bluuebunny/crossref_metadata_2025 huggingface dataset:
Reticulocyte lipoxygenase, ingensin, and ATP‐dependent proteolysis
Authors: Ishiura Shoichi , Yoshimoto Tanihiro , Villee Claude A.
Published: July 2002

Lipoxygenase purified from rabbit reticulocyte lysate has a molecular mass of 68 kDa on SDS gel and a pI of 5.97. Lipoxygenase is inhibited by nordihydroguaiaretic acid (NDGA), 3‐amino‐1‐(m‐(trifluoromethyl)phenyl)‐2‐pyrazoline (BW755C), 5,8,11,14‐eicosatetraynoic acid (ETYA), salicylhydroxamate (SHAM) or hemin. Metal ions or nucleotides do not affect its activity. The addition of certain of these inhibitors to the reticulocyte extract also inhibited the ATP‐dependent proteolysis of casein, one of the distinct characteristics of reticulocytes. No clear correlation between lipoxygenase activity and ATP‐dependent proteolysis could be detected. Hemin and NDGA inhibited both processes, but the concentrations necessary for inhibition were quite different. SHAM completely inhibited lipoxygenase, but not proteolysis. o‐Phenanthroline inhibited ATP‐dependent proteolysis, but had no effect on lipoxygenase activity. We have also purified a high‐molecular‐mass protease, ingensin, from reticulocyte extract. This protease accounted for more than 90% of the casein‐degrading activity in reticulocyte extract. NDGA inhibited ingensin at the same concentrations required for inhibition of ATP‐dependent proteolysis. These results suggest that lipoxygenase is not indispensable for the ATP‐dependent proteolysis and the novel high‐molecular‐mass protease, ingensin, may be involved in the process.
Inhibition of catalytic unit of adenylate cyclase and activation of GTPase of Ni protein by βγ‐subunits of GTP‐binding proteins
Authors: Enomoto Keiichi , Asakawa Takeo
Published: July 2002

A protein factor which inhibited adenylate cyclase was purified to apparent homogeneity from rat brain and identified as the βγ‐subunits of the GTP‐binding regulatory proteins of adenylate cyclase. (i) The βγ‐subunits (protein factor) inhibited the partially purified catalytic unit of adenylate cyclase in the presence of an activator, forskolin or the stimulative regulatory protein (Ns), to 60 and 40% of the control, respectively; inhibition of the catalytic unit in the presence of forskolin required no guanine nucleotides. (ii) The subunits enhanced the GTPase activity of the purified α‐subunit of the inhibitory regulatory protein (Niα) 3.8‐fold, (iii) The subunits stimulated ADP‐ribosylation of niα catalyzed by islet‐activating protein (pertussis toxin). ADP‐ribosylation had no effect on the GTPase activity of Niα in the presence of the βγ‐subunits. The results suggest that direct inhibition of the catalytic unit by the βγ‐subunits liberated from Ni is essential for the receptor‐mediated inhibition of adenylate cyclase.
Detection and partial characterization of an amidating enzyme in skin secretion of Xenopus laevis
Authors: Mollay Christa , Wichta Jitka , Kreil Günter
Published: July 2002

Peptidyl‐glycine‐α‐amidating monooxygenase has been purified about 150‐fold from skin secretion of Xenopus laevis by a three‐step procedure. The enzyme converts [14C]succinyl‐Ala‐Phe‐Gly to [14C]succinyl‐Ala‐Phe‐amide at both pH 5.5 and 8.4 in the presence of different metal ions and ascorbate or tetrahydropterin. In contrast to the mammalian enzyme, higher activities were detected at pH 5.5.
Tumour promotor 12‐O‐tetradecanoylphorbol 13‐acetate inhibits angiotensin II‐induced inositol phosphate production and cytosolic Ca2+ rise in rat renal mesangial cells
Authors: Pfeilschifter Josef
Published: July 2002

Preincubation of rat renal mesangial cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) strongly inhibited the increases of inositol phosphates and of free cytosolic Ca2+ induced by angiotensin II (10−7 M). TPA had no significant effect on the basal values of inositol phosphates and of free cytosolic Ca2+. Inhibition appeared already after 1 min and was maximal after 5 min. These effects occur without significant changes on angiotensin II binding in intact cells. The concentration of TPA needed (10−9–10−7 M) was in the range believed to cause specifically an activation of protein kinase C. Furthermore the biologically inactive phorbol ester 4α‐phorbol 12,13‐didecanoate was without effect. From the entirety of these results it is likely that protein kinase C inhibits angiotensin II activation of phospholipase C at a stage distal to receptor occupancy.
Phorbol ester treatment impairs hormone‐ but not stable GTP analog‐induced inhibition of adenylate cyclase
Authors: Bauer Silvia , Jakobs Karl H.
Published: July 2002

Treatment of intact human platelets and S49 lymphoma cyc− cells with the phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate, impairs GTP‐dependent and hormone‐induced inhibition of adenylate cyclase, an action mediated by the inhibitory coupling protein Ni. In contrast, receptor‐independent activation of Ni with subsequent adenylate cyclase inhibition induced by the stable GTP analog, guanosine 5'‐[γ‐thio]triphosphate, was affected in neither the potency nor onset of Ni activation by the stable GTP analog, in both membrane systems studied. The data indicate that modification of Ni following phorbol ester treatment does not impair its activation by stable GTP analogs.
A gene in Paracoccus for subunit III of cytochrome oxidase
Authors: Saraste Matti , Raitio Mirja , Jalli Tuulikki , Perämaa Anja
Published: July 2002

The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa
3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.
Muteins of human interleukin‐1 that show enhanced bioactivities
Authors: Huang James J. , Newton Robert C. , Horuk Richard , Matthew James B. , Covington Maryanne , Pezzella Kathleen , Lin Yuan
Published: July 2002

Using recombinant DNA techniques, we have made a series of amino‐terminal muteins of human interleukin‐1 (IL‐1). Two of the muteins demonstrated 4–7‐fold increase in bioactivity as compared to that of the native IL‐1. The enhanced biological potency coincides with an increase in both receptor binding affinity and in vivo tumor inhibitory activity. By site specific mutagenesis, we have shown that the arginine at the fourth position of IL‐1 is one of the key residues in the function of IL‐1. Circular dichroism studies of the amino‐terminus analogs showed little structural rearrangement. The change in bioactivity might be due to a change in the stability of the muteins, in the side chain interactions with receptors or in the minor change in folding near the receptor binding site.
Different abilities of two distinct Fcγ receptors on guinea pig polymorphonuclear leukocytes to trigger the arachidonic acid metabolic cascade
Authors: Sato Masaki , Nakamura Tohoru , Koyama Jiro
Published: July 2002

The nature of signals transmitted by two Fcγ receptors (Fcγ2R for IgG2 and Fcγ1/γ2R for both IgG2 and IgG1) on guinea pig polymorphonuclear leukocytes was investigated. The specific binding of hen ovalbumin (OA)‐complexed IgG1 antibody to Fcγ1/γ2R did not seem to trigger the release of [3H]arachidonic acid from the cells prelabeled with [3H]arachidonic acid. In contrast, marked release occurred on the binding of OA‐complexed IgG2 antibody to the cells. This reaction was not inhibited, but rather enhanced, by the Fab′ of a monoclonal antibody to Fcγ/γ2R. Therefore, a signal for the activation of the arachidonic acid metabolic cascade was found to be transmitted by Fcγ2R, but scarcely by Fcγ1/γ2R.
Clathrin β‐light chain of rat liver coated vesicles is phosphorylated in vitro and in vivo
Authors: Cantournet Benoit , Creuzet Claudine , Komano Odile , Loeb Jacques
Published: July 2002

Clathrin β‐light chain of rat liver coated vesicles is phosphorylated in vitro in the presence of poly(L‐lysine) by an endogenous protein kinase which appears to be similar to casein kinase II. Clathrin β‐light chain is also phosphorylated in vivo. After injection of [32P]phosphate into rats and preparation of purified coated vesicles in the presence of phosphatase inhibitors, electrophoretic analysis showed the presence of several labeled polypeptides including clathrin β‐light chain. A polypeptide of 50 kDa, which may correspond to the major polypeptide phosphorylated in vitro of coated vesicles, is also labeled in vivo.
Chromosomal localization of the gene coding for α‐subunit of Na+,K+‐ATPase in the American mink (Mustela vison)
Authors: Matveeva N.M. , Khlebodarova T.M. , Karasik G.I. , Rubtsov N.B. , Serov O.L. , Sverdlov E.D. , Broude N.E. , Modyanov N.N. , Monastyrskaya G.S. , Ovchinnikov Yu.A.
Published: July 2002

The gene coding for the α‐subunit of Na+,K+‐ATPase has been localized on chromosome 2 of the American mink (Mustela vison) using the somatic cell hybrids mink‐Chinese hamster and pig cDNA clones as hybridization probes.
The primary structure of histone H3 from the nematode Caenorhabditis elegans
Authors: Vanfleteren Jacques R. , Van Bun Stefaan M. , Van Beeumen Jozef J.
Published: July 2002

The complete amino acid sequence of histone H3 (135 residues) from the nematode Caenorhabditis elegans has been established. Microheterogeneity occurs at positions 96 and 100 of the chain. The sequences of the nematode H3 isoforms are very similar to the major chain of calf thymus H3 with which they show 4 substitutions in total. The major variant has cysteine in position 96. This is the first report of cysteine in this position in H3 from non‐mammalian tissue. An exceptional methylation site has been detected at position 79. Various other sites of secondary modification are of a conservative nature.
Ribose‐5‐phosphate isomerase and ribulose‐5‐phosphate kinase show apparent specificity for a specific ribulose 5‐phosphate species
Authors: Anderson Louise E.
Published: July 2002

Ribose‐5‐phosphate isomerase and ribulose‐5‐phosphate kinase appear to show specificity for a particular ribulose 5‐phosphate species. The effect of this specificity will be channeling of ribulose 5‐phosphate from the isomerase to the kinase during photosynthesis.
Vitamin E and selenium regulate balance between β‐adrenergic and muscarinic responses in rat lungs
Authors: Doelman C.J.A. , Kramer K. , Timmerman H. , Bast A.
Published: July 2002

The effects of hydrogen peroxide on the β‐adrenergic and muscarinic responses of the rat trachea muscle were studied in vitro, after feeding rats, for 6 weeks, either a diet deficient in vitamin E and selenium or a control diet. In the control situation after incubation with 1 mM hydrogen peroxide for 30 min, a reduction of the maximal response to methacholine of 39% occurred whereas no pD
2 shift could be demonstrated. Moreover, no response to isoprenaline after precontraction with 3 × 10−7 M methacholine was left. In the deficient situation, we found a reduction to 64% of the response to methacholine after incubation with 1 mM hydrogen peroxide. Again isoprenaline became inactive, i.e. no relaxation with isoprenaline was observed after precontraction with 3 × 10−7 M methacholine. We therefore conclude that vitamin E and selenium protect against oxidative stress in lung tissue and thus regulate the (patho‐) physiological balance between adrenergic and muscarinic responses.
Characterization of yeast fructose‐2,6‐bisphosphate 6‐phosphatase
Authors: Plankert Uwe , Purwin Claudio , Holzer Helmut
Published: July 2002

To obtain information on the biological significance of yeast fructose‐2,6‐bisphosphate 6‐phosphatase, kinetic data of the purified enzyme [(1987) Eur. J. Biochem. 164, 27–30] have been measured. Maximal activity was found between Ph 6 and 7, the apparent Michaelis constant with fructose 2,6‐bisphosphate was 7.2 μM at Ph 6.0 and 79 μm at Ph 7.0. Concentrations required for 50% inhibition of the enzyme at Ph 6.0 were 8 μm Fru2P, 45 μm G1c6P, 80 μm Fru6P and 200 μm inorganic phosphate. The known intracellular steady‐state level of about 10 μm fructose 2,6‐bisphosphate in the presence of glucose is likely to be the result of a balance between the rapid synthesis of fructose 2,6‐bisphosphate catalyzed by 6‐phosphofructose 2‐kinase and a fructose 2,6‐bisphosphate degrading activity. The biological function of fructose‐2,6‐bisphosphate 6‐phosphatase with an apparent Michaelis constant between 7 and 79 μm fructose 2,6‐bisphosphate at Ph 6–7 is therefore suggested to participate in the maintenance of a steady‐state level of fructose 2,6‐bisphosphate in a concentration range that fits well with the Michaelis constant of the enzyme.
Processing by inverted plasma membrane vesicles of in vitro synthesized major lipoprotein from Escherichia coli
Authors: Krishnabhakdi Siegfried S. , Müller Matthias
Published: July 2002

Synthesis, lipid modification and proteolytic processing of the major lipoprotein from Escherichia coli is shown to occur in a homologous in vitro transcription‐translation system containing inverted plasma membrane vesicles. The primary translation product (cross‐reacting with anti‐lipoprotein antiserum) is a precursor which is converted into a lower molecular mass species of the size of mature lipoprotein by the addition of inverted membrane vesicles from E. coli. Conversion is prevented by globomycin, a specific inhibitor of the unique lipoprotein‐signal peptidase II, which is active only on lipid‐containing precursors. The inverted plasma membrane vesicles used here must therefore contain active lipid‐modifying enzymes and signal peptidase II.
Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase in human erythrocytes
Authors: Kimura Hirokazu , Nakano Minoru
Published: July 2002

Superoxide dismutase in human erythrocytes was assayed by the inhibition of highly diluted erythrocyte lysates on 2‐methyl‐6‐(p‐methoxyphenyl)‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one‐dependent luminescence induced by the hypoxanthine‐xanthine oxidase system. Our chemiluminescence procedure gave 95‐times higher sensitivity than the cytochrome c method. The concentration of superoxide dismutase in erythrocytes of Down's syndrome patients was approx. 1.8‐times higher than that of normal humans.
Activation of human pepsinogens
Authors: Foltmann B.
Published: July 2002

Human pepsinogen A3 and A5 have been purified to chromatographic and electrophoretic homogeneity. At pH 2 pepsinogen A3 activates at a much faster rate than pepsinogen A5. Leu‐23 — Lys‐24 is the first bond cleaved during activation of pepsinogen A3. This bond is also cleaved in pepsinogen A5, but together with the cleavage of Asp‐25 — Phe‐26. Amino acid sequencing shows that pepsinogen A3 has Glu at position 43, whereas pepsinogen A5 has Lys.
Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells
Authors: Rexin Martin , Busch Willi , Segnitz Bernd , Gehring Ulrich
Published: July 2002

Mouse lymphoma cells contain a nonactivated glucocorticoid receptor of M
r∼330000 which is heteromeric in nature and is unable to bind to DNA. Following affinity labeling of the steroid‐binding subunit and subsequent cross‐linking with dimethyl suberimidate at various times either in cell extracts or in intact cells, a series of labeled bands was detected in SDS gels. From the molecular masses of completely and partially cross‐linked complexes we conclude that the large nonactivated receptor is a tetramer composed of two 90 kDa subunits, one 50 kDa polypeptide and one steroid‐binding subunit.
Expression of human leukotriene A4 hydrolase cDNA in Escherichia coli
Authors: Minami Michiko , Minami Yasufumi , Emori Yasufumi , Kawasaki Hiroshi , Ohno Shigeo , Suzuki Koichi , Ohishi Nobuya , Shimizu Takao , Seyama Yousuke
Published: July 2002

The cDNA clone encoding human leukotriene A4 hydrolase was inserted into a vector pUC9 and expressed in Escherichia coli as a fusion protein containing the first 10 amino acid residues derived from a vector. The leukotriene A4 hydrolase activity was recovered in the soluble fraction of the transformants. The purified enzyme showed kinetic properties similar to the native enzyme, including inactivation by the substrate and sulfhydryl‐modifying reagents. The results demonstrate that a protein with an M
r of 70000 was expressed in Escherichia coli with a full enzyme activity and structural fidelity. Acquisition of the expression system makes it feasible to elucidate the reaction mechanism of the enzyme.
Cytochrome c‐550 mediates electron transfer from inducible periplasmic c‐type cytochromes to the cytoplasmic membrane of Paracoccus denitrificans
Authors: Davidson Victor L. , Kumar M.Arun
Published: July 2002

Electron transfer from periplasmic cytochromes c to the membrane‐bound respiratory chain has been studied with the isolated cytochromes and membrane preparations from Paracoccus denitrificans. When reduced cytochromes were incubated with spheroplasts only the constitutive cytochrome c‐550 was rapidly oxidized. The inducible cytochromes c‐551i and c‐553i were not oxidized at appreciable rates. Cytochrome c‐550 was able to mediate the transfer of electrons from either cytochrome c‐551i or cytochrome c‐553i to the membrane preparation.
Identity of GA1, GM1a and GD1b synthase in Golgi vesicles from rat liver
Authors: Iber Heinrich , Kaufmann Reinhard , Pohlentz Gottfried , Schwarzmann Günter , Sandhoff Konrad
Published: July 2002

Synthesis of ganglioside GDD1b from ganglioside GD2 was demonstrated using Golgi membranes isolated from rat liver. Competition experiments using gangliosides GA2, GM2 and GD2 as substrates, and as mutual inhibitors for ganglioside galactosyltransferase activity in preparations of Golgi vesicles derived from rat liver, suggested that galactosyl transfer to these three compounds, leading to gangliosides GA1, GM1a and GD1b respectively, is catalyzed by one enzyme. These results strengthen the hypothesis that the main site for the regulation of ganglioside biosynthesis occurs within the reaction sequence LacCer→GA3→GD3→GT3.
Addition of a methyl group changes both the catalytic velocity and thermostability of the neutral protease from Bacillus stearothermophilus
Authors: Takagi Masahiro , Imanaka Tadayuki
Published: July 2002

Specific activity was compared between wild‐type (WT) neutral protease from Bacillus stearothermophilus and mutant protease (M1; Gly144 replaced by Ala144) with enhanced thermostability. When casein was used as a substrate, M1 showed 1.5‐times higher specific activity than that of WT. In contrast, the specific activities of M1 for soluble reduced lysozyme and insulin B chain were lower than those of WT by 17.2 and 13.2 %, respectively. After digestion of the insulin A chain by these enzymes, the peptide products were purified and the N‐terminal amino acid sequences were determined. WT enzyme cleaved insulin A chain at three sites, whereas no digestion was observed with M1. Using Z‐Gly‐Leu‐NH2 as a substrate, the kinetic parameters were determined. The K
m values are nearly equal for both enzymes, whereas the k
cat of M1 (240 min−1) was much smaller compared to the WT (830 min−1). The data indicate that the mutation (addition of a methyl group) exerts an effect by changing both the catalytic velocity and thermostability.
Calmodulin‐independent bovine brain adenylate cyclase Amino acid sequence and nucleotide sequence of the corresponding cDNA
Authors: Lipkin V.M. , Khramtsov N.V. , Andreeva S.G. , Moshnyakov M.V. , Petukhova G.V. , Rakitina T.V. , Feshchenko E.A. , Ishchenko K.A. , Mirzoeva S.F. , Chernova M.N. , Dranytsyna S.M.
Published: July 2002

An individual catalytic component of calmodulin‐independent adenylate cyclase has been isolated from bovine brain cortex. Affinity chromatography on an immunosorbent was used. The amino acid sequence of adenylate cyclase as well as the corresponding nucleotide sequence of the cDNA has been determined. cDNA of adenylate cyclase encodes a protein consisting of 834 amino acid residues and the signal peptide (19 amino acid residues). A series of adenylate cyclase isoforms has been found. A homology between adenylate cyclases from bovine brain, E. coli and Bordetella pertussis has been revealed.
Neutralizing monoclonal antibody specific for α‐bungarotoxin: Preparation and characterization of the antibody, and localization of antigenic region of α‐bungarotoxin
Authors: Kase Ryoichi , Kitagawa Hisayo , Hayashi Kyozo , Tanoue Kenjiro , Inagaki Fuyuhiko
Published: July 2002

We prepared an α‐bungarotoxin‐specific monoclonal antibody that neutralizes the biological activity of the toxin in vivo. The antigenic determinant combining specifically with this antibody was determined on the basis of cross‐reaction experiments using three other long neurotoxins and peptide fragments of α‐bungarotoxin. The antigenic determinant was located on the peptide fragment containing S34‐S35‐R36‐G37‐K38, which forms a part of the expected site that binds to the acetylcholine receptor proteins
Immunological evidence that insulin activates protein kinase C in BC3H‐1 myocytes
Authors: Acevedo-Duncan Mildred , Cooper Denise R. , Standaert Mary L. , Farese Robert V.
Published: July 2002

Effects of insulin on immunoreactive protein kinase C were examined in BC3H‐1 myocytes. Insulin provoked rapid dose‐dependent decreases in cytosolic enzyme, and transient increases and subsequent decreases in membrane‐associated enzyme. Phorbol esters provoked similar changes. Our findings suggest that insulin provokes the translocative activation of protein kinase C.
Effects of solute conditions on the relative affinities of the oligonucleotides d(G‐C)5 and d(A‐T)5 for the anthracycline drug 2‐fluoro‐4‐demethoxydaunomycin
Authors: Hammer Brendon C. , Russell Richard A. , Warrener Ronald N. , Collins J.Grant
Published: July 2002

19F NMR has been used to show that changes in NaCl concentration, as well as the presence of lysine or arginine, affect the equilibrium distribution of the synthetic anthracycline 2‐fluoro‐4‐demethoxydaunomycin (2FD) between binding sites on d(G‐C)5 and d(A‐T)5 in a 1:1:1 molar aqueous system: 2FD/d(G‐C)5/d(A‐T)5. Varying the pH between 6.2 and 7.7 had no effect. NaCl concentrations below 0.1 M led to a d(G‐C)5 preference while above 0.1 M a preference for d(A‐T)5 was observed. At comparable solute concentrations, use of either lysine or arginine resulted in a significant drug preference for d(G‐C)5 compared to systems containing only NaCl.
Trametes versicolor ligninase: Isozyme sequence homology and substrate specificity
Authors: Jönsson Leif , Karlsson Olov , Lundquist Knut , Nyman Per Olof
Published: July 2002

The substrate specificity of three ligninase isozymes from the white‐rot fungus Trametes versicolor has been investigated using stereochemically defined synthetic dimeric models for lignin. The isozymes have been found to attack non‐phenolic β‐O‐4 as well as β‐1 lignin model compounds. This finding confirms the classification of the isozymes from T. versicolor as ligninases. The amino‐terminal residues of the three isozymes from T. versicolor have been determined using Edman degradation. Minor differences found between the sequences suggest the existence of several structural genes for ligninase in T. versicolor. Comparisons have been made with the sequences of three previously reported ligninases from Phanerochaete chrysosporium, another lignin‐degrading fungus. One of the sequences from P. chrysosporium is distinctly more similar to the T. versicolor isozymes than to the other two sequences from P. chrysosporium.
Lipocortin I and lipocortin II inhibit phosphoinositide‐ and polyphosphoinositide‐specific phospholipase C The effect results from interaction with the substrates
Authors: Machoczek Kathrin , Fischer Manfred , Söling Hans-Dieter
Published: July 2002

Lipocortins I and II, known to inhibit phospholipase A2, have been purified from bovine lung and tested with respect to their ability to affect the enzymatic activities of phosphoinositide‐ and polyphosphoinositide‐specific phospholipase C from human platelets, rat liver cytosol or rat brain membranes. At 0.67,μM, both lipocortins led to complete inhibition of phospholipase C activity with either phosphatidylinositol or phosphatidylinositol 4,5‐bisphophate as substrate. The inhibition could be overcome by increasing the substrate concentration. Ultracentrifugation studies with lipocortin II showed a direct interaction between phosphatidylinositol and the lipocortin, indicating that the lipocortins inhibit phospholipase C not directly but by interacting with the substrate. In experiments with plasma membranes from [3H]inositol‐labeled HL‐60 cells, lipocortin II did not affect PI‐specific phospholipase C activity in the absence or presence of calcium plus or minus GTP‐γ‐S.
Estrogen synthetase in the horse
Authors: Vibet A. , Dintinger T. , Maboundou J.C. , Gaillard J.L. , Divoux D. , Silberzahn P.
Published: July 2002

NADPH‐cytochrome c (P‐450) reductases from horse placenta and rat liver were purified and their biological activities compared using cytochrome c as substrate. Rat liver reductase was purified to electrophoretic homogeneity in one Chromatographic step on 2',5'‐ADP agarose, and had a relative mass of 85 000 Da as estimated by SDS‐PAGE. Equine placental reductase was separated from cytochrome P‐450 on aminohexyl‐Sepharose 4B and further purified on 2',5‐ADP agarose; this preparation exhibited two bands, one of 85 000 and one of 80 000 Da, on SDS‐PAGE. The lower molecular weight form was assumed to be a proteolytic product of the higher molecular weight form. A high retention of activity was obtained in both preparations. Equine placenta and rat liver enzymes were found to exhibit very similar Vmax
and Km
, suggesting that they are not species specific.
Structure and expression of rat muscle‐specific enolase gene
Authors: Sakimura Kenji , Kushiya Etsuko , Ohshima-Ichimura Yuka , Mitsui Hiromi , Takahashi Yasuo
Published: July 2002

The structure of rat muscle‐specific enolase (ββ enolase) gene was determined. It comprises 12 exons of various lengths (59–223 bp) spanning about 6 kbp and its exon‐intron organization is similar to that of neuron‐specific enolase (γγ enolase) gene. A transcriptional start site was identified by a combination of S1 nuclease mapping and primer extension analyses. In the 5'‐flanking region we found a TATA‐box‐like sequence and several MyoD‐binding motifs. The in vitro cell free transcription of the truncated genomic DNA fragment using HeLa cell extract showed that the transcription start site has been correctly identified and the promoter sequences work well.
Interaction of smooth muscle caldesmon with phospholipids
Authors: Vorotnikov Alexander V. , Gusev Nikolai B.
Published: July 2002

Taking into account the perimembrane localization of caldesmon [(1986) Nature 319, 68] and its ability to participate in the regulation of receptor clusterization [(1989) J. Biol. Chem. 264, 496], we studied the interaction of duck gizzard caldesmon with soybean phospholipids (azolectin). By using four independent methods, i.e. light scattering, gel‐electrophoresis, gel‐filtration and ultracentrifugation, we showed a Ca‐independent complex formation between caldesmon and azolectin. Interacting with caldesmon, calmodulin is shown to dissociate the caldesmon‐azolectin complex. It is supposed that the caldesmon‐phospholipid interaction may affect caldesmon phosphorylation by Ca‐phospholipid‐dependent protein kinase. This effect may be important for various cell motility processes.
Assembly of an adult type acetylcholine receptor in a mouse cell line transfected with rat muscle ϵ‐subunit DNA
Authors: Criado Manuel , Koenen Michael , Sakmann Bert
Published: July 2002

The mouse muscle cell line BC3H‐1 expresses an acetylcholine receptor (AChR) composed of α‐,β‐, and δ‐subunits [1]. The functional characteristics of this AChR are comparable to the non‐synaptic AChR subtype in mouse muscle [2,3]. To investigate the role of the ϵ‐subunit, which is believed to replace the γ‐subunit in forming the adult AChR subtype [4], BC3H‐1 cells were stably transfected with cDNA encoding the rat muscle AChR ϵ‐subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced ϵ‐subunit specific RNA after heat induction. Single‐channel current recording from cells which contained abundant ϵ‐subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle ϵ‐subunit may assemble with mouse muscle α‐, β‐ and δ‐subunits to form a mouse‐rat hybrid AChR with properties similar to that of end‐plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in ϵ‐subunit mRNA. Thus, the notion that replacement of the γ‐subunit by the ϵ‐subunit during development is the result of the postnatal rise in the level of ϵ‐subunit specific mRNA is further supported.
Cholesterol oxidation on fluorocarbon emulsion surface leads to the formation of 7‐peroxycholesterol
Authors: Beriozov Alan T. , Ivanov Aleksey S. , Ivkov Vladimir G. , Obraztsov Victor V. , Khalilov Elvis M. , Archakov Alexander I.
Published: July 2002

Formation of biologically active oxidized derivatives of cholesterol as a result of its oxidation on the surface of fluorocarbon emulsions was studied. A single product of cholesterol oxidation, 7‐peroxycholesterol, was found. It was shown that 7‐peroxycholesterol and its derivative 7‐keto‐cholesterol inhibit the rosette formation between human T‐lymphocytes and sheep erythrocytes. These substances exert a strong cytostatic action on the growth of procaryotic and eucaryotic cell cultures. Thus, oxidative modification of blood plasma components on the surface of fluorocarbon emulsion particles with the formation of highly active compounds must be taken into account when using the fluorocarbon emulsions in medicine.
Pericentral expression pattern of glucokinase mRNA in the rat liver lobulus
Authors: Moorman Antoon F.M. , de Boer Piet A.J. , Charles Robert , Lamers Wouter H.
Published: July 2002

The spatial distribution of glucokinase mRNA (GK mRNA) in rat liver was studied by in situ hybridization under normal and inducing conditions. GK mRNA was first detectable in the liver parenchyma of neonatal rats of 1.5 days. The density of grains decreases in a central portal direction. This pattern remains essentially unchanged up to 15 days, after which the adult type of distribution gradually starts to develop, i.e. low density of grains indicating low levels of GK mRNA, in which no gradient of expression could be visualized. Within 2 h after an oral glucose load to starved animals, the GK mRNA expression pattern changed from hardly detectable to a clear gradient with the highest grain density around the terminal central venules. Within 6 h relatively high levels of grains, almost homogeneously distributed across the liver lobule, were observed. Glucocorticosteroid treatment also induced GK mRNA in the pericentral area. It is concluded that the observed induction pattern qualifies GK mRNA as a pericentral mRNA suggesting that the pericentral expression pattern of the protein is primarily regulated at the pretranslational level.
Molecular resolution imaging of polyglucose by scanning tunneling microscopy
Authors: Yang Jie , Takeyasu Kunio , Somlyo Andrew P. , Shao Zhifeng
Published: July 2002

We have obtained atomic resolution images of poly‐α‐D‐glucose by scanning tunneling microscopy. The oxygen atoms near the scanning tip were imaged, but the carbon and hydrogen atoms were not visible. The measured inter‐atomic distances are consistent with the molecular structure of poly‐α‐D‐glucose deduced from chemical and X‐ray diffraction studies. The results also demonstrate that is 1% feasible to image surface atomic structures of a relatively thick non‐conducting specimen, suggesting that the technique may be applied to the study of other macromolecules of biological importance.
High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor G proteins interactions
Authors: Horn Valerie J. , Ambudkar Indu S. , Baum Bruce J.
Published: July 2002

The binding of the non‐selective muscarinic antagonists [3H] quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions. [3H]QNB bound to a homogenous population of muscarinic receptors (Kd
. 118±19 pM; B
max 572±42 fmol/mg membrane protein. n = 12). The addition of G protein activators AIF−
4 or guanosine‐5′‐O‐(3‐thiotriphospate) (GTPγS) + Mg2+ increased the K
d by 77 ± 7% (n=4. P < 0.05) and 83 ± 27% (n=7, P < 0.05), respectively, without a change in the B
max or homogeneity of the binding site. GTPγS added without exogenous Mg2+ did not affect [3H] QNB binding requires a muscarinic receptor/G protein interaction.
Probing DNA triple helix structure by chemical ligation
Authors: Dolinnaya Nina G. , Pyatrauskene Olga V. , Shabarova Zoe A.
Published: July 2002

A series of oligomeric double and triple helical DNAs with irregular sequences of homopurine and homopyrimidine strands were prepared. DNA triplexes were identified by CD spectroscopy and thermal denaturation profiles (biphasic helix‐coil transition). Condensation of oligonucleotides on single and double‐stranded DNA templates was performed using water‐soluble carbodiimide, phosphodiester and pyrophosphate internucleotide bonds being newly formed. Such chemical ligation proved to be a sensitive monitor of changes in the sugar‐phosphate backbone resulting from conversion of double to triple helix and of third‐strand binding.
Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endorenous kinase
Authors: Donovan Maura G. , Bodley James W.
Published: July 2002

Mammalian cells contain a Ca2+/calmodulin‐dependent protein kinase that specifically phosphorylates and inactivates elongation factor 2 (EF‐2) in response to hormones and other agents which increase intracellular Ca2+ concentrations. Therefore, it has been proposed that the rate of translation in mammals is regulated by EF‐2 phosphorylation. In the present study, EF‐2 purified from the yeast Saccharomyces cerevisiae is shown to be a substrate for the mammalian EF‐2 kinase. Furthermore, evidence was obtained using two‐dimensional gel electrophoresis and peptide mapping which suggests that yeast EF‐2 is a substrate for an endogenous kinase which phosphorylates the same site as the mammalian EF‐2 kinase. Based on these findings, we propose that in yeast as in higher eukaryotes, the protein synthesis elongation cycle is regulated by phosphorylation of EF‐2.
Functional properties of human thyroid hormone receptor β1 overexpressed using baculovirus
Authors: Collingwood T.N. , Sydenham M. , Page M.J. , Chatterjee V.K.K.
Published: July 2002

We have overexpressed the human β1 thyroid hormone receptor in insect cells using a recombinant baculovirus to a level of 5–10% of total cellular protein. The recombinant protein migrates as a 50 kDa band by SDS‐PAGE and Western blot analysis. The expressed receptor binds to L‐T3 with a K
d of 1.3±0.4 × 10−10 M and to thyroid hormone analogues with an affinity hierarchy of TRIAC>L‐T3>L‐T4>rT3. Gel retardation assays show highly specific receptor binding to a TRE which is modified by the presence of ligand and avidin—biotin complex DNA analysis shows a K
d of 6.2±2.0 × 10−10 M for this interaction. These results indicate high level expression of hTRβ with authentic hormone and DNA binding properties.
Over‐expression and refolding of β‐subunit from the chloroplast ATP synthase
Authors: Chen Zugen , Wu Inmin , L. Richter Mark , Gegenheimer Peter
Published: July 2002

We established a bacterial system for high‐level over‐expression of the spinach chloroplast atpB gene which encodes the ATP Synthase β subunit. Upon induction, atpB was expressed as at least 50% to 70% of total cell protein. Although the over‐expressed β polypeptide formed insoluble inclusion bodies, more than fifty percent of it was restored to a functional form by solubilizing the inclusion bodies with 4 M urea and slowly removing the urea by stepwise dialysis. The resulting β subunit exhibited specific and selective nucleotide binding properties identical to those of the native β subunit.
Functional control of gastrin releasing peptide (GRP) mRNA in rat stomach
Authors: Dimaline R. , Sandvik A.K. , Barclay G. , Dockray G.J.
Published: July 2002

The gastric factors controlling abundance of mRNA encoding the important neuropeptide, gastrin releasing peptide (GRP) in rat stomach, were examined by Northern and slot blot analysis. Withdrawal of food increased antral GRP mRNA, as did treatment of fed rats with the acid inhibitory drug, omeprazole. There was no change in GRP mRNA abundance in gastric corpus. The data indicate functionally distinct populations of GRP neurons in different regions of the stomach, and control of antral neuropeptide biosynthesis by the gastric luminal contents.
Kinetic analysis of translocation into isolated chloroplasts of the purified ferredoxin precursor
Authors: Pilon Marinus , Weisbeek Peter J. , de Kruijff Ben
Published: July 2002

Time courses of the import into isolated chloroplasts of the purified ferredoxin precursor were measured at different protein concentrations. Analysis of the initial import rates indicates the presence of one saturable import system with an apparent K
m value of approximately 100 nM and a V
max corresponding to the uptake of approximately 2.5·104 precursor proteins per minute per chloroplast. We conclude that the in vitro observed activity of the chloroplast protein import machinery, functioning independently from cytosolic factors. would be enough to allow chloroplast developmental at physiological rate.
Structural analysis of the putative regulatory region of the rat gene encoding poly(ADP‐ribose) polymerase
Authors: Potvin Frédéric , Thibodeau Jacques , Kirkland James B. , Dandenault Brigitte , Duchaine Caroline , Poirier Guy G.
Published: July 2002

A γ EMBL3 clone containing the first three exons along with part of the 4th exon of the rat poly(ADP‐ribose) polymerase gene was isolated from a genomic DNA library. This clone also contains 6.6 kbp of upstream sequences. Nucleotide sequence analysis of the proximal 5′ 670 nucleotides flanking the major RNA start site of the rat gene does not reveal significant global homology with the same region of the human gene, but a series of short sequences are identical. Among these sequences are found two putative Spl binding sites along with a decanucleotide sequence responsible for the attachment of the transcription factor AP‐2.
Transcription efficiency of human apolipoprotein A‐I promoter varies with naturally occurring A to G transition
Authors: Tuteja Renu , Tuteja Narendra , Melo Carlos , Casari Giorgio , Baralle Francisco E.
Published: July 2002

In human, the gene coding for apolipoprotein A‐I (apo A‐I), a protein of the plasma lipid transport system, is expressed only in the liver and the intestine. A naturally occurring A to G substitution in the promoter at position −78 was shown to be associated with high density lipoproteins (HDL) in females. We have studied the effect of this mutation on promoter activity using various lengths of promoter sequences and the CAT reporter gene system. Transient expression studies after introduction of these constructs into Hep 3B cells revealed that in the region spanning −330 to +1 of the promoter an A to G substitution increases the activity approximately twofold. On the other hand, when further upstream region (−1469 to +397) is also included, the promoter activity seems comparable in both alleles. Our results show how minimal sequence variations can affect the in vitro analysis of promoter activity.
Identification of cholesterol‐bound aldehydes in copper‐oxidized low density lipoprotein
Authors: Kamido H. , Kuksis A. , Marai L. , Myher J.J.
Published: July 2002

Lipid‐soluble cholesteryl ester core aldehydes (aldehydes still bound to the cholesterol ring) were identified among the products of copper‐catalyzed peroxidation of human low density lipoprotein (LDL). The LDL was exposed to oxygenated buffer and 5 μM CuSO4 for 24 h. The core aldehydes were isolated as the dinitrophenylhydrazones, and were identified by reverse‐phase HPLC with mass spectrometry. The major components were the C4–C10 oxoalkanoyl esters of cholesterol and 7‐ketecholesterol, and accounted for 1–2% of the cholesteryl linoleate and arachidonate consumed.
Isoproterenol‐induced Mg2+ uptake in liver
Authors: Günther Theodor , Vormann Jürgen , Höllriegl Vera
Published: July 2002

Isoproterenol increased the Mg2+ content of hepatocytes after injection into rats or after addition to collagenase‐dispersed hepatocytes. cAMP also the increased cellular Mg2+ content of isolated hepatocytes. This effect was prevented by staurosporine. Phorbol ester had no effect on the Mg2+ content of isolated hepatocytes, and after injection of isoproterenol into rats, protein kinase C of liver was not affected. It was concluded that isoproterenol induced long‐term Mg2+ influx via the activation of protein kinase A which can be inhibited by staurosporine.
Leukotriene A4 hydrolase, a bifunctional enzyme Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site‐directed mutagenesis at Glu‐297
Authors: Minami Michiko , Bito Haruhiko , Ohishi Nobuya , Tsuge Hideaki , Miyano Masashi , Mori Mikiro , Wada Hiroo , Mutoh Hiroyuki , Shimada Shinobu , Izumi Takashi , Abe Keiko , Shimizu Takao
Published: July 2002

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu‐297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His‐296, His‐300 and Glu‐319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site‐directed mutagenesis experiments were carried out. Glu‐297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu‐297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.
Cellobiose oxidase from Phanerochaete chrysosporium Stopped‐flow spectrophotometric analysis of pH‐dependent reduction
Authors: Samejima Masahiro , Phillips Robert S. , Eriksson Karl-Erik L.
Published: July 2002

Cellobiose oxidase (CBO) from Phanetochaete chrysosporium can utilize dichlorphenol—indophenol (Cl2Ind) and cytochrome c as effective electron acceptors for the oxidation of cellobiose. However, the pH dependencies of activity for these electron acceptors are significantly different. Both compounds act as effective electron acceptors at pH 4.2, whereas only dichlorophenol‐indophenol is active at pH 5.9. To explain this discrepancy, the pH dependencies of the reduction rates of FAD and heme, respectively, in CBO by cellobiose have been investigated by stopped‐flow spectrophotometry. Both FAD and heme are reduced with a high rate constant at pH 4.2. In contrast, at pH 5.9, only FAD reduction is fast, while the reduction of the heme is extremely slow. As a conclusion, the reduction of cytochrome c by CBO is dependent on heme, which functions at a lower pH range compared to reduction of FAD.
Identification of actin kinase activity in purified fragmin—actin complex
Authors: Furuhashi Kiyoshi , Hatano Sadashi
Published: July 2002

Actin kinase phosphorylates actin of fragmin—actin complex, resulting in the inactivation of the nucleation and capping activities of the complex. Fragmin—actin complex was prepared by a new purification procedure. Incubation with ATP caused inactivation of the purified complex and phosphorylation of actin of fragmin—actin complex. The detailed analysis of the complex by SDS‐gel electrophoresis showed that actin kinase was co‐purified with the fragmin—actin complex. Formation of such an association between actin kinase and substrate suggests that the kinase is localized on the fragmin—actin complex to efficiently regulate actin cytoskeletons.
Cloning of the PABA peptide hydrolase alpha subunit (PPHα) from human small intestine and its expression in COS‐1 cells
Authors: Dumermuth Eric , Eldering Joyce A. , Grünberg Jürgen , Jiang Weiping , Sterchi Erwin E.
Published: July 2002

PABA peptide hydrolase (PPH) from human enterocytes is comprised of two submits, alpha and beta. PPHα is over 70% identical to meprin, a protease isolated from mouse and rat kidney. The enzyme shows a modular organization in that it contains an astacin protease domain, an adhesive domain, an EGF‐like domain, and a putative C‐terminal membrane spanning domain. Expression of a chimeric meprin‐PPHα cDNA in COS‐1 cells led to the synthesis of immature, transport‐incompetent homodimers. In addition, complex glycosylated forms were detected in the culture medium, suggesting that the enzyme is secreted after proteolytic removal of the membrane anchor.
Nucleoside triphosphatase activity associated with the N‐terminal domain of mammalian tryptophanyl‐tRNA synthetase
Authors: Kovaleva Galina , Nikitushkina Tatyana , Kisselev Lev
Published: July 2002

Bovine tryptophanyl‐tRNA synthetase (EC 6.1.1.2) deprived of Zn2+ by chelation with the phosphonate analog of Ap4A hydrolized ATP(GTP) to ADP(GDP) although its ability to form tryptophanyl adenylate was impaired. This hydrolytic activity is stimulated by Mg2+ and Mn2+ ions and inhibited by Zn2+. Monoclonal antibody Aml against the N‐terminal domain of the enzyme completely abolished ATP(GTP)ase activity. The core peptide generated after proteolytic splitting of the N‐domain lacks this activity. We suggest that the nucleotide binding site(s) different from ATP sites involved in aminoacylation reaction reside(s) at the N‐terminal domain(s) of the enzyme.
Ultrastructure of membrane‐bound Na,K‐ATPase after extensive tryptic digestion
Authors: Ning Gang , Maunsbach Arvid B. , Esmann Mikael
Published: July 2002

Membrane‐bound Na,K‐ATPase was digested with trypsin in the presence of Rb+ to form the stable 19‐kDa and smaller fragments of the α‐chain known to preserve occlusion of Rb+ (K+) or Na+. The trypsinized membranes obtained from pig kidney and shark rectal gland were analyzed by electron microscopy. Tryptic digestion preserved general membrane structure but removed both the surface particles observed by negative staining and the protruding cytoplasmic portion of the α‐subunit identified in thin sections. However, intramembrane particles defined by freeze‐fracture were preserved after trypsinization suggesting that the remaining membrane spanning protein fragments retain the native structure within the lipid bilayer after proteolysis.
Phosphorylation of elongation factor‐2 from the lepidopteran insect, spodoptera frugiperda
Authors: Oldfield Susan , Proud Christopher G.
Published: July 2002

In mammalian cells, protein synthesis can be regulated at the level of elongation by the phosphorylation of elongation factor 2 (eEF‐2) by a highly specific Ca2+/calmodulin‐dependent kinase. In this report, we show that eEF‐2 from a cell line derived from the insect, Spodoptera frugiperda, is a substrate for mammalian eEF‐2 kinase and that phosphorylation is Ca2+‐dependent. Furthermore, two‐dimensional peptide mapping shows that the kinase phosphorylates the same sites in Spodoptera eEF‐2 as those phosphorylated in the rabbit protein. However, we were unable to detect an eEF‐2 kinase in Spodoptera cells.