bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute and long‐term effects of insulin‐like growth factor I on glucose transporters in muscle cells Translocation and biosynthesis
Authors: Bilan Philip J. , Mitsumoto Yasuhide , Ramlal Toolsie , Klip Amira
Published: July 2002

Insulin‐like growth factor I (IGF‐I) rapidly (<10 min) stimulated glucose uptake into myotubes of the L6 muscle cell line, at concentrations that act specifically on IGF‐I receptors. Uptake remained stimulated at a steady level for 1–2 h, after which a second stimulation occurred. The first phase was insensitive to inhibition of protein synthesis. Subcellular fractionation demonstrated that it was accompanied by translocation of glucose transporters (both GLUT1 and GLUT4) to the plasma membrane from intracellular membranes. Translocation sufficed to explain the first phase increase in glucose transport, and there was no change in the total cellular content of GLUT1 or GLUT4 glucose transporters. The second phase of stimulation was inhibitable by cycloheximide, and involved a net increase in either GLUT1 or GLUT4 transporter content, which was reflected in an increase in transporter number in plasma membranes. These results define a cellular mechanism of metabolic action of IGF‐I in muscle cells; furthermore, they suggest that IGF‐I has acute metabolic effects that mimic those of insulin, bypassing action on the insulin receptor.
Studies on the reaction coordinates of the water oxidase in PS II membrane fragments from spinach
Authors: Renger G. , Hanssum B.
Published: July 2002

The temperature dependency of the rate constants of the univalent redox steps Yz
oxS
i
→ YzS
i+1
(i = 0,1,2) and Yz
oxS3 → (YzS4) → YzSo + O2 in the water oxidase was investigated by measuring time resolved absorption changes at 355 nm induced by a laser flash train in dark adapted PS II membrane fragments from spinach. Activation energies of 5.0, 12.0 and 36.0 kJ/mol were obtained for the reactions Yz
oxS
i
→ YzS
i+1
with i = 0,1 and 2, respectively. The reaction Yz
oxS3 → (YzS4) → YzS0 + O2 exhibits a temperature dependence with a characteristic break point at 279 K with activation energies of 20 kJ/mol (T > 279 K) and 46 kJ/mol (T > 279 K). Evaluation of the data within the framework of the classical Marcus theory of nonadiabatic electron transfer [(1985) Biochim. Biophys. Acta 811, 265–322] leads to the conclusion that the S2 oxidation to S3 is coupled with significant structural changes. Furthermore, the water oxidase in S3 is inferred to attain two different conformational states with populations that markedly change at a characteristic transition temperature.
The binding of nonmuscle caldesmon from brain to microtubules Regulations by Ca2+‐calmodulin and cdc2 kinase
Authors: Ishikawa Ryoki , Kagami Osamu , Hayashi Chihiro , Kohama Kazuhiro
Published: July 2002

Nonmuscle caldesmon from bovine brain bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with values of K
A 4.5×105 M−1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. The phosphorylation of caldesmon by cdc2 kinase also eliminated the microtubule‐binding activity. These results suggest that caldesmon may play a physiological role in the functions of microtubules.
Cloning, expression and modulation of a mouse NMDA receptor subunit
Authors: Yamazaki Makoto , Mori Hisashi , Araki Kazuaki , Mori Kazuhiro J. , Mishina Masayoshi
Published: July 2002

The primary structure and presence of two forms of the mouse N‐methyl‐d‐aspartate (NMDA) receptor channel subunit ζl have been disclosed by cloning and sequencing the cDNAs. The ζl subunit shows −20% amino acid sequence identities with the rodent α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA)‐ or kainate‐selective GluR subunits and has structural features common to neurotransmitter‐gated ion channels. Functional homomeric ζl channels expressed in Xenopus oocytes by injection of the subunit specific mRNA exhibit current responses characteristics for the NMDA receptor channel such as activation by glycine, Ca2+ permeability, blocking by Mg2+ and activation by polyamine. It has been found that the ζl channel activity is positively modulated by treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA).
Production of the cysteine proteinase inhibitor cystatin C by rat Sertoli cells
Authors: Esnard Annick , Esnard Frédéric , Guillou Florian , Gauthier(xaa) Francis
Published: July 2002

The Sertoli cells of the rat testis produce cystatin C, a cysteine proteinase inhibitor. Primary culture of Sertoli cells secreted both unglycosylated and glycosylated forms of rat cystatin C. Despite the low concentration of cystatin C in rete testis fluid, equilibrium dissociation constants (K
1) for the interaction between cystatin C and lysosomal cathepsins indicate that this molecule could be involved in the local regulation of testicular cysteine proteinase activity which may be necessary for spermatogenesis and spermiogenesis.
The consensus sequences for cdc2 kinase and for casein kinase‐2 are mutually incompatible A study with peptides derived from the β‐subunit of casein kinase‐2
Authors: Marin Oriano , Meggio Flavio , Draetta Giulio , Pinna Lorenzo A.
Published: July 2002

Two series of synthetic peptides that reproduce the amino‐ and carboxyl‐terminal segments of the β‐subunit of casein kinase‐2, including the sites phosphorylated by CK2 and cdc2 kinase, respectively, have been used as model substrates for these enzymes. The N‐terminal peptide β(1–9), MSSSEEVSW, is readily phosphorylated by CK2 but not at all by cdc2. The opposite is true of the C‐terminal peptide β(206–215), NFKSPVKTIR. whose Ser‐4 is a good target for cdc2 while being unaffected by CK2. The individual substitutions of Pro‐5 and Lys‐7 in the latter peptide with Gly and Ala (or Glu), respectively, prevent its phosphorylation by cdc2, whereas the substitution of Lys‐3 with Ala is well tolerated and the substitution of the target Ser with Thr actually improves phosphorylation. Thus the consensus sequence for cdc2 is shown to be X‐S‐P‐X‐K. Such a requirement of a basic residue at Position +3 is opposite to that of CK2 whose consensus sequence (S‐X‐X‐E/D/Yp/Sp) includes an acidic residue at the same position. Moreover the motif Ser‐Pro is detrimental for CK2, preventing the phosphorylation of otherwise suitable peptides. These observations would rule out the possibility that the site specificity of CK2 might overlap with that of cdc2 and possibly of other Pro‐directed protein kinases.
Structure of holo‐chaperonin studied with electron microscopy Oligomeric cpn10 on top of two layers of cpn60 rings with two stripes each
Authors: Ishii Noriyuki , Taguchi Hideki , Sumi Masato , Yoshida Masasuke
Published: July 2002

A structural model of holo‐chaperonin, known as a protein‐folding control protein comprising 60 kDa (cpn60) and 10 kDa polypeptides (cpn10), is proposed based on the electron microscopic images of holo‐chaperonin from Thermus thermophilus and cpn60 from Paracoccus denitrificans. Isolated Paracoccus cpn60 shows very similar images to those of Escherichia coli tetradecameric cpn60, a seven‐membered ring in the top view and a rectangular shape with four stripes in the side view. However, a small number of half‐thick rectangles with two stripes are also seen which indicates that a single cpn60‐heptamer ring has two stripes parallel to the plane of the ring. Thermus holo‐chaperonin shows a bullet‐like shape in the side view, and antibory against cpn10 binds only to the round side of the bullet. We conclude that a single cpn60‐heptamer ring with two stripes stacks into two layers, and a cpn10 oligomer binds to one side of the layers.
Two sites of primary degradation of the D1‐protein induced by acceptor or donor side photo‐inhibition in photosystem II core complexes
Authors: De Las Rivas Javier , Andersson Bertil , Barber James
Published: July 2002

Depending on experimental conditions we have found that photo‐inhibitory treatment of photosystem II (PSII) core complexes, isolated from wheat, can generate two fragments of about 23–24 kDa that contain either the C‐terminal or N‐terminal regions of the D1‐protein. A 24 kDa C‐terminal fragment appears when the water splitting reaction is not functional and an electron acceptor is present. This ‘donor’‐side inhibition also generates an N‐terminal fragment of about 10 kDa and is suggested to be due to the cleavage of a peptide bond in the region connecting transmembrane segments I and II of the D1‐protein. In contrast, an N‐terminal 23 kDa D1‐protein fragment is detected when the water splitting reactions of the isolated complex are active, and occurs in the absence of an added electron acceptor. This ‘acceptor’‐side photo‐inhibition also generates a C‐terminal fragment of about 10 kDa.
Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves
Authors: Seseke Florian G. , Gardemann Andreas , Jungermann Kurt
Published: July 2002

Cell‐to‐cell communication via gap junctions has been proposed to be involved in the metabolic actions of sympathetic liver nerves in the rat. The effects of hepatic nerve stimulation and noradrenaline‐, PGF2α‐ and glucagon infusion on glucose metabolism and perfusion flow were studied in perfused rat liver in the absence and presence of the gap junctional inhibitors, heptanol, carbenoxolone and (4β)phorbol 12‐myristate 13‐acetate (4βPMA). (i) Stimulation of the hepatic nerve plexus increased glucose output, decreased flow and caused an overflow of noradrenaline into the hepatic vein. (ii) Heptanol completely inhibited not only the nerve stimulation‐dependent metabolic and hemodynamic alterations but also the noradrenaline overflow. Thus the heptanol‐dependent inhibitions were caused primarily by a strong impairment of transmitter release. (iii) Carbenoxolone inhibited the effects of neurostimulation on glucose metabolism partially by about 50%, whereas it left perfusion flow and noradrenaline overflow essentially unaltered. (iv) 4βPMA reduced the nerve stimulation‐dependent enhancement of glucose release by about 80% but the noradrenaline‐dependent increase in glucose output only by about 30%; the increase in glucose release by PGF2α and by glucagon remained essentially unaltered. 4βPMA reduced the nerve stimulation‐dependent decrease in portal flow by about 35% but did not affect the noradrenaline‐and PGF2α‐elicited alterations, nor did it alter noradrenaline overflow. The results allow the conclusion that gap junctional communication plays a major role in the regulation of hepatic carbohydrate metabolism by sympathetic liver nerves, but not by circulating noradrenaline, PGF2α or glucagon.
A Ca2+‐dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize
Authors: Ogawa Noriyuki , Okumura Satoru , Izui Katsura
Published: July 2002

In C4 plants the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is regulated by phosphorylation/dephosphorylation which is mediated by light/dark signals. The study using protein kinase inhibitors showed that the inhibition pattern of maize PEPC‐protein kinase (PEPC‐PK) is similar to that of myosin light chain kinase, a Ca2+‐calmodulin‐dependent PK. The kinase activity was also inhibited by EGTA and the inhibition was relieved by Ca2+. These results suggest that PEPC‐PK is Ca2+‐dependent in contrast with previous observations by other research groups.
The role of adenylate kinase in dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space
Authors: Gellerich Frank Norbert
Published: July 2002

To investigate the existence of a dynamic adenine nucleotide (AdN) compartment in the mitochondrial intermembrane space, we used reconstituted systems consisting of (i) functional intact liver and heart mitochondria and (ii) pyruvate kinase plus phosphoenolpyruvate, both competing for ADP either formed in the intermembrane space by adenylate kinase or added directly into, or regenerated by ATPase within, the extramitochondrial space. It is shown that ADP formation in the mitochondrial intermembrane space is a prerequisite for a dominating oxidative phosphorylation in reconstituted systems, suggesting dynamic ADP compartmentation in that space.
The pleiotypic response to amino acid deprivation is the result of interactions between components of the glycolysis and protein synthesis pathways
Authors: Rabinovitz Marco
Published: July 2002

Several diverse metabolic events become compromised when mammalian cells are made deficient in essential amino acids or when charging of their tRNA is blocked by amino acid analogs. This rapid general demise of cell function can be due to inhibition of phosphofructokinase (PFK) by uncharged tRNA. It has now been demonstrated that when tRNA is added to PFK in an assay dependent upon the reassociation of inactive, dissociated enzyme subunits, nanomolar concentrations cause complete inhibition. The model for control suggests that charged tRNA becomes associated with EF‐1, which is specific for aminoacyl‐tRNAs and is present in sufficiently high concentrations in cells to sequester the charged forms from an inhibitory role. Support for this model include: (1) the rapid onset of inhibition of glycolysis and glucose uptake upon amino acid deficiency; (2) the unique role of the product of PFK activity, fructose‐1,6‐diphosphate, in reactions of peptide chain initiation, particularly its role as a co‐factor for purified elF‐2B, the GDP/GTP exchange factor; (3) the correlations of this interaction with the cellular and molecular lesions of insulin insufficiency; (4) the recognition that the anomalous role of high concentrations of cAMP as a stimulant of peptide chain initiation in energy depleted or gel‐filtered cell lysates correlates with its stimulatory action on PFK as an analog for the positive effector, adenosine‐5′‐monophosphate; and (5) the role of fructose‐1,6‐diphosphate in the formation of glyceraldehyde‐3‐phosphate, a substrate for synthesis of ribose‐5‐phosphate via the non‐oxidative portion of the pentose phosphate pathway, which, as a precursor of phosphoribosylpyrophosphate, is essential for nucleic acid synthesis.
Characterization of the yeast BMH1 gene encoding a putative protein homologous to mammalian protein kinase II activators and protein kinase C inhibitors
Authors: van Heusden G.Paul H. , Wenzel Thibaut J. , Lagendijk Ellen L. , de Steensma H.Y. , van den Berg Johan A.
Published: July 2002

We describe the identification and characterization of the BMH1 gene from the yeast Saccharomyces cerevisiae. The gene encodes a putative protein of 292 amino acids which is more than 50% identical with the bovine brain 14‐3‐3 protein and proteins isolated from sheep brain which are strong inhibitors of protein kinase C. Disruption mutants and strains with the BMH1 gene on multicopy plasmids have impaired growth on minimal medium with glucose as carbon source, i.e. a 30–50% increase in generation time. These observations suggest a regulatory function of the bmh1 protein. In contrast to strains with an intact or a disrupted BMH1 gene, strains with the BMH1 gene on multicopy plasmids hardly grew on media with acetate or glycerol as carbon source.
Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main β‐neurotoxin from the venom of the South American scorpion Tityus serrulatus
Authors: Martin-Eauclaire M.F. , Céard B. , Ribeiro A.M. , Diniz C.R. , Rochat H. , Bougis P.E.
Published: July 2002

A cDNA encoding the main Tityus serrulatus β‐neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues: (ii) the amino acid sequence of the mature toxin: and (iii) an extra Gly‐Lys‐Lys tail at the C‐terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post‐translational cleavage by a carboxypeptidase specific for basic residues and the action of an α‐amidating enzyme. These results also show that the biosynthetic pathway for β‐toxins of ‘New World’ scorpion venoms is similar to that already described for α‐toxins of ‘Old World’ scorpion venoms.
The importance of C‐terminal amino acid residues of actin to the inhibition of actomyosin ATPase activity by caldesmon and troponin I
Authors: Makuch Robert , Kołakowski Janusz , Dąbrowska Renata
Published: July 2002

Proteolytic elimination of three C‐terminal amino acid residues from actin weakens its interaction with caldesmon and troponin I and, in consequence, lowers the inhibitory effects of both proteins on actomyosin ATPase activity. These results prove the importance of C‐terminal extremity of actin to the overall interaction of this protein with caldesmon and troponin I.
Inhibitors of polyamine biosynthesis affect the expression of genes encoding cytoskeletal proteins
Authors: Kamińska Bożena , Kaczmarek Leszek , Grzelakowska-Sztabert Barbara
Published: July 2002

The polyamines are ubiquitous components of mammalian cells. Those compounds have been postulated to play an important role in different cellular functions including the reorganization of cytoskeleton associated with the cell cycle. In the studies reported here, it was found that inhibitors of polyamine biosynthesis, methylglyoxal‐bis[quanylhydrazone] (MGBG) and difluoromethylornithine (DFMO), prevent mitogen‐induced accumulation of mRNAs encoding major cytoskeletal components, β‐actin and α‐tubulin, in mouse splenocytes. These findings suggest mechanisms through which polyamines may exert their effects on the cytoskeleton integrity.
Cloning of a human choline kinase cDNA by complementation of the yeast cki mutation
Authors: Hosaka Kohei , Tanaka Susumu , Nikawa Jun-ichi , Yamashita Satoshi
Published: July 2002

A human choline kinase cDNA was cloned by complementation of the yeast choline kinase mutation, cki, from a human glioblastoma cDNA expression library. The deduced sequence of the human enzyme comprised 456 amino acids with a calculated relative molecular mass of 52.065. The human enzyme resembled the rat liver enzyme over the entire sequence. It also resembled the yeast enzyme in the carboxy‐terminal region, but not much in the amino‐terminal region.
Is bupivacaine a decoupler, a protonophore or a proton‐leak‐inducer?
Authors: Schönfeld P. , Sztark F. , Slimani M. , Dabadie P. , Mazat J.-P.
Published: July 2002

This paper deals with the mechanism of bupivacaine uncoupling of oxidative phosphorylation in rat heart mitochondria. By comparison with the effects of QX 572, a permanently charged quarternary amine‐type local anesthetic, it is concluded that the effects of bupivacaine and QX 572 may be explained by classical uncoupling behaviour. In the case of bupivacaine this uncoupling effect is mediated through a protonophore‐like mechanism, whereas that of QX 572 is simply explained by an electrophoretic uptake.
Isolation of a new ligand‐carrying casein fragment from bovine mammary gland microsomes
Authors: Neuteboom Ben , Giuffrida Maria Gabriella , Conti Amedeo
Published: July 2002

Whilst looking for components involved in retinol metabolism in secreting mammary gland cells, a 12 kDa Protein was isolated. This protein had bound a ligand with characteristics of retinol. N‐Terminal sequencing and amino acid analysis showed that this protein is highly homologous with an α‐sl‐casein fragment. No ligand was found for β‐lactoglobulin, previously thought to be involved in retinol metabolism.
Chlorpheniramine inhibits the ornithine decarboxylase induction of Ehrlich carcinoma growing in vivo
Authors: Urdiales J.L. , Matés J.M. , de Castro I.Núñez , Sánchez-Jiménez F.M.
Published: July 2002

The antihistaminic (±)‐chlorpheniramine significantly reduced the progression of Ehrlich carcinoma when it was administered at 0.5 mg/mouse/day from the third day on, after tumour inoculation. The ODC activity of tumour cells was diminished by 70% on day 7 after tumour transplantation, when maximum ODC activity is detected in non‐treated tumour growing ‘in vivo’. Northern blot analyses indicated that the inhibitory effect of this 1,4‐diamine takes place at a post‐transcriptional level. Results obtained from serum‐free cultured cells indicated that chlorpheniramine inhibits the ODC synthesis rate.
Electrical potential dissipation induced by free fatty acids in pea stem mitochondria
Authors: Petrussa E. , Braidot E. , Nagy G. , Vianello A. , Macri F.
Published: July 2002

Linolenic, linoleic, oleic, palmitic and stearic acids (FFA) collapse the electrical potential of pea stem mitochondria in the absence or in the presence of 0.5 mM Mg2+, Higher concentrations of this cation (5 mM) lower the rate or dissipation caused by linoleic, oleic and palmitic acids, while abolishing that induced by stearic acid. Carboxyatractyloside and ADP do not reverse the FFA‐induced collapse both in the presence or absence of Mg2+. EDTA, EGTA or BHT do not influence the dissipation caused by FFA that, in addition, is not linked to lipid peroxidation evaluated as malondialdehyde or conjugated diene formation. Only linolenic acid sustains a peroxidation which, however, appears to be caused by its owm oxidation catalysed by lipoxygenases rather than by membrane lipoperoxidation induced by this free fatty acid. These results suggest that neither the ATP/ADP exchanger nor lipid peroxidation appear to be involved in FFA‐induced uncoupling in pea stem mitochondria.
Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B
Authors: Joao Heidi C. , Scragg Ian G. , Dwek Raymond A.
Published: July 2002

Assignment of most of the proton NMR resonances of bovine pancreatic RNase B has been achieved using standard NMR techniques and by comparison with the published assignments for RNase A. A comparison of the NMR spectra of RNase B with RNase A shows that glycosylation of the enzyme has little overall effect on the conformation of the protein in solution. Comparisons of hydrogen–deuterium solvent exchange rates for the NH protons of RNase A and RNase B were made using two‐dimensional 1H correlation spectroscopy. In the case of the glycosylated enzyme the exchange rates decreased for the NH protons of residues 9–14, 23–24, 32, 34–35, 39–40, 43–44, 48–49, 60, 71, 75–76, 80, 83–85, 100–101, 107, 111 and 112, relative to the unglycosylated RNase A. These results are consistent with the presence of the oligosaccharide inducing enhanced global dynamic stability and consequent changes to the unfolding equilibrium of the enzyme. The enhanced stability is observed not only for residues in the vicinity of the glycosylation site, asparagine‐34, but also at residues remote from this site, as much as 30 Å away.
Tetrahydro‐6‐biopterin is associated with tetrahydro‐7‐biopterin in primary murine mast cells
Authors: Ziegler Irmgard , Hültner Lothar
Published: July 2002

Murine bone marrow‐derived mast cells proliferate in response to interleukin 3. In addition to 6‐biopterin, 7‐biopterin was identified in these cells by HPLC analysis of iodine oxidized extracts and by alkaline permanganate oxidation to the 6‐ and 7‐carboxylic acids. 7‐Biopterin comprised 31.9 (± 7.7)% of the total biopterin. It was absent in cells which were grown with of l‐p‐chlorophenylalanine, an inhibitor of tryptophan 5‐monooxygenase. Both 6‐ and 7‐biopterin were present in the cell as their tetrahydro forms. From these data we conclude that 7‐biopterin, in contrast to e.g. brain tissue, regularly occurs as a normal metabolite in primary mast cells and that it is generated during hydroxylation of tryptophan.
A model of the structure of human annexin VI bound to lipid monolayers
Authors: Driessen H.P.C. , Newman R.H. , Freemont P.S. , Crumpton M.J.
Published: July 2002

Annexin VI is an eight repeat member of the annexin family of proteins which are both water soluble and bind to negatively charged phospholipids in a calcium‐dependent manner. Here we present a model for annexin VI based on fitting the three‐dimensional structure of two annexin V molecules (Huber (1990) EMBO J. 9, 3867–23;874) to the two‐dimensional stain‐excluding density of lipid‐bound annexin VI (Newman (1989) J. Mol. Biol. 206, 213–219). Both annexin VI lobes could only be fitted with their convex faces closest to the lipid monolayer. This supports the hypothesis that annexin—lipid binding is mediated by the interaction between calcium bound to the loops protruding from the convex protein surface and phospholipid headgroups.
A large domain common to sperm receptors (Zp2 and Zp3) and TGF‐β type III receptor
Authors: Bork Peer , Sander Chris
Published: July 2002

A new family of mosaic proteins is defined by sequence analysis. The family is characterized by a 260 residue domain common to proteins of apparently diverse function and tissue specificity; sperm receptors Zp2 and Zp3, betaglycan (also called TGF‐β type III receptor), uromodulin, as well as the major zymogen granule membrane protein (GP‐2). The location of the common domain is similar with respect to putative transmembrane regions. The results lead to the hypothesis that this type of domain has a common tertiary structure and that there is a functional similarity in the recognition mechanism of the sperm receptor system and the TGF‐β receptor complex.
Crystallographic binding studies with triosephosphate isomerases: Conformational changes induced by substrate and substrate‐analogues
Authors: Wierenga R.K. , Borcher T.V. , Noble M.E.M.
Published: July 2002

TIM catalyses the interconversion of a triosephosphate aldehyde into a triosephosphate ketone. This is a simple chemical reaction in which only protons are transferred, The crystallographic studies of TIM from chicken, yeast and trypanosome complexed with substrate and substrate analogues are discussed. The substrate binds in a deep pocket. On substrate binding, large conformational changes are induced in three loops. As a result of these conformational changes in the liganded Structure, the active site pocket is sealed off from bulk solvent and the sidechain of the catalytic glutamate becomes optimally positioned for catalysis.
Identification, characterization, and molecular cloning of a novel transporter‐like protein localized to the central nervous system
Authors: Gingrich Jay A. , Andersen Peter H. , Tiberi Mario , Mestikawy Salah El , Jorgensen Per N. , Fremeau Robert T. , Caron Marc G.
Published: July 2002

During the course of large scale purification of the D1 dopamine receptor from a rat brain, a protein of∼87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2225 base pairs encoding a protein or 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton‐driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of ∼4.8 kb in cortex. hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence from Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class transporter‐like proteins.
Production of an antiserum specific to the ADP‐ribosylated form of elongation factor 2 from archaebacteria and eukaryotes
Authors: Siegmund Klaus-Detlef , Klink Friedrich
Published: July 2002

An antiserum to ADP‐ribosylated elongation factor 2 (ADPR‐EF‐2) from S. acidocaldarius was raised in rabbits using stained, homogenized, ADPR‐EF‐2‐containing slices from SDS‐gels as a source of antigen. Elongation factor 2 (EF‐2) from S. acidocaldarius was cloned in E. coli and the expressed gene product was used in order to adsorb all anti‐EF‐2 antibodies which do not contain the ADP‐ribosyl group within their epitopes, as E. coli is unable to synthesize the ADP‐ribosyl acceptor diphthamide. The remaining antibodies were specific to ADP‐ribosylated EF‐2 from Thermoplasma acidophilum, S. acidocaldarius and Desulfurococcus mucosus. ADP‐ribosylated EF‐2 from eukaryotic sources also reacted with the adsorbed antiserum as shown for EF‐2 isolated from the killi‐fish Cynolebias whitei, the mouse species BALB/c and Han/Wistar rats. The adsorbed antiserum did not cross‐react with ADP‐ribosylated actin or rho protein or with FAD‐containing D‐amino acid oxidase.
Amiloride at pH 7.0 inhibits the Na+‐driven flagellar motors of Vibrio alginolyticus but allows cell growth
Authors: Atsumi Tatsuo , Maekawa Yukiyo , Tokuda Hajime , Imae Yasuo
Published: July 2002

Amiloride, a specific inhibitor for the Na+‐driven flagellar motors of alkalophilic Bacillus, is known to inhibit secondarily the growth of alkalophiles. The motility of a marine Vibrio, V. alginolyticus, was almost completely inhibited by 2 mM amiloride either at pH 7.0 or 8.5. We found that this concentration of amiloride inhibited the cell growth completely at pH 8.5 but only slightly at pH 7.0. Kinetic analysis of the inhibition of motility by amiloride at pH 7.0 showed that the inhibition was competitive with Na+ in the medium. Thus, amiloride at pH 7.0 is really a specific and useful tool for the analysis of the Na+‐driven flagellar motors of Vibro.
γ‐COP, a coat subunit of non‐clathrin‐coated vesicles with homology to Sec21p
Authors: Stenbeck Gudrun , Schreiner Rupert , Herrmann Doris , Auerbach Sylvia , Lottspeich Friedrich , Rothman James E. , Wieland Felix T.
Published: July 2002

Constitutive secretory transport in eukaryotes is likely to be mediated by non‐clathrin‐coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329–336; (1991) Nature 349, 215–220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248–250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, γ‐COP, is a true constituent of non‐clathrin‐coated vesicles, and that γ‐COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.
Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus
Authors: Kurihara Hiroyuki , Mitsui Yukio , Ohgi Kazuko , Irie Masachika , Mizuno Hiroshi , Nakamura Kazuo T.
Published: July 2002

The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 Å resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R‐factor is 0.200 in the 10—2.5 Å resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six α‐helices and seven β‐strands, belonging to the α+β type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site‐directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.
The hepatic interleukin‐6 receptor Down‐regulation of the interleukin‐6 binding subunit (gp80) by its ligand
Authors: Zohlnhöfer Dietlind , Graeve Lutz , Rose-John Stefan , Schooltink Heidi , Dittrich Elke , Heinrich Peter Claus
Published: July 2002

Interleukin‐6 (IL6) exerts its action via a cell surface receptor composed of an 80 kDa IL6‐binding protein (gp80) and a 130 kDa polypeptide involved in signal transduction (gp13O). We studied the role of gp80 in binding, internalization and down‐regulation of the hepatic IL6‐receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of transfected HepG2 cells overexpressing gp8O with parental cells indicate that gp80 is responsible for low affinity binding (Kd= 500 pM) of IL6. Furthermore, gp80 is rate‐limiting in internalization and degradation of IL6. Internalization resulted in a rapid down‐regulation (t≈ 15–30 min) of IL6‐binding sites at the cell surface. More than 80% of the internalized [125I]rhIL6 was degraded. The reappearance of IL6‐binding sites at the cell surface required >8 h and was sensitive to cycloheximide, suggesting that gp80 is not recycled after internalization. The down‐regulation of the hepatic IL6R by its ligand might play an important role as a protection against overstimulation.
Two different Ca2+ ion binding sites in factor VIIa and in des(1–38) factor VIIa
Authors: Schiødt Jakob , Harrit Niels , Christensen Ulla , Petersen Lars C.
Published: July 2002

The Ca2+ ion binding of factor VIIa and the derivative lacking the γ‐carboxyglutamic acid domain, des(1–38) factor VIIa, was investigated using intrinsic protein fluorescence and Tb3+ ion phosphorescence methods. Binding of Ca2+ ions giving rise to a decrease in the intrinsic protein fluorescence (approximately 50% at saturating conditions) is seen with both proteins. Each of the saturation curves is in accordance with the formation of a 1:1 complex of factor VIIa‐Ca2+ (K
D∼30 μM) and des(1–38) factor VIIa‐Ca2+ (K
D∼40 μM)). Yet another Ca2+ ion binding site reveals itself in each protein in Tb3+ ion phosphorescence experiments. Ca2+ ion competition studies have showed 1:1 complexes (K
D′s∼2 mM). The results are interpreted in terms of two different Ca2+ ion binding sites, one in the EGF‐1 domain and one in the Gly‐209‐Gin‐221 loop of the serine proteinase part.
A novel role for calcite in calcium homeostasis
Authors: Anderson Shawna , Appanna Vasu D. , Huang John , Viswanatha Thammaiah
Published: July 2002

Calcium carbonate (CaCO3) minerals are known to be deposited in a wide array of different organisms, ranging from microbes to vertebrates [(1989) On Biomineralization, Oxford University Press, New York]. Calcite, aragonite and vaterite are the major crystalline structural polymorphs of CaCO3 associated with living systems, and participate in a variety of biological functions [(1989) Biomineralization: Chemical and Biochemical Perspectives. VCH Publishers, Weinham, Germany; (1991) Advances in Inorganic Chemistry 36, 137–200]. Here we report on the ability of a soil bacterium to synthesize calcite in a calcium‐stressed environment. The elaboration of this exocellular crystalline residue enables the organism to regulate its calcium content. The attainment of calcium homeostasis via the exocellular deposition of bacterial calcite with unique crystal habits is a novel biological phenomenon.
The complete primary structure of abrin‐a B chain
Authors: Chen Yung-Liang , Chow Lu-Ping , Tsugita Akira , Lin Jung-Yaw
Published: July 2002

The complete 267 amino acid sequence of abrin‐a B chain was determined by analysis of peptides obtained by digestion with trypsin, chymotrypsin, lysyl endopeptidase, Staphylacaccus aureus V8 protease and thermolysin. The sequence is not identical with that predicted previously by nucleotide sequencing, indicating the presence of isoforms of abrin. Comparison of the amino acid sequence of abrin‐a B chain with that of ricin‐D B chain reveals a high degree of sequence identity (59%). Abrin‐a B chain appears to consist of two domains, each domain with subdomains (α, β, γ) of about 40 amino acid residues.
A protective role for nitric oxide in the oxidative modification of low density lipoproteins by mouse macrophages
Authors: Yates Mark T. , Lambert Laurie E. , Whitten Jeffrey P. , McDonald Ian , Mano Masayuki , Ku George , Mao Simon J.T.
Published: July 2002

Low density lipoproteins (LDL) oxidatively modified by macrophages have been shown to be atherogenic in ex vivo studies. We studied the potential role of nitric oxide (NO), a free radical produced by macrophages, in LDL modification. Human LDL (1 mg/ml) were incubated with mouse peritoneal macrophages in Ham's F‐10 medium. The cells were then stimulated by interferon‐γ and tumor necrosis factor‐α to increase their production of NO from 1.3 to 12.2 μM in 24 h, as measured by nitrite. Lipid peroxidation of LDL, as measured by thiobarbituric acid‐reactive materials (TBARS), was reduced in stimulated cells in a time‐dependent manner. At 24 h, the decrease was about 27%. In the presence of an NO synthase inhibitor (No‐aminophomoarginine), the generation of NO was diminished and the protection against LDL lipid peroxidation was reversed. The extent of LDL protein modification was also assessed by examining its electrophoretic mobility. It was found that macrophage NO reduced the change in LDL electromobility. These data indicate that the production of NO may inhibit the oxidative modification of LDL with cytokine‐stimulated macrophages. We suggest that NO plays a protective role in limiting macrophage‐induced LDL modification.
The microtubule binding repeats of tau protein assemble into filaments like those found in Alzheimer's disease
Authors: Crowther R.A. , Olesen O.F. , Jakes R. , Goedert M.
Published: July 2002

The paired helical filament, which comprises the major fibrous element of the neurofibrillary tangle in Alzheimer's disease, contains abnormally phosphorylated microtubule‐associated protein tau as its principal constituent. The repeat region of tau protein, which represents the microtubule binding domain, forms the core of the filament. Here we show that an expressed fragment of tau protein spanning the repeat region can assemble in vitro into filaments like those found in Alzheimer's disease
Structural relationship between lipases and peptidases of the prolyl oligopeptidase family
Authors: Polgár László
Published: July 2002

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three‐dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10‐residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.
Utrophin, the autosomal homologue of dystrophin, is widely‐expressed and membrane‐associated in cultured cell lines
Authors: thi Man Nguyen , Thanh LeThiet , Blake D.J. , Davies K.E. , Morris G.E.
Published: July 2002

Utrophin, the autosomal dystrophin‐related protein (DRP), is expressed in HeLa cells, smooth muscle‐like BC3HI cells from mouse brain, COS monkey kidney cells, the P38BD1 monocyte‐macrophage cell line and untransformed human skin fibroblasts, as well as in rat C6 glioma and Schwannoma cells. It was undetectable, however, in the Sp2/O mouse myeloma cell line and in hybridoma lines derived from it. Dystrophin was not detected in any of these cell lines. Although all utrophin‐containing cells were capable of forming monolayers in culture, no major effects of either attachment to substratum or length of time in culture (2–17 days) on utrophin levels were observed. After subcellular fractionation ofBC3HI or glioma cells, nearly all of the utrophin was found in the Triton‐soluble fraction, suggesting an association with cell membranes.
Stilbene synthase from Scots pine (Pinus sylvestris)
Authors: Schanz Sigrid , Schröder Gudrun , Schröder Joachim
Published: July 2002

Stilbene synthases are named according to their substrate preferences. By this definition, enzymes preferring cinnamoyl‐CoA are pinosylvin synthases, and proteins with a preference for phenylpropionyl‐CoA are dihydropinosylvin synthases. We investigated the assignment of a stilbene synthase cloned from Scots pine (Pinus sylvestris) as dihydropinosylvin synthase and the proposal of an additional pinosylvin synthase [1992, Plant Mol. Biol. 18, 439–503]. The results show that the previous interpretation was misled by several unexpected factors. Firstly, we found that the substrate preference and the activity of the plant‐specific protein expressed in E. coli was influenced by bacterial factors. This was reduced by improvement of the expression system, and the subsequent kinetic analysis revealed that cinnamoyl‐CoA rather than phenylpropionyl‐CoA is the preferred substrate of the cloned stilbene synthase. Secondly, mixing experiments showed that extracts from P. sylvestris contain factor(s) which selectively influenced the substrate preference, i.e. the activity was reduced with phenylpropionyl‐CoA, but not with cinnamoyl‐CoA. This explained the apparent differences between plant extracts and the cloned enzyme expressed in E. coli. Taken together, the results indicate that the cloned enzyme is a pinosylvin synthase, and there is no evidence for a second stilbene synthase. This study cautions that factors in the natural and in new hosts may complicate the functional identification of cloned sequences.
Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins
Authors: Stokoe David , Engel Katrin , Campbell David G. , Cohen Philip , Gaestel Matthias
Published: July 2002

MAP kinase‐activated protein kinase‐2 (MAPKAP kinase‐2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser40 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Se86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co‐purifies with MAPKAP kinase‐2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase‐2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.
Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria
Authors: Lapidus Rena G. , Sokolove Patricia M.
Published: July 2002

The effect of spermine on the permeability transition of the inner mitochondrial membrane of isolated rat heart mitochondria was evaluated. The permeability transition was triggered using a series of agents (t‐butyl hydroperoxide, phenylarsine oxide, carboxyatractylate, and elevated Ca2+ and inorganic phosphate concentrations), and was monitored via Ca2+‐release, mitochondrial swelling and pyridine nucleotide oxidation. By all three criteria, spermine inhibited the transition. A C50 of 0.38 ± 0.06 (SD) mM was measured for inhibition.
ISK, a slowly activating voltage‐sensitive K+ channel Characterization of multiple cDNAs and gene organization in the mouse
Authors: Lesage Florian , Attali Bernard , Lazdunski Michel , Barhanin Jacques
Published: July 2002

mISK is a protein consisting of 129 amino acids with a single putative transmembrane domain. The injection of mISK cRNA into Xenopus oocytes directs the expression of a voltage‐gated K+ current. A heart mRNA blot probed with mISK, DNA revealed at least two transcripts. The messenger diversity of mISK was investigated by cloning and characterization of multiple cDNAs of one genomic clone, and by performing primer extension experiments. All cDNAs characterized have the same protein‐coding sequence, and heterogeneity of the transcripts arises from alternative splicing, and multiple sites of transcription start and polyadenylation. ISK is encoded by a single gene in the mouse genome. The gene organization reveals the existence of an exon containing the whole protein‐coding sequence and of two alternative exons corresponding to the 5′ untranslated sequences. We failed to detect the presence of another exon capable of extending the protein‐coding sequence. The diversity of mISK messengers is not associated with a diversity of the mISK protein.
Curcumin inhibits nitrite‐induced methemoglobin formation
Authors: Unnikrishnan M.K. , Rao M.N.A.
Published: July 2002

Curcumin protects hemoglobin from nitrite‐induced oxidation to methemoglobin. The protection is not observed when curcumin is added after the autocatalytic stage of the oxidation of hemoglobin by nitrite. The ability of curcumin to scavenge superoxide may be responsible since superoxide is implicated in promoting the autocatalytic stage of oxidation of hemoglobin by nitrite.
The purification and amino acid sequences of four Tx2 neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer (Keys)
Authors: Cordeiro Marta do Nascimento , Diniz Carlos Ribeiro , do Carmo Valentim Ana , von Eickstedt Vera Regina D. , Gilroy John , Richardson Michael
Published: July 2002

Four neurotoxic polypeptides (Tx2.1, Txt2‐5, Tx2‐6 and Tx2‐9) were purified from the venom of the South American ‘armed’ spider Phoneutria nigriventer (Keys) by gel filtration and reverse phase FPLC and HPLC. These cysteine‐rich polypeptides exhibited different levels of neurotoxicity in mice after intracerebroventricular injection. Tx2‐1, Tx2‐5 and Tx2‐6 caused spastic paralysis and death, but the less toxic Tx2‐9 produced only tail erection and scratching. The molecular weights of the polypeptides as determined by desorption mass spectroscoopy were 5838.8 for Tx2‐1, 5116.6 (Tx2‐5), 5291.3 (Tx2‐6) and 3742.1 (Tx2‐9). The complete amino acid sequences of the neurotoxins were determined by automated Edman degradation and by manual DABITC‐PITC microseqeunce analysis of peptides obtained after digestions with various proteases. The amino acid sequences of Tx2‐1 (53 residues), Tx2‐5 (48 residues) and Tx2‐6 (48 residues) were homologous, but had only limited similarities to the less toxic Tx2‐9 (32 residues). All four polypeptides had varying sequence identities with other neurotoxins from different spider species and biologically active peptides from scorpions, a sea snail and seeds of Mirabilis jalapa.
Suppression of peroxisomal lipid β‐oxidation enzymes by TNF‐α
Authors: Beier Konstantin , Völkl Alfred , Fahimi H.Dariush
Published: July 2002

TNF‐α is a potent cytokine which induces marked hyperlipidemia. Because of the important role of peroxisomes in lipid metabolism we investigated the effects of human recombinant TNF‐α upon rat liver peroxisomal enzymes. Sixteen hours after the administration of a single dose of 25 μg of TNF‐α to male rats the activity of peroxisomal fatty acyl‐CoA oxidase was reduced by 50%. This was confirmed also by immunoblotting and by quantitative immunoelectron microscopy which in addition revealed substantial reduction of the trifunctional protein (hydratase—dehydrogenase—isomerase) in peroxisomes. These observations suggest that the suppression of peroxisomal β‐oxidation may contribute to the perturbation of the lipid metabolism induced by TNF‐α.