bluuebunny/crossref_metadata_2025 huggingface dataset:
Methicillin resistance in Staphylococcus epidermidis
Authors: GAISFORD Wendy C. , REYNOLDS Peter E.
Published: March 2005

The penicillin‐binding proteins (PBP) of a methicillin‐resistant strain of Staphylococcus epidermidis, 100604 p+ m+ and a non‐isogenic sensitive strain, p−m− were characterised. The presence of a novel PBP, produced by the methicillin‐resistant strain of S. epidermidis, with an Mr identical to that of PBP2′ in Staphylococcus aureus 13136 p−m+, was revealed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and subsequent fluorography of solubilised membrane proteins isolated from cells labelled with [3H]benzylpenicillin. This novel PBP was only detected in cells which had been grown at 30°C, in media containing β‐lactam antibiotic and 5% NaCl. The sensitivity of an attachment transpeptidation reaction measured under non‐growing conditions in the sensitive and resistant strains indicated that the novel PBP catalysed this reaction. The similarity of radiolabelled peptides resulting from partial proteolytic digestion of the novel PBP in S. epidermidis 100604 p+m+ and from PBP2′ in S. aureus 13136 p+m+ lends support to the theory that the additional DNA encoding PBP2′ in S. aureus and the same protein in S. epidermidis has been passed to both species from an unknown source. Studies of the development and loss of resistance of attachment transpeptidase activity, and the appearance and disappearance of the novel protein when cultures of the resistant strain were transferred from conditions allowing the expression of resistance to those not allowing such expression and vice‐versa, indicated that there was a strong correlation between the presence of PBP2′ and the degree of resistance of the attachment transpeptidation reaction and that the production of this protein was affected by temperature at a regulatory or genetic level. Studies on the induction and loss of β‐lactamase activity and of the novel PBP when the resistant strain was grown in the presence or absence of β‐lactam antibiotics at either 40°C or 30°C suggests that there is little relationship between the production of this enzyme and of PBP2′ other than the fact that β‐lactam antibiotics are common inducers of both.
Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption
Authors: KODAKI Tsutomu , YAMASHITA Satoshi
Published: March 2005

PEM1 and PEM2 are structural genes for the yeast phosphatidylethanolamine methylation pathway which mediates the three‐step methylation of phosphatidylethanolamine to phosphatidylcholine. Selective disruption of each locus in the yeast genome was performed using the in‐vitro‐inactivated gene with insertion of yeast LEU2 or HIS3. Complementation test and spore analysis indicated that the disruptants were allelic with our previous mutants that were isolated by chemical mutagenesis and used for the cloning of PEM1 and PEM2.

The methyltransferase activities of the disruptants were assayed using their membrane fractions. When the PEM1 locus was disrupted, the activity for the first methylation was greatly decreased but was still detectable, while the activities for the second and third methylations were well retained. The remaining three activities exhibited nearly identical pH optima and apparent Km values for S‐adenosyl‐L‐methionine. The disruptant incorporated radioactivity from L‐[methyl‐14C]Met into phosphatidylcholine at a low but measurable rate and required choline for optimal growth. When choline was omitted from the culture medium, the phosphatidylcholine content of the cells significantly decreased, but was restored by the addition of N‐monomethylethanolamine or choline.

When the PEM2 locus was disrupted, the activities for the second and third methylations were totally lost, but that for the first methylation remained. This activity could be distinguished from those remaining in the pemIdisruptant by its different pH optimum and apparent Km for S‐adenosyl‐L‐methionine. When incubated with [methyl‐14C]Met, the pem2 disruptant accumulated the radioactivity in phosphatidylmonomethylethanolamine. This disruptant also required choline for optimal growth. In the absence of choline, it accumulated phosphatidyl‐monomethylethanolamine with a concomitant decrease in phosphatidylcholine and phosphatidylethanolamine. When both loci were disrupted, all phospholipid‐methylating activities were lost and cells absolutely required choline for growth. The flux through the pathway became negligible. Thus, the PEM1‐encoded methyltransferase was strictly specific to the first step while the PEM2‐encoded methyltransferase exhibited a somewhat broader specificity with a preference for the second and third steps of the pathway. These two enzymes accounted for all the activities in the yeast phosphatidylethanolamine methylation pathway.
Two arylamine N‐acetyltransferases from chicken pineal gland as identified by cDNA cloning
Authors: OHTOMI Michiko , SASAKI Masato , DEGUCHI Takeo
Published: March 2005

A cDNA library prepared from the poly(A)‐rich RNA of the chicken pineal gland obtained at night was screened with the 32P‐labeled cDNA of arylamine N‐acetyltransferase from the chicken liver recently isolated in this laboratory. Two positive clones (p‐NAT‐3 and p‐NAT‐10) that cross‐hybridized with the liver cDNA were isolated. The cDNAs did not cross‐hybridize each other under a high stringency, indicating that they corresponded to different mRNAs. When the cDNAs were inserted into an expression vector pcDLl under the control of the early promoter of simian virus 40 and introduced into Chinese hamster ovary cells, both cDNAs expressed arylamine N‐acetyltransferase activity in the transfected cells. The nucleotide sequences of the cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p‐NAT‐3, p‐NAT‐10 and liver N‐acetyltransferases. Poly(A)‐rich RNA blot hybridization analysis indicated that p‐NAT‐3 cDNA detected a 2.2‐kb band with the poly(A)‐rich RNAs from the brain, gut and, less intensively, spleen, liver and kidney, while p‐NAT‐10 cDNA hybridized only with the poly(A)‐rich RNA from the kidney. Neither cDNA detected any hybridization band with the poly(A)‐rich RNA from the pineal gland, suggesting that the contents were low. Genomic Southern blot hybridization analysis showed that p‐NAT‐3, p‐NAT‐10 and liver N‐acetyltransferases were encoded in a separate single gene.

The properties of the enzymes expressed in the transfected cells were compared with N‐acetyltransferases from the pineal gland, brain and kidney. On a DEAE‐cellulose column, the kidney and p‐NAT‐10 enzymes appeared in the effluent fraction, whereas the brain and p‐NAT‐3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. The supernatant of the pineal gland obtained in the daytime showed two peaks appearing in the effluent fraction and the eluate fraction at 0.08 M NaCl. The substrate specificity of these enzymes were examined with p‐phenetidine, 2‐aminofluorene, tryptamine and phenylethylamine as substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p‐NAT‐3 and p‐NAT‐10 cDNAs encoded arylamine N‐acetyltransferases.
Characterization of mononuclear‐phagocyte terminal maturation by mRNA phenotyping using a set of cloned cDNA probes
Authors: GANTER Ursula , BAUER Joachim , MAJELLO Barbara , GEROK Wolfgang , CILIBERTO Gennaro
Published: March 2005

The terminal step in the maturation of mononuclear cells from circulating monocytes to resident macrophages is accompanied by dramatic changes in cell morphology and physiology. Applying a cultivation system which allows peripheral monocytes to undergo terminal maturation in vitro under absolutely endotoxin‐free conditions, we have determined the pattern of expression of a set of eight genes by mRNA phenotyping. The results can be summarized as follows: the two protease inhibitors α1‐antitrypsin and α2‐macroglobulin show a inverse pattern of expression. α1‐Antitrypsin mRNA is repressed, α2‐macroglobulin mRNA is strongly induced during maturation to macrophages. Therefore, these two genes are excellent markers of the terminal maturation. In addition, ferritin‐light‐chain mRNA progressively increases during the course of differentiation, providing a further marker for maturation. Gene expression as a function of activation was studied in mononuclear cells stimulated with bacterial endotoxin (lipopolysaccharide). In monocytes, complement‐factor‐B, interleukin‐1 and interleukin‐6 mRNAs are drastically induced upon lipopolysaccharide activation whereas lysozyme RNA is strongly repressed. However, the ability of all four genes to respond to endotoxin was markedly diminished or abolished in mature macrophages, indicating that susceptibility to a certain type of activation may be restricted to a specific stage of maturation. Our data show that mRNA phenotyping is excellently suited for the characterization of the differentiation and activation state of mononuclear phagocytes.
Tissue specificity of nucleo‐cytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions
Authors: MOSEVITSKY Mark I. , NOVITSKAYA Vera A. , IOGANNSEN Michael G. , ZABEZHINSKY Mark A.
Published: March 2005

The levels and distribution between nucleus and cytoplasm of HMG1 and HMG2 proteins have been investigated in different tissues of mammals. In lymphoid tissues and testis high amounts of these proteins are present in both nuclei and cytoplasm, while in the hepatic tissues and brain they accumulate in cytoplasm, mainly in the cytosol. In particular, very low amounts, if any, of HMG1 and 2 are present in the nuclei active for DNA replication (rat regenerating liver and primary hepatoma) or transcription (adult liver and brain). Therefore, it appears that HMG1 and 2 are not necessary for these processes. On the other hand, nuclear (chromosomal) HMG1 and 2 are characteristic for the tissues containing undifferentiated cells: lymphoid tissues, testis, neonatal liver. These proteins are bound to the chromatin regions solubilized early by sonication or DNase action. Comparison of the data obtained for different tissues shows an inverse correlation between the amounts of chromosomal HMG1 and 2, on the one hand, and of histone H1°, on the other hand. These results suggest that chromosomal HMG1 and 2 take part in the processes that occur during cell differentiation, while histone H1° is induced to preserve differentiated cells from dedifferentiation.
The ribosomal genes of the mosquito, Aedes aegypti
Authors: GALE Kevin , CRAMPTON Julian
Published: March 2005

The characterisation of the ribosomal genes of the mosquito, Aedes aegypti, is described. Preliminary experiments using a cloned Drosophila ribosomal DNA (rDNA) repeat to probe Southern transfers of Ae. aegypti genomic DNA has indicated that the rDNA repeat of Ae. aegypti is 9.0 kb in length and that individual rDNA repeats exhibit a high degree of homogeneity with respect to length and the position of restriction enzyme recognition sites within the rDNA. The preliminary mapping data together with partial digestion experiments demonstrate that, as in all other higher eukaryotes, the rDNA repeats are arranged in a head‐to‐tail, tandemly repeating manner.

The restriction mapping of cloned rDNA repeats confirmed the largely uniform length of the Ae. aegypti rDNA repeat and provided a more detailed physical map of the DNA. A restriction site polymorphism was detected in one clone (Aar9) which contains an extra HincII site, which is not present in three other clones studied (Aar1, Aar3, or Aar7).

Transcription mapping has allowed the allocation of identities to the various restriction fragments and the approximate positioning of the transcription unit. The estimate of rDNA repeat copy number in Ae. aegypti (approximately 500 copies per haploid genome) is similar to the estimate reported for the closely related species. Aedes albopictus, of 430 copies per haploid genome. Ribosomal DNA thus comprises approximately 0.6% of the total Ae. aegypti genome. Analysis of the variation of the rDNA repeat unit both within individual mosquitoes and between strains of Ae. aegypti, has severed to confirm the remarkable homogeneity of the rDNA repeat unit in this insect.
Study of the fast‐reacting cysteines in phosphoglycerate kinase using chemical modification and site‐directed mutagenesis
Authors: MINARD Philippe , DESMADRIL Michel , BALLERY Nathalie , PERAHIA David , MOUAWAD Liliane , HALL Len , YON Jeannine M.
Published: March 2005

Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5′‐dithio‐bis(2‐nitrobenzoate) (Nbs2) following second‐order kinetics (k= 640 M−1· s−1). Selective cyanylation of the fast‐reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site‐directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97 → Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast‐reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast‐reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.
Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose‐1,6‐bisphosphatase
Authors: STEIN Mariana , LAZARO Juan J. , WOLOSIUK Ricardo A.
Published: March 2005

The incubation of chloroplast fructose‐1,6‐bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1‐14C]iodoacetamide (10‐12 mol iodoacetamide incorporated/mol enzyme). Digestion of S‐carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C‐labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose‐1,6‐bis‐phosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6‐bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6‐bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2‐propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin‐f (0.5 μM) and subsequently subjected to proteolysis and electrophoresis, S‐carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose‐1,6‐bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose‐1,6‐bisphosphatase constituted the underlying mechanism in light‐activation by the ferredoxin‐thioredoxin system.
Interleukin 2 stimulates tyrosine phosphorylation in T cell membrane fractions
Authors: PIAU Jean‐Pierre , WAKASUGI Hiro , BERTOGLIO Jacques , TURSZ Thomas , FRADELIZI Didier , GACON Gérard
Published: March 2005

Interleukin 2 is a growth factor secreted by T lymphocytes upon antigenic stimulation and inducing the proliferation of T cells bearing at their surface the heterodimeric high‐affinity form of its receptor. No enzymatic function has so far been demonstrated in the recptor subunits. In an attempt to elucidate the biochemical pathway of signal transduction, we investigated the capacity of interleukin 2 to modulate tyrosine phosphorylation in T cell membranes. Membrane‐rich fractions from T cells were tested for their ability to phosphorylate tyrosine in the presence or absence of added recombinant interleukin 2. Using as substrate a synthetic polymer of glutamic acid and tyrosine, we demonstrated a 3‐4‐fold stimulation of tyrosine phosphorylation in the presence of interleukin 2; this stimulating effect appeared to be well correlated with interleukin 2 function since (a) it was not observed in insensitive cells, (b) it required the presence of the high‐affinity form of the receptor and (c) it was dose‐dependent. Confirmatory results were obtained by phosphorylating membrane‐rich fractions with [y‐32P]ATP and by analysing the resulting phosphoproteins: only in fractions from cells with the high‐affinity form of the receptor were several membrane proteins specifically phosphorylated on tyrosine residues in response to interleukin 2. At least two proteins of 115 and 58 kDa were consistently hyperphosphorylated on tyrosine in an interleukin‐2‐dependent manner. This stimulation was strongly dependent on the presence of the protein tyrosine phosphatase inhibitor, sodium orthovanadate.

Thus, we propose that interleukin 2 enhances tyrosine phosphorylation by stimulating a tyrosine kinase activity. The nature of the enzyme involved remains to be determined.
Biosynthesis of dolichol by rat liver peroxisomes
Authors: APPELKVIST Eeva‐Liisa , KALÉN Anders
Published: March 2005

The ability of peroxisomes and microsomes to synthesize dolichol from [3H]mevalonate, [3H]isopentenyl‐P2 or [3H]farnesyl‐P2in vitro was investigated. It was found that isoprenoid biosynthesis also occurs in peroxisomes and that this process demonstrates properties differing from those of isoprenoid biosynthesis by microsomes. The pH optimum in peroxisomes was 8.0 and, in contrast to microsomes, the peroxisomal biosynthesis was largely insensitive to detergents. After treatment with proteolytic enzymes, microsomes lost their capacity to incorporate [3H]mevalonate into dolichol, whereas proteolysis of intact peroxisomes did not influence their corresponding rate of incorporation. The soluble content of peroxisomes was separated from the membranes and found to demonstrate half of the biosynthetic capacity of the intact organelle. Fasting and cholestyramine treatment decreased only the microsomal incorporation of [3H]mevalonate into dolichol, while treatment with clofibrate, di‐2‐ethylhexyl phthalate or phenobarbital increased microsomal, but decreased peroxisomal labeling. After injection of [3H]mevalonate into the portal vein of rats, high initial labeling of dolichol was recovered both in isolated microsomes and peroxisomes, whereas when [3H]glycerol was administered, peroxisomal phospholipids became labeled later than the corresponding microsomal constituents. These results support the conclusion that dolichol is synthesized both in peroxisomes and the endoplasmic reticulum, but that the biosynthetic processes at these two locations have different properties.
Evidence that 31P NMR is a sensitive indicator of small conformational changes in the coenzyme of aspartate aminotransferase
Authors: SCHNACKERZ Klaus D. , WAHLER Gudrun , VINCENT Michael G. , JANSONIUS Johan N.
Published: March 2005

The pH dependence of 31P‐NMR spectra of pig cytosolic aspartate aminotransferase, containing either N‐(5′‐phosphopyridoxyl)‐L‐aspartate or pyridoxal 5′‐deoxymethylenephosphonate in place of the normal coenzyme pyridoxal 5′‐phosphate, has been analysed. The chemical shifts of phosphopyridoxylaspartate and of pyridoxal 5′‐deoxymethylenephosphonate model Schiff base in free solution show pK values of 6.3 and 7.4, attributable to the second deprotonation step of phosphate and phosphonate, respectively. However, these compounds behave very differently when bound to apoaspartate aminotransferase. 31P‐NMR spectra of these enzyme derivatives indicate that the phosph(on)ate group remains dianionic throughout the pH range 4–8.5. A clear correlation between apparent pK values obtained from spectrophotometric titration of the coenzyme chromophore and those obtained by 31P NMR indicates that the same ionisation is being reported by both methods. The data are interpreted, on the basis of available crystallographic structures of chicken mitochondrial aspartate aminotransferase, to indicate that in each case the alteration in 31P chemical shift results from a conformational change in the coenzyme 5′ side chain, in which one of the structures involves a near‐eclipsed pair of bonds. Such a stressed conformation produces slight alterations in bond angles around the phosphorus atom, which in turn cause the observed change in 31P chemical shift. The evidence is taken to indicate that in this case 31P NMR is a sensitive reporter of stress in enzyme‐bound pyridoxal 5′‐phosphate and its derivatives.
Purification and characterization of three (1→4)‐β‐d‐xylan endohydrolases from germinated barley
Authors: SLADE Amanda M. , HØJ Peter B. , MORRICE Nick A. , FINCHER Geoffrey B.
Published: March 2005

Three (1→4)‐β‐D‐xylan xylanohydrolases (xylan endohydrolases, EC 3.2.1.8) have been purified 1200–2800‐fold from extracts of germinated barley (Hordeum vulgare L. cv. Clipper) by a sequence of ammonium sulphate fractionation, Procion‐blue‐dye chromatography, ion‐exchange and gel filtration chromatography. The enzymes are likely to function in the depolymerization of cell wall arabinoxylans during mobilization of the starchy endosperm. They are classified as endohydrolases on the basis of analyses of products released during hydrolysis of a (1→4)‐β‐xylan. The three xylan endohydrolases are monomeric proteins of apparent Mr 41000 and all have isoelectric points of 5.2. The sequences of the 30 NH2‐terminal amino acids of the three enzymes are the same, but it is not yet known whether they represent the products of separate genes or originate by differences in post‐translational modification of a single gene product.
Induction and stability of the adult myosin phenotype in striated muscles of dwarf mice after chronic thyroid hormone treatment
Authors: PRULIÈRE Gérard , BUTLER‐BROWNE Gillian S. , CAMBON Natalie , TOUTANT Madeleine , WHALEN Robert G.
Published: March 2005

It is known that a deficiency in thyroid hormone delays the post‐natal maturation of several mammalian tissues. In striated muscle tissue, hypothyroidism delays or inhibits some of the isoform transitions of myosin heavy chains which would occur during normal development. In this paper, using the mouse mutant dwarf, we demonstrate an influence of thyroid hormone on expression of the myosin phenotype in cardiac and skeletal muscle of dwarf mice. Myosin isoforms were identified by gel electrophoresis of native myosin, localised within muscle cells by indirect immunofluorescence and quantified using an ELISA technique. We show that an adult phenotype can be established in both cardiac and skeletal muscle following a treatment involving multiple injections of thyroxine although cardiac musle responds more rapidly. The skeletal myosin phenotype remains stable until at least five weeks after the last injection. In contrast, the fetal form of cardiac myosin reaccumulates upon cessation of thyroxine treatment. Thus, cardiac and skeletal muscles are not only affected differently by the dwarf mutation but also they respond differently to thyroxine treatment and thyroxine withdrawal.
Complete amino acid sequence of pyrazine‐binding protein from cow nasal mucosa
Authors: TIRINDELLI Roberto , KEEN Jeffrey N. , CAVAGGIONI Andrea , ELIOPOULOS Elias E. , FINDLAY John B. C.
Published: March 2005

The sequence is reported of the pyrazine‐binding protein from cow olfactory/respiratory mucosa. The protein consists of 159 amino acids and clearly belongs to the retinol‐binding protein family. It is most closely related to the urinary proteins from mice and rats and to the odour‐binding protein from rat nasal epithelium. It is unique however, in that only one of the otherwise conserved features of the family is still present – namely a single tryptophan. Most surprisingly the protein contains no cysteine and, therefore, does not rely for its structural stability on the disulphide bond(s) present in other members of this group. A model for the protein has been constructed based on the co‐ordinates of β‐lactoglobulin. From this, it is possible to identify residues which may line the binding site. The impression gained is of a much larger pocket than occurs with retinol‐binding protein or β‐lactoglobulin. The character of the binding pocket remains essentially hydrophobic but with a significant reduction in its aromatic content and an increase in H‐bonding side chains.
NADPH‐dependent H2O2 generation catalyzed by thyroid plasma membranes
Authors: DUPUY Corinne , KANIEWSKI Jacques , DÈME Danièle , POMMIER Jacques , VIRION Alain
Published: March 2005

Hog thyroid plasma membrane preparations containing a Ca2+‐regulated NADPH‐dependent H2O2‐generating system were studied. The Ca2+‐dependent reductase activities of ferricytochrome c, 2,6‐dichloroindophenol, nitroblue tetrazolium, and potassium ferricyanide were tested and the effect of these scavengers on H2O2 formation, NADPH oxidation and O2 consumption were measured, with the following results.

Thyroid plasma membrane Ca2+‐independent cytochrome c reduction was not catalyzed by the NADPH‐dependent H2O2‐generating system. This activity was superoxide‐dismutase‐insensitive.

Of the three other electron scavengers tested, only K3Fe(CN)6 was clearly, but partially reduced in a Ca2+‐dependent manner.

Though the NADPH‐dependent reduction of nitroblue tetrazolium was very low and superoxide‐dismutase‐insensitive, nitroblue tetrazolium inhibited O2 consumption, H2O2 formation and NADPH oxidation, indicating that nitroblue tetrazolium inhibits the H2O2‐generating system.

We conclude that the thyroid plasma membrane H2O2‐generating system does not form or liberate O−2 and that Ca2+ controls the first step (NADPH oxidation) of the H2O2‐generating system.
2‐Aminobenzoyl‐CoA monooxygenase/reductase, a novel type of flavoenzyme
Authors: BUDER Reiner , FUCHS Georg
Published: March 2005

A novel aerobic mechanism of 2‐aminobenzoate metabolism was proposed in a denitrifying Pseudomonas species. 2‐Aminobenzoic acid is activated in a coenzyme‐A‐ligase reaction to 2‐aminobenzoyl‐CoA and this intermediate is dearomatized by a unique enzyme, tentatively named 2‐aminobenzoyl‐CoA monooxygenase/reductase. This paper describes the purification and some molecular, kinetic and spectral properties of this flavoenzyme which catalyzes the hydroxylation and reduction of 2‐aminobenzoyl‐CoA to an unknown non‐aromatic compound.

2‐Aminobenzoyl‐CoA monooxygenase/reductase was purified 25‐fold to a specific activity of 25 μmol · min−1· mg−1 protein using ammonium sulfate precipitation, DEAE‐cellulose anion‐exchange, hydroxylapatite and Mono Q FPLC anion‐exchange chromatography. Superose 6 gel filtration for estimation of molecular mass resulted in one symmetrical protein peak corresponding to a molecular mass of 170 kDa. Several experimental data suggest that the protein is probably an α2 dimer; however, it may exist in three dimeric forms, αα, αα′ and α′α′, where α′ may be a subunit with a different conformation. Approximately 2 mol noncovalently bound FAD/mol enzyme was found, which in the absence of O2 was reduced by NADH. The enzyme was specific for the substrates 2‐aminobenzoyl‐CoA (Km≤ 25 μM) and O2 (Km≤ 5 μM), but less specific for the reduced pyridine nucleotides NADH (Km= 42 μM) or NADPH [Km= 500 μM; Vmax (NADH)/Vmax (NADPH) = 1.7:1]. The turnover number was 4250 min−1. The enzyme also reduced N‐ethylmaleimide and maleimide with NAD(P)H. The substrate, the products and the reaction stoichiometry are described in two following papers.
2‐Aminobenzoyl‐CoA monooxygenase/reductase, a novel type of flavoenzyme
Authors: BUDER Reiner , ZIEGLER Klaus , FUCHS Georg , LANGKAU Bernd , GHISLA Sandro
Published: March 2005

The reaction catalyzed by 2‐aminobenzoyl‐coenzyme‐A monooxygenase/reductase from a denitrifyingPseudomonas sp.has been investigated. 2‐Aminobenzoyl‐CoA and 2‐amino[carboxy‐14C]benzoyl‐CoA were synthesized enzymatically using 2‐aminobenzoyl‐CoA synthetase from the same organism. The product was purified by chromatography and characterized by ultraviolet/visible and1H‐NMR spectroscopy. The conversion of 2‐aminobenzoyl‐CoA catalyzed by the monooxygenase/reductase requires NADH and oxygen, and yields at least two different products depending on the relative concentration of NADH. At [NADH] <Km(40 μM), i.e. [NADH]/[2‐aminobenzoyl‐CoA] ∼ 0.02–0.05, the main product is probably a hydroxylated derivative of 2‐aminobenzoyl‐CoA, which is characterized by an absorbance maximum around 375 nm. When [NADH]/[2‐aminobenzoyl‐CoA] ∼ 2–5, the predominant product is a non‐aromatic coenzyme A thioester (λmax∼ 320 nm). The stoichiometry in this case is 2.1–2.4 mol NADH oxidized (mol oxygen consumed)−1(mol 2‐aminobenzoyl‐CoA metabolized)−1. The product is extremely unstable in the acidic pH range and undergoes decarboxylation in a few minutes at pH < 5. Some degree of stabilisation is obtained upon reduction with sodium borohydride, probably resulting in a further reduced non‐aromatic coenzyme‐A thioester.
Differential effects of α‐, β‐ and γ‐cyclodextrins on human erythrocytes
Authors: OHTANI Yoshiro , IRIE Tetsumi , UEKAMA Kaneto , FUKUNAGA Kazuhiro , PITHA Josef
Published: March 2005

α‐, β‐ and γ‐cyclodextrins are cyclic hexamers, heptamers, and octamers of glucose, respectively, and thus are hydrophilic; nevertheless, they have the ability to solubilize lipids through the formation of molecular inclusion complexes. The volume of lipophilic space involved in the solubilization process increases with the number of glucose units in the cyclodextrin molecule and, consequently, cyclodextrins were found to have different effects on human erythrocytes: (a) in the induction of shape change from discocyte to spherocyte the potency was observed to be α > γ, but with β‐cyclodextrin hemolysis occurred before the change was complete; (b) in the increase of fluorescence intensity of 1‐anilinonaphthalene‐8‐sulfonate in cyclodextrin‐pretreated membranes, the observed potency was β≫γ > α; (c) in the release of potassium and hemoglobin, the potency was β > α > γ. The potencies of cyclodextrin for solubilizing various components of erythrocytes were α > β≫γ for phospholipids, β≫γ > α for cholesterol and β≫γ > α for proteins. The solubilization potencies were derived from concentration/final‐effect curves. The above processes occurred without entry of solubilizer into the membrane, since (a) β‐[14C]cyclodextrin did not bind to erythrocytes and (b) cyclodextrins did not enter the cholesterol monolayer. A study of the [3H]cholesterol in erythrocytes indicated that β‐cyclodextrin extracted this lipid from membrane into a new compartment located in the aqueous phase which could equilibrate rapidly with additional erythrocytes. Therefore, the effects of cyclodextrins differ from those of detergents which first incorporate themselves into membranes then extract membrane components into supramolecular micelles.
Efficient tissue‐specific expression of bovine α‐lactalbumin in transgenic mice
Authors: VILOTTE Jean‐Luc , SOULIER Solange , STINNAKRE Marie‐George , MASSOUD Micheline , MERCIER Jean‐Claude
Published: March 2005

Transgenic mice carrying a 3.2‐kb genomic fragment comprising the bovine α‐lactalbumin gene transcription unit with 750 bp and 336 bp of 5′ and 3′ flanking regions, respectively, were generated. Tissue‐specific expression of the transgene was observed in five out of the six lines analyzed at levels close to the endogenous gene in two of them.
Pike eel (Muraenesox cinereus) gonadotropin
Authors: LIU Chen‐Sheng , HUANG Fore‐Lien , CHANG Yea‐Sha , LO Tung‐Bin
Published: March 2005

The amino acid sequences of pike eel gonadotropin α and β subunits have been determined by standard sequencing analytical methods. The α subunit is composed of 93 amino acid residues while the β subunit comprises 113 amino acid residues. All the invariant half‐cystine residues are in the same positions as those found in other gonadotropins.

It is noteworthy that the first, putative glycosylation site (Asn56) found in the α subunit of other gonadotropins was replaced by Asp56 in the α subunit of pike eel gonadotropin. Similarity analyses indicate that both subunits are structurally more similar to other known fish gonadotropin subunits than to those of the mammalian gonadotropins.
Occurrence of free and esterified lipoxygenase products in leaves of Glechoma hederacea L. and other Labiatae
Authors: KÜHN Hartmut , WIESNER Rainer , ALDER Lutz , SCHEWE Tankred
Published: March 2005

Leaves of Glechoma hederacea L. and other Labiatae contain (9S,10E,12Z,15Z)‐9‐hydroxy‐10,12,15‐octadecatrienoic acid, (10E,12Z,15Z)‐9‐oxo‐10,12,15‐octadecatrienoic acid, (9S,10E,12Z)‐9‐hydroxy‐10,12‐octadecadienoic acid and (10E,12Z)‐9‐oxo‐10,12‐octadecadienoic acid in a ratio of 71/14/12/3 (by mass), predominantly esterified in the membrane ester lipids. The leaves contain the highest level of these products, whereas only small amounts were found in the stalk and the roots. The chemical structures of these compounds were established by ultraviolet and infrared spectroscopy, by co‐chromatography with authentic standards on various types of HPLC columns including chiral‐phase HPLC and gas chromatography/mass spectrometry. The stereochemical specificity indicates the enzymatic origin of the products, most probably via a lipoxygenase reaction. Freshly harvested specimens of G. hederacea L. contain only small amounts of hydroxy‐polyenoic fatty acids. Air‐drying causes a strong increase in the content of free and esterified (9S,10E,12Z,15Z)‐9‐hydroxy‐10,12,15‐octadecatrienoic acid. Up to 80% of the hydroxy fatty acids of the total lipid extracts were esterified in the cellular lipids.

The data presented indicate that lipoxygenase products occur in the cellular ester lipids of G. hederacea L. and other Labiatae. The results are discussed in the light of a possible involvement of the lipoxygenase pathway in the natural senescence of leaves.
Solubilization of somatostatin receptors in hamster pancreatic beta cells
Authors: MARIE Jean‐Claude , COTRONEO Patrizia , de CHASSEVAL Régina , ROSSELIN Gabriel
Published: March 2005

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I‐[Leu8,d‐Trp22,Tyr25]somatostatin‐28 (125I‐somatostatin‐28) and solubilized with the non‐denaturing detergent Triton X‐100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I‐somatostatin‐28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.‐C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219–224]. The major labelled complex (128 kDa) was adsorbed to a wheat‐germ‐agglutinin agarose column and eluted by N‐acetylglucosamine.

Also, the binding of 125I‐somatostatin‐28 to plasma membranes was specifically inhibited by the GTP analog, guanosine‐5′‐O‐(3‐thiotriphosphate) (GTP[S]) in a dose‐dependent manner. Furthermore, when somatostatin‐28 receptors were solubilized by Triton X‐100 as a reversible complex with 125I‐somatostatin‐28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma‐membrane‐embedded receptors, the radioactivity remaining bound after 15 min at 37°C being 30% and 83% respectively. After pertussis‐toxin‐induced [32P]ADP‐ribosylation of pancreatic membranes, a 41‐kDa [32P]ADP‐ribose‐labelled inhibitory guanine nucleotide binding protein coeluted with the 128‐kDa and 45‐kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228‐kDa band was inconsistent.

These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128‐kDa band and is a glycoprotein. The pancreatic membrane contains a 41‐kDa GTP‐binding protein that can complex with somatostatin receptors.
Specificity of Bacillus thuringiensisδ‐endotoxins
Authors: VAN RIE Jeroen , JANSENS Stefan , HÖFTE Herman , DEGHEELE Danny , VAN MELLAERT Herman
Published: March 2005

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringiensisδ‐endotoxins, we have performed binding studies with three δ‐endotoxins on membrane preparations from larval insect midgut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I‐labeled Bt2 toxin, a recombinant B. thuringiensisδ‐endotoxin, to brush border membrane vesicles of Manduca sexta.

The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens.

Equilibrium binding studies revealed saturable, high‐affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity.

Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three‐site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non‐toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I‐labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non‐susceptible organisms.

Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.
Evidence that 1,3‐bisphosphoglycerate dissociation from phosphoglycerate kinase is an intrinsically rapid reaction step
Authors: KVASSMAN Jan , PETTERSSON Gösta
Published: March 2005

The steady‐state kinetics of 1,3‐bisphosphoglycerate formation through the action of phosphoglycerate kinase on 3‐phosphoglycerate and ATP have been examined. The results show that initial velocities determined by the standard method of coupling bisphosphoglycerate production to NADH reduction in the presence of glyceraldehyde‐3‐phosphate dehydrogenase do not differ significantly from those determined in the absence of the latter enzyme. This observation invalidates the proposal that bisphosphoglycerate dissociation from phosphoglycerate kinase is much too slow to account for the high rates of phosphoglycerate turnover observed in the coupled two‐enzyme system. The capacity for rapid bisphosphoglycerate production and release is an intrinsic catalytic property of phosphoglycerate kinase that does not require the presence of other enzymes or the involvement of a mechanism of channelized (non‐diffusional) transfer of bisphosphoglycerate from the producing enzyme to the consuming one.
Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+‐ATPase of sarcoplasmic reticulum
Authors: MONTERO‐LOMELI Mónica , DE MEIS Leopoldo
Published: March 2005

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady‐state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady‐state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low‐affinity Ca2+‐binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady‐state level of phosphoenzyme formed by Pi.
Purification and properties of the 3‐deoxy‐d‐arabino‐heptulosonate‐7‐phosphate synthase (phenylalanine‐inhibitable) of Saccharomyces cerevisiae
Authors: PARAVICINI Gerhard , SCHMIDHEINI Tobias , BRAUS Gerhard
Published: March 2005

The phenylalanine‐inhibitable 3‐deoxy‐d‐arabino‐heptulosonate‐7‐phosphate (dHplP) synthase from Saccharomyces cerevisiae has been purified to apparent homogeneity by a 1250‐fold enrichment of the enzyme activity present in wild‐type crude extracts, employing an overproducing strain. The estimated molecular mass of 42 kDa corresponds to the calculated molecular mass of 42.13 kDa deduced from the previously determined primary sequence. Gel filtration indicates that the active enzyme is a monomer. The enzyme is an Fe protein and is inactivated by EDTA in a reaction which is reversible by several bivalent metal ions. The Michaelis constant of the enzyme is 18 μM for phosphoenolpyruvate (P‐pyruvate) and 130 μM for erythrose 4‐phosphate (Ery4P) and the rate constant was calculated as 10 s−1. Inhibition by phenylalanine is competitive with respect to erythrose 4‐phosphate and non‐competitive to phosphoenolpyruvate, with a Ki of 10 μM.
Cloning and sequencing of the gene coding for aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus
Authors: CUBELLIS Maria Vittoria , ROZZO Carla , NITTI Gianpaolo , ARNONE Maria Immacolata , MARINO Gennaro , SANNIA Giovanni
Published: March 2005

The gene coding for aspartate aminotransferase (EC 2.6.1.1) has been cloned from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus strain MT4. Partial sequence data obtained directly from the purified protein and from the two cyanogen‐bromide‐generated peptides confirm the primary structure of aspartate aminotransferase inferred from the nucleotide sequence of its gene.

A comparison of the enzyme with other aminotransferases revealed an interesting similarity with tyrosine aminotransferase from rat liver (EC 2.6.1.5) and allowed some tentative assignments of the residues implied in the catalysis.

The aspartate aminotransferase gene‐flanking regions were compared to those of other archaebacterial genes already described in the literature with the aim of identifying potential regulatory sites.
Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium
Authors: MITAMURA Toshihide , URABE Itaru , OKADA Hirosuke
Published: March 2005

Three glucose dehydrogenases (GlcDH) from Bacillus megaterium, GlcDH‐I, GlcDH‐II and GlcDH‐IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDK1 and pGDK2 contain two isozyme genes, gdhI and gdhII, respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH‐IWG3 is a variant of GlcDH‐I. GlcDH‐I and GlcDH‐II have similar pH/activity profiles and the profile for GlcDH‐IWG3 is identical to that of GlcDH‐I. The pH/stability profiles of these enzymes show that GlcDH‐IWG3 is the most stable enzyme in the acidic region, while GlcDH‐II is the most stable in the alkaline region, and GlcDH‐I is the most unstable throughout the entire pH range examined. As for thermostability, GlcDH‐II is the most resistant against heat inactivation at pH 6.5. The values of the first‐order rate constant for heat inactivation at 50°C are 0.27 min−1, 0.05 min−1 and 0.11 min−1 for GlcDH‐I, GlcDH‐II and GlcDH‐IWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in Kia for NAD(P) and Ka (the limiting Michaelis constant) for NAD; the values of the ratio of Kia for NAD and NADP are 11340 and 8.7 for GlcDH‐I, GlcDH‐II and GlcDH‐IWG3, respectively. GlcDH‐I and GlcDH‐IWG3 have very similar substrate specificities and GlcDH‐II has a slightly higher specificity for D‐glucose and 2‐deoxy‐D‐glucose than the others. The results are discussed on the basis of the amino acid substitutions between the enzymes.
Structure of sea‐urchin arylsulfatase gene
Authors: YAMADA Kazumi , AKASAKA Koji , SHIMADA Hiraku
Published: March 2005

The gene encoding arylsulfatase (Ars; EC 3.1.6.1) as well as two Ars pseudogenes were isolated from sea urchin genomic libraries. The Ars gene was 20‐kbp long and contained six exons interrupted by five introns. Four polypyrimidine repetitive sequences were dispersed in its upstream‐flanking region.

Comparison of the amino acid sequence of sea‐urchin arylsulfatase with those of human sterol sulfatase, human arylsulfatase A and bacterial arylsulfatase revealed that they have two similar sequences in common.

The position of the transcription‐start site of the Ars gene was determined to be approximately 40‐bp upstream from the 5′ end of the protein‐coding region, and the nucleotide sequence of the 5′‐flanking region was determined up to 3.3 kbp upstream from the transcription start point. Putative TATAA box and CCAAT consensus sequences were located at positions ‐28 and ‐82, respectively. A highly conserved hexamer motif, CTCTTT, localized near the transcription‐start site of the sea‐urchin Ars gene, was also detected in similar regions of other sea urchin genes such as CyIIIa, Spec 1, Spec 2a, Spec 2c, Spec 2d, and SM50, but not in the histone genes.
Short‐chain basement membrane collagen
Authors: DIXIT Saryu N.
Published: March 2005

The paper describes further characterization of the 55‐kDa short‐chain collagen from lens capsule. Lens capsules were extracted with 5.5 M guanidine · HCl and the extracted material was fractionated on agarose A‐5M followed by high‐pressure liquid chromatography (HPLC). By amino acid composition, the major fraction obtained from HPLC was found to be different than type‐IV collagen fragments. The 55‐kDa short‐chain collagen on pepsin digestion produced a 45‐kDa pepsin‐resistant fragment. The undifferentiated embryonal carcinoma (F‐9) cells were found to synthesize increased amounts of 55‐kDa short‐chain collagen. The identity of this biosynthesized molecule with 55‐kDa short‐chain collagen from lens capsules was established by immunoprecipitation experiments. The results indicated a close similarity or identical nature of the short‐chain collagens from these two sources.