bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Turnover of the D1 Polypeptide and Photoactivation of Electron Transfer in Novel Herbicide Resistant Mutants ofSynechocystissp. PCC 6803
Authors: Chjesa Marta Dalla , Friso Giulia , DeÁK Zsuzsanna , Vass Imre , Barber James , Nixon Peter J.
Published: July 2004

Two missense mutants, A263P and S264P, and a deletion mutant des‐Ala263, Ser264, have been constructed in the D1 protein of the cyanobacteriumSyneclzocystissp PCC 6803. All were expected to induce a significant conformational change in the QB‐binding region of photosystem II (PSII). Although the des‐Ala263, Ser264‐D1 mutant accumulated some D1 protein in the thylakoid membrane it was unable to grow photoautotrophically or evolve oxygen. Thermoluminescence and chlorophyll fluorescence studies confirmed that this deletion mutant did not show any functional PSII activity. In contrast, [S264P]D1 was able to grow photoautotrophically and give light‐saturated rates of oxygen evolution at 60% of the rate of the wild‐type control strain, TC31. The A263P missense mutant was also able to evolve oxygen at 50% of TC31 rates although it did not readily grow photoautotrophically. Thermoluminescence, flash oxygen yield and chlorophyll fluorescence measurements indicated that in both missense mutants electron transfer from QAto QBwas significantly impaired in dark adapted cells. However, QAto QBelectron transfer could be photoactivated in the mutants by background illumination. Both the A263P and S264P mutants also showed an increase in resistance to thes‐triazine family of herbicides although this feature did not hold for the phenolic herbicide, ioxynil. Of particular interest was that the two missense mutants, especially S264P, possessed a slower rate of turnover of the D1 protein compared with TC31 andin vivocontained detectable levels of a 41‐kDa adduct consisting of D1 and the α subunit of cytochrotne B559. When protein synthesis was blocked by the addition of lincomycin, D1 degradation was again slower in S264P than TC31. The results are discussed in terms of structural changes in the QB‐binding region which affect herbicide and plastoquinone binding and perturb the normal regulatory factors that control the degradation of the D1 protein and its synchronisation with the synthesis of a replacement D1 protein.
Purification, Characterisation and cDNA Cloning of an Antimicrobial Peptide from Macadamia Integrifolia
Authors: Marcus John P. , Goulter Ken C. , Green Jodie L. , Harrison Stuart J. , Manners John M.
Published: July 2004

An antimicrobial peptide with no significant amino acid sequence similarity to previously described peptides has been isolated from the nut kernels of Macadamia integrifolia. The peptide, termed MiAMP1, is highly basic with an estimated pI of 10.1, a mass of 8.1 kDa and contains 76 amino acids including 6 cysteine residues. A cDNA clone containing the entire coding region corresponding to the peptide was obtained. The deduced amino acid sequence of the cDNA indicated a 26‐amino‐acid signal peptide at the N‐terminus of the preprotein. Purified MiAMP1 inhibited the growth of a variety of fungal, oomycete and gram‐positive bacterial phytopathogens in vitro. Some pathogens exhibited close to 100% inhibition in less than 1 μM peptide (5 μg/ml). Antimicrobial activity was diminished against most, but not all, microbes in the presence of calcium and potassium chloride salts (1 mM and 50 mM, respectively). MiAMPl was active against bakers yeast, was inactive against Escherichia coli and was non‐toxic to plant and mammalian cells. Analysis of genomic DNA indicated that MiAMP1 was encoded on a single copy gene containing no introns. The MiAMP1 gene may prove useful in genetic manipulations to increase disease resistance in transgenic plants.
Derepression of Gene Expression Mediated by the 5′ Upstream Region of the Isocitrate Lyase Gene of Candida Tropicalis is Controlled by two Distinct Regulatory Pathways in Saccharomyces Cerevisiae
Authors: Umemura Ken , Atomi Haruyuki , Kanai Tamotsu , Takeshita Shigeru , Kanayama Naoki , Ueda Mitsuyoshi , Tanaka Atsun
Published: July 2004

The 5′ upstream region of the gene encoding isocitrate lyase of Candida taropicalis (UPR‐ICL) is functional as a promoter in Saccharomyces cerevisiae, and it is regulated by carbon source; the expression of the gene is repressed when cells are grown on glucose, while it increases to a higher level in acetate‐grown cells. Therefore, we have investigated regions in UPR‐ICL responsible for gene expression in glucose‐grown and acetate‐grown cells. In glucose‐grown cells, a deletion of the region between –801 and –569 (region G1) significantly decreased gene expression compared with that observed with the complete UPR‐ICL. The region from –421 to –379 (region G2) also repressed gene expression in glucose‐grown cells. In acetate‐grown cells, two regions were found to strongly enhance gene expression, one between –728 and –569 (region A1) and the other between –370 and –356 (region A2). Whereas region A2 contained a sequence motif similar to the carbon‐source‐responsive element (CSRE), which mediates regulation by carbon source of S. cerevisiae ICL1, region A1 did not show similarity to any reported cis‐acting elements. Deletion mutants of UPR‐ICL containing only one of these regions showed that each region could independently activate gene expression to a similar level when the cells were grown on acetate. The influences of null mutations in the MIG1, SNF1 and CAT8 genes on regulation of UPR‐ICL–mediated gene expression were examined. Expression of the ICL gene with full‐length UPR‐ICL increased about tenfold in mig1 cells grown on glucose, while little difference was observed in acetate‐grown cells. The effects of snf1 and cat8 mutations were different between region‐A1‐mediated and region‐A2‐mediated gene expression in acetate‐grown cells. Region‐A2‐mediated expression decreased 9.5% and 84% in snf1 and cat8 cells, respectively, while region‐A1‐mediated expression decreased 72% in snf1 cells and was not affected by the cat8 mutation. This finding indicates that region‐A1‐mediated gene expression is regulated by a pathway independent of CAT8, which is necessary for derepression of CSRE‐mediated gene expression in S. cerevisiae.
C‐Terminal Ladder Sequencing by an Approach Combining Chemical Degradation with Analysis by Matrix‐Assisted‐Laser‐Desorption Ionization Mass Spectrometry
Authors: Thiede Bernd , Salnikow Johann , Wittmann‐Liebold Brigitte
Published: July 2004

A chemical procedure for the degradation of peptides and analysis by matrix‐assisted‐laser‐desorption ionization mass spectrometry (MALDI‐MS) was used for C‐terminal sequence determination. The method allowed us to determine up to eight amino acid residues in the lower picomole range by mass analysis without any repetitions of the degradation nor any extraction or purification of the truncated peptide chains. The C‐terminal degradation of all 20 common amino acid residues was proved by applying this method. Extended C‐terminal sequence information from enzymatic digests using carboxypeptidase P was obtained by combining the enzymatic with the chemical mass spectrometric approach. Furthermore the amino acids lysine and glutamine, with the same masses, were distinguishable due to the formation of acetyl‐lysine in the chemical process.
Effect of Phosphorylated Rat Fetuin on the Growth of Hepatocytes in Primary Culture in the Presence of Human Hepatocyte‐Growth Factor
Authors: Ohnishi Tomokazu , Nakamura Osamu , Arakaki Naokatu , Daikuhara Yasushi
Published: July 2004

Rat fetuin, a counterpart of human α2,‐HS glycoprotein and bovine fetuin, that is synthesized and secreted by hepatocytes is mostly phosphorylated, though rat fetuin isolated from bone matrix does not contain phosphorus. A rat 63‐kDa phosphorylated N‐glycoprotein (pp63) is the phosphorylated form of rat fetuin and pp63 has been shown to inhibit insulin‐receptor tyrosine kinase activity. Therefore, we examined the effect of phosphorylated rat fetuin (phosphofetuin) on DNA synthesis in rat hepatocytes in culture in the presence of human hepatocyte‐growth factor (HGF), since the human receptor of HGF, c‐Met, is known to contain a tyrosine‐kinase domain in its intracellular domain. We found that phosphofetuin from conditioned medium of rat‐hepatocyte cultures dose‐dependently decreased HGF‐stimulated DNA synthesis in hepatocytes, whereas addition of non‐phosphorylated rat fetuin had no effect. Addition of anti‐(rat fetuin) Ig to the culture medium increased HGF‐stimulated DNA synthesis by hepatocytes. Immunoprecipitation and cross‐linking experiments showed that phosphofetuin bound to human HGF. We found that phosphofetuin interfered with binding of HGF to its specific receptor(s). These observations suggest that phosphofetuin synthesized by hepatocytes may be a natural modulator of HGF as a chalone, and that regulation of expression of phosphofetuin by growth factors and cytokines may be involved in liver regeneration under inflammatory conditions, such as in hepatitis.
The Complete Sequence, Expression in Escherichia Coli, Purification and Some Properties of Carbonic Anhydrase from Neisseria Gonorrhoeae
Authors: Chirică Laura C. , Elleby Björn , Jonsson Bengt‐Harald , Lindskog Sven
Published: July 2004

The complete nucleotide sequence of the carbonic anhydrase gene from Neisseria gonorrhoeae has been determined. The gene encodes a 252‐residue polypeptide with a molecular mass of 28085 Da. The gene has been cloned and overexpressed in Escherichia coli, and the enzyme has been purified. A 26‐residue signal peptide is cleaved off by the E. coli processing machinery. Thus, the isolated enzyme contains 226 amino acid residues with a molecular mass of 25314 Da. Most of the enzyme seems to be produced as a soluble protein located in the periplasm of E. coli. The enzyme is homologous to carbonic anhydrases from the animal kingdom; it is an α‐carbonic anhydrase. A comparison with the amino acid sequences of human carbonic anhydrases I and II suggests that the secondary structures are essentially intact in the bacterial enzyme but that several loops are much shorter than in the human forms. Most of the active‐site residues are identical to those found in the high‐activity human isozyme II. The bacterial enzyme has a high CO2 hydration activity with a kcat of 1.1 · 106 s−1 and Km of 20 mM at pH 9 and 25°C. The enzyme also catalyzes the hydrolysis of 4‐nitrophenyl acetate. The pH/rate profile can be described as a titration curve with pKa of 6.7 and a maximal value of the catalytic second‐order rate constant, kenz, of 130 M−1 s−1.
Identification and Characterization of a Sequence Related to Human Sorbitol Dehydrogenase
Authors: Carr Ian M. , Markham Alexander F. , Coletta P. Louise
Published: July 2004

The polyol pathway comprises the enzymes aldose reductase and sorbitol dehydrogenase which convert glucose to fructose via sorbitol. Accumulation of sorbitol within the cell has been suggested to contribute to the progression of secondary complications of diabetes. High levels of sorbitol accumulate within the cell due to inadequate regulation of blood glucose levels. It has also been suggested that polymorphism in either the aldose reductase or sorbitol dehydrogenase genes might contribute to sorbitol accumulation. The human sorbitol dehydrogenase gene (SORD) has been described previously and a range of putative polymorphic variants were identified. Further analysis of human SORD yeast artificial chromosome clones has now shown that there is a second SORD‐like sequence in man, which is extremely similar in sequence to SORD itself and which also maps to chromosome 15. Detailed sequence analysis suggests that this SORD‐related gene cannot be expressed as a full‐length sorbitol dehydrogenase isoenzyme. However, knowledge of the presence of this highly similar sequence in the human genome is essential to ensure that sequence variations identified during genetic analysis of SORD are not attributed to polymorphisms within that gene itself.
Cloning, Bacterial Expression and Biological Characterization of Recombinant Human Granulocyte Chemotactic Protein‐2 and Differential Expression of Granulocyte Chemotactic Protein‐2 and Epithelial Cell‐Derived Neutrophil Activating Peptide‐78 mRNAs
Authors: Froyen Guy , Proost Paul , Ronsse Isabelle , Mitera Tania , Haelens Annemie , Wuyts Anja , Opdenakker Ghislain , van Damme Jo , Billiau Alfons
Published: July 2004

Human osteosarcoma cells secrete a novel C‐X‐C chemokine called granulocyte chemotactic protein‐2 (GCP‐2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP‐2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin‐1β and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP‐2 was isolated. This sequence was cloned into the bacterial expression vector pHENl and, after induction, GCP‐2 was secreted into the periplasm of Escherichia coli. Recombinant GCP‐2 (rGCP‐2) was purified and characterized by SDS/PAGE as a monomeric 6.5‐kDa protein and by amino‐terminal sequencing. The chemoattractive potency of GCP‐2 for neutrophilic granulocytes was about 10‐times less than that of interleukin‐8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin‐8. Both characteristics correspond with those of natural GCP‐2. In addition, intracellular calcium release in neutrophils by recombinant GCP‐2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP‐2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial‐cell‐derived neutrophil‐activating peptide‐78 (ENA‐78) mRNA, the GCP‐2 mRNA levels were higher in all cell lines tested. In addition, GCP‐2 and ENA‐78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin‐I was demonstrated to be a general inducer for both chemokines, while interferon‐)) down‐regulates their mRNA expression. The availability of recombinant GCP‐2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP‐2 during the inflammatory response.
Two Ways of Legumin‐Precursor Processing in Conifers
Authors: Häger Klaus‐Peter , Wind Claudia
Published: July 2004

Subunit monomers and oligomers of crystalloid‐type legumins are major components of SDS‐soluble fractions from Metasequoia glyptostroboides (Dawn redwood, Taxodiaceae) seed proteins. The subunits are made up of disulfide linked α‐polypeptides and β‐polypeptides with molecular masses of 33 kDa and 23–25 kDa, respectively. Unusually for legumins, those from Metasequoia are glycosylated and the carbohydrate moieties are residing in the C‐terminal region of the respective β‐polypeptides. A Metasequoia endosperm cDNA library has been constructed and legumin‐encoding transcripts representing two divergent gene subfamilies have been characterized. Intersubfamily comparisons reveal 75 % identity at the amino acid level and the values range from 53–34% when the legumin precursors deduced were compared with those from angiosperms. The predicted sequences together with data from amino acid sequencing prove that post‐translational processing of Metasequoia prolegumins is directed to two different processing sites, each of them specific for one of the legumin subfamilies. The sites involved differ in their relative position and in the junction to be cleaved: Metasequoia legumin precursors MgLeg18 and MgLeg26 contain the conventional post‐translational Asn‐Gly processing site, which is generally regarded as highly conserved. In contrast, the MgLeg4 precursor is lacking this site and post‐translational cleavage is directed to an unusual Asn‐Thr processing site located in its hypervariable region, causing N‐terminal extension of the β‐polypeptide relative to those hitherto known. Evidence is given that the unusual variant of processing also occurs in other conifers. Phylogenetic analysis reveals the precursors concerned as representatives of a distinct legumin subfamily, originating from duplication of an ancestral gene prior to or at the beginning of Taxodiaceae diversification.
Bov‐B Long Interspersed Repeated DNA (LINE) Sequences are Present in Vipera Ammodytes Phospholipase A2 Genes and in Genomes of Viperidae Snakes
Authors: Kordiš Dušan , Gubenšek Franc
Published: July 2004

Ammodytin L is a myotoxic Ser49 phospholipase A2 (PLA2) homologue, which is tissue‐specifically expressed in the venom glands of Vipera ammodytes. The complete DNA sequence of the gene and its 5′ and 3′ flanking regions has been determined. The gene consists of five exons separated by four introns. Comparative analysis of the ammodytin L and ammodytoxin C genes shows that all intron and flanking sequences are considerably more conserved (93–97%) than the mature protein‐coding exons. The pattern of nucleotide substitutions in protein‐coding exons is not random but occurs preferentially on the first and the second positions of codons, which suggests positive Darwinian evolution for a new function. An Ruminantia specific ART‐2 retroposon, recently recognised as a S′‐truncated Bov‐B long interspersed repeated DNA (LINE) sequence, was identified in the fourth intron of both genes. This result suggests that ammodytin L and ammodytoxin C genes are derived by duplication of a common ancestral gene. The phylogenetic distribution of Bov‐B LINE among vertebrate classes shows that, besides the Ruminantia, it is limited to Viperidae snakes (Vipera ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3′ end of Bov‐B LINE in the Vipera ammodytes genome is between 62000 and 75000. The absence of Bov‐B LINE at orthologous positions in other snake PLA2 genes indicates that its retrotransposition in the V. ammodytes PLA2 gene locus has occurred quite recently, about 5 My ago. The amplification of Bov‐B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it may be a valuable phylogenetic marker. The neighbor‐joining phylogenetic tree shows two clusters of truncated Bov‐B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.
Tie2 Receptor Expression and Phosphorylation in Cultured Cells and Mouse Tissues
Authors: Koblizek Thomas I. , Runttng Andrew S. , Stacker Steven A. , Wilks Andrew F. , Risau Werner , Deutsch Urban
Published: July 2004

Accumulating experimental evidence indicates that endothelial cell growth and blood vessel morphogenesis are processes that are governed by the activity of specifically expressed receptor tyrosine kinases (RTKs). We have used two new rat monoclonal antibodies (mAbs) to study the expression and phosphorylation of one such receptor, mouse Tie2 (tyrosine kinase that contains immunoglobulin‐like loops and epidermal‐growth‐factor‐similar domains 2]), in transfected cells, endothelioma cell lines and mouse tissues. The Tie2 receptor was found to be constitutively autophosphorylated when over‐expressed in COS7 cells. In contrast, the endogenous Tie2 protein was not phosphorylated in endothelioma cell lines. However, in these cell lines, Tie2 could be induced to become tyrosine phosphorylated, and this activation was found to be independent of Tiel. Studying Tie2 receptor activity during angiogenesis in mouse development, the receptor was only weakly phosphorylated in the early postnatal mouse brain whereas a stronger activation could be detected in mouse embryos at day 10.5 post coitum.
A Heterodimer of the Zn2Cys6 Transcription Factors Pip2p and Oaf1p Controls Induction of Genes Encoding Peroxisomal Proteins in Saccharomyces Cerevisiae
Authors: Rottensteiner Hanspeter , Kal Arnoud J. , Hamilton Barbara , Ruis Helmut , Tabak Henk F.
Published: July 2004

In the yeast Saccharornyces cerevisiae, two transcriptional activators belonging to the Zn2Cys6 protein family, Pip2p and Oaf1p, are involved in fatty‐acid‐dependent induction of genes encoding peroxisomal proteins. This induction is mediated via an upstream activation sequence called the oleate‐response element (ORE). DNA‐bandshift experiments with ORE probes and epitope‐tagged proteins showed that two binary complexes occurred: in wild‐type cells the major complex consisted of a Pip2p · Oaf1p heterodimer, but in cells in which Oaf1p was overexpressed an Oaf1p homodimer was also observed. The genes encoding Oaf1p and Pip2p were controlled in different ways. The OAF1 gene was constitutively expressed, while the PIP2 gene was induced upon growth on oleate, giving rise to positive autoregulatory control. We have shown that the Pip2p · Oaf1p heterodimer is responsible for the strong expression of the genes encoding peroxisomal proteins upon growth on oleate. Pip2p and Oaf1p form an example of a heterodimere of yeast Zn2Cys6 zinc‐finger proteins binding to DNA.
Ionic‐Strength‐Dependent Transition of Hen Egg‐White Lysozyme at Low PH to a Compact State and its Aggregation on Thermal Denaturation
Authors: Babu Kodali Ravindra , Bhakuni Vinod
Published: July 2004

Equilibrium acid‐induced unfolding of hen egg‐white lysozyme has been investigated by a combination of optical methods, size‐exclusion chromatography, and differential scanning calorimetry. The results showed the presence of a partially folded state of hen egg‐white lysozyme at pH 1.5, characterized by a substantial secondary structure, a large solvent exposure of non‐polar clusters, and significantly disrupted tertiary structure. A large enthalpy was also associated with the conversion of the acid‐unfolded state to a fully unfolded state. Size‐exclusion chromatography and 8‐anilino‐1‐naphthalenesulphonic acid‐binding studies showed an ionic‐strength‐induced transition of the partially folded state to a compact conformation. Furthermore, an ionic‐strength‐dependent aggregation on thermal unfolding of the partially folded intermediate was also observed. These observations provide insights into the possible features responsible for the stabilization of intermediates in the folding of hen egg‐white lysozyme.
Structural Transitions of a GG‐Platinated DNA Duplex Induced by pH, Temperature and Box A of High‐Mobility‐Group Protein 1
Authors: Berners‐Price Susan J. , Corazza Alessandra , Guo Zijian , Barnham Kevin J. , Sadler Peter J. , Ohyama Yumiko , Leng Marc , Locker Daniel
Published: July 2004

[1H,15N] and1H NMR, and CD spectroscopy are used to show that the duplex d(A‐T‐A‐C‐A‐T‐Pt7G‐Pt7G‐T‐A‐C‐A‐T‐A) ˙ d(T‐A‐T‐G‐T‐A‐C‐C‐A‐T‐G‐T‐A‐T), where Pt7G is platinated guanine, containing thecis‐[Pt(NH3)2]2+adduct undergoes reversible temperature‐induced (T0.5310 K) and pH‐induced (pKa≈ 4.8) transitions between kinked‐duplex and distorted forms, with the latter forms predominating at high temperature and low pH. A related pH‐induced structural change was observed for the unplatinated duplex (pKa4.69, Hill coefficientn= 1.4) but was less cooperative than for the platinated duplex (n= 2). The pH‐induced transition is attributed to protonation of cytosine residues and has wider implications, since many reported NMR studies of DNA are carried out near pH 5 to minimize NH‐exchange rates. The [Pt(en)]2+(where en is 1,2‐ethanediamine) GG chelate of the same duplex is shown to exist in kinked and distorted forms, and the [1H,15N]‐NMR shifts for the kinked form are indicative of the presence of highly stereospecific interactions with the Pt‐NH protons. On binding of the duplex platinated with [Pt(NH3)2]2+to high‐mobility‐group protein 1 (HMG1) box A, similar changes in shifts of the Pt‐NH3resonances to those induced by raising the temperature or lowering the pH were observed. The specific changes in1H‐NMR chemical shifts of HMG1 box A are consistent with binding of the platinated duplex (intermediate exchange rate on the1H‐NMR time‐scale) to the concave face of the protein via helices I and II and the intervening loop.
Binding of 1‐Anilinonaphthalene‐8‐Sulfonic Acid to α‐crystallin
Authors: Stevens Arthur , Augusteyn Robert C.
Published: July 2004

α‐Crystallin was found to exhibit a time‐dependent uptake of the hydrophobic probe, 1‐anilinonaph‐thalene‐8‐sulfonic acid (ANS), similar to that typically observed with lipid membranes. Analysis of the interaction of ANS with α‐crystallin revealed two types of interactive processes, partitioning and binding. The predominant process involved partitioning, with a coefficient of 300 M−1. The binding component had the following characteristics: 1 binding site/24 subunits and a Kd of about 9 μM. The binding was unaffected by the number of subunits used in the assembly of the α‐aggregate, since both the αm‐ and αc‐forms had similar binding characteristics. No discernible differences were observed in the binding of ANS to homopolymers of αA and αB subunits, suggesting that the hydrophobic sites to which ANS bound were similar in both the A and B subunits. The majority of the fluorescence was lost when the protein was incubated in 3 M urea, a concentration of denaturant where the protein is still intact, suggesting that the ANS binding sites are located near the surface of the protein. The decrease was attributed to a decrease in the quantum yield of the bound dye.
Liposome Solubilization and Membrane Protein Reconstitution Using Chaps and Chapso
Authors: Cladera Josep , Ricaud Jean‐Louis , Verde Joaquim Villa , DuÑach Mireia
Published: July 2004

The processes of liposome solubilization and reconstitution of two transport proteins have been studied using Chaps and Chapso (3‐[(3‐cholamidopropyl)dimethylammonio]‐2‐hydroxy‐l‐propanesulfonate). The solubilization of unilamellar liposomes was followed by absorption experiments and the process was shown to fit well to the three‐stage model previously proposed for other detergents. The solubilization parameters have been determined and the detergent to phospholipid ratios at which the lamellar‐to‐micellar transition initiates and ends were estimated to be 0.21 mol/mol and 0.74 mol/mol, for Chapso and 0.4 mol/mol and 1.04 molhol for Chaps, respectively. The best conditions for the incorporation of two membrane proteins, bacteriorhodopsin and the H+‐ATP synthase from chloroplasts, were analyzed at each step of the solubilization process. After detergent removal, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitutions were obtained by addition of the proteins to completely solubilized lipid‐detergent micelles. The use of Chapso and Chaps for membrane protein reconstitution studies provides a reproducible method of achieving active proteoliposomes, homogeneous in size with a low permeability and thus, well suited for transport measurements.
Molecular Cloning of the Gene for Mouse Leukotriene‐C4 Synthase
Authors: Penrose John F. , Baldasaro Mathew H. , Webster Matthew , Xu Kongyi , Austen K. Frank , Lam Bing K.
Published: July 2004

Leukotriene C4 (LTC4) synthase (LTC4S), an integral membrane protein, catalyzes the conjugation of leukotriene A4 with reduced glutathione to form LTC4, the biosynthetic parent of the additional cysteinyl leukotriene metabolites. An XmnI‐digested fragment of a P1 clone from a 129 mouse ES library contained the full‐length gene of 2.01 kb for mouse LTC4S. The mouse LTC4S gene is comprised of 5 exons of 122, 100, 71, 82 and 241 nucleotides, with intron sizes that range from 76 nucleotides to 937 nucleotides. The intron/exon boundaries are identical to those of the human genes for LTC4S and 5′‐lipoxygenase‐activating protein (FLAP). Primer extension demonstrated a single transcription‐initiation site 64 bp 5′ of the ATG translation‐start site. Nucleotide sequencing of 1.2 kb of the 5′ flanking region revealed multiple putative sites for activating protein‐2, CCAAT/enhancer‐binding protein, and polyoma virus enhancer‐3. Fluorescent in situ hybridization mapped the mouse LTC4S gene to mouse chromosome 11, in a region containing the genes for interleukin 13 and granulocyte/macrophage‐colony‐stimulating factor, and orthologous to the chromosomal location of 5q35 for the human LTC4S gene. Thus, the mouse LTC4S gene is similar in size, introdexon organization and chromosomal localization to the human LTC4S gene. Recent mutagenic analysis of the conjugation function of human LTC4S has identified R51 and Y93 as critical for acid and base catalysis of LTA4 and reduced glutathione, respectively. A comparison across species for proteins that possess LTC4S activity reveals conservation of both of these residues, whereas R51 is absent in the FLAP molecules. Thus, within the glutathione S‐transferase superfamily of genes, alignment of specific residues allows the separation of LTC4S family members from their most structurally similar counterparts, the FLAP molecules.
The Three‐Dimensional Solution Structure of the Lantibiotic Murein‐Biosynthesis‐Inhibitor Actagardine Determined by NMR
Authors: Zimmermann Norbert , Jung Günther
Published: July 2004

The three‐dimensional solution structure of the lantibiotic actagardine was determined at high resolution by homonuclear and heteronuclear two‐dimensional and three‐dimensional NMR spectroscopy in [2H3]acetonitrile/H2O (7:3). 133 non‐trivial distance and 22 torsional‐angle constraints were derived from the NMR data. An ensemble of 15 low‐energy structures was calculated by distance geometry followed by an iterative relaxation‐matrix‐refinement procedure. The rmsd of the backbone coordinates with respect to the average structure was 17 pm. The two distinct thioether ring systems 1–6 and 7–19 were even better defined, with backbone rmsd of 10 pm and 14 pm, respectively. Actagardine shows a rigid compact globular shape based on the constraining bridging pattern, which is composed of an N‐terminal lanthionine ring from residues 1–6 and three intertwined C–terminal methyllanthionine rings comprising residues 7–12, 9–17 and 14–19. In addition, this C–terminal ring system is stabilised by a short antiparallel β sheet. A feature of the actagardine structure is the presence of two putative binding pockets. A pocket is generated by the covalent constraints of the C‐terminal thioether ring system. The rim of this pocket is built up by a loop structure comprising residues 12–19, whose backbone amide protons are all directed to the centre of the pocket. The second pocket is formed by an L‐shaped orientation of the N‐terminal and C‐terminal thioether ring systems. The only two hydrophilic amino acid residues of actagardine, Glu11 and Ser2, are directed to this pocket. A region of high sequence similarity with the related lantibiotic mersacidin is located exactly at the position of the second pocket (residues 3–12). This suggests that the second pocket is responsible for the antibiotic mode of action of actagardine and mersacidin as inhibitors of the murein biosynthesis of gram‐positive bacteria.
Inactivation of Tritrichomonas Foetus and Schistosoma Mansoni Purine Phosphoribosyltransferases by Arginine‐Specific Reagents
Authors: Kanaani Jamil , Maltby David , Somoza John R. , Wang Ching C.
Published: July 2004

The arginine‐specific reagents phenylglyoxal and butane‐2,3‐dione irreversibly inactivate the Tritrichomonas foetus hypoxanthine‐guanine‐xanthine phosphoribosyltransferase (HGXPRT) and Schistosoma mansoni hypoxanthine‐guanine phosphoribosyltransferase (HGPRT). The inactivation of the tritrichomonal enzyme by phenylglyoxal follows time‐dependent and concentration‐dependent pseudo‐first‐order kinetics. Complete protection against inactivation is afforded by the addition of 25 μM GMP, whereas 5‐phosphoribosyl‐1‐diphosphate (PRibPP) at 50–250 μM can only slow down the inactivation, without being protective. Digestion of [7−14C] phenylglyoxal‐modified enzyme with trypsin and separation of the peptides by reverse‐phase HPLC shows that only one radioactive peak is greatly diminished by incubation with 25 μM GMP or 1 mM PRibPP. Mass‐spectral analysis identifies Arg155 as the target site of two molecules of phenylglyoxal that is protected by the substrates. This amino acid residue is positioned next to Tyr156, which is a highly conserved aromatic residue among all the purine phosphoribosyltransferases (PRT) and is always found stacked on top of the purine substrate. This may explain why phenylglyoxal labeling of Arg155 inactivates the enzyme and why GMP can protect Arg155 more effectively than PRibPP. Among the purine PRT in our possession, only schistosomal HGPRT, the only other enzyme that contains an arginine residue at the corresponding location (Arg187), was susceptible to phenylglyoxal and butane‐2,3‐dione. The presence of Lys185‐Phe186 and Ser179‐Trp180 at the corresponding locations in human HGPRT and Giardia lamblia GPRT, respectively, may explain their resistance to phenylglyoxal. Thus, Arg155 in T. foetus HGXPRT and Arg187 in S. mansoni HGPRT will be attractive targets for future studies.
The Absence of the Mitochondrial ATP Synthase  Subunit Promotes a Slow Growth Phenotype of Rho− Yeast Cells by a Lack of Assembly of the Catalytic Sector F1
Authors: Giraud Marie‐France , Velours Jean
Published: July 2004

In the yeast Saccharomyces cerevisiae, inactivation of the gene encoding the  subunit of the ATP synthase led to a lack of assembly of the catalytic sector. In addition a slow‐growth phenotype was observed on fermentable medium. This alteration appears in strains lacking intact mitochondrial DNA and showing a defect in the assembly of the catalytic sector, such as the yeast strain inactivated in the gene encoding the ε subunit. In rho− mitochondria having an intact F1, the ion movement resulting from the exchange of ADP formed in the organelle and ATP entering the mitochondrial compartment led to a mitochondrial transmembranous potential Ψ that was sensitive to carboxyactractyloside. This ion movement was dramatically decreased in rho− mitochondria lacking the  subunit and thus the F1 sector, whereas a cell devoid of  subunit and complemented with a plasmid harboring the ATP gene displayed an assembled F1, a normal generation time and a fully restored mitochondrial potential. This result could be linked to the involvement of the membrane potential Ψ which is indispensible for mitochondrial biogenesis.
Structural Studies of the O‐Antigenic Polysaccharide of the Lipopolysaccharide from Acinetobacter (DNA Group 11) Strain 94 Containing 3‐amino‐3,6‐dideoxy‐d‐Galactose Substituted by the Previously Unknown Amide‐Linked l‐2‐acetoxypropionic Acid or l‐2‐hydroxypropionic Acid
Authors: Haseley Simon R. , Holst Otto , Brade Helmut
Published: July 2004

A polysaccharide containing D‐Gal, D‐GalNAc, 3‐(L‐2‐acetoxypropionamido)‐3,6‐dideoxy‐D‐galac‐tose (≈80%) and 3‐(L‐2‐hydroxypropionamido)‐3,6‐dideoxy‐D‐galactose (≈20%) was isolated by mild acid hydrolysis, followed by gel‐permeation chromatography, from the phenol‐soluble lipopolysaccharide (phenol/water extracted) derived from Acinetobacter strain 94. The polysaccharide, characterised by means of monosaccharide analyses, partial acid hydrolysis, and NMR studies, consisted of a branched tetrasaccharide repeating unit, as depicted below, in which Fucp3Nacyl represents 3‐(L‐2‐hydroxypropion‐amido)‐3,6‐dideoxy‐D‐galactose, in which approximately 80% of the acyl residues are O‐acetylated. These Fucp3N derivatives and an O‐acetylated acyl group are therefore constituents of bacterial LPS, but to our knowledge are not present in any other natural carbohydrates.
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Envelope Protein of Parasitic Wasp Symbiont Virus, Polydnavirus, Protects the Wasp Eggs from Cellular Immune Reactions by the Host Insect
Authors: Hayakawa Yoichi , Yazak Kazumori
Published: July 2004

Cotesia kariyai polydnavirus (CkPDV) virions are present in the oviducts of C. kariyai wasp and are injected with eggs into the hemocoel of the host armyworm Pseudaleda separata larvae during parasitization. Evidence that the presence of polydnavirus particles on the surface of the wasp eggs may be essential for prevention of cellular immune reactions by the host hemocytes was obtained by isolating an immunoevasive factor from CkPDV virions. The purified proteinaceous factor protects foreign materials from adhesion and encapsulation by hemocytes of the host P. separata larvae but hot by those of common cutworm Spodoptera litura larvae which is an incompatible host for the C. kariyai wasp. Purification procedures consisted of extraction with ethanol/trifluoroacetic acid and reverse‐phase high performance liquid chromatography. A factor with a molecular mass of approximately 50 kDa is demonstrated to be present on the envelope of CkPDV virion by immunoelectronmicroscopic observations. Furthermore, immunoreactive proteins are found in plasma of the armyworm larvae but not in the common cutworm larvae, indicating that only the natural host of C. kariyai wasp shares a similar epitope with CkPDV. The sequence of 23 amino acid residues at the amino terminus of the factor was determined to be Ile‐Ser‐Val‐Glu‐Asn‐Val‐Xaa‐Thr‐Thr‐Gly‐Ile‐Phe‐Leu‐Asp‐Ser‐Gly‐Glu‐Xaa‐Val‐Pro‐Tyr‐Ala‐Thr‐Lys‐Pro.
Cloning and Biochemical Characterization of an Anionic Peroxidase from Zea Mays
Authors: Teichmann Thomas , Guan Changhui , Kristoffersen Peter , Muster Gerhard , Tietz Olaf , Palme Klaus
Published: July 2004

We have isolated, cloned and characterized a cDNA from Zea mays L., denoted ZmAP1, coding for an anionic peroxidase. The open reading frame of ZmAP1 starting 72 residues from the 5′ end of the cDNA predicts a 37778 dalton protein of 356 amino acid residues. The protein has high similarity to other peroxidases and contains two peroxidase motifs that carry two highly conserved histidines in the active center. We expressed recombinant ZmAP1 protein in E. coli as a fusion with maltose‐binding protein. The fusion protein was biochemically active after addition of hemin to the apoprotein. The maize peroxidase ZmAP1 has a pH optimum at pH 4.0 and a km of 0.2 mM for the substrate 2,2′‐azinobis‐(3‐ethyl‐benzothiazolin‐6‐sulfonic acid) at this pH. In maize seedlings the ZmAP1 gene is expressed predominantly in roots, the mesocotyl, the coleoptile and to a lower extent in the node, whereas no expression in the primary leaf was found. In situ hybridization shows that the expression of ZinAPl in the young maize root is confined to the epidermis, hypodermis and the pericycle.
Relocation of Syk Protein‐Tyrosine Kinase to the Actin Filament Network and Subsequent Association with Fak
Authors: Sada Kiyonao , Minami Yasuhiro , Yamamura Hirohei
Published: July 2004

Previous studies demonstrated that Syk protein‐tyrosine kinase (Syk) is activated by thrombin in platelets. To elucidate the function of Syk in platelets, we have biochemically examined the intracellular location of Syk and the molecules associated with Syk, following platelet activation. In human platelets, thrombin induces the relocation of Syk to the cytoskeletal fraction presumably via Syk tyrosine phosphorylation. Relocated Syk is associated with the actin filament network, and the early phase (10–90 s) of this association can be partially inhibited by the pretreatment of platelets with cytochalasin D, an inhibitor of actin polymerization. Upon thrombin stimulation, Syk becomes associated with Fak as demonstrated by co‐immunoprecipitation. The association of both kinases can be inhibited by pretreatment of platelets with cytochalasin D. Interestingly, reconstitution experiments, using COS cells transfected with various porcine Syk mutants, revealed that the kinase domain, but not the kinase activity, of Syk is required for the association of Syk with the actin filament network. These findings suggest that thrombin‐induced association of Syk with Fak correlates with the state of actin polymerization, and may play an important role in platelet activation.
Purification and Functional Characterization of a Low‐Molecular‐Mass Na+ K+‐ATPase Inhibitor Protein from Rat Brain Cytosol
Authors: Bhattacharyya Dipankar , Sen Parimal C.
Published: July 2004

A number of low‐molecular mass (12–13 kDa) Na+, K+‐ATPase inhibitor proteins have been purified from rat brain cytosol by gel filtration followed by FPLC fractionation on a Mono Q anion‐exchange column. Eight peaks were obtained using 0.1 M NaCl eluent of which one peak was found to be the most potent inhibitor of Na+, K+‐ATPase. The molcular mass of the inhibitor was about 13 kDa on 16.5% SDS/PAGE. The concentration at which 50% inhibition (I50) was found was in the nanomolar range. The inhibitor seems to bind to Na+, K+‐ATPase at a site distal from the ATP‐binding site. The binding to the ATPase is non‐competitive. The CD analysis suggests an unordered secondary structural element. It also inhibits p‐nitrophenyl phosphatase activity from rat brain with comparable I50 value to that for Na+, K+‐ATPase. The protein does not contain any Trp as evident from Trp fluorescence and amino acid analysis. Amino acid analysis shows that glycine and serine, derivatives of tyrosine and phenylalanine are the predominant amino acids. The data suggests that it is a negatively charged protein in which the contribution of the hydrophobic part is 27%.
Characterization of Gelsolin Truncates that Inhibit Actin Depolymerization by Severing Activity of Gelsolin and Cofilin
Authors: Fujita Hisakazu , Allen Philip G. , Janmey Paul A. , Azuma Toshifumi , Kwiatkowski David J. , Stossel Thomas P. , Furu‐Uchi Keiji , Kuzumaki Noboru
Published: July 2004

Gelsolin is a calcium‐activated actin‐binding protein with six subdomains. The N‐terminal (G1) domain is essential for actin‐filament‐severing activity while other domains within G2–3 position the protein on the filament side allowing G1 to sever. In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin‐binding proteins, gelsolin and cofilin. A CA2+‐sensitive truncate containing G2–6 inhibited the F‐actin‐depolymerizing activities of both gelsolin and cofilin, while a G2–3 truncate was less effective. Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics. Cosedimentation assays in the presence of calcium demonstrate that the G2–6 truncate binds to F‐actin more strongly than the G2–3 truncate consistent with a protection mechanism by conformational change of F‐actin and/or competitive binding to actin filaments which depends upon the presence of actin filament binding domains.
Electron Transfer to Cytochrome P‐450scc Limits Cholesterol‐Side‐Chain‐Cleavage Activity in the Human Placenta
Authors: Tuckey Robert C. , Woods Stephen T. , Tajbakhsh Mayamin
Published: July 2004

The aim of this study was to determine whether electron transfer from adrenodoxin reductase and adrenodoxin limits the activity of cytochrome P‐450scc in mitochondria from the human placenta. Mitochondria were disrupted by sonication to enable exogenous adrenodoxin and adrenodoxin reductase to deliver electrons to cytochrome P‐450scc. After sonication, the rate of pregnenolone synthesis was greatly decreased relative to that by intact mitochondria, due to dilution of endogenous adrenodoxin and adrenodoxin reductase into the incubation medium. The addition of saturating concentrations of bovine or human adrenodoxin and bovine adrenodoxin reductase to the disrupted mitochondria gave an initial rate of pregnenolone synthesis that was 6.3‐fold higher than that for intact mitochondria. Similar results were observed when 20α‐hydroxycholesterol was used as substrate rather than endogenous cholesterol. The turnover number of cytochrome P‐450scc in sonicated placental mitochondria supplemented with adrenodoxin and adrenodoxin reductase was comparable to that for the purified enzyme assayed under conditions where electron transfer was not limiting. Addition of exogenous adrenodoxin and adrenodoxin reductase to sonicated mitochondria from the pig corpus luteum and rat adrenal had a much smaller effect on pregnenolone synthesis compared with intact mitochondria, than observed for the placenta. We conclude that in the human placenta, electron transfer to cytochrome P‐450scc is limiting, permitting pregnenolone synthesis to proceed at only 16% maximum velocity.
Benzoyl‐CoA Reductase (Dearomatizing), A Key Enzyme of Anaerobic Aromatic Metabolism
Authors: Boll Matthias , Albracht Simon S. P. , Fuchs Georg
Published: July 2004

An enzyme was recently described, benzoyl‐CoA reductase (dearomatizing), which catalyses the ATP‐driven reduction of the aromatic ring of benzoyl‐CoA yielding a non‐aromatic CoA thioester, ADP and phosphate [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921–933]. The 170‐kDa enzyme consists of four different subunits and contains approximately 12 Fe and acid‐labile sulfur/mol. Benzoyl‐CoA reductase exhibits ATPase activity in the absence of substrate. It is shown that only the reduced form of this iron‐sulfur protein has ATPase activity. ATPase activity is reversibly lost when the enzyme is oxidized by thionine; reduction of the enzyme fully restores ATPase and ring‐reduction activity. 2 mol ATP are hydrolyzed/2 mol electrons transferred in the course of the reaction. The product ADP acts as competitive inhibitor (Ki= 1.1 mM) for ATP in benzoyl‐CoA reduction; ADP inhibits ATPase activity to the same extent as ring‐reduction activity. EPR investigation of the dithionite‐reduced enzyme suggested the presence of two separate [2Fe‐2S] clusters and two interacting [4Fe‐4S] clusters. Addition of MgATP to the reduced enzyme resulted in a new isotropic signal at g= 5.15 and a weak signal at g= 12; in controls with MgADP only a minor signal at g= 5.15 was observed. The positions, shapes and temperature dependencies of these MgATP‐induced signals are indicative for excited states of a S= 7/2 spin multiplet. The [2Fe‐2S] signals were not affected by ATP, but one of the [4Fe‐4S] clusters became slowly oxidized. Addition of both benzoyl‐CoA and MgATP resulted in a major oxidation of the iron‐sulfur clusters accompanied by the appearance of some minor signals of unknown origin in the g= 2.037 ‐ 1.96 region. Neither the benzoyl‐CoA plus MgATP‐oxidized nor the thionine‐oxidized enzyme showed the ATP‐dependent formation of the high‐spin signals of the reduced enzyme. At present we hypothesize that the S= 7/2 signal is due to an ATP‐induced change of one of the [4Fe‐4S] clusters.

The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates. MgATP‐driven formation of this excited state of the reduced enzyme rather than transfer of electrons from the reduced enzyme to the aromatic substrate appears to be the rate‐limiting step in the catalytic cycle. We suggest that the excited state is required to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible.