bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of L‐carnitine transport into rat skeletal muscle plasma membrane vesicles
Authors: Berardi Simona , Stieger Bruno , Hagenbuch Bruno , Carafoli Ernesto , Krähenbühl Stephan
Published: March 2003

Transport ofl‐carnitine into skeletal muscle was investigated using rat sarcolemmal membrane vesicles. In the presence of an inwardly directed sodium chloride gradient,l‐carnitine transport showed a clear overshoot. The uptake ofl‐carnitine was increased, when vesicles were preloaded with potassium. When sodium was replaced by lithium or cesium, and chloride by nitrate or thiocyanate, transport activities were not different from in the presence of sodium chloride. However,l‐carnitine transport was clearly lower in the presence of sulfate or gluconate, suggesting potential‐dependent transport. An osmolarity plot revealed a positive slope and a significant intercept, indicating transport ofl‐carnitine into the vesicle lumen and binding to the vesicle membrane. Displacement experiments revealed that approximately 30% of thel‐carnitine associated with the vesicles was bound to the outer and 30% to the inner surface of the vesicle membrane, whereas 40% was unbound inside the vesicle. Saturable transport could be described by Michaelis–Menten kinetics with an apparentKmof 13.1 µmand aVmaxof 2.1 pmol·(mg protein−1)·s−1.l‐Carnitine transport could betrans‐stimulated by preloading the vesicles withl‐carnitine but not with the carnitine precursor butyrobetaine, and wascis‐inhibited byl‐palmitoylcarnitine,l‐isovalerylcarnitine, and glycinebetaine. On comparing carnitine transport into rat kidney brush‐border membrane vesicles and OCTN2, a sodium‐dependent high‐affinity human carnitine transporter, cloned recently from human kidney also expressed in muscle, theKmvalues are similar but driving forces, pattern of inhibition and stereospecificity are different. This suggests the existence of more than one carnitine carrier in skeletal muscle.
Formation of a new class of oxylipins from N‐acyl(ethanol)amines by the lipoxygenase pathway
Authors: van der Stelt Marcelis , Noordermeer Minke A. , Kiss Tünde , van Zadelhoff Guus , Merghart Ben , Veldink Gerrit A. , Vliegenthart Johannes F. G.
Published: March 2003

N‐Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase‐1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both α‐N‐linolenoylethanolamine and γ‐N‐linolenoylethanolamine (18:3), as well as α‐N‐linolenoylamine and γ‐N‐linolenoylamine were converted into their (13S)‐hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of α‐N‐linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z)‐hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12‐Oxo‐N‐(9Z)‐dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S)‐hydroperoxy‐α‐N‐linolenoylethanolamine and the (13S)‐hydroperoxide of linoleic acid and its ethanolamine derivative. α‐Ketols and γ‐ketols were characterized as products. In addition, cyclized products, i.e. 12‐oxo‐N‐phytodienoylamines, derived from (13S)‐hydroperoxy‐α‐N‐linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.
Identification of tudor repeat associator with PCTAIRE 2 (Trap)
Authors: Hirose Takashi , Kawabuchi Masahiro , Tamaru Teruya , Okumura Nobuaki , Nagai Katsuya , Okada Masato
Published: March 2003

PCTAIRE 2 is a Cdc2‐related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two‐hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor‐like domain), conserved in Drosophila tudor protein. Trap associates with the N‐terminal domain of PCTAIRE 2 through its C‐terminal domain, which contains two tudor‐like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons.
Aprotinin binding to amyloid fibrils
Authors: Cardoso Isabel , Pereira Pedro J. Barbosa , Damas Ana M. , Saraiva Maria J. M.
Published: March 2003

Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the β‐sheet structure arranged in a cross‐β conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium‐99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra‐abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, β‐amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot‐blot ligand‐binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 µm−1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of β‐sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand β‐sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils.
Bacterial‐injection‐induced syntheses of N‐β‐alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae
Authors: Kim Mi Hee , Joo Chang Hun , Cho Mi Young , Kwon Tae Hyuk , Lee Kwang Moon , Natori Shunji , Lee Tae Ho , Lee Bok Luel
Published: March 2003

Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine‐like substance on the electrochemical detector. To characterize this dopamine‐like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N‐β‐alanyldopamine using the liquid chromatographic/tandem mass spectrometric method. Chemically synthesized N‐β‐alanyldopamine showed the same retention time on HPLC as the purified N‐β‐alanyldopamine from immunized larvae. To elucidate the molecular mechanism of N‐β‐alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E. coli‐injected hemolymph of T. molitor larvae. The enzyme activity of Dopa decarboxylase increased dramatically ≈ 8 h after injection; Dopa decarboxylase activity of injected larvae being 10‐times higher than naive larvae after 24 h. To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated. RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3–8 h after E. coli challenge. Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E. coli‐injected larval extract. Thus, bacterial injection into T. molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N‐β‐alanyldopamine. The synthesized N‐β‐alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response.
N‐Carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea
Authors: Bartoschek Stefan , Vorholt Julia A. , Thauer Rudolf K. , Geierstanger Bernhard H. , Griesinger Christian
Published: March 2003

N‐Carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from ‘MFR’(MFR + MFRH+) and ‘CO2’ (CO2 + H2CO3 + HCO3−+ CO32–) was found to be 7 m−1·s−1 (v1 = k1*[‘MFR’][‘CO2’]) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s−1 (v2 = k2*[carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/[‘MFR’][‘CO2’] was 0.6 m−1 at pH 7.0 corresponding to a free energy change ΔG°′ of + 1.3 kJ·mol−1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3− in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3− which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N‐carboxymethanofuran to N‐formylmethanofuran.

From the concentrations of ‘CO2’ and of ‘MFR’ determined by 1D‐NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1[MFR][CO2]). The second order rate constant k1 was found to be approximately 1000 m−1·s−1 at 300 K and pH values between 7.0 and 8.0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.
Molecular cloning and characterization of murine and human N‐acetylglucosamine kinase
Authors: Hinderlich Stephan , Berger Markus , Schwarzkopf Martina , Effertz Karin , Reutter Werner
Published: March 2003

N‐Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N‐acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino‐acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N‐acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N‐acetylglucosamine kinase activity as well as N‐acetylmannosamine kinase activity.

The complete cDNA sequence of human N‐acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N‐acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino‐acid level. Amino‐acid sequences of murine and human N‐acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present.

Tissue distribution of murine N‐acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.
Studies on the recycling of the transferrin receptor in Trypanosoma brucei using an inducible gene expression system
Authors: Kabiri Mostafa , Steverding Dietmar
Published: March 2003

Uptake of host transferrin in bloodstream forms of Trypanosoma brucei is mediated by a heterodimeric, glycosylphosphatidylinositol‐anchored receptor. After endocytosis, transferrin is delivered to lysosomes where it is proteolytically degraded. Whether the heterodimeric transferrin receptor is returned to mediate several cycles in ligand uptake is undefined. By using an inducible gene expression system we provide evidence for recycling of the transferrin receptor in bloodstream forms of T. brucei. The metabolic half‐life of the transferrin receptor in bloodstream‐form trypanosomes is determined to be 7 h which is comparable to the half‐lives of recycling receptors in mammalian cells. The cycling time of the trypanosomal transferrin receptor is calculated to be 11 min. By means of the half‐life and the cycling time, we calculated that each receptor is recycled 60 times before being degraded on average.
A DNA ligase from the psychrophilePseudoalteromonas haloplanktisgives insights into the adaptation of proteins to low temperatures
Authors: Georlette D. , Jónsson Z. O. , Van Petegem F. , Chessa J.‐P. , Van Beeumen J. , Hübscher U. , Gerday C.
Published: March 2003

The cloning, overexpression and characterization of a cold‐adapted DNA ligase from the Antarctic sea water bacteriumPseudoalteromonas haloplanktisare described. Protein sequence analysis revealed that the cold‐adaptedPhDNA ligase shows a significant level of sequence similarity to other NAD+‐dependent DNA ligases and contains several previously described sequence motifs. Also, a decreased level of arginine and proline residues inPhDNA ligase could be involved in the cold‐adaptation strategy. Moreover, 3D modelling of the N‐terminal domain ofPhDNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue. The recombinantPhDNA ligase was overexpressed inEscherichia coliand purified to homogeneity. Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74 593 Da.PhDNA ligase shows similar overall catalytic properties to other NAD+‐dependent DNA ligases but is a cold‐adapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpartE. coliDNA ligase. A kinetic comparison of three enzymes adapted to different temperatures (P. haloplanktis,E. coliandThermus scotoductusDNA ligases) indicated that an increasedkcatis the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreasedKmfor the nicked DNA substrate seems to allowT. scotoductusDNA ligase to work efficiently at high temperatures. Besides being useful for investigation of the adaptation of enzymes to extreme temperatures,P. haloplanktisDNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.
Targeting of single‐stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides
Authors: Maksimenko Andrei V. , Volkov Evgueny M. , Bertrand Jean‐Rémi , Porumb Horea , Malvy Claude , Shabarova Zoe A. , Gottikh Marina B.
Published: March 2003

The aim of this work was to construct an anti‐messenger targeted to the pim‐1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single‐stranded targets of both DNA (5′‐CCCTCCTTTGAAGAA‐3′) and RNA type (5′‐CCCUCCUUUGAAGAA‐3′). The third, ‘Hoogsteen’ strand of the triplex was represented by G,T‐rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3′‐GGGAGGAAACTTCTTTT‐TTGTTGTTT‐TT‐GGTGGG‐5′, where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the ‘Hoogsteen’ strand switches from one strand of the Watson–Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti‐messenger (underlined). The portion of the ‘Hoogsteen’ sequence of the triplex between the two TT dinucleotides binds to the 3′ extremity of the target strand and runs parallel to it. The portion situated at the 5′ end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5′‐TTCTTCAAAGGAGGG‐3′^3′‐GGGTGGTTT‐T‐GTTGTT‐5′) the portion of the ‘Hoogsteen’ sequence that bound to the 3′ extremity of the target strand had to be antiparallel to it.
Human herpes virus 8 interleukin‐6 homologue triggers gp130 on neuronal and hematopoietic cells
Authors: Hoischen Susanne H. , Vollmer Petra , März Pia , Özbek Suat , Götze Katharina S. , Peschel Christian , Jostock Thomas , Geib Till , Müllberg Jürgen , Mechtersheimer Sabine , Fischer Martina , Grötzinger Joachim , Galle Peter R. , Rose‐John Stefan
Published: March 2003

Human herpes virus‐8 (HHV8) encodes a cytokine named viral interleukin‐6 (vIL‐6) that shares 25% amino‐acid identity with its human homologue. Human IL‐6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL‐6 is expressed in HHV8‐associated‐diseases including Kaposi's sarcoma, Body‐cavity‐based‐lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B‐cell associated diseases and other malignant tumours. We expressed vIL‐6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL‐6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL‐6 protein. vIL‐6 turned out to be active on cells that expressed gp130 but no IL‐6 receptor (IL‐6‐R) suggesting that, in contrast to human IL‐6, vIL‐6 stimulated gp130 directly. Accordingly, vIL‐6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL‐6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL‐6 exhibits biologic activity that has only been observed for the IL‐6/soluble IL‐6‐R complex but not for IL‐6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL‐6.
The effect of tryptophanyl substitution on folding and structure of myoglobin
Authors: Sirangelo Ivana , Tavassi Simona , Martelli Pier Luigi , Casadio Rita , Irace Gaetano
Published: March 2003

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A‐5) and 14 (A‐12) in the N‐terminal region (A helix) of the protein molecule. The crucial role of tryptophanyl residues has been investigated by site‐directed mutagenesis and molecular dynamics simulation. The apomyoglobin mutants with a double W→F substitution were found to be not correctly folded and therefore not expressed as holoprotein. The introduction of a tyrosyl residue at position 7, that is, W7YW14F, resulted in the expression of a correctly folded myoglobin. Not correctly folded apomyoglobins were found with the following mutants: W7FW14Y, W7EW14F, W7FW14E, W7KW14F, W7FW14K. Moreover, in all these cases, very low levels of expression were observed.

The acid‐induced denaturation curves of wild‐type and folded mutant W7YW14F, obtained following the fluorescence variation of the extrinsic fluorophore 1‐anilino‐8‐naphthalenesulfonate, revealed that the stability of the native state of mutant apoprotein is decreased, thus indicating that the replacement W→Y in position 7 is able to restore a correct folding but not the same stability. Molecular dynamics simulation indicated that both tryptophans are involved in forming favorable, specific tertiary interactions in the native apomyoglobin structure. The lack of some of these interactions caused by tryptophanyl replacement affects the overall protein structure and may provide an explanation for the observed stability decrease. In the case of the double W→F substitution, the simulated structure shows conclusively the domain formed by helices A, G and H to be not correctly folded. This effect is attenuated if at least one of the two residues is conserved or a tyrosyl residue replaces W7.
A heparin‐binding growth factor, midkine, binds to a chondroitin sulfate proteoglycan, PG‐M/versican
Authors: Zou Kun , Muramatsu Hisako , Ikematsu Shinya , Sakuma Sadatoshi , Salama Ragaa H. M. , Shinomura Tamayuki , Kimata Koji , Muramatsu Takashi
Published: March 2003

Midkine is a heparin‐binding growth factor with survival‐promoting and migration‐enhancing activities. In order to understand the regulation of midkine signaling, we isolated midkine‐binding proteoglycans from day 13 mouse embryos, when midkine is intensely expressed. Deglycosylation followed by SDS/PAGE revealed various protein bands; one of these was identified as PG‐M/versican by in gel trypsin digestion and sequencing the resulting peptides. PG‐M/versican isolated from day 13 mouse embryos bound midkine with aKdof 1.0 nm. Pleiotrophin/heparin‐binding growth‐associated molecule, which has a structure related to midkine, was also bound similarly. Digestion with chondroitinase ABC, AC‐I or B abolished the binding to midkine. Heparin as well as chondroitin sulfate D and E inhibited the binding. After chondroitinase ABC digestion, the midkine‐binding PG‐M/versican released 4‐sulfated, 6‐sulfated, 2,6‐disulfated and 4,6‐disulfated unsaturated disaccharides. These results suggest that midkine binds to a polysulfated domain in the chondroitin sulfate chain with a region of dermatan sulfate structure. This proteoglycan may modulate the midkine activity, as binding to midkine can enhance midkine action by concentrating it to the cell periphery or inhibit the action by competing with the binding to a signaling receptor.
Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture
Authors: Chigorno Vanna , Palestini Paola , Sciannamblo Mariateresa , Dolo Vincenza , Pavan Antonio , Tettamanti Guido , Sonnino Sandro
Published: March 2003

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X‐100 and fractionated by sucrose‐gradient centrifugation to prepare a detergent‐insoluble membrane fraction known to be enriched in sphingolipid and caveolin‐1, i.e. of caveolae.

The detergent‐insoluble membrane fraction prepared after feeding [1‐3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18‐fold). By feeding cells photoactivable radioactive GM3, after 2 h‐chase, caveolin‐1, CAV1, and proteins of high molecular mass became cross‐linked to GM3, the cross‐linking complexes being highly concentrated in the detergent‐insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time‐dependent, transient process so that CAV1 cross‐linking to GM3 was hardly detectable after a 24‐h chase followed the pulse time. After a 24‐h chase, only the high molecular mass proteins cross‐linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized.

Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold‐conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti‐GM3 and anti‐CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals.

Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.
Substrates enhance autophosphorylation and activation of p21‐activated protein kinase γ‐PAK in the absence of activation loop phosphorylation
Authors: Jakobi Rolf , Huang Zhongdong , Walter Barbara N. , Tuazon Polygena T. , Traugh Jolinda A.
Published: March 2003

The p21‐activated protein kinase γ‐PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPγS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13‐fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild‐type γ‐PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPγS). This substrate‐level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild‐type γ‐PAK. Basic proteins or peptides which are not substrates of γ‐PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by γ‐PAK following activation by Cdc42(GTPγS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPγS), by activating γ‐PAK directly.
Specific Ser‐Pro phosphorylation by the RNA‐recognition motif containing kinase KIS
Authors: Maucuer Alexandre , Le Caer Jean‐Pierre , Manceau Valérie , Sobel André
Published: March 2003

We present here a first appraisal of the phosphorylation site specificity of KIS (for ‘kinase interacting with stathmin’), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA‐recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser‐Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks.
Expression of gelatinases A and B in the ovary of the medaka fish Oryzias latipes
Authors: Matsui Hitoshi , Ogiwara Katsueki , Ohkura Ryuich , Yamashita Masakane , Takahashi Takayuki
Published: March 2003

We cloned cDNAs for gelatinase A and gelatinase B from an ovary cDNA library of the medaka fish Oryzias latipes. The gelatinase A clone encodes a protein of 657 amino acids, whereas the gelatinase B clone encodes a protein of 690 amino acids. Gelatinase A mRNA was expressed in the testis, ovary, intestine, heart, spleen and kidney of the animal. In contrast, gelatinase B mRNA was detected in the ovary. Localization of the respective mRNAs in the ovary was examined using in situ hybridization. Gelatinase A mRNA was found only in the oocytes of small and middle‐sized follicles. In contrast, gelatinase B was expressed exclusively in follicular tissues that had ovulated. In situ zymographic analysis revealed that gelatinolytic activity, presumably due to matrix metalloproteinase activity, was detectable in the areas surrounding small and middle‐sized follicles, interstitial stromal tissues and the cytoplasm of oocytes. Using extracts of the whole ovary and of ovulated oocytes, several gelatin‐degrading enzymes, which probably represent the intermediate and active forms of medaka fish gelatinase A and gelatinase B, were detected by gelatin zymographic analysis. These results clearly indicate that gelatinase A and gelatinase B play a discrete role in the ovary of this lower vertebrate animal.
Genetic organization of A chain and B chain of β‐bungarotoxin from Taiwan banded krait (Bungarus multicinctus)
Authors: Wu Pei‐Fung , Chang Long‐Sen
Published: March 2003

β‐Bungarotoxin, the main presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains, the A chain and the B chain, cross‐linked by an interchain disulfide bond. In this study, A and B chain genes isolated from the liver of B. multicinctus encoded the A and B chain precursors, respectively. Analyses of the coding regions of the A and B chain genes revealed that both consist of three exons and two introns. The sequences of all exon/intron junctions agree with the GT/AG rule. However, sequence alignment and phylogenetic analysis did not support that the evolution of A and B chain genes are closely related. Comparative analysis of A chain genes with Viperinae and Crotalinae phospholipase A2 genes indicated that genetic divergence of the A chain and phospholipase A2s was in accordance with their family. Moreover, evolutionary divergence of the intron and exon regions of the A chain, as observed for phospholipase A2 genes, was not consistent. Noticeably, the transcription of A and B chain genes may be regulated under different transcription factors as revealed by analyses of their promoter sequences. In terms of the finding that A and B chains are encoded separately by different genes, this strongly supports the view that the intact β‐bungarotoxin molecules should be derived from the pairing of A and B chains after their mRNAs are translated.
Relating multidrug resistance phenotypes to the kinetic properties of their drug‐efflux pumps
Authors: Westerhoff H. V. , Riethorst A. , Jongsma A. P. M.
Published: March 2003

The simplest model for pump‐mediated multidrug resistance is elaborated quantitatively. The way in which toxicity data should be evaluated to characterize most effectively the drug‐efflux pump is then examined. The isotoxic drug dose (D10) depends on too many unrelated properties. The D10 of a cell line taken relative to that of the parental (nonresistant) cell line has been called the relative resistance (RR). This is inappropriate for characterizing the drug pump, as it depends on the extent of amplification of the latter. The reduced RR (RRR) is newly defined as the ratio of the (RR − 1) for one drug to the (RR – 1) for a different drug. This RRR should be independent of both the drug–target affinity and the extent of amplification of the drug pump in cell lines belonging to a family. The RRR depends on the avidities with which the pump extrudes the drugs relative to the passive membrane permeabilities of the latter. In plots of RRR for one drug combination vs. that for a second drug combination, cell lines that have the same pump amplified should cluster, whereas those with amplification of (functionally) different drug‐efflux pumps should segregate. Both a set of new experimental data and literature results are discussed in terms of RRR. RRRs discriminate between human MDR1 and mouse mdr1a and mdr1b, between hamster pgp1 and a mutant thereof, as well as between human MDR1 and a mutant thereof. RRRs are not affected by changes in membrane surface area. Our results indicate that RRR may be used to (a) characterize drug‐resistance mechanisms and (b) determine which drug‐resistance mechanism is operative. Moreover, our analysis suggests that some of the reported phenotypic diversity among multidrug‐resistant cell lines may not be due to diversity in the resistance mechanism.
A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides
Authors: Velayos Antonio , Eslava Arturo P. , Iturriaga Enrique A.
Published: March 2003

Using functional analyses in Escherichia coli and Mucor circinelloides, it has been shown that a single M. circinelloides gene (carRP) codes for a protein with two different enzymatic activities, lycopene cyclase and phytoene synthase, which are encoded by independent genes in organisms other than fungi. This gene was identified using complementation tests among different classes of carotenoid mutants of M. circinelloides. The carRP gene product contains two domains: the R domain is located at the N‐terminus and determines lycopene cyclase activity; the P domain is located at the C‐terminus and displays phytoene synthase activity. The R domain is functional even in the absence of the P domain, while the latter needs the proper R domain conformation to carry out its function. The carRP gene is closely linked to the phytoene dehydrogenase (carB) gene, and the promoter regions of both genes are located within only 446 bp. Northern analyses show a co‐ordinated regulation of the expression of both genes by blue light. Several motifs found in this promoter region suggest a bi‐directional mode of transcription control.
Human brain GABA transaminase
Authors: Jeon Seong Gyu , Bahn Jae Hoon , Jang Joong Sik , Park Jinseu , Kwon Oh‐Shin , Cho Sung‐Woo , Choi Soo Young
Published: March 2003

Human brain γ‐aminobutyrate transaminase is differentially expressed in a tissue‐specific manner. mRNA master dot‐blot analysis for 50 different human tissues, including different brain regions and fetal tissues, provided a complete map of the tissue distribution. Genomic Southern analysis revealed that the γ‐aminobutyrate transaminase gene is a single copy, at least 15 kb in size. In addition, human brain γ‐aminobutyrate transaminase cDNA was expressed inEscherichia coliusing a pGEX expression vector system. Catalytically active γ‐aminobutyrate transaminase was expressed in large quantities and the purified recombinant enzyme had kinetic parameters that were indistinguishable from those isolated from other mammalian brains. The human enzyme was inactivated by a well‐known antiepileptic drug vigabatrin. Values ofKiandkinactwere 1 mmand 0.35 min−1, respectively. Results from inactivation kinetics suggested that human γ‐aminobutyrate transaminase is more sensitive to the vigabatrin drug than the enzyme isolated from bovine brain.
Proteomics of Synechocystis sp. strain PCC 6803
Authors: Fulda Sabine , Huang Fang , Nilsson Fredrik , Hagemann Martin , Norling Birgitta
Published: March 2003

Periplasmic proteins isolated by cold osmotic shock of Synechocystis sp. PCC 6803 cells were identified using 2D PAGE, MS and genome analysis. Most of the periplasmic proteins represent ‘hypothetical proteins’ with unknown function. A number of proteases of different specificity, and several enzymes involved in cell wall biosynthesis were also found. In salt‐adapted cells, six proteins were greatly enhanced and three proteins were newly induced. Most of the salt‐enhanced proteins are involved in the alteration of cell wall structure of salt‐adapted cells. The precursors of all 57 periplasmic proteins identified have a signal peptide; 47 of them contain a typical Sec‐dependent signal peptide, whereas 10 contain a putative twin‐arginine signal peptide.
ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase
Authors: Hastings Stuart F. , Heathcote Peter , Ingledew W. John , Rigby Stephen E. J.
Published: March 2003

The putative oxidation of ubiquinol by the cytochrome bo3 terminal oxidase of Escherichia coli in sequential one‐electron steps requires stabilization of the semiquinone. ENDOR spectroscopy has recently been used to study the native ubisemiquinone radical formed in the cytochrome bo3 quinone‐binding site [Veselov, A.V., Osborne, J.P., Gennis, R.B. & Scholes, C.P. (2000) Biochemistry39, 3169–3175]. Comparison of these spectra with those from the decyl‐ubisemiquinone radical in vitro indicated that the protein induced large changes in the electronic structure of the ubisemiquinone radical. We have used quinone‐substitution experiments to obtain ENDOR spectra of ubisemiquinone, phyllosemiquinone and plastosemiquinone anion radicals bound at the cytochrome bo3 quinone‐binding site. Large changes in the electronic structures of these semiquinone anion radicals are induced on binding to the cytochrome bo3 oxidase. The changes in electronic structure are, however, independent of the electronic structures of these semiquinones in vitro. Thus it is shown to be the structure of this binding site in the protein, not the covalent structure of the bound quinone, that determines the electronic structure of the protein‐bound semiquinone.
Changes to the length of the flexible linker region of the Rieske protein impair the interaction of ubiquinol with the cytochrome bc1 complex
Authors: Nett Jürgen H. , Hunte Carola , Trumpower Bernard L.
Published: March 2003

Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron–sulfur protein, which carries the [2Fe−2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c1. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol‐cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent Km for ubiquinol to decrease from 9.3 to 2.6 µm. Addition of two alanine residues lowers the activity by 90% and the apparent Km decreases to 1.9 µm. Deletion of an alanine residue lowers the activity by ≈ 40% and the apparent Km decreases to 5.0 µm. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol‐cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc1 complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron–sulfur protein and cytochrome b.
Expression and characterization of Edg‐1 receptors in rat cardiomyocytes
Authors: Nakajima Nobuko , Cavalli Amy L. , Biral Donatella , Glembotski Christopher C. , McDonough Patrick M. , Ho Peter D. , Betto Romeo , Sandoná Doriana , Palade Philip T. , Dettbarn Christine A. , Klepper Robert E. , Sabbadini Roger A.
Published: March 2003

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G‐protein‐coupled receptors of the Edg family. In the present study, it was found that sphingosine 1‐phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg‐1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg‐1 protein expression and reduced the Sph1P‐mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid‐sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg‐mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.
Characterization of RCI‐1, a chloroplastic rice lipoxygenase whose synthesis is induced by chemical plant resistance activators
Authors: Schaffrath Ulrich , Zabbai Frauke , Dudler Robert
Published: March 2003

A full‐length lipoxygenase cDNA (RCI‐1) has been cloned from rice (Oryza sativa) whose corresponding transcripts accumulate in response to treatment of the plants with chemical inducers of acquired resistance such as benzo(1,2,3)thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH), 2,6‐dichloroisonicotinic acid (INA), and probenazol. In contrast, RCI‐1 transcript levels did not increase after inoculation with compatible and incompatible races of the rice blast fungus Magnaporthe grisea and the nonhost pathogen Pseudomonas syringae pv. syringae. RCI‐1 transcript levels also increased after exogenous application of jasmonic acid, but not upon wounding. Dose–response and time course experiments revealed a similar pattern of transcript accumulation and lipoxygenase activity in BTH‐treated rice leaves. Enzymatic analysis of recombinant RCI‐1 protein produced in Escherichia coli revealed that 13‐hydroperoxy‐octadecanoic acids were the predominant reaction products when either linoleic or linolenic acid used as a substrate. The RCI‐1 sequence features a putative chloroplast targeting sequence at its N‐terminus. Indeed, a protein consisting of the putative chloroplast transit peptide fused to green fluorescent protein was exclusively localized in chloroplasts, indicating that RCI‐1 is a chloroplastic enzyme.
Activation of cyanobacterial RuBP‐carboxylase/oxygenase is facilitated by inorganic phosphate via two independent mechanisms
Authors: Marcus Yehouda , Gurevitz Michael
Published: March 2003

Orthophosphate (Pi) modulates the activity and activation of ribulose 1,5‐bis‐phosphate carboxylase/oxygenase (RuBisCO) via a mechanism that is still controversial. Whereas its effects on the higher plant enzyme have been described, little is known about Pi regulation of the structurally similar, yet kinetically different cyanobacterial enzyme. We found that RuBisCO of Synechocystis PCC6803 was affected by Pi in a paradoxical fashion. On the one hand, Pi inhibited catalysis by competing with the substrate RuBP, and on the other hand it stimulated enzyme activation in a dual manner manifested by multiphasic kinetics, which differed from the effect on activation of the higher plant enzyme. Pi concentrations > 5 m
m promoted the carbamylation of the cyanobacterial enzyme and the binding of Mg2+ to the carbanion at suboptimal concentrations of CO2 and Mg2+. Surprisingly, Pi also increased the activation level of the carbamylated enzyme via another putative site of interaction. In contrast with the higher plant RuBisCO, RuBP did not inhibit the stimulatory effect of phosphate on activation of the cyanobacterial enzyme, suggesting a Pi effect through a site other than the sugar binding site. The dual effect on activation could be distinguished by the phosphate analogue vanadate, which inhibited only the stimulation achieved at high phosphate concentrations. The elevation of RuBisCO activation at suboptimal levels of CO2 and high concentrations of RuBP suggests that in cyanobacteria Pi may have a role analogous to that of RuBisCO activase in higher plants.
A masquerade‐like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect,Holotrichia diomphalialarvae
Authors: Kwon Tae Hyuk , Kim Moon Suk , Choi Hye Won , Joo Chang Hun , Cho Mi Young , Lee Bok Luel
Published: March 2003

Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor‐I (PPAF‐I) that encoded a member of the serine proteinase group with a disulfide‐knotted motif at the N‐terminus and a trypsin‐like catalytic domain at the C‐terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S., Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998)Eur. J. Biochem.257, 615–621]. PPAF‐I is directly involved in the activation of pro‐phenoloxidase (pro‐PO) by limited proteolysis and the overall structure is highly similar to that ofDrosophilaeaster serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45‐kDa novel PPAF from the hemocyte lysate ofHolotrichia diomphalialarvae. The gene encodes a serine proteinase homologue consisting of 415 amino‐acid residues with a molecular mass of 45 256 Da. The overall structure of the 45‐kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development inDrosophila melanogaster. The 45‐kDa protein contained a trypsin‐like serine proteinase domain at the C‐terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide‐knotted domain at the N‐terminus. A highly similar 45‐kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran,Tenebrio molitor. Byin vitroreconstitution experiments, we found that the purified 45‐kDa serine proteinase homologue, the purified active PPAF‐I and the purified pro‐PO were necessary for expressing phenoloxidase activity in theHolotrichiapro‐PO system. However, incubation of pro‐PO with either PPAF‐I or 45‐kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45‐kDa protein was incubated with PPAF‐I and pro‐PO in the absence, but not in the presence of Ca2+, the 45‐kDa protein was cleaved to a 35‐kDa protein. RNA blot hybridization revealed that expression of the 45‐kDa protein was increased in theHolotrichiahemolymph afterEscherichia colichallenge.