bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a tyrosine residue in the saccharide binding site of ricin B‐chain using N‐[14C]acetylimidazole
Authors: Wawrzynczak Edward J. , Falasca Anna , Jeffery William A. , Watson Graham J. , Thorpe Philip E.
Published: July 2002

The binding of ricin B‐chain to Sepharose, a galactose‐based adsorbent, was reversibly inactivated by acetylation of tyrosine residues in the absence of lactose. In the presence of lactose, two tyrosine residues were protected against modification and the B‐chain retained its binding ability. Analyses of tryptic peptides from B‐chain modified with N‐[14C]acetylimidazole in the presence and absence of lactose showed that Tyr‐248 is present in one of the galactose‐binding sites.
The developmental regulation of phosphatidylinositol kinase in Dictyostelium discoideum
Authors: Varela Isabel , Van Lookeren Campagne Michiel M. , Alvarez Jose F. , Mato Jose M.
Published: July 2002

Phosphatidylinositol kinase was examined in Dictyostelium discoideum since this organism offers molecular and genetic advantages to study the role of phosphatidylinositol metabolism during cell growth and development. D. discoideum homogenates phosphorylated phosphatidylinositol to form phosphatidylinositol 4‐phosphate in a reaction which was found to be linear with time and cell concentration. Optimal activity was obtained in the presence of 1 mM MgCl2 and pH 7.6 and has an apparent K
m for ATP of about 250 μM. Changes in phosphatidylinositol kinase were examined during D. discoideum development. Activity increased about 2‐fold, 4 h after removal of the food source, to decline to almost no activity at late aggregation. During slug formation the activity increased about 15‐fold and remained constant during further development. These results suggest a role for D. discoideum phosphatidylinositol kinase during development.
Epidermal growth factor‐induced phosphoinositide hydrolysis Modulation by protein kinase C
Authors: Vicentini L.M. , Cervini R. , Zippel R. , Mantegazza P.
Published: July 2002

A short‐term treatment with phorbol 12,13‐dibutyrate (PDBu) was found to inhibit totally the epidermal growth factor (EGF)‐stimulated phosphoinositide hydrolysis in A431 cells, whereas long‐term pretreatment with PDBu, which is known to down regulate protein kinase C, induced a greater accumulation of the EGF‐triggered inositol phosphate accumulation, particularly of Ins(1,3,4,5)P4. The increased Ins(1,4,5)P3/Ins(1,3,4,5)P4 formation in the PDBu long‐term pretreated cells was coincident with the increased Ca2+ influx stimulated by EGF in the same cells. Since long‐term pretreatment with PDBu was found to enhance the EGF signals, an explanation for the synergism between EGF and phorbol esters in the induction of DNA synthesis is provided.
Enzymic properties of intestinal aminopeptidase P: A new continuous assay
Authors: Lasch Jürgen , Koelsch Regine , Steinmetzer Torsten , Neumann Ulf , Demuth Hans-Ulrich
Published: July 2002

A continuous photometric assay of aminopeptidase P activity was developed which is based on a coupled enzymic assay with the substrate Gly‐Pro‐Pro‐pNA and DPP IV as auxiliary enzyme. This assay was used to evaluate the kinetic parameters and inhibitory profile of intestinal brush border aminopeptidase P.
Proton nuclear magnetic resonance study of the ferrous derivatives of the dimeric and tetrameric hemoglobin from the mollusc Scapharca inaequivalvis
Authors: Inubushi Toshiro , Yonetani Takashi , Chiancone Emilia
Published: July 2002

Proton NMR spectra have been measured for the two hemoglobins from the mollusc Scapharca inaequivalvis: HbI, a homodimer, and HbII, a heterotetramer. These hemoglobins are endowed with a unique subunit assembly, since the heme carrying E and F helices are involved in the major intersubunit contact. In the far‐downfield region of hyperfine‐shifted resonances the spectra of HbI and HbII in the deoxy state show respectively one (66.7 ppm) and two (67.8 and 63.6 ppm) exchangeable signals of the proximal histidine NδH groups, the resonance position being indicative of a significant strain in the iron‐imidazole interaction. In the hydrogen‐bonded proton region, inter‐ and intrasubunit hydrogen‐bonded proton signals have been detected for both hemoglobins. Deoxy‐HbI shows two unique downfield resonances at 11.83 and 11.51 ppm which disappear in the oxygenated state, suggesting that the corresponding hydrogen bonds are involved in the stabilization of the tertiary and/or quaternary structure of the deoxy form. HbII shows even smaller changes in this region upon changes in ligation state. These results therefore provide further proof that, at variance with the vertebrate hemoglobin tetramer, the unique subunit assembly of these proteins is stabilized mainly by hydrophobic interactions.
[3H]AHN 086 acylates peripheral benzodiazepine receptors in the rat pineal gland
Authors: McCabe R.Tyler , Schoenheimer Joyce A. , Skolnick Phil , Newman Amy Hauck , Rice Kenner C. , Reig Juan-Antonio , Klein David C.
Published: July 2002

AHN 086, an isothiocyanato derivative of Ro 5‐4864 (4′‐chlorodiazepam), inhibits radioligand binding to peripheral benzodiazepine receptors with characteristics of an irreversible (acylating) ligand. We now report that [3H]AHN 086 labels a ≈ 30 kDa protein in the rat pineal gland determined by both SDS‐polyacrylamide gel electrophoresis and gel filtration high‐performance liquid chromatography of digitonin‐solubilized membranes. Specific incorporation of [3H]AHN 086 into this protein was inhibited by preincubating membranes with excess AHN 086. Moreover, significant specific binding of [3H]AHN 086 was not observed in either bovine pineal gland (which does not possess high‐affinity binding sites for Ro 5‐4864) or ovalbumin. These findings suggest that the ≈ kDa protein labeled by [3H]AHN 086 in rat pineal gland is associated with peripheral benzodiazepine receptors in this tissue.
Cloning and expression of a functional fucose‐specific lectin from an orange peel mushroom, Aleuria aurantia
Authors: Fukumori Fumiyasu , Takeuchi Naomi , Hagiwara Toshihiko , Ito Katumi , Kochibe Naohisa , Kobata Akira , Nagata Yoshiho
Published: July 2002

Aleuria aurantia lectin (AAL) shows sugar‐binding specificity for L‐fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3‐kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino‐terminal amino acids. The 5′‐terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA‐1. Escherichia coli carrying pKA‐1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.
Synergistic stimulatory effect of glucagon‐like peptide‐1 (7–36) amide and glucose‐dependent insulin‐releasing polypeptide on the endocrine rat pancreas
Authors: Fehmann H.-C. , Göke B. , Göke R. , Trautmann M.E. , Arnold R.
Published: July 2002

The interaction of glucagon‐like peptide‐1 (7–36)amide (GLP‐1) and glucose‐dependent insulin‐releasing polypeptide (GIP) on insulin release from the perfused rat pancreas was studied. The GLP‐ 1 stimulated (0.5 nmol/l), glucose‐induced (6.7 mmol/l) insulin secretory answer was enhanced by GIP (0.1, 1.0 and 10.0 nmol/l) to the arterial perfusate. This effect was maximal at 1 nmol/l GIP and smaller but still significant at 0.1 nmol/l GIP. The high GIP concentration of 10 nmol/l GIP did not further increase insulin secretion compared to the stimulation by 1 nmol/l GIP. Our data demonstrate an additive synergistic effect of GLP‐ 1 and GIP on the glucose‐induced insulin release. This supports the concept of an action ‘in concert’ of gastrointestinal incretin hormones postprandially released on the endocrine pancreas to guarantee adequate insulin answers after meals.
The protonic shortcircuit by DCCD in photosystem II A common feature of all redox transitions of water oxidation
Authors: Jahns Peter , Junge Wolfgang
Published: July 2002

Photosystem II of green plants oxidizes water at an Mn centre and reduces bound plastoquinone to supply electrons to photosystem I. Owing to the vectorial properties of the electron transfer (water‐)protons are released into the lumen of thylakoids, while (quinole‐)protons are taken up from the medium. The proton‐pumping activity can be shortcircuited by covalent modification of two polypeptides with N,N′dicyclohexylcarbodiimide (DCCD). Then (water‐)protons are no longer released but, within milliseconds, they are funnelled through the protein to the reduced quinone [(1988) EMBO J. 7,589‐594]. When dark‐adapted thylakoids are excited by a series of light flashes water oxidation to dioxygen is synchronised to follow a stepped reaction pattern with period four, which is apparent in the pattern of proton release. We asked whether or not the above 'protonic shortcircuit' was restricted to certain reactions out of the four‐stepped cycle. This was not observed. Instead, each step was equally affected and the complement of the period four pattern was observed for proton uptake at the site of quinone reduction. This proved that seemingly non‐released protons were transferred to the quinone and, for unmodified membranes, it suggested that protons produced during the partial reactions of water oxidation used one and the same DCCD‐sensitive outlet to the lumen.
Identification of a phosphorylation site of the rat insulin receptor catalyzed by protein kinase C in an intact cell
Authors: Koshio Osamu , Akanuma Yasuo , Kasuga Masato
Published: July 2002

In two‐dimensional tryptic phosphopeptide mapping, the β‐subunit of the insulin receptor phosphorylated by 12‐O‐tetra‐decanoylphorbol‐13‐acetate in rat hepatoma cells (H‐35) was separated into one phosphothreonine‐containing peptide and several phosphoserine‐containing peptides. The synthetic peptide coding residues 1327–1343 in the C‐terminal region of the rat insulin receptor was phosphorylated at the threonine residue by protein kinase C in a phosphatidylserine and oleoylacetylglycerol dependent manner. Tryptic digest of this phosphopeptide migrated to the same position as the phosphothreonine containing peptide obtained from the β‐subunit in two‐dimensional phosphopeptide mapping. These data suggested that Thr 1336 of the insulin receptor is the site of phosphorylation by protein kinase C in intact cells.
P‐glycoproteins encoded by mdr 1b in murine gravid uterus and multidrug resistant tumor cell lines are differentially glycosylated
Authors: Greenberger Lee M. , Croop James M. , Horwitz Susan Band , Arceci Robert J.
Published: July 2002

There are 3 members of the multidrug‐resitance gene family expressed in mouse. Only one of these, mdr lb, and its gene product P‐glycoprotein are induced to high levels in the mouse endometrium during pregnancy. It is shown here that P‐glycoprotein in the gravid uterus is significantly larger (M
r 155000) compared to P‐glycoprotein encoded by mdr lb in a murine multidrug‐resistant cell line (M
r 140000). However, both species co‐migrate after enzymatic removal of N‐linked sugars (M
r 125000). These results demonstrate that differential glycosylation of the mdr lb gene product contributes to molecular heterogeneity found in P‐glycoprotein from normal and multidrug‐resistant cells.
Identification of four lipocortin proteins and phosphorylation of lipocortin I by protein kinase C in cytosols of porcine thyroid cell cultures
Authors: Antonicelli F. , Omri B. , Breton M.F. , Rothhut B. , Russo-Marie F. , Pavlovic-Hournac M. , Haye B.
Published: July 2002

Four proteins of the lipocortin family, lipocortin I (35 kDa), lipocortin II (36 kDa), lipocortin V (32 kDa) and lipocortin VI (67–70 kDa), were identified in the cytosols of 2‐day‐old cultures of thyroid cells. Only lipocortin I was phosphorylated in vitro in fully differentiated, thyroid stimulating hormone‐treated cells (0.1 mU/ml). Protein kinase C was the only kinase activity which phosphorylated lipocortin I. Phosphorylation shifted its pI from 6.9 to 6.6. The in vitro phosphorylation of lipocortin I was impaired in cultures exposed for 2 days to phorbol ester (10−7 M), although it was present in both the cytosol and the particulate fraction of these cells.
In vivo activity of the most proximal promoter of the human aldolase A ene and analysis of transcriptional control elements
Authors: Izzo Paola , Costanzo Paola , Lupo Angelo , Rippa Emilia , Salvatore Francesco
Published: July 2002

Transcriptional control; DNA‐binding factor; Aldolase A promoter; Footprinting analysis
Fructose‐6‐phosphate modifies the pathway of the urea‐induced dissociation of the allosteric phosphofructokinase from Escherichia coli
Authors: Teschner Wolfgang , Deville-Bonne Dominique , Garel Jean-Renaud
Published: July 2002

Phosphofructokinase from Escherichia coli binds fructose‐6‐phosphate with the sugar moiety of the substrate interacting with one subunit and the phosphate group with another one, so that bound fructose‐6‐phosphate lies across the interface between the subunits [(1988) J. Mol. Biol. 204, 973‐994]. When this interface is ‘cross‐linked’ by fructose‐6‐phosphate. it becomes more stable because of the extra interactions between subunits: inactivation upon dissociation occurs only above 5 M urea, instead of 1 M urea for the free protein. At saturation in fructose‐6‐phosphate. this interface is no longer the first to dissociate as in the free protein [(1989) Biochemistry 28, 6836‐6841]: instead, the addition of urea to phosphofructo‐kinase in the presence of fructose‐6‐phosphate induces a conformational change within the tetramer which alters the environment of Trp‐311 and distorts the regulatory site.
The two oxygen‐regulated subunits of cytochrome c oxidase in Dictyostelium discoideum derive from a common ancestor
Authors: Capaldi Roderick A. , Zhang Yu-Zhong , Rizzuto Rosario , Sandonà Dorianna , Schiavo Giampietro , Bisson Roberto
Published: July 2002

The two smallest polypeptide components of D. discoideum cytochrome c oxidase, whose alternative expression depends on oxygen concentration [Schiavo G. and Bisson R. (1989) J. Biol. Chem. 264, 7129‐7134], have been partially sequenced. They show 45$ homology and are isoforms of the same subunit, which must be encoded on two different genes.
Substrate specificity of the autocatalytic protein that primes glycogen synthesis
Authors: Lomako Joseph , Lomako Wieslawa M. , Whelan William J.
Published: July 2002

The autocatalytic protein that primes muscle‐glycogen synthesis, and which glucosylates itself from UDPglucose, is inhibited by maltose. Investigation of the reason for the inhibition led to the finding that the protein will glucosylate substrates other than itself. p‐Nitrophenyl αglucoside, αmaltoside, αmaltotrioside and αmaltotetraoside each inhibit self‐glucosylation of the protein by acting as alternative acceptor substrates. The αmaltoside is the best acceptor. The αmaltohexaoside did not act as an acceptor but was an effective inhibitor. These findings help to explain the self‐limiting nature of the autocatalytic extension of the maltosaccharide chain of the protein and suggest that protein self‐glucosylation may be an intennolecular event. They may also point to the mechanism by which the autocatalytic protein is initially glycosylated.
Interactions with tRNALys induce important structural changes in human immunodeficiency virus reverse transcriptase
Authors: Robert Dominique , Sallafranque-Andreola Marie-Line , Bordier Bruno , Sarih-Cottin Leila , Tarrago-Litvak Laura , Graves Pierre Vincent , Barr Philip J. , Fournier Michel , Litvak Simon
Published: July 2002

Retroviral RNA‐dependent DNA polymerase (reverse transcriptase or RT) uses the 3'OH end of a cellular tRNA as primer to initiate DNA synthesis. Previous work with avian retrovirus has shown that reverse transcriptase is implicated in the selection of cellular virion‐encapsidated tRNAs and has shown that the primer tRNA is positioned on the primer binding site near the 5' end of the viral RNA. These mechanisms support the idea that the retroviral polymerase should form complexes with primer tRNA and the specific encapsidated ones. The genomic sequence of human immunodeficiency virus (HIV) allows the prediction that tRNALys
3 is the natural primer. In this article we show, using the mobility shift assay, that recombinant HIV reverse transcriptase is able to form a complex with bovine tRNALys. By fluorescence studies and α‐chymotrypsin analysis we have observed a modification of the enzyme conformation when reverse transcriptase is bound to the putative primer tRNA. This structural change is specific for tRNALys although the retroviral polymerase is able to interact with other tRNAs.
Atrial natriuretic factor recognizes two receptor subtypes in endothelial cells cultured from bovine pulmonary artery
Authors: Redmond Eileen M. , Cahill Paul A. , Keenan Alan K.
Published: July 2002

In this study specific high affinity binding sites for atrial natriuretic factor (rANF(99–126)) have been identified on cultured endothelial cells of bovine pulmonary artery origin (BPAEC). A time‐dependent rise in cellular cGMP levels stimulated by rANF(99–126) was followed by release of the nucleotide into the incubation medium. The use of truncated, ring‐deleted and linear atrial peptide analogs in competitive displacement analysis and measurement of cGMP accumulation indicated that only a minor proportion (5–11%) of the available receptor pool was of the ANF‐B receptor subtype, linked to guanylate cyclase, with the remaining major proportion possibly of the ANF‐C (clearance) receptor subtype. The existence of two ANF receptor subtypes in this cell culture model would suggest a significant role for the circulating peptide in modulation of pulmonary endothelial cell function, which would influence or complement its direct actions on the underlying vasculature of the pulmonary circulation.
Bile acid N‐acetylglucosaminides
Authors: Matern H. , Bolz R. , Marschall H.-U. , Sjövall J. , Matern S.
Published: July 2002

Bile acid N‐acetylglucosaminyltransferase activity has been identified in microsomes from human liver and kidney. In both organs the transferases required UDP‐N‐aeetylglucosamine as sugar donor and were mainly active towards ursodeoxycholic acid. Minor activities were observed towards amidated ursodeoxycholic, hyodeoxycholic and β‐muricholic acids. No N‐acetylglucosaminidation was detectable with the major primary and secondary bile acids suggesting a specific requirement of the enzymes for bile acids containing 7β‐ or 6α‐hydroxyl groups. Kinetic parameters and other catalytic properties of liver and kidney microsomal N‐acetylglucosaminyltransferase activities towards ursodeoxycholic acid are described.
Irreversible transitions in a model substrate cycle An experimental illustration
Authors: Cimino Alain , Hervagault Jean-François
Published: July 2002

In a previous article [(1987) J. Theor. Biol, 127, 439‐449], the dynamic behavior of a simple substrate cycle, bounded by moiety conservation, and in which one of the two antagonist enzymes is subjected to a destabilizing factor, was investigated. Depending upon the control parameter chosen, that is, the total interconverted substrate concentration and the ratio of the interconverting enzyme maximal activities, monostability, reversible (hysteresis) and/or irreversible transitions could be observed. In the present work, we report experiments dealing with the moiety ATP/ADP interconverted by enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). The cycle operates under conditions where (1) PFK is inhibited by excess of its substrate, ATP, and (2) both enzymes are working under zero‐order kinetics for their respective cosubstrates F6P and PEP. Under conditions where the PK maximal activity is lower than the PFK optimal activity, irreversible transitions from a high ATP (resp. low ADP) steady‐state concentration to a lower (resp. higher) one, are observed when varying the total moiety (ATP + ADP) concentration. A graphical interpretation of the observed behavior is given. Plausible biochemical consequences of this phenomenon are also emphasized.
Left handed α‐helix formation by a bacterial peptide
Authors: Mortishire-Smith Russel J. , Drake Alex F. , Nutkins Jennifer C. , Williams Dudley H.
Published: July 2002

The α‐helix is a common element of secondary structure in proteins and peptides. In eukaryotic organisms, which exclusively incorporate L‐amino acids into such molecules, stereochemical interactions make such α‐helices, invariably right‐handed. Pseudomonas tolaasii Paine is the causal organism of the economically significant brown blotch disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach. P. tolaasii proceduces an extracellular lipodepsipeptide toxin, tolaasin, which causes the brown pitted lesions on the mushroom cap. Circular dichroism studies tolaasin in a membrane‐like environment indicate the presence of a left‐handed α‐helix, probably formed by a sequence of 7 D‐amino acids in the peptide. P. tolaasii represents the first reported example of an organism which has evolved the ability to biosynthesize a left‐handed α‐helix.
Phosphorylation and activation of hormone‐sensitive lipase in isolated macrophages
Authors: Small Catherine A. , Rogers M.Perenna , Goodacre John A. , Yeaman Stephen J.
Published: July 2002

Hormone‐sensitive lipase (HSL) is responsible for the neutral cholesterol ester hydrolase activity in macrophages. Incubation of intact WEHI macrophages or mouse peritoneal macrophages leads to phosphorylation of HSL, which is increased by incubation with either dibutyryl cyclic AMP and 3‐isobutyl‐1‐methylxanthine or okadaic acid. Correspondingly, these agents also activate neutral cholesterol ester hydrolase activity in intact WEHI cells. Regulation of mobilisation of esterified cholesterol in macrophages may be of antiatherogenic value, which this model system now allows us to investigate further.
Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components
Authors: Tokatlidis Kostas , Salamitou Sylvie , Béguin Pierre , Dhurjati Prasad , Aubert Jean-Paul
Published: July 2002

The function of the non‐catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross‐reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I‐labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit. 125I‐labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.
Purification and spectral characterization of a b‐type cytochrome from the plasma membrane of the archaebacterium Sulfolobus acidocaldarius
Authors: Becker Marita , Schäfer Günter
Published: July 2002

For the first time the purification of a heme‐b containing cytochrome from the plasma membrane of an extremely thermoacidophilic archaebacterium is described. The detergent solubilized 30 kDa polypeptide contains two heme‐b centers and one copper ion. According to its low temperature spectra and CO‐binding properties, it is likely to function as a cytochrome‐o like terminal oxidase in the membrane. The purified cytochrome does not retain catalytic activity, however.
Minor‐groove width and accessibility in B‐DNA drug and protein complexes
Authors: Neidle Stephen
Published: July 2002

A new definition is presented for minor‐groove width in double‐helical B‐DNA structures. This uses interstrand H4′…H5′ rather than P…P distances. It is shown by examination of various oligonucleotide crystal structures that these H4′…H5′ distances are a sensitive measure of minor‐groove drug and protein binding, since these hydrogen atoms are in direct non‐bonded contact with such bound ligands.
A novel super‐secondary structure of β‐proteins A triple‐strand corner
Authors: Efimov A.V.
Published: July 2002

A novel super‐secondary structure of β‐proteins, denoted here as a triple‐strand corner, is considered in this paper. This structure can be represented as an antiparallel triple‐strand β‐sheet folded on itself so that the two β‐β‐hairpins are packed approximately orthogonally in different layers and the central strand bends by 90° in the right‐handed direction when passing from one layer to the other. In all the triple‐strand corners observed in proteins, the first β‐β‐hairpins are right‐handed and the second ones are left‐handed when viewed from the concave sides of the corners. Arrangement of other β‐strands in the proteins involving the triple‐strand corners is also examined.
NADH:ubiquinone oxidoreductase from bovine heart mitochondria A fourth nuclear encoded subunit with a homologue encoded in chloroplast genomes
Authors: Arizmendi Jesús M. , Runswick Michael J. , Skehel J.Mark , Walker John E.
Published: July 2002

The amino acid sequence has been determined of the precursor of a nuclear encoded 20 kDa subunit of complex I from bovine heart mitochondria. The sequence of the mature protein is related to a protein of uncertain function, hitherto known as psbG, encoded in the chloroplast genomes of higher plants. Open reading frames encoding homologues of psbG have also been detected in bacteria and in the mitochondrial genome of Paramecium tetraurelia. The chloroplast psbG gene is found between ndhC and ndhJ, which encode homologues of ND3, a hydrophobic subunit of complex I encoded in the bovine mitochondrial genome, and of the nuclear encoded 30 kDa subunit of complex 1. This 20 kDa protein is the eleventh out of the forty or more subunits of bovine complex I with a chloroplast encoded homologue, and its sequence provides further support for the presence in chloroplasts of a multisubunit enzyme related to complex I that could be involved in chlororespiration. The strict conservation of three cysteines suggests that the subunit might be an iron‐sulphur protein.
Spectroscopic identification of the haem ligands of cellobiose oxidase
Authors: Cox Mark C , Rogers Melanie S , Cheesman Myles , Jones Gareth D , Thomson Andrew J , Wilson Michael T , Moore Geoffrey R
Published: July 2002

A spectroscopic study of the flavocytochrome b enzyme, cellobiose oxidase, employing optical, NMR, EPR and near infra‐red MCD techniques, has identified the axial ligands of the b‐type haem. These are a histidine and a methionine, and this ligation set is discussed in relation to the functional role of the haem group.
Molecular cloning of the α‐subunit of rat endopeptidase‐24.18 (endopeptidase‐2) and co‐localization with endopeptidase‐24.11 in rat kidney by in situ hybridization
Authors: Corbeil Denis , Gaudoux Florence , Wainwright Sandra , Ingram Jean , Kenny A.John , Boileau Guy , Crine Philippe
Published: July 2002

Endopeptidase‐24.18 (endopeptidase‐2, EC 3.4.24.18, E‐24.18) is a Zn‐ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligometric structure, α2‐β2. The primary structure of the α‐subunit of E‐24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9‐kb cDNA coding for the α‐subunit was isolated and sequenced. It had an open reading frame of 2.244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo‐endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, ‘PABA‐peptide hydrolase’. Northern blot analysis revealed the α‐subunit to be encoded by a single mRNA species of 3.2‐kb. In situ hybridization performed on rat kidney showed a co‐localization of E‐24.18 with endopeptidase‐24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.
Human interleukin‐1 receptor antagonist is expressed in liver
Authors: Steinkasserer Alexander , Estaller Cornelia , Weiss Elisabeth H. , Sim Robert B.
Published: July 2002

Using PCR and Northern blot analysis, an IL‐1 receptor antagonist specific transcript was amplified from HepG2‐ and liver mRNA, cDNA clones coding for IL‐1 receptor antagonist were isolated from a liver cDNA library and sequence comparison revealed complete identity with the secreted, monocytic form of IL‐1 receptor antagonist.
Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli
Authors: Rogers W.John , Hodges Michael , Decottignies Paulette , Schmitter Jean-Marie , Gadal Pierre , Jacquot Jean-Pierre
Published: July 2002

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N‐ and C‐termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr‐7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.
Effect of phosphoramidon on big endothelin‐2 conversion into endothelin‐2 in human renal adenocarcinoma (ACHN) cells Analysis of endothelin‐2 biosynthetic pathway
Authors: Yorimitsu Kazuyuki , Shinmi Osamu , Nishiyama Mariko , Moroi Kayoko , Sugita Yoshiki , Saito Toshihiro , Inagaki Yoshiaki , Masaki Tomoh , Kimura Sadao
Published: July 2002

The biosynthetic pathway of endothelin (ET)‐2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET‐2‐related peptides, ET‐2, big ET‐2(1–38) and big ET‐2(22–28). Phosphoramidon decreased the amount of ET‐2 and increased that of big ET‐2(13̄8)dose‐dependently. The amount of big ET‐2(1–37) did not significantly change. These results suggest that big ET‐2 is composed of 38 and not 37 amino acid residues, and that a putative ET‐2‐converting enzyme (ECE‐2) should be classified as a phosphoramidon‐sensitive neutral metalloprotease, bearing a resemblance to the putative ET‐1‐converting enzyme (ECE‐1) in endothelial cells.
Distribution and second messenger coupling of four somatostatin receptor subtypes expressed in brain
Authors: Kaupmann Klemens , Bruns Christian , Hoyer Daniel , Seuwen Klaus , Lübbert Hermann
Published: July 2002

The mRNA distribution in the brain and the coupling to cellular effector systems of four somatostatin receptors (SSTR1‐4) was studied. All four SRIF receptor subtypes were expressed in cortex and hippocampus. In addition, SSTR1 mRNA was relatively abundant in the spinal cord whereas SSTR2 mRNA was also present in the striatum. The SSTR3 gene was predominantly expressed in the olfactory bulb and in the cerebellum. Conflicting results about the effector coupling of SSTR1‐3 have been published previously. We have stably expressed human SSTR1‐4 in HEK 293 human embryonal kidney cells. Agonist binding to the receptor subtypes, including the recently cloned SSTR4, inhibited the formation of forskolin‐induced cAMP. Is is concluded that, in an appropriate cellular environment, all four receptor subtypes can functionally couple to the inhibition of adenylyl cyclase.
Characterization of the alternative oxidase protein in the yeast Hansenula anomala
Authors: Sakajo Shigeru , Minagawa Nobuko , Yoshimoto Akio
Published: July 2002

The cyanide‐resistant respiratory pathway is induced by respiratory inhibitors in the yeast Hansenula anomala. A monoclonal antibody against the alternative oxidase in the higher plant Sauromatum guttatum cross‐reacted with a 36‐kDa mitochondrial protein induced by antimycin A in H. anomala and with a protein encoded by a cDNA which was previously cloned for an antimycin A‐inducible mRNA in the yeast. There was a similarity in the amino acid sequence between the cDNA‐encoded protein and the plant alternative oxidase protein. We propose that the 36‐kDa mitochondrial protein encoded by the cDNA is a component of alternative oxidase in H. anomala.
Lipopolysaccharide promoted opening of the porin channel
Authors: Ishii Junko , Nakae Taiji
Published: July 2002

We show here that the imipenem (a carbapenem, β‐lactam antibiotic)‐permeable porin channels (protein D2 or OprD2) of Pseudomonas aeruginosa were closed mostly in the lipopolysaccharide (LPS)‐free membrane and were openable by adding LPS to the membrane as assayed by ion conductivity measurements using planar lipid bilayers. Open and closed states of the OprD2 channels exhibited conductivities of about 400 and 30 pS, respectively, in 1 M NaCl. The OprD2 channel in the LPS‐containing membrane showed very rapid opening and closing events in a second order and the duration of closure became longer at low membrane potentials.
A thioredoxin‐independent fully active NADP‐malate dehydrogenase obtained by site‐directed mutagenesis
Authors: Issakidis Emmanuelle , Decottignies Paulette , Miginiac-Maslow Myroslawa
Published: July 2002

A triple cysteine mutant of sorghum leaf NADP‐malate dehydrogenase has been constructed by site‐directed mutagenesis, combining the previously obtained mutation of the two N‐terminal cysteines with the mutation of the most internal of the two C‐terminal cysteines. The construct, over‐expressed in E. coli, yielded an always active, dithiol‐insensitive enzyme. It can be concluded that the dithiol activation of the unmodified enzyme involves a maximum of two different disulfides per subunit, and that none of the mutated cysteines is implicated in catalysis.
Effect of modification of carbohydrate component on properties of glucoamylase
Authors: Neustroev K.N. , Golubev A.M. , Firsov L.M. , Ibatullin F.M. , Protasevich I.I. , Makarov A.A.
Published: July 2002

In this study, we investigated enzymatic deglycosylation of glucoamylase from Aspergillus awamori X 100/D27, a glycoprotein which has two N‐linked and about forty short mannose‐bearing O‐linked sugars per molecule. O‐Linked sugars were modified by treatment with α‐mannosidase and N‐linked sugars were removed using endo‐β‐N‐acetylglucosaminidase F. Analysis ofconformational changes following deglycosylation suggests that O‐linked sugars essentially contribute to the stabilization of glucoamylase domains. Modification of the carbohydrate component by adding 1‐deoxymannojirimycin to the culture medium induced inhibition of α‐mannosidases involved in the processing, leading to a more complete glycosylation and, consequently, to a higher stability of the enzyme.
Location of tolerated insertions/deletions in the structure of the maltose binding protein
Authors: Betton Jean-Michel , Martineau Pierre , Saurin William , Hofnung Maurice
Published: July 2002

In a previous study [(1987) J. Mol. Biol. 194, 663‐673], we isolated ten insertion/deletion mutants (indels) of the maltose binding protein for which the maltose binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three‐dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications.
Interaction between a membrane‐associated serine proteinase of U‐937 monocytes and peptides from the V3 loop of the human immunodeficiency virus type 1 (HIV‐1) gp120 envelope glycoprotein
Authors: Avril Laurence-Emmanuelle , Di Martino-Ferrer Michèle , Barin Francis , Gauthier Francis
Published: July 2002

A trypsin‐like proteinase which is inhibited by recombinant gp120 and by synthetic peptides of various lengths spanning the conserved sequence of the V3 loop has been purified and partially characterized from a U‐937 cell membrane extract. V3 loop peptides behave as competitive inhibitors of the enzyme, while gp 120 exerts a tight‐binding inhibition, reacting in stoichiometric amounts with the proteinase to provide significant inhibition. Though the properties of the U‐937 membrane proteinase towards gp 120 and synthetic peptides of the V3 loop resemble those of the Molt‐4 T‐cell tryptase TL2, these two proteinases differ by their physicochemical properties and their susceptibility to other inhibitors of serine proteinases. These results give support to the concept of a membrane‐associated proteinase as a complementary or alternative receptor to the CD4, for allowing virus to enter host cells and thus spreading HIV infection.
Combined effects of diffusional hindrances, electrostatic repulsion and product inhibition on the kinetic properties of a bound acid phosphatase
Authors: Thiébart-Fassy Isabelle , Hervagault Jean-François
Published: July 2002

The kinetic properties of a soybean cell wall phosphatase were studied and compared under different environmental conditions. The native enzyme isolated from the wall exhibits classical Michaelis Menten kinetics. When buried into the cell wall, its natural environment, the enzyme mimics an apparent negative cooperativity. This deviation from hyperbolic dependence of the activity vs. the substrate concentration may result from an heterogeneous distribution of the enzyme molecules between the surface and the inside of the wall, where an electrostatic partition effect takes place. Cell wall fragments compacted on a Millipore‐type membrane allow the study of the dual effects of diffusional hindrances and electrostatic interactions on the global kinetic behavior. By increasing the ionic strength, partition effects can be suppressed and therefore diffusional effects alone can be taken into account. On the one hand, diffusion and partition act individually and synergetically to decrease the apparent global affinity of phosphatase with respect to glucose 6‐phosphate. On the other hand, product inhibition by inorganic phosphate is subjected to dual effects from diffusion through accumulation and partition through repulsion. A simple diffusion—partition reaction model accounts qualitatively and quantitatively for the experimental observations.
Modulation of AP‐1 activity by nitric oxide (NO) in vitro: NO‐mediated modulation of AP‐1
Authors: Tabuchi Akiko , Sano Kuniaki , Oh Esther , Tsuchiya Tomofusa , Tsuda Masaaki
Published: July 2002

To understand the role of nitric oxide (NO) in controlling the specific DNA‐binding activities of transcriptional factors, we investigated the in vitro effect of the NO‐donor sodium nitroprusside (SNP) on the AP‐1 activity of cultured mouse cerebellar granule cells. A gel‐mobility assay showed that SNP inhibited AP‐1 activity in the presence, but not the absence of dithiothreitol (DTT). This DTT‐dependent inhibition of AP‐1 activity by SNP corresponded with the activation of the chemical reactivity of SNP with DTT, which can be monitored by the production of nitrite (NO−
2). In contrast, diamide, a typical sulfhydryl oxidizing agent, inhibited AP‐1 activity in the absence of DTT and its inhibitory effect was reversed competitively by DTT. Studies using structurally or functionally related analogues of SNP demonstrated that S‐nitrosylation of the AP‐1 moiety mediated by some NO‐carriers but not by free NO, which can be produced by the chemical reaction of SNP with DTT, was responsible for the inhibition of AP‐1 activity, suggesting NO‐mediated regulation of the AP‐1 transcriptional factor.
Inactivation of the Na,K‐ATPase by radiation‐induced free radicals Evidence for a radical‐chain mechanism
Authors: Hitschke K. , Bühler R. , Apell H.-J. , Stark G.
Published: July 2002

Free radicals produced by water radiolysis were used to study the inactivation of the enzymatic activity of the Na,K‐ATPase. A decrease of the activity to virtually zero with a mono‐exponential dependence on the radiation dose was observed. The inactivation process is initiated by hydroxyl radicals. This was shown by the effect of appropriate radical scavengers such as t‐butanol, formate and vitamin C. In all cases a significant increase in the characteristic D
37 dose of inactivation was observed. Inactivation was found to show a so‐called inverse dose‐rate effect, i.e, the sensitivity of the enzyme to radical attack is increased if the dose rate is reduced. The data were found to agree with the relationship 1/D
371̃/D1/2, which is known to be a strong indicator of a radical chain mechanism. This means that the inactivation, after initiation by single radicals, is amplified by a subsequent chain mechanism.
A synthetic peptide with anti‐platelet activity derived from a CDR of an anti‐GPIIb‐IIIa antibody
Authors: Jarrin Annie , Andrieux Annie , Chapel Agnès , Buchou Thierry , Marguerie Gérard
Published: July 2002

A monoclonal antibody, AC7, directed against the RGD (Arg‐Gly‐Asp) binding site on the GpIIIa subunit of the platelet fibrinogen receptor, interacts only with activated platelet. In order to identify the regions of AC7 that interact with the receptor, cDNA sequences of AC7 immunoglobulin heavy and light chain variable regions were determined. Among the six complementarity‐determining regions (CDRS) of AC7, the CDR3 heavy chain (H3) contains homology to the RGDF sequence within fibrinogen. A synthetic peptide encompassing the H3 region (H3, RQMIRGYFDV) inhibited platelet aggregation and fibrinogen binding to platelet (IC50 = 700μM). The inhibitory potencies of modified H3 peptides suggest that the RGYF sequence within the H3 peptide mimic the receptor recognition sequence in fibrinogen.
The 20S proteasome mediates the degradation of mouse and yeast ornithine decarboxylase in yeast cells
Authors: Mamroud-Kidron Emanuelle , Rosenberg-Hasson Yael , Rom Eran , Kahana Chaim
Published: July 2002

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is one of the most rapidly degraded proteins in mammalian cells. Recently it has been demonstrated that mammalian ODC is degraded in vitro by the 26S protease that contains the 20S proteasome as its catalytic core, in a reaction that does not require ubiquitin. Here, we show that yeast and mouse ODC are both rapidly degraded in yeast cells and that their degradation severely inhibited in a mutant yeast cell line defective in the chymotryptic activity of proteinase yscE, the yeast 20S proteasome. These results provide compelling genetic support to previous biochemical studies suggesting the involvement of the 20S proteasome in the degradation of ornithine decarboxylase.
Escherichia coli PII protein: purification, crystallization and oligomeric structure
Authors: Vasudevan Subhash G. , Gedye Craig , Dixon Nicholas E. , Cheah Eong , Carr Paul D. , Suffolk Peter M. , Jeffrey Peter D. , Ollis David L.
Published: July 2002

The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix‐assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 × 0.2 × 0.3 mm were grown and analysed by X‐ray diffraction. The crystals belong to space group P63 with a=b=61.6Å,c= 56.3 Å and V
m of 2.5 for one subunit in the asymmetric unit. A low‐resolution electron density map showed electron density concentrated around a three‐fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 ± 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.
The amino acid sequences of human and pig l‐arginine:glycine amidinotransferase
Authors: Humm Andreas , Huber Robert , Mann Karlheinz
Published: July 2002

We have isolated and sequenced the l‐arginine:glycine amidinotransferase of pig kidney mitochondria. Due to endogenous proteolysis, the purified molecules showed some heterogeneity at the N terminus. The longest form recovered had 386 amino acids. Part of the pig amidinotransferase sequence information was used to isolate cDNA clones coding for the human enzyme. The deduced amino acid of the human amidinotransferase was 37 amino acids longer due to the presence of a signal sequence. The mature proteins were 94% identical to each other and 36% identical to the sequences of bacterial l‐argininednosamine phosphate amidinotransferases.
The functional roles of disulfide bonds in the β‐subunit of (Na,K)ATPase as studied by site‐directed mutagenesis
Authors: Noguchi Shunsuke , Mutoh Yoshinobu , Kawamura Masaru
Published: July 2002

The β‐subunit of Torpedo californica (Na,K)ATPase contains seven cysteine residues; one (Cys46) is in the single transmembrane segment and the other six (Cys127, Cys150, Cys160, Cys176, Cys215 and Cys278) are in the extracellular domain and form three highly conserved disulfide bonds. Aβ‐subunit mutant with replacement of Cys46 by Ser could assemble with the α‐subunit, and the resulting αβ‐complex was catalytically active. Mutants in which either the N‐terminal side or both Cys residues of the Cys127‐Cys150 bond were replaced by Ser could also tightly assemble with the α‐subunit, but the resulting αβ‐complex was catalytically inactive. On the other hand, disruption of either the Cys160‐Cys176 or Cys215‐Cys278 bond by substituting the N‐terminal side only or both Cys residues with Ser led to a β‐subunit that could not assemble with the α‐subunit. We conclude that the structure of the β‐subunit around the Cys160‐Cys176 and Cys215‐Cys278 loops is indispensable for assembly with the α‐subunit, whereas the Cys127‐Cys150 loop is not essential for assembly but is required for enzyme activity.
Formation of 22 and 24 carbon 6‐desaturated fatty acids from exogenous deuterated arachidonic acid is activated in THP‐1 cells at high substrate concentrations
Authors: Caruso D. , Risé P. , Galella G. , Regazzoni C. , Toia A. , Galli G. , Galli C.
Published: July 2002

Deuterated arachidonic acid (AA, [2H820 :4 n‐6) 1–25 μM, is converted to other fatty acids, as evaluated by gas chromatography‐mass spectrometry, in THP‐1 cells. The major products, in the 1 to 10 μM range, are 22:4 (elongated) and 20:3 (reduced in 5). At 25 μM, 24:4, 24:5 and 22:5 accumulate, with [2H8]/[2H0] ratios higher than in AA. At high AA concentration preferential conversion to elongated fatty acids with 5 unsaturations, through a 6 desaturase takes place and the 4‐desaturated 22:5 appears to be formed through β‐oxidation of 24:5.
The human HIP gene, overexpressed in primary liver cancer encodes for a C‐type carbohydrate binding protein with lactose binding activity
Authors: Christa Laurence , Felin Murielle , Morali Olivier , Simon Marie-Thérèse , Lasserre Chantal , Brechot Christian , Sève Annie-Pierre
Published: July 2002

HIP was originally identified as a gene expressed in primary liver cancers, and in normal tissues such as pancreas and small intestine. Based on gene data base homologies, the HIP protein should consist of a signal peptide linked to a single carbohydrate recognition domain. To test this hypothesis HIP and the putative carbohydrate recognition domain encoded by the last 138 C‐terminal amino acids, were expressed as glutathione‐S ‐transferase proteins (GST‐HIP and GST‐HIP‐142, respectively). Both recombinant proteins were purified by a single affinity purification step from bacterial lysates and their ability to bind saccharides coupled to trisacryl GF 2000M were tested. Our results show that HIP and HIP‐142 proteins bind to lactose, moreover the binding requires divalent cations. Thus the HIP protein is a lactose‐binding lectin with the characteristics of a C‐type carbohydrate recognition domain of 138 amino acids in the C‐terminal region.