys288 or Lys284. Amino acids are numbered by reference to the sequence of the Drosophilaβ subunit. The 28‐kDa fragment also binds to the low‐molecular‐mass subunit of neurofilaments; brain spectrin heterodimer disrupts this binding. Hence, spectrin binds to F‐actin and to neurofilaments via a common binding domain.
Amphibian lutropin from the bullfrog Rana catesbeiana
Authors: HAYASHI Hiroaki , HAYASHI Tomoko , HANAOKA Yoichi
Published: March 2005

The amino acid sequence of lutropin (LH) β subunit of an amphibian, the bullfrog Rana catesbeiana, has been determined. The primary structure was determined by sequencing the intact protein (residues 1–44) and peptides originated by cyanogen bromide cleavage and lysyl endopeptidase digestion. 12 cysteine residues are conserved in the bullfrog and mammalian LH β subunit. One sugar‐chain‐binding site at Asn‐8 is also conserved in the bullfrog and in all mammals except humans. This glycoprotein is composed of 112 amino acid residues with a molecular mass of 12675 Da, considering the six cystine bridges and excepting the sugar chain. The bullfrog β subunit has approximately 50% sequence identity with that of mammals and with the fish gonadotropin β subunit, and about 40% with bullfrog follicle‐stimulating hormone β subunit.
The third subunit of desulfoviridin‐type dissimilatory sulfite reductases
Authors: PIERIK Antonio J. , DUYVIS Martina G. , van HELVOORT Joop M. L. M. , WOLBERT Ronnie B. G. , HAGEN Wilfred R.
Published: March 2005

In addition to the 50‐kDa (α) and 40‐kDa (β) subunits, an 11‐kDa polypeptide has been discovered in highly purified Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted α2β2 tetrameric subunit composition. Purification, high‐ionic‐strength gel‐filtration, native electrophoresis and isoelectric focussing do not result in dissociation of the 11‐kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel‐filtration indicate a stoichiometric occurrence. A similar 11‐kDa polypeptide is present in the desulfoviridin of D. vulgaris oxamicus (Monticello), D. gigas and D. desulfuricans ATCC 27774. We attribute an α2β2γ2 subunit structure to desulfoviridin‐type sulfite reductases. N‐terminal sequences of the α, β and γ subunits are reported.
Characterization of a recombinant chimeric plasminogen activator composed of a fibrin fragment‐d‐dimer‐specific humanized monoclonal antibody and a truncated single‐chain urokinase
Authors: VANDAMME Anne‐Mieke , DEWERCHIN Maria , LIJNEN H. Roger , BERNAR Hilde , BULENS Frank , NELLES Luc , COLLEN Désiré
Published: March 2005

A recombinant chimeric plasminogen activator, MA‐15C5Hu/scu‐PA‐32k, composed of a humanized fibrin fragment‐d‐dimer‐specific monoclonal antibody (MA‐15C5Hu) and a recombinant low‐molecular‐mass single‐chain urokinase‐type plasminogen activator, comprising amino acids Leu144–Leu411 (scu‐PA‐32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA‐15C5Hu light‐chain sequence and the cDNA encoding the MA‐15C5Hu heavy‐chain sequence fused with the cDNA encoding scu‐PA‐32k. Purified MA‐15C5Hu/scu‐PA‐32k migrated as a 215‐kDa band on non‐reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu‐PA‐32k moieties. However, the chimera was obtained as a mixture of single‐chain u‐PA‐32k (37%) and amidolytically inactive (50%) and active (13%) two‐chain u‐PA‐32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two‐chain urokinase. The fragment‐d‐dimer affinity and enzymatic properties of MA‐15C5Hu/scu‐PA‐32k were similar to those of MA‐15C5Hu or of scu‐PA‐32k. In an in vitro system composed of a 125I‐fibrin‐labeled human plasma clot submerged in citrated human plasma, MA‐15C5Hu/scu‐PA‐32k had a 12‐fold higher fibrinolytic potency than scu‐PA‐32k: 50% lysis in 2 h required 0.43 ± 0.12 μg u‐PA‐32k equivalent of the chimera/ml versus 5.4 ± 0.3 μg/ml of scu‐PA‐32k (mean ± SEM, n= 4). Addition of purified fibrin fragment‐D dimer reduced the fibrinolytic potency of MA‐15C5Hu/scu‐PA‐32k in a concentration‐dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u‐PA chimera has been obtained in which only the variable domains of the antibody moiety are of non‐human origin. The chimera has intact antigen‐binding capacity, u‐PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.
Use of sialic acid analogues to define functional groups involved in binding to the influenza virus hemagglutinin
Authors: KELM Sørge , PAULSON James C. , ROSE Ursula , BROSSMER Reinhard , SCHMID Walther , BANDGAR Babasaheb P. , SCHREINER Erwin , HARTMANN Michael , ZBIRAL Erich
Published: March 2005

The initial step of influenza infection is binding of the virus particles via their hemagglutinin to cell‐surface sialic acids. This study was initiated to elucidate the functional groups of the nine‐carbon sialic acid molecule which interact with the hemagglutinin and contribute to the affinity of this sugar to the protein. In order to address this question, synthetic sialic acid analogues were tested in a virus adsorption inhibition assay for their inhibitory potency. Modifications in three regions of the sialic acid molecule were evaluated: the glycerol side chain (C7 – C9), the N‐acetyl group at C5, and the carboxy group (C1). In the glycerol side chain, the hydroxy groups at C7 and C8 appear to be important for binding through hydrogen bonds, whereas the hydroxyl at C9 does not appear to be involved. The N‐acetyl group is critical for the interaction of sialic acid with the hemagglutinin. The results suggest that its contribution is mediated through hydrophobic interactions of the methyl group. Finally, the orientation of the carboxy group is essential for the binding of sialic acid to the hemagglutinin. The information gained in this study will be useful in developing novel compounds which bind more avidly to the influenza virus hemagglutinin. Such a strategy may contribute to the design of new anti‐influenza drugs.
Effect of benzyl alcohol on phospholipid transverse mobility in human erythrocyte membrane
Authors: BASSÉ François , SAINTE‐MARIE Josette , MAURIN Luc , BIENVENÜE Alain
Published: March 2005

The effect of benzyl alcohol on the transverse mobility and repartition of phospholipids in the human erythrocyte membrane was investigated using electron spin resonance and morphological modification of red blood cells. Transmembrane internalization rates and equilibrium distribution in red blood cells of short‐chain spin‐labeled phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were strongly modified by treatment with 10–70 mM benzyl alcohol. A dual effect was observed: (a) at 4°C and 37°C there was an N‐ethylmaleimide‐sensitive, long lasting and fully reversible increase in the spin‐labeled phosphatidylserine and phosphatidylethanolamine internalization rate; (b) at 37°C, an enhancement of N‐ethylmaleimide‐insensitive fluxes of all the labeled phospholipids through the membrane occurred. Both effects were dose‐dependent. Erythrocytes submitted to benzyl alcohol incubation also showed dose‐dependent shape changes: an immediate one from discocytes to echinocytes, followed by a slower N‐ethylmaleimide‐ and ATP‐dependent change to stomatocytes. Moreover, benzyl alcohol treatment was shown to lead to enhanced hydrolysis of intracellular ATP. All the effects of benzyl alcohol can be described as an accumulation of labeled phosphatidylethanolamine (and labeled phosphatidylcholine at 37°C) in the inner leaflet. This can be interpreted as a perturbation of the erythrocyte membrane, leading to an energy‐consuming specific increase in aminophospholipid translocase activity, in addition to a slow and passive bidirectional flux of all phospholipids at 37°C.