bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.
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.
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.
Regulation of protein phosphatase‐1G from rabbit skeletal muscle
Authors: HUBBARD Michael J. , COHEN Philip 
Published: March 2005

The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) comprises a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit. In the preceding paper in this issue of the journal we showed that the C subunit is released from PP‐1G in response to phosphorylation of the G subunit by cAMP‐dependent protein kinase. We now show that at 0.15–02 M KCl the phosphorylase phosphatase activity of glycogen‐bound PP‐1G is 5–8 times higher than that of released C subunit or unbound PP‐1G, which are strongly inhibited at these ionic strengths. The activity of glycogen‐bound PP‐1G towards glycogen synthase was about 5‐fold higher than that of released C subunit at 0.15M KCl. Studies with glycogen‐bound substrates and myosin P‐light chain (which does not interact with glycogen) indicated that PP‐1G activity is only enhanced compared to free C subunit at near physiological ionic strength and when both PP‐1G and substrate are glycogen‐associated.The inhibition by increasing ionic strength and enhanced activity upon binding to glycogen reflected changes in K′m, but not Vmax. From the determined specificity constant, k′cat/K′m∼ 4 × 106 s−1 M−1, it was calculated that at physiological levels of glycogen‐bound PP‐1G (200 nM) and phosphorylase (70 μM). dephosphorylation of the latter could occur with a half time of 15 s, sufficient to account for inactivation rates in vivo.The much higher catalytic efficiency of glycogen‐bound PP‐1G toward the glycogen‐metabolising enzymes at physiological ionic strength compared to free C subunit substantiates the role of PP‐1G in the regulation of these substrates, and establishes a novel mechanism for selectively regulating their phosphorylation states in response to adrenalin and other factors affecting phosphorylation of the G subunit.