bluuebunny/crossref_metadata_2025 huggingface dataset:
Mössbauer studies on iron(II)‐substituted yeast metallothionein
Authors: DING Xiao‐Qi , BILL Eckhard , TRAUTWEIN Alfred Xaver , HARTMANN Hans‐Jürgen , WESER Ulrich
Published: March 2005

Iron(II)‐substituted yeast metallothionein has been studied with Mössbauer spectroscopy. The iron in the protein is in the high‐spin ferrous state. A maximum metal content of four iron(II)/molecule has been determined, with the four metal ions forming a diamagnetic cluster due to the antiferromagnetic exchange interaction between Fe2+ via bridging thiolates. In the case where the iron titration gives a value of less than four iron(II)/apoprotein, the metal ions are magnetically non‐interacting, with each individual iron(II) behaving like iron(II) in reduced rubredoxin.
Deletion variants of l‐hydroxyisocaproate dehydrogenase
Authors: FEIL Ingeborg K. , LERCH Hans Philipp , SCHOMBURG Dietmar
Published: March 2005

The substrate specificity and catalytic activity of the dinucleotide‐dependent l‐2‐hydroxyisocaproate dehydrogenase from Lactobacillus confusus (l‐HicDH) have been altered by modifying an enzyme region which is assumed to be involved in substrate recognition. The design of the variant enzymes was based on an amino acid alignment of the modified region with the functionally related l‐lactate dehydrogenases. The best absolute sequence similarity for a protein with known tertiary structure was found for l‐lactate dehydrogenase from dogfish (23%). In this study, the coenzyme loop, a functional element which is essential for catalysis and substrate specificity, was modified in order to identify the residues involved in the catalytic reaction and observe the effect on the substrate specificity. Deletions were introduced into the l‐hydroxyisocaproate gene by site‐directed mutagenesis. Several deletion‐variant enzymes Ile100AΔ, Lys100BΔ, Leu101Δ, Asn105AΔ and Pro105BΔ showed an altered substrate specificity. For the variant enzyme with the deletion of Asn/Pro105A/B, 2‐oxo carboxylic acids branched at C4 proved to be better substrates than 2‐oxocaproate, the substrate with the best kcat/KM ratio known for the wild‐type enzyme. The mutation resulted in a 5.2‐fold increased catalytic efficiency towards 2‐oxoisocaproate compared to the wild‐type enzyme. After deleting Ile/Lys100A/B, 2‐phenylpyruvate is the only substrate which is still converted at a significant catalytic rate. The Kcat ratios of 2‐oxocaproate versus 2‐phenylpyruvate changed by a factor of 6500 when comparing wild‐type enzyme and deletion‐variant enzyme data. The single amino acid deletions in position 100A and 100B caused drastic reductions in the catalytic activity for all tested substrates, whereas the deletion of Lys100B, Leu101, Asn105A as well as Pro105B showed more specific modifications in catalytic rates and substrate recognition for each tested substrate.
Lipid modulation of glycolipid receptor function
Authors: BOYD Beth , MAGNUSSON Göran , ZHIUYAN Zhang , LINGWOOD Clifford A.
Published: March 2005

Verotoxins bind to glycosphingolipids containing terminal Gal(α1‐4)Gal residues. Globotriaosylceramide is the most effective receptor for verotoxin‐1 in vitro and is the functional plasma‐membrane receptor which mediates cytopathology for most sensitive cells. Binding of verotoxin‐1 to a series of galabiose‐containing or globotriaose‐containing synthetic glycolipids with monoalkylsulfides and bisalkylsulfides or sulfones as the lipid moiety, have been studied for toxin binding by TLC overlay and in solid phase in the presence of auxiliary lipids. The results demonstrate that for an identical carbohydrate, binding is dramatically altered according to the nature of the lipid moiety. The close proximity of the galabiose sequence and the hydrophobic species also compromised recognition.

The lipid environment is also a major determinant of receptor function, since species that were effective, even preferred toxin receptors as monitored by TLC overlay, were not necessarily recognized in the presence of auxiliary lipids. Certain glycolipids, which were not recognized by TLC overlay, were nevertheless found to be effective receptors in an auxiliary lipid matrix.

These results demonstrate the crucial role of the lipid moiety in verotoxin/glycolipid recognition and are discussed in relation to toxin pathogenesis and glycolipid receptor function.
Cartilage oligomeric matrix protein and thrombospondin 1
Authors: DiCESARE Paul E. , MÖRGELIN Matthias , MANN Karlheinz , PAULSSON Mats
Published: March 2005

Cartilage oligomeric matrix protein (COMP) and thrombospondin 1 (TSP1) were purified in a native form from normal bovine articular cartilage. The key step in the purification scheme was selective extraction with EDTA‐containing buffer. Final separation of these two molecules was achieved by heparin affinity chromatography. Particles viewed by electron microscopy after rotary shadowing and negative staining revealed structures similar to their prototype molecules; from the Swarm rat chondrosarcoma for COMP, or from platelets for TSP1. Attachment of primary bovine chondrocytes to purified matrix proteins was investigated. Cells attached to COMP but not to the structurally related TSP1 indicating separate functions for these proteins in cartilage.
High affinity of ergopeptides for cytochromes P450 3A
Authors: PEYRONNEAU Marie‐Anne , DELAFORGE Marcel , RIVIERE René , RENAUD Jean‐Paul , MANSUY Daniel
Published: March 2005

The interaction between rat and human liver cytochromes P450 with a series of lysergic acid derivatives and ergopeptide alkaloids was studied by difference visible spectroscopy. Ergopeptides, like bromocriptine, ergocryptine and dihydroergotamine, strongly interacted with rat liver microsomes with the appearance of a difference spectrum which is characteristic of their binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P450s 3A in the microsomes and was at its maximum in dexamethasone‐treated rat microsomes. All the ergopeptides studied exhibited a high affinity for rat P450s 3A (Ks around 1 μM), although lysergic acid derivatives not bearing the tripeptide moiety failed to give significant interactions with these P450s. A cyclic azatripeptide exhibiting a structure very similar to that of the tripeptide moiety of ergopeptides also interacted with P450s 3A with appearance of an intense type I difference spectrum. Very similar results were observed with two allelic forms of human liver P450 3A4, P450 NF25 and P450 hPCN1, produced in yeast. In both cases all the ergopeptides studied showed high affinities for the P450s (Ks 0.6–2.2 μM) and an intense shift from the low‐spin to the high‐spin state upon substrate binding (60–100% spin shift). Lysergic acid derivatives not bearing the tripeptide group of ergopeptides also completely failed to interact with P450s 3A4.

Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P450 3A, were found to be particularly active for the hydroxylation of bromocriptine, which occurs at the level of its tripeptide moiety. Human liver microsomes as well as P450 NF25 and P450 hPCN1 also exhibited a high activity for bromocriptine hydroxylation at this level.

These results show that ergopeptides exhibit a particularly high affinity for P450s of the 3A subfamily. The tripeptide moiety of ergopeptides is essential for their recognition by P450s 3A and binds at a site close to P450 heme, producing type‐I difference spectra. Accordingly, at least one of the studied ergopeptides, bromocriptine, is hydroxylated by P450s 3A at the proline ring of the cyclopeptide moiety. As cyclosporine is known to be a good substrate of P450s 3A, these results suggest that P450s 3A may be especially prone in a general manner to recognize and oxidize peptides or pseudopeptides.
Isolation and characterization of new phosphatidylglycerol acetals of plasmalogens
Authors: JOHNSTON Norah C. , GOLDFINE Howard
Published: March 2005

A new phosphatidylglycerol acetal of cardiolipin plasmalogen has been isolated from Clostridium innocuum. The structure was derived from the results of quantitative group analyses, the identification of the products of acid hydrolysis, alkaline methanolysis, hydrolysis by a cardiolipin‐specific phospholipase D and by one‐ and two‐dimensional proton NMR. Two other minor ether phospholipids: the lyso form of the phosphatidylglycerol acetal of cardiolipin plasmalogen, and the phosphatidylglycerol acetal of plasmenylglycerol have been identified in C. innocuum lipid extracts.
Various vectorial behaviours of a spatially structured substrate cycle
Authors: THIÉBART‐FASSY Isabelle , HERVAGAULT Jean‐François
Published: March 2005

The dynamic properties of the glucose/glucose‐6‐phosphate cycle are studied under conditions where the phosphatase and kinase interconverting enzymes are spatially distributed. A semi‐artificial membrane made of compacted plant cell walls bearing active phosphatase in its natural state, separates two compartments, one of these compartments containing soluble hexokinase. Depending only upon the two enzyme activity levels and the initial distribution of the substrates, numerous asymmetrical and vectorial behaviours can be observed, such as facilitated glucose 6‐phosphate diffusion, active transport of either glucose or glucose 6‐phosphate and sequential (alternative) transport between glucose and glucose 6‐phosphate. A diffusion‐partition reaction coupling can account for these oriented mass transfers. The possibility of such a coupling in this model system is clearly dictated by the global analog of the Curie principle. These results may provide new insight on (a) the still obscure role played by the cell‐wall phosphatase activities, particularly their involvement in the transport of exogenous phosphomonoesters, and (b) the actual in vivo operation of substrate and protein cycles in view of the heterogeneity and anisotropy of the cellular milieu.
Genomic organization of the human catechol O‐methyltransferase gene and its expression from two distinct promoters
Authors: TENHUNEN Jukka , SALMINEN Marjo , LUNDSTRÖM Kenneth , KIVILUOTO Tuula , SAVOLAINEN Raija , ULMANEN Ismo
Published: March 2005

Human genomic DNA fragments containing catechol O‐methyltransferase (COMT) sequences were isolated and the exon‐intron structure analysed by sequencing, PCR and comparing to the human COMT cDNA sequences. The gene contains six exons, of which exons 1 and 2 are non‐coding. MB‐ATG and S‐ATG codons, responsible for the initiation of translation of the membrane‐bound (MB) and soluble (S) forms of the enzyme, are located in exon 3. Two distinct COMT‐specific transcripts, 1.3 kb and 1.5 kb, were detected in various human tissues and cell lines. Different quantities of the shorter COMT‐specific mRNA in the tissues studied suggest a tissue‐specific regulation of the COMT gene at transcriptional level. Mapping of the 5′ ends of the COMT mRNAs showed that transcription initiates at multiple sites in two separate DNA regions, which are preceded by functional promoter sequences. The proximal promoter (P1), located between the two translation initiation codons and extending approximately 200 bp upstream of the MB‐ATG initiation codon, apparently gives rise to the 1.3‐kb S‐COMT mRNA (S‐mRNA). The distal promoter (P2) is located in a DNA fragment in front of and partly overlapping the transcription‐start region of the 1.5‐kb transcript, suggesting that it controls the expression of this MB‐mRNA. Similarities between the rat and human COMT gene promoters are analyzed.
Inhibition and catalysis of carbonic anhydrase
Authors: LILJAS Anders , HÅKANSSON Kjell , JONSSON Bengt Harald , XUE Yafeng
Published: March 2005

CONTENTS–Sulfonamide inhibitors–Metal coordination in carbonic anhydrase–Binding of anions–Regular tetra‐coordination–Distorted tetra‐coordination–Penta‐coordination–Uncoordinated–Mutations of the doorkeeper function–Substrate binding–The catalytic mechanism
Cloning and expression of three abrin A‐chains and their mutants derived by site‐specific mutagenesisin Escherichia coli
Authors: HUNG Chih‐Hung , LEE Ming‐Chih , CHEN Jeen‐Kuan , LIN Jung‐Yaw
Published: March 2005

DNAs encoding of three abrin A‐chains were obtained from the cDNA library of Abrus precatorius by polymerase chain reaction and ligated into the expression vector, pGEX‐2T. The mature A‐chains of abrins a, b and d have been expressed in the cytoplasm of Escherichia coli, and the yield of the soluble recombinant proteins was 7 mg/l induced culture. Three recombinant abrin A‐chains were purified to be homogeneity and their N‐glycosidase ability to inhibit protein biosynthesis in a cell‐free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The recombinant abrin‐a A‐chain had the highest N‐glycosidase activity among three recombinant abrin A‐chains while the recombinant abrin‐b A‐chain, the least.

Three mutants, glutamic‐acid‐to‐alanine replacement (E164A), arginine to leucine (R167L) or double mutation (E164A and R167L) were constructed and expressed. The protein‐biosynthesis‐inhibitory activity of mutant (E164A), mutant (R167L) and the double mutant was found to be 25‐fold, 625‐fold and 1250‐fold lower than that of wild type, respectively. The results indicated that Arg167 was essential for abrin toxin A‐chain catalysis.
Solution structure of synthetic peptides corresponding to the C‐terminal helix of interleukin‐6
Authors: MORTON Craig J. , SIMPSON Richard. J. , NORTON Raymond S.
Published: March 2005

Two synthetic peptides corresponding to the C‐terminal 19 residues of human and murine interleukin‐6, respectively, have been synthesized and their structures in solution investigated using high‐resolution 1H‐NMR spectroscopy. Both peptides show a marked dependence of chemical‐shift dispersion on pH, with a greater degree of structure apparent above pH 4.5, where their glutamate carboxyl groups are ionised. In purely aqueous solution, neither peptide adopts a well‐defined structure, although the murine peptide has characteristics of a nascent helix. Titration of the murine peptide with trifluoroethanol produced a significant increase in structure, which was then investigated using two‐dimensional NMR. In 50% (by vol.) trifluoroethanol the murine peptide consists of a well‐defined central helix of 12 residues with unstructured N‐terminal and C‐terminal regions. These observations lend experimental support to the current model of the interleukin‐6 structure, which proposes a four‐helical bundle with the last helix encompassing the C‐terminal 20–30 residues. Furthermore, the fact that synthetic peptides corresponding to part of the putative receptor‐binding surface of interleukin‐6 are able to adopt a similar conformation in solution to that proposed for the intact protein suggests that such peptide analogues should be useful starting points in the design of peptide agonists and antagonists of interleukin‐6.
Quantitative stoichiometry of the proteins of the stimulatory arm of the adenylyl cyclase cascade in neuroblastoma x glioma hybrid, NG108‐15 cells
Authors: KIM Gun‐Do , ADIE Elaine J. , MILLIGAN Graeme
Published: March 2005

To understand the details of regulation of guanine‐nucleotide‐binding‐protein‐linked transmembrane cellular‐signalling cascades, it is important to know the absolute levels of each polypeptide component and the stoichiometry of their interactions. Amounts of the IP prostanoid receptor, the stimulatory G protein of the adenylyl cyclase cascade (Gsα) and the functional complex of Gsα with adenylyl cyclase, which acts as the cyclic AMP generator, were measured in membranes of neuroblastoma x glioma hybrid, NG108‐15, cells. As measured by the specific binding of [3H]prostaglandin E1, the IP prostanoid receptor was present in some 100000 copies/cell. Gsα assessed by quantitative immunoblotting with recombinantly expressed protein, was present in considerably higher levels (1250000 copies/cell). However, the maximal formation of a complex of Gsα and adenylyl cyclase represented only some 17500 copies/cell. The previously established 8:1 stoichiometry of concurrent downregulation of Gsα and the IP prostanoid receptor in these cells [Adie, E. J., Mullaney, I., McKenzie, F. R. & Milligan, G. (1992) Biochem. J. 285, 529–536] indicates that full‐agonist occupation of the receptor should be able to activate some 65% of the expressed Gs. Despite the potential 70‐fold excess of Gsα over the Gsα/adenylyl cyclase complex, IP prostanoid‐receptor‐agonist‐mediated reduction of Gsα levels by some 35% resulted in a 25% reduction in the maximal formation of the Gsα/adenylyl cyclase complex. Such results demonstrate that adenylyl cyclase is quantitatively the least highly expressed component of this signalling cascade and suggests that much of the cellular Gsα may not have access to adenylyl cyclase.
Trp279 is involved in the binding of quaternary ammonium at the peripheral site of Torpedo marmorata acetylcholinesterase
Authors: SCHALK Isabelle , EHRET‐SABATIER Laurence , BOUET Françoise , GOELDNER Maurice , HIRTH Christian
Published: March 2005

Specific photoaffinity labelling of purified acetylcholinesterase from Torpedo marmorata by p‐N,N‐[3H]dimethylamino benzenediazonium and p‐N,N‐[3H]dibutylamino benzenediazonium derivatives was demonstrated. This occured at the active site of the enzyme for lower concentrations of the probes and at the peripheral ammonium binding site for higher concentrations. The affinities and the rate constants of alkylation for each probe on both sites have been established. Specific labelling at the peripheral site of the enzyme with both probes allowed the identification of radio‐labelled peptides having the common sequence K270PQELIDVEW. The radioactivity was always associated with the residue Trp279 indicating the preferential ammonium complexation with this aromatic residue.
Low‐frequency stimulation of rat fast‐twitch muscle enhances the expression of hexokinase II and both the translocation and expression of glucose transporter 4 (GLUT‐4)
Authors: HOFMANN Sabine , PETTE Dirk
Published: March 2005

This study followed changes in the capacities of uptake and phosphorylation of glucose in response to contractile activity in low‐frequency stimulated (10Hz, 24 h/d) rat fast‐twitch muscle. We investigated the intracellular distribution of GLUT‐4, the major glucose transporter isoform in muscle, changes in the amounts of its specific mRNA and total cellular protein, as well as changes in its relative synthesis rate. These analyses were complemented by measurements of total hexokinase activity and hexokinase II (HKII) expression at the levels of mRNA content and protein synthesis. Changes in protein synthesis were determined by in vivo labeling with [35S]methionine. Translocation of GLUT‐4 into the sarcolemma was an immediate response to contractile activity, whereas changes in its total amount were observed only with ongoing stimulation (5 d and longer). A twofold increase in GLUT‐4 content after 5 d and longer stimulation periods was preceded by elevations of its mRNA and by enhanced [35S]methionine incorporation. Conversely, increases in HKII expression with a rise in total hexokinase activity occurred soon after the onset of stimulation (30‐fold elevations of HKII mRNA after 12 h and 20‐fold increases in [35S]methionine incorporation after 24 h). With ongoing stimulation, HKII mRNA and synthesis returned to lower levels (fivefold elevations). Nevertheless, hexokinase activity continued to rise, stabilizing at fivefold‐elevated levels after 3 d. These observation suggested that posttranscriptional mechanisms contributed to the upregulation of HKII, e.g. stabilization by elevated intracellular glucose and mitochondrial binding of the enzyme. This suggestion was supported by experiments with cessation after 24 h where hexokinase activity continued to increase, although the mRNA content and, especially, the [35S]methionine incorporation decayed steeply. The increase in HKII prior to GLUT‐4 suggests that phosphorylation may be rate limiting in glucose utilization of glycolytic fibers under conditions of sustained contractile activity. Taken together, the changes in distribution and content of GLUT‐4, as well as in HKII represent early metabolic adaptations. In addition, they are related to the overall process of stimulation‐induced fiber type transformation.
Contribution of phospholipases A2 and D to arachidonic acid liberation and prostaglandin D2 formation with increase in intracellular Ca2+ concentration in rat peritoneal mast cells
Authors: ISHIMOTO Tsuyoshi , AKIBA Satoshi , SATO Takashi , FUJII Tatsuzo
Published: March 2005

The contribution of phospholipases A2 (PLA2) and D (PLD) activation to arachidonic acid liberation and prostaglandin D2 (PGD2) formation was studied in stimulated rat peritoneal mast cells. Stimulation of the cells with ionomycin induced time‐dependent and Ca2+‐concentration‐dependent increase in arachidonic acid liberation and PGD2 formation, and the Ca2+‐dependent increase was especially remarkable at extracellular Ca2+ concentration higher than 200 μM. Staurosporine did not induce any effect on the arachidonic acid liberation, indicating that protein kinase C is not involved in the liberation. Addition of ethanol to the cells decreased the ionomycin‐stimulated arachidonic acid liberation to 40% of the control, while it decreased the PGD2 formation almost completely, with the increase in phosphatidylethanol formation. Propranolol, a phosphatidate phosphohydrolase inhibitor, caused similar effects. p‐Bromophenacyl bromide, a PLA2 inhibitor, inhibited partially the arachidonic acid liberation. The inhibition of the liberation by combination of p‐bromophenacyl bromide and ethanol was additive and reached approximately 90%. Under the conditions used p‐bromophenacyl bromide did not influence significantly the PLD activity assessed by the phosphatidylethanol formation. Histamine release was decreased by ethanol treatment to 35% of the control. These results suggest that more than half of the total arachidonic acid liberation is mediated by the sequential pathway of PLD/phosphatidate phosphohydrolase/diacylglycerol lipase and more than half of histamine release is also dependent on PLD activation, while the PGD2 formation is fully mediated by the pathway. PLA2 also contributes to arachidonic acid liberation but to a lower extent.
Hepatocyte growth factor elevates the activity levels fo glycolipid sulfotransferases in renal cell carcinoma cells
Authors: KOBAYASHI Takahiko , HONKE Koichi , GASA Shinsei , MIYAZAKI Tamotsu , TAJIMA Hisao , MATSUMOTO Kunio , NAKAMURA Toshikazu , MAKITA Akira
Published: March 2005

Accumulation of sulfoglycolipids associated with markedly elevated activity levels of glycolipid sulfotransferases has previously been demonstrated in the human renal cell carcinoma cell line, SMKT‐R3. To elucidate the regulatory mechanisms of sulfoglycolipid synthesis in SMKT‐R3 cells, the effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated. Hepatocyte growth factor (HGF) significantly increased the activity levels of the sulfotransferases in a dose‐dependent manner, but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. Scatchard analysis of 125I‐HGF binding to SMKT‐R3 cells indicated that the cells expressed high‐affinity receptors for HGF with a Kd of 36 pM and 750 sites/cell. Furthermore, metabolic labeling with [35S]sulfate revealed that the addition of HGF to the culture medium of the cells resulted in an increment of sulfoglycolipid synthesis. Therefore, these observations suggest that HGF can function as a regulatory factor in sulfoglycolipid synthesis through the modulation of the sulfotransferase activity levels in renal cell carcinoma cells. In addition, HGF stimulated the proliferation and motility of SMKT‐R3 cells, suggesting that HGF has multiple biological activities in renal cell carcinoma cells.
Mechanisms involved in serum‐dependent inactivation of the immunotoxin enhancers monensin and carrier‐protein—monensin
Authors: FRANCESCHI Antonia , DOSIO Franco , ANSELMI Cristina , CHIGNOLA Roberto , CANDIANI Carola , PASTI Marcella , TRIDENTE Giuseppe , COLOMBATTI Marco
Published: March 2005

The immunotoxin‐enhancing properties of monensin and of human‐serum‐albumin—monensin conjugates are severely impaired in the presence of human serum. In this study we have therefore investigated the interaction between serum proteins and monensin leading to the inactivation of monensin function as immunotoxin potentiator. We found that the binding of monensin‐specific mAb to thioether‐cross‐linked or disulfide‐cross‐linked protein‐monensin conjugates is negatively affected by serum, as indicated by immunoenzymic (ELISA) and radioimmunobinding analysis. Size‐exclusion chromatography of serum samples indicated that the greatest blocking effect is due to protein components of 40–90 kDa eluting as a broad peak (peak 4). Analysis of the proteins contained within peak 4 by ion‐exchange chromatography followed by microsequencing revealed that the major components of peak no. 4 were transferrin, human serum albumin and immunoglobulin fragments. Investigations on the nature of the interactions between serum proteins and monensin leading to monensin inactivation were conducted by affinity chromatography of serum on immobilized human‐serum‐albumin—monensin conjugates, size‐exclusion chromatography, SDS/PAGE analysis of serum‐treated human‐serum‐albumin—monensin conjugates, and evaluation of the stability of immobilized human‐serum‐albumin‐bound 125I‐monensin following treatment with serum. Addition of esterase inhibitors (e.g. EDTA, 4‐nitrophenyl phosphate) or prior treatment of the serum at 56°C partially reversed the serum effects observed. We conclude that serum proteins block the immunotoxin‐enhancing effect of monensin and of human‐serum‐albumin—monensin conjugates by multiple mechanisms involving hydrophobic and covalent interactions and enzyme‐mediated cleavage of protein‐bound monensin.
Expression and characterization of recombinant human and rat liver 6‐pyruvoyl tetrahydropterin synthase
Authors: BÜRGISSER Daniel M. , THÖNY Beat , REDWEIK Udo , HUNZIKER Peter , HEIZMANN Claus W. , BLAU Nenad
Published: March 2005

6‐Pyruvoyl‐tetrahydropterin synthase is the rate‐limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. The human and rat liver cDNAs encoding the 16‐kDa subunit of 6‐pyruvoyl tetrahydropterin synthase were expressed as maltose‐binding–6‐pyruvoyl‐tetrahydropterin‐synthase fusion proteins. After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity. Apparent Km for the substrate dihydroneopterin triphosphate (8.5 μM for the human and 8.0 μM for the rat enzyme), pI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes. The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification. The modification of the only cysteine residue in rat 6‐pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80%. Modification under non‐reducing conditions of both cysteine residues of the human enzyme by N‐ethylpyridine resulted in a 95% loss of enzyme activity. This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis. Cross‐linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits.
Purification and characterization of erythrocyte caldesmon
Authors: DER TERROSSIAN E. , DEPRETTE C. , LEBBAR I. , CASSOLY R.
Published: March 2005

We have previously shown that in human or pig whole erythrocytes, only a single 71‐kDa polypeptide cross‐reacts with the affinity‐purified antibody to pig platelet caldesmon (der Terrossian et al., 1989). In the present paper, we demonstrate that this polypeptide represents a genuine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreactivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other components of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regulate platelet actin‐activated rabbit skeletal muscle myosin ATPase activity. The stoichiometry of 1 caldesmon/1 tropomyosin/7–9 actin molecules indicates that the amount of caldesmon, in the red cell membrane, corresponds precisely to the amount of tropomyosin. Immunofluorescent labelling of whole erythrocytes gave similar punctate patterns with purified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. Together, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhibitory component, so far uncharacterized, of a thinfilament‐like system in erythrocyte.
Cloning and characterization of the gene for the catalytic subunit of cAMP‐dependent protein kinase in the aquatic fungus Blastocladiella emersonii
Authors: DE OLIVEIRA Julio Cezar FRANCO , CANTISANI BORGES Ana Cláudia , VALLE MARQUES Marilis do , LOPES GOMES Suely
Published: March 2005

We have isolated and characterized cDNA and genomic DNA clones encoding the catalytic subunit (C) of cAMP‐dependent protein kinase in the aquatic fungus Blastocladiella emersonii. The C‐subunit amino acid sequence derived from the nucleotide sequence predicts a basic polypeptide of 424 residues, excluding the initiator methionine, which by amino‐terminal sequence analysis has been shown to be absent from the mature protein. The Blastocladiella C presents a 70‐amino‐acid extension at the amino terminus, when aligned to the mouse Cα subunit, being one of the largest C subunits already characterized. The B. emersonii C‐gene‐coding region is interrupted by three introns, ranging in size over 57–69 bp. The positions of the introns are quite different from those found in other species, suggesting a considerable amount of evolutionary drift in the gene structure. The 5′‐flanking region lacks recognizable TATA or CCAAT sequences, is remarkably high in GC content (70%), and primer extension experiments indicate that transcription initiates from multiple sites. Several sequence motifs were identified in the promoter region which could be involved in the developmental control of this gene.
Proteinase yscD (oligopeptidase yscD)
Authors: BÜCHLER Markus , TISLJAR Ursula , WOLF Dieter H.
Published: March 2005

The yeast PRD1 gene, encoding proteinase yscD, was cloned by complementation of the prd1‐6 point mutation. Sequencing of the gene revealed an open reading frame of 2.136 kb, encoding a protein of 712 amino acids with a calculated molecular mass of 81.8 kDa. The sequence HEGLG beginning at residue 501 represents the HEXXH motif, unique for the zinc metallo‐peptidases. Sequence comparison revealed complete identity of the proteinase yscD gene with a recently published open reading frame of yeast chromosome III. We found 34.8% identity between proteinase yscD and rat metalloendopeptidase (thimet oligopeptidase, EP 24.15). Proteinase yscD hydrolyzes several chromogenic and fluorogenic peptides that are substrates of thimet oligopeptidase. N‐[1‐(RS)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Phe‐p‐aminobenzoic acid, a compound designed as specific inhibitor of EP 24.15, is also a strong inhibitor of the yeast enzyme. Proteinase yscD is a non‐vacuolar enzyme. 3–5% of the total enzyme activity can be detected in the intermembrane space of mitochondria. In a mutant carrying a deletion of the PRD1 gene no proteinase yscD activity is detectable in the cytoplasm and in mitochondria of these cells. They do not show any grossly altered phenotype but exhibit a decrease in the intracellular degradation of peptides. This suggests a function of proteinase yscD in the late stages of protein degradation.
Secretion, purification and characterization of a soluble form of the yeast KEX1‐encoded protein from insect‐cell cultures
Authors: LATCHINIAN‐SADEK Lilian , THOMAS David Y.
Published: March 2005

The Saccharomyces cerevisiae KEX1 gene encodes a carboxypeptidase involved in the C‐terminal processing of the lysine and arginine residues from the precursors of K1 and K2 killer toxins and α‐factor (mating pheromone). In order to produce large quantities of this unique carboxypeptidase for structural studies, a functional soluble form was obtained by deleting 224 amino acids from the C‐terminus of the KEX1‐encoded protein which includes a putative membrane‐spanning domain. The resulting truncated KEX1 gene (KEX1Δ) has been expressed in the baculovirus/insect cell system. The protein (Kex1Δp) is efficiently secreted into the culture medium and was purified to apparent homogeneity with a yield of approximately 4 mg/l culture. Kex1Δp is a glycoprotein with a molecular mass of 56 kDa, its N‐terminal sequence is identical to that of the full‐length membrane‐associated form of the enzyme [Latchinian‐Sadek, L. & Thomas, D. Y. (1993) J. Biol. Chem. 268, 534–540], and like the full‐length enzyme it is not made as a proenzyme. For the soluble enzyme form, the optimum pH for activity was 5.5–6.0, and the apparent pI value of the protein determined by isoelectric focusing was 4.2. The enzyme cleaves arginine from the C‐terminus of the synthetic peptide benzoyl‐Phe‐Ala‐Arg with Km 335 μM and Vmax 282 μmol · min−1· mg protein−1. Insect‐cell‐derived Kex1Δp processes α‐factor‐Lys‐Arg, a known natural substrate, to mature active α‐factor in a manner similar to the membrane‐associated full‐length enzyme. This secreted form of the enzyme is a convenient source for the isolation of substantial quantities of the pure enzyme for detailed kinetic and structural studies.
The structure of the O‐specific polysaccharide of Citrobacter O16 containing glycerol phosphate
Authors: KOCHAROVA Nina A. , THOMAS‐OATES Jane E. , KNIREL Yurity A. , SHASHKOV Alexander S. , DABROWSKI Ursula , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , KHOLODKOVA Elena V.
Published: March 2005

The O‐specific polysaccharide, obtained by mild acid degradation of Citrobacter O16 lipopolysaccharide, consists of D‐glucose, D‐galactose, 2‐acetamido‐2‐deoxy‐D‐galactose, glycerol and phosphate in the ratios 2:2:2:1:1. Selective cleavage of the polysaccharide was carried out by Smith degradation, N‐deacetylation‐deamination and dephosphorylation with 48% hydrofluoric acid, which was accompanied by unexpected splitting of one of the glycosidic linkages. The structures of the oligosaccharides thus obtained were established using 1H‐ and 13C‐NMR spectroscopy, including one‐dimensional NOE, two‐dimensional rotating‐frame NOE, homonuclear and heteronuclear 13C,1H correlation spectroscopy, and, for the Smith degradation product, positive‐ and negative‐ion‐mode fast‐atom‐bombardment MS and MS/MS with collision‐induced dissociation. On the basis of these data and the results of methylation analysis, it was concluded that the O‐specific polysaccharide has the following repeating unit structure:

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The derivation of the formyl‐group oxygen of chlorophyll b in higher plants from molecular oxygen
Authors: PORRA Robert J. , SCHÄFER Wolfram , CMIEL Edmund , KATHEDER Ingrid , SCHEER Hugo
Published: March 2005

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7‐formyl‐group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark‐grown plants, by illumination under white light in the presence of either H218O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7‐formyl substituent by reversible formation of chlorophyll b‐71‐gem‐diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process.

Mass spectra of chlorophyll a and [7‐hydroxymethyl]‐chlorophyll b extracted from leaves greened in the presence of either H218O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7‐formyl group of chlorophyll b but also for the well‐documented incorporation into the 131‐oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599–602]. The incorporation of isotope led to as much as 77% enrichment of the 131‐oxo group of chlorophyll a: assuming identical incorporation into the 131 oxygen of chlorophyll b, then enrichment of the 7‐formyl oxygen was as much as 93%. Isotope dilution by re‐incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea.

The high enrichment using 18O2, and the absence of labelling by H218O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7‐formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase‐type enzyme.
Effect of acclimation temperature on the concentration of uncoupling protein and GDP binding in rat brown fat mitochondria
Authors: FEIL Sabine , RAFAEL Johannes
Published: March 2005

The effect of the acclimation temperature on the concentration of the uncoupling protein (UCP) and specific GDP binding in rat brown adipose tissue mitochondria was investigated. UCP was measured by competition ELISA using purified UCP as a standard and antiserum developed against the C‐terminus of the protein. UCP was purified by means of specific polyclonal antibodies immobilized on protein‐A–agarose. It represented 2.4% of the total protein in brown fat mitochondria from rats acclimated to 4°C and 1.1% in mitochondria from rats acclimated to 29°C. No UCP was found in liver mitochondria. The molar ratio of bound GDP and UCP was 0.5 in mitochondria from warm‐acclimated rats and 1.0 in cold‐acclimated rats. The GDP/UCP ratio was increased from 0.5 to 1.0 after a 90‐min exposure to 4°C of warm‐acclimated rats; it was decreased from 1.0 to 0.5, when cold‐acclimated rats were transferred to 29°C for 24 h. Treatment of mitochondrial membranes from warm‐acclimated rats with 3 M urea at pH 10.0 increased the GDP/UCP ratio from 0.5 to 1.0. Specific GDP binding was a direct measure of the UCP concentration during maximally activated or inactivated thermogenesis in brown adipose tissue. We suggest that variable GDP binding reflects the functional activity of UCP based on different protein conformations.
[Ala4]Surfactin, a Novel Isoform from Bacillus subtilis Studied by Mass and NMR Spectroscopies
Authors: Peypoux Françoise , Bonmatin Jean‐Marc , Labbe Henri , Grangemard Isabelle , Das Bhupesh C. , Ptak Marius , Wallach Jean , Michel Georges
Published: March 2005

When Bacillus subtilis S 499 was grown on a culture medium containing L‐alanine as nitrogen source, a mixture of surfactins was obtained. Suitable chromatographic conditions allowed the separation of isoforms. Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry. It contains a peptide sequence which differs from that of standard surfactin by the replacement of the L‐valine residue by L‐alanine residue in position 4. The folding mode of [Ala4]surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed.
A Second Terminal Oxidase in Sulfolobus acidocaldarius
Authors: Lübben Mathias , Arnaud Sylvain , Castresana Jose , Warne Antony , Albracht Simon P. J. , Saraste Matti
Published: March 2005

We previously found that the soxABCD operon encodes a quinol oxidase complex in Sulfolobus acidocaldarius and this enzyme was purified and characterized. In this study, we have used a cloning procedure based on the conservation of oxidase sequences and the polymerase chain reaction to isolate a new gene (soxM) encoding a subunit of another terminal oxidase. This terminal oxidase is a fusion between two central components of cytochrome oxidases, subunits I and III. soxM forms a transcriptional unit which is expressed under heterotrophic growth conditions. The corresponding protein was detected by direct protein sequencing in a preparation enriched with a cytochrome absorbing light at 562 nm. This preparation contains a terminal oxidase which is able to oxidize the artificial substrate N,N,N′,N′ ‐tetramethyl‐p ‐phenylenediamine. This preparation also contains SoxC, a protein homologous to the mitochondrial cytochrome b, and a Rieske iron‐sulphur center. We suggest that SoxM is the core component of a second terminal oxidase complex and that this complex may share a subunit (SoxC) with the SoxABCD complex.
A Peptide that Stimulates Phosphorylation of the Plant Insulin‐Binding Protein
Authors: Watanabe Yoshihiro , Barbashov Sergei F. , Komatsu Setsuko , Hemmings Andrew M. , Miyagi Masaru , Tsunasawa Susumu , Hirano Hisashi
Published: March 2005

The soybean seed basic 7S globulin (Bg) is capable of binding bovine insulin and insulin‐like growth factors, and has protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor. A 4‐kDa peptide named leginsulin, which can bind to Bg and compete with insulin for binding to Bg, was isolated from radicles of germinated soybean seeds. The leginsulin had a stimulatory effect on the phosphorylation activity of Bg, suggesting that it is involved in cellular signal transduction. The leginsulin was sequenced by automated Edman degradation and electrospray ionization mass spectrometry. It consisted of 37 amino acid residues with six half‐cystines in three disulfide bridges. The mass spectrometric analysis revealed that a portion of the peptide is processed to delete the C‐terminal glycine like a number of animal peptide hormones, but not C‐terminally amidated. The cDNA encoding the leginsulin was cloned, sequenced and considered to code for a precursor polypeptide consisting of a putative signal peptide, the leginsulin, a linker peptide, a 6‐kDa peptide and a C‐terminal peptide. Although there is no sequence similarity between the leginsulin and insulin or insulin‐like growth factors, the leginsulin is a possible candidate for plant peptide hormones.
A Specific Association Between the Glyoxylic‐Acid‐Cycle Enzymes Isocitrate Lyase and Malate Synthase
Authors: Beeckmans Sonia , Khan A. Salam , Van Driessche Edilbert , Kanarek Louis
Published: March 2005

There is accumulating evidence that metabolic pathways are organized in vivo as multienzyme clusters or metabolons. To assess interactions between consecutive enzymes of a pathway in vitro, it is usually essential to modify the physical properties of water around the enzymes, e.g. by immobilizing the latter onto a solid support. Such immobilized enzyme preparations can be embedded in agarose gels and used for affinity electrophoresis [Beeckmans, S., Van Driessche, E. & Kanarek, L. (1989) Eur. J. Biochem. 183, 449–454; Beeckmans, S., Van Driessche, E. & Kanarek, L. (1990) J. Cell. Biochem. 43, 297–306].

In this study we use the aforementioned technique to investigate the association between two plant glyoxylic acid cycle enzymes, i.e. isocitrate lyase and malate synthase. A specific histochemical staining technique is described for both enzymes. Affinity electrophoresis using either isocitrate lyase or malate synthase as the immobilized enzyme clearly shows that associations are formed between both enzymes. Moreover, experiments with metabolically unrelated enzymes prove that the observed interaction is specific.
Evidence that Two Non‐Overlapping High‐Affinity Calmodulin‐Binding Sites are Present in the Head Region of Synapsin I
Authors: Goold Robert , Baines Anthony J.
Published: March 2005

Calmodulin is an important element in the regulation of nerve terminal exocytosis by Ca2+. Calmodulin has been shown to interact with the synaptic vesicle phosphoproteins synapsins Ia and Ib [Okabe, T. & Sobue, K. (1987) FEBS Lett. 213, 184–188; Hayes, N. V. L., Bennett, A. F. & Baines, A. J. (1991) Biochem. J. 275, 93–97]. These proteins are thought to provide regulated linkages between synaptic vesicles and cytoskeletal elements. It is well established that calmodulin modulates synapsin I activities via calmodulin‐dependent protein‐kinase‐II‐catalysed phosphorylation. The direct binding of calmodulin to synapsin I suggests a second mode of regulation in addition to phosphorylation. In this study, we present evidence indicating that two sites for calmodulin binding exist in the N‐terminal head region of synapsins Ia and Ib. In unphosphorylated synapsin I, these sites had a Kd value of = 36±14 nM for binding to calmodulin labelled with acetyl‐N′‐(5‐sulpho‐1‐naphthyl)ethylene diamine. The Kd values for synapsin I phosphorylated at various sites were as follows: site I 18% 11 nM; sites II and III 35±14nM; sites I–III 16±9nM. The fluorescence data indicated a stoichiometry of not less than 2 mol calmodulin bound to 1 mol synapsin I at saturation in each case. Consistent with this stoichiometry, two chemically cross‐linked species (96 kDa and 116 kDa) containing calmodulin and synapsin I were generated in vitro, corresponding to one and two calmodulin molecules bound/synapsin I. Defined fragments of synapsin I were generated with the reagent 2‐nitro‐5‐thiocyanobenzoic acid, which cleaves at cysteine residues. Cysteine‐specific cleavage of whole synapsin I after cross‐linking to biotinylated calmodulin generated a pair of polypeptide complexes (approximately 46 kDa and 38 kDa), the masses of which indicated cross‐linking of calmodulin to the N‐terminal and middle regions of synapsin I. Purified N‐terminal and middle fragments each showed a Ca2+‐dependent interaction with calmodulin affinity columns. Two calmodulin‐binding fragments (7.4 kDa and 6.5 kDa) were generated using Staphylococcus aureus V8 protease digestion of synapsin I. These fragments were isolated by calmodulin affinity chromatography and reverse‐phase HPLC. N‐terminal sequence analysis indicated that the fragments originated from two non‐overlapping areas of the synapsin I head region, and that each was contained within one of the 2‐nitro‐5‐thiocyanobenzoic‐acid‐derived calmodulin‐binding fragments. The origins of these fragments are close to the putative sites of actin and tubulin interaction. Addition of calmodulin in the presence of Ca2+ to mixtures of F‐actin and synapsin I reduced both the binding of synapsin I to F‐actin and bundling of F‐actin by synapsin I. Direct binding of calmodulin to synapsin I may represent a non‐covalent mode of regulation of synapsin I activity in addition to covalent regulation by phosphorylation.