bluuebunny/crossref_metadata_2025 huggingface dataset:
Tropomodulin binding to tropomyosins
Authors: SUSSMAN Mark A. , FOWLER Velia M.
Published: March 2005

Tropomodulin is a human erythrocyte membrane cytoskeletal protein that binds to one end of tropomyosin molecules and inhibits tropomyosin binding to actin filaments [Fowler, V. M. (1990) J. Cell Biol. 111, 471–482]. We have characterized the interaction of erythroid and non‐erythroid tropomyosins with tropomodulin by non‐denaturing gel electrophoresis and by solid‐phase binding assays using 125I‐tropomyosin. Non‐denaturing gel analysis demonstrates that all tropomodulin molecules are able to bind tropomyosin and that tropomodulin forms complexes with tropomyosin isoforms from erythrocyte, brain, platelet and skeletal muscle tissue. Scatchard analysis of binding data using tropomyosin isoforms from these tissues indicate that tropomodulin binds preferentially to erythrocyte tropomyosin. Specificity is manifested by decreases in the apparent affinity or the saturation binding capacity of tropomodulin for non‐erythrocyte tropomyosins. Erythrocyte tropomyosin saturates tropomodulin at approximate stoichiometric ratios of 1:2 and 1:4 tropomyosin/tropomodulin (apparent Kd= 14 nM−1 and 5 nM−1, respectively). Brain tropomyosin saturates tropomodulin at a 1:2 ratio of tropomyosin/tropomodulin, but with a threefold lower affinity than erythrocyte tropomyosin. Platelet tropomyosin saturates tropomodulin at a tropomyosin/tropomodulin ratio of 1:4, but with a sevenfold lower affinity than erythrocyte tropomyosin at the 1:4 ratio. These results correlate with oxidative cross‐linking data which indicate that tropomodulin can self‐associate to form dimers and tetramers in solution. Since tropomodulin interacts with one of the ends of tropomyosin, varying interactions of tropomyosin isoforms with tropomodulin probably reflect the heterogeneity in N‐terminal or C‐terminal sequences characteristic of the different tropomyosin isoforms. Isoform‐specific interactions of tropomodulin with tropomyosins may represent a novel mechanism for selective regulation of tropomyosin/actin interactions.
Plant mitochondrial F0F1 ATP synthase
Authors: HAMASUR Beston , GLASER Elzbieta
Published: March 2005

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N‐terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28‐kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N‐terminal Pro‐Val‐Pro‐ which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N‐terminal 32 amino acids of the 23‐kDa protein reveals that this protein is similar to mammalian oligomycin‐sensitivity‐conferring protein and corresponds to the F1δ subunit of the chloroplast and E. coli ATPases. The 18.5‐kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N‐terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N‐terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15‐kDa protein and the 10.5‐kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1‐ATPase, respectively. The 9.5‐kDa protein is an N,N′‐dicyclohexylcarbodiimide‐binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5‐kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin‐sensitive ATPase activity of 3.5 μmol · mg−1· min−1. The enzyme catalyzes also hydrolysis of GTP (7.5 μmol · mg·1· min−1) and ITP (4.4 μmol · mg−1· min−1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.
Nutritional and hormonal regulation of lipogenic‐enzyme gene expression in rat liver
Authors: IRITANI Nobuko
Published: March 2005

The present paper reviews recent advances which have been made in studies, predominantly in rat liver, on the nutritional and hormonal regulation of gene expression of lipogenic enzymes (acetylCoA carboxylase, fatty acid synthase, malic enzyme and glucose‐6‐phosphate dehydrogenase).
Mutants of human insulin‐like growth factor II
Authors: LÜTHI Christine , ROTH Birgit V. , HUMBEL René E.
Published: March 2005

Insulin‐like growth factor II (IGF II) and four structural analogs, constructed by site‐directed mutagenesis, were expressed as protein A fusion proteins in Escherichia coli BL21pLysS cells, cleaved with cyanogen bromide and purified by affinity chromatography and HPLC. Two mutants (Ser29 substituted by Arg‐Leu‐Pro‐Gly, and Ser33 substituted by Cys‐Gly‐Asp) represent two naturally occurring variants of IGF II. The other two mutants, (7–67)IGF II and (9–67)IGF II, are truncated at the amino‐terminus in analogy to the naturally occurring des(1–3)IGF I (‘truncated IGF I’). These mutants were tested for their binding affinities to type‐1 and type‐2 IGF receptors, to IGF binding protein‐3 (IGFBP‐3) and for their stimulation of thymidine incorporation into DNA. The affinities of the Ser29 and Ser33 mutants to the type‐1 IGF receptor were 85% and 39%, respectively, compared to wild‐type IGF II, those of (7–67)IGF II and (9–67)IGF II 96% and 15%, respectively. The potencies of the Ser33 and the (9–67) mutant to stimulate thymidine incorporation into DNA correlated closely with the affinities to the type‐1 IGF receptor, whereas the bioavailability of the Ser29 mutant was lower and that of the (7–67) mutant higher than the type‐1 receptor binding, possibly due to interferences with endogenously secreted IGFBPs. The affinities of the Ser29 and Ser33 mutants to the type‐2 IGF receptor were 110% and 71%, respectively, those of the two truncated mutants 25% and 23%, respectively. The affinity of the Ser29 mutant to IGFBP‐3 was increased to 171%, whereas those of the Ser33 mutant and the two truncated mutants were reduced (34%, 10% and 19%, respectively).
Enzyme activation by denaturants in organic solvent systems with a low water content
Authors: GARZA‐RAMOS Georgina , FERNÁNDEZ‐VELASCO D. Alejandro , RAMÍREZ Leticia , SHOSHANI Liora , DARSZON Alberto , DE GÓMEZ‐PUYOU Marietta TUENA , GÓMEZ‐PUYOU Armando
Published: March 2005

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of heart lactate dehydrogenase, glycerol‐3‐phosphate dehydrogenase, hexokinase, inorganic pyrophosphatase, and glyceraldehyde‐3‐phosphate dehydrogenase was studied in low‐water systems. Most of the experiments were made in a system formed with toluene, phospholipids, Triton X‐100, and water in a range that varied over 1.0–6.5% (by vol.) [Garza‐Ramos, G., Darszon, A., Tuena de Gómez‐Puyou, M. & Gómez‐Puyou, A. (1990) Biochemistry 29, 751 – 757]. In such conditions at saturating substrate concentrations, the activity of the enzymes was more than 10 times lower than in all‐water media. However the activity of the first four aforementioned enzymes was increased between 4 and 20 times by the denaturants. The most marked activating effect was found with lactate dehydrogenase; with 3.8% (by vol.) water maximal activation was observed with 1.5 M GdmCl (about 20‐fold); 4 M urea activated, but to a lower extent. Activation by guanidine thiocyanate was lower than with GdmCl. The activating and inactivating effects of GdmCl on lactate dehydrogenase depended on the amount of water; as the amount of water was increased from 2.0% to 6.0% (by vol.), activation and inactivation took place with progressively lower GdmCl concentrations. When activity was measured as a function of the volume of 1.5 M GdmCl solution, a bell‐shaped activation curve was observed. In a low‐water system formed with n‐octane, hexanol, cetyltrimethylammonium bromide and 3.0% water, a similar activation of lactate dehydrogenase by GdmCl and urea was observed. The water solubility diagrams were modified by GdmCl and urea, and this could reflect on enzyme activity. However, from a comparison of denaturant concentrations on the activity of the enzymes studied, it would seem that, independently of their effect on the characteristics of the low‐water systems, denaturants bring about activation through their known mechanism of action on the protein. It is suggested that the effect of denaturants is due to the release of constraints in enzyme catalysis imposed by a low‐water environment.
Structural studies on fetal mucins from human amniotic fluid
Authors: HANISCH Franz‐Georg , PETER‐KATALINIC Jasna
Published: March 2005

Mucins in human amniotic fluid are represented by two distinct molecular species, FM‐1 and FM‐2, with apparent molecular masses of 700 and 570 kDa, respectively, in SDS/polyacrylamide gradient gels. FM‐1 and FM‐2 were isolated by preparative SDS/PAGE to apparent homogeneity and subjected to structural studies on their carbohydrate portions. The carbohydrate compositions of the mucin species differed only marginally and exhibited significant amounts of mannose.

O‐linked core‐region glycans on human amniotic mucin‐derived pronase‐stable glycopeptides were analyzed after reductive β‐elimination and purification on HPLC by a combination of methylation analysis, electron‐impact mass spectrometry of permethylated oligosaccharide alditols and fast‐atom‐bombardment mass spectrometry of acetylated or methylated alditols (positive‐ion mode) or alditol‐derived neoglycolipids (negative‐ion TLC‐MS). The primary structures of major monosaccharides to tetrasaccharides have been established which exhibit at their reducing termini core 1, core 2 and core 3 sequences, as follows.

GalNAc‐ol  (N1)

Galβ(1–3)GalNAc‐ol  (N3)

Fucα(1–2)Galβ(1–3)GalNAc‐ol  (N4)

Galβ(1–3)GlcNAcβ(1–3)GalNAc‐ol  (N5)

Galβ(1–4)GlcNAcβ(1–3)GalNAc‐ol  (N5)

Galβ(1–3)[GlcNAcβ(1–6)]GalNAc‐ol  (N6)

Galβ(1–4)[Fucα(1–3)]GlcNAcβ(1–3)GalNAc‐ol  (N7)

Galβ(1–3)[Galβ(1–4)GlcNAcβ(1–6)]GalNAc‐ol  (N9)
Comparison of human tenascin expression in normal, Simian‐virus‐40‐transformed and tumor‐derived cell lines
Authors: CARNEMOLLA Barbara , BORSI Laura , BANNIKOV Gregory , TROYANOVSKY Sergey , ZARDI Luciano
Published: March 2005

Tenascin is a polymorphic high‐molecular‐mass extracellular‐matrix glycoprotein composed of six similar subunits. Using two‐domain‐specific anti‐tenascin monoclonal antibodies, we have studied the expression and distribution of tenascin in four cultured normal human fibroblasts, two simianvirus‐40‐(SV40)‐transformed and three tumor‐derived (melanoma, rhabdomyosarcoma and fibrosarcoma) cell lines. We found that (a) cultured normal human fibroblasts accumulate considerable amounts of tenascin and retain 60–90% in the extracellular matrix, while they release the remainder into the tissue‐culture medium; (b) of the two SV40‐transformed counterparts we have tested, the AG‐280 cell line accumulates no detectable amounts of tenascin and the WI‐38‐VA cell line accumulates about 10‐times less tenascin than its normal counterpart and releases about 90% of it into the culture medium; (c) some tumor‐derived cell lines accumulate considerable amounts of tenascin, but in theses cases, more than 90% is released into the culture media; (d) in normal human fibroblasts, two major tenascin isoforms, generated by alternative splicing of the mRNA precursor, are detectable (280 kDa and 190 kDa, respectively) and the lower‐molecular‐mass tenascin isoform is accumulated preferentially in the extracellular matrix; (e) in SV40‐transformed or tumor‐derived cell lines, only the higher‐molecular‐mass isoform is detectable and it is more sialylated than the tenascin produced by the normal human fibroblast cell lines.
Enzymatic characterisation of the multifunctional enzyme δ‐(l‐α‐aminoadipyl)‐l‐cysteinyl‐d‐valine synthetase from Streptomyces clavuligerus
Authors: SCHWECKE Torsten , AHARONOWITZ Yair , PALISSA Harriet , von DÖHREN Hans , KLEINKAUF Horst , van LIEMPT Henk
Published: March 2005

δ‐(l‐α‐Aminoadipyl)‐l‐cysteinyl‐d‐valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700‐fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on l‐cysteine and l‐ and l‐valine, but no l‐α‐aminoadipic‐acid‐dependent ATP/PPi exchange could be detected. Other l‐ and l‐cysteine‐ and l‐valine‐activating enzymes present in crude extracts were identified as aminoacyl‐tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for l‐valine ligase and 50 kDa for l‐cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with α‐aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non‐protein amino acid.
Changes in the fluorescence of bound nucleotide during the reaction catalysed by glucose‐fructose oxidoreductase from Zymomonas mobilis
Authors: HARDMAN Michael J. , TSAO Marlene , SCOPES Robert K.
Published: March 2005

The reduction of gluconolactone by glucose‐fructose oxidoreductase containing tightly bound NADPH (enzyme‐NADPH) is biphasic in nucleotide fluorescence. The initial rapid decrease, which represents quenching of the fluorescence by bound lactone, is followed by a slower decrease which corresponds to the change in absorbance. At low glucose concentrations, the oxidation of glucose by enzyme‐NADP+ involves a single first‐order process with similar rate constants in fluorescence and absorbance. At higher glucose concentrations, the apparent first‐order rate constants for the fluorescence change are less than those for the absorbance change. This is consistent with a mechanism in which the fluorescence change occurs during the lactone dissociation step, which is slower than the hydrogen transfer step during which the absorbance change occurs. The rate constant for gluconolactone dissociation is 360 ± 10 s−1 and this step is therefore rate‐determining for the overall reaction. Reduction of fructose by enzyme‐NADPH is first order with a limiting rate constant of at least 2000 s−1.
Novel aerobic 2‐aminobenzoate metabolism
Authors: ALTENSCHMIDT Uwe , FUCHS Georg
Published: March 2005

A new pathway for the aerobic metabolism of 2‐aminobenzoate which proceeds via 2‐amino‐benzoyl‐CoA has recently been revealed in a Pseudomonas strain KB 740−. The enzyme catalyzing the first step, the formation of the coenzyme A (CoA) thioester of 2‐aminobenzoate, is 2‐aminobenzoate‐CoA ligase. It was purified from cells aerobically grown with 2‐aminobenzoate as sole carbon, energy, and nitrogen source and characterized. It is rather specific for 2‐aminobenzoate, but activates also benzoate and fluorobenzoates. ATP was cleaved into AMP and pyrophosphate. The ligase is a monomer of Mr, 65000, as determined by gel filtration and SDS/PAGE. The N‐terminal amino acid sequence was determined and the gene locus of the enzyme was identified by Southern blot hybridization on a small 8‐kbp plasmid pKB 740. The 1.8‐kb nucleotide sequence of the 2‐aminobenzoate‐CoA ligase gene and the derived amino acid sequence of the native enzyme (597 residues) are reported.
Glutamate mutase from Clostridium cochlearium
Authors: LEUTBECHER Ulrich , BÖCHER Reinhard , LINDER Dietmar , BUCKEL Wolfgang
Published: March 2005

Both components, E and S, of the adenosylcobalamin‐(coenzyme B12)‐dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50‐kDa subunit. The enzyme proved to be very similar to that of C. tetanomorphum as described by Barker et al. [Barker, H. A., Rooze, V., Suzuki, F. & Iodice, A. A. (1964) J. Biol. Chem. 239, 3260–3266] but component E of C. cochlearium was more stable and led to the first pure preparation. The pink component E showed a cobamide‐like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Coα‐[α‐(aden‐9‐yl)]‐cob(II)amide. A typical cob(II)amide signal at g= 2.23 with hyperfine and superhyperfine splitting was observed by EPR pectroscopy. A cobamide content of about 0.43 mol/mol 50‐kDa subunit was determined by cyanolysis.

Substitution of the migrating hydrogen at C‐4 of glutamate by fluorine yielded the0potent competitive inhibitor (2S,4S)‐4‐fluoroglutamate (Ki= 70 μM). (2R,3RS)‐3‐Fluoroglutamate (Ki= 600 μM) was also inhibitory. The competitive inhibition by 2‐methyleneglutarate (Ki= 400 μM) and (S)‐3‐methylitaconate (Ki= 100 μM) but not by (RS)‐2‐methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N‐terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.
Biosynthesis and excretion of gangliosides by the isolated perfused rat liver
Authors: SENN Hans‐Jürgen , SELLIN Susanne , FITZKE Edith , STEHLE Thomas , HÄUSSINGER Dieter , WIELAND Heinrich , GEROK Wolfgang
Published: March 2005

De novo synthesis and excretion into perfusate and bile fluid of hepatic gangliosides were studied in isolated perfused rat livers. Addition of N‐acetyl‐[6‐3H(n)]d‐mannosamineto the perfusate resulted in radioactive synthesis of at least eight gangliosides labeled in their sialic acid residues. About 10% of total de novo synthesized gangliosides were excreted into the perfusate, less than 1% into the bile fluid. Labeled gangliosides were tentatively identified by cochromatography with known standards. All of them are known to occur in rat liver and sera. The results indicate that most, if not all, normal serum gangliosides are synthesized in the liver; excretion with bile fluid is negligible. They explain previous observations, and indicate clinical implications, whch are discussed.
Overexpression of the multidrug resistance gene product in adult rat hepatocytes during primary culture
Authors: FARDEL Olivier , RATANASAVANH Damrong , LOYER Pascal , KETTERER Brian , GUILLOUZO André
Published: March 2005

Expression of P‐glycoprotein (P‐gp), the product of multidrug resistance gene(s), was investigated in primary cultures of normal adult rat hepatocytes. Levels of P‐gp mRNAs determined by Northern blotting and of P‐gp measured by immunoblotting increased in parallel with time in culture. As in normal liver, P‐gp was found to be localized on the membrane of bile canaliculus‐like structures. This increased expression of P‐gp was associated with decreased intracellular retention of doxorubicin, which could be restored by compounds such as verapamil and cyclosporin; doxorubicin (and also vincristine) was more cytotoxic to early than to late cultures. As in preneoplastic and neoplastic liver, overexpression of P‐gp in cultured hepatocytes was associated with differential changes in drug‐metabolizing enzymes, including increased glutathione S‐transferase 7‐7. Functional P‐gp over‐expression was observed in the absence of xenobiotic exposure or cell division; it could be linked to cellular stress occurring during cell isolation and plating. Increased expression of P‐gp was blocked by actinomycin D, indicating its dependence on increased transcription, while cycloheximide led to a superinduction suggesting negative regulation by a protein factor.
H8 chemical shifts in oligonucleotides cross‐linked at a GpG Sequence by cis‐Pt(NH3)22+: a clue to the adduct structure
Authors: KOZELKA Jir̆i , FOUCHET Marie‐Hélène , CHOTTARD Jean‐Claude
Published: March 2005

The origin of the anomalous H8 chemical shifts observed in 1H‐NMR spectra of oligonucleotides cross‐linked at a GpG sequence with cis‐[Pt(NH3)2]2+ has been investigated and clarified. The main contributions that distinguish the H8 resonances of the two platinum‐ligating guanines from other GH8 signals and from each other are: (a) the inductive effect of platinum binding which we have recently quantified as a downfield shift of 0.48 ± 0.07 ppm (M. H. Fouchet, D. Lemaire, J. Kozelka and J.‐C. Chottard, unpublished results); (b) the ring‐current effect of one GpG guanine on the H8 resonance of the other guanine, which is negative (shielding) for the 5′‐H8 and positive (deshielding) for the 3′‐H8 in single‐stranded adducts, but has the opposite sign in double‐stranded adducts; (c) a deshielding polarization effect of the phosphate 5′ to the GpG unit. The different signs of the ring‐current effects in single‐stranded and double‐stranded oligonucleotides originate from the orientation of the guanines in the cis‐[Pt(NH3),(Gua)2]2+ moiety (Gua, guanine), which is left‐handed helicoidal in single strands and right‐handed helicoidal in double strands. In the platinated dinucleotides (cis‐ [Pt(NH3)2(GpG)]+, cis‐[Pt(NH3)2{d(GpG)}]+ and cis‐[Pt(NH3)2{d(pGpG)}]), the guanines assume either the left‐handed or the right‐handed arrangement, depending on the sugar moiety (ribose or deoxyribose), protonation state at N1 and, in the solid state, on crystal forces. This work shows that chemical shifts contain valuable structural information which is complementary to that extracted from correlated spectroscopy and nuclear Overhauser spectroscopy data.
Purification and properties of a recombinant sulfur analog of murine selenium‐glutathione peroxidase
Authors: ROCHER Corinne , LALANNE Jean‐Louis , CHAUDIÈRE Jean
Published: March 2005

We previously constructed plasmids for synthesis of glutathione‐peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active‐site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [CysIGPx and [SerIGPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [CysIGPx mutant exhibited a significant GPx activity, unlike the [SerIGPx mutant.

Purification of [Cys]GPx was performed in two steps of ion‐exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel‐filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000‐fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa, of 8.3, presumably that of the active‐site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.
Phosphorylation of the inhibitory guanine‐nucleotide‐binding protein as a possible mechanism of inhibition by protein kinase C of agonist‐induced Ca2+ mobilization in human platelet
Authors: YATOMI Yutaka , ARATA Yoichiro , TADA Shusuke , KUME Shoji , UI Michio
Published: March 2005

Increases in the intracellular Ca2+ concentration of human platelets caused by receptor agonists, such as thrombin, 9,11‐epitho‐l1,12‐methanothromboxane A2 (STA2), platelet‐activating factor (PAF) and arginine‐vasopressin, were inhibited by prior addition of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in time‐dependent and concentration‐dependent manners. The inhibitions were mostly reversed by staurosporine, and inhibitor of protein kinase C, added 1 min before TPA. Prior treatment of platelets with thrombin or STA2, the efficacious Ca2+ mobilizer, suppressed the increase in the intracellular Ca2+ concentration of the cells to other agonists, but treatment with less efficacious PAF or vasopressin did not. The heterologous receptor desensitizations were also reversed by staurosporine. The antibody, directed against the carboxy‐terminal region of the α subunits 1 and 2 of the inhibitory guanine‐nucleotide‐binding proteins (Gi1α and Gi2α), was raised in rabbit and was used to immunoprecipitate Giα in 32P‐labeled platelets. The radioactivity was detected in Giα after incubation of 32P‐labeled platelets with TPA, thrombin or STA2, but not in the cells incubated with PAF or vasopressin. The time‐dependency or concentration‐dependency of TPA‐induced phosphorylation of Giα was similar to the dependency of its inhibitory action on agonist‐induced Ca2+ mobilization. Thus, strong activation of Ca2+/phospholipid‐dependent protein kinase C by phorbol ester or agonists of certain Ca2+‐mobilizing receptors leads to phosphorylation of the α subunit of guanine‐nucleotide‐binding protein, thereby impairing the coupling of the G protein to receptors as a feedback regulatory component of the receptor‐triggered intracellular Ca2+‐mobilizing system.
Translational regulation of ribosomal protein synthesis in Xenopus cultured cells: mRNA relocation between polysomes and RNP during nutritional shifts
Authors: LORENI Fabrizio , AMALDI Francesco
Published: March 2005

Translational control of ribosomal protein mRNA was analyzed in a Xenopus cell line during growth‐rate changes induced by serum deprivation and readdition. After being transferred into serum‐free medium, the cells rapidly decrease their DNA, RNA and protein synthesis, while addition of serum to the culture after a few hours of deprivation causes a rapid recovery. During these growth‐rate changes, we observed a shift in ribosomal protein mRNA distribution between polysomes and RNP. The proportion of mRNA on polysomes for the four ribosomal proteins analyzed changed from 70–80% during rapid growth to 25–35% during the downshift and back to 70–80% after the upshift. Northern blot analysis showed that ribosomal protein mRNA level was constant during the shifts even in the presence of the transcriptional inhibitor actinomycin D. This indicates that the distribution changes were due to a reversible transfer of ribosomal protein mRNA between polysomes and RNP without altering mRNA stability. We have also compared the kinetics of ribosomal protein mRNA distribution changes with the kinetics of the changes in the partition of ribosomes between free monomers and polysomes. The results obtained show that the change in ribosomal protein mRNA localization is very fast, allowing short‐term adjustments of ribosome synthesis rate. Moreover, our observations are consistent with the hypothesis that the amount of free ribosomes present in the cell could affect ribosomal protein mRNA utilization.
cDNA and gene sequences of wheat chloroplast sedoheptulose‐1,7‐bisphosphatase reveal homology with fructose‐1,6‐bisphosphatases
Authors: RAINES Christine A. , LLOYD Julie C. , WILLINGHAM Nicola M. , POTTS Susan , DYER Tristan A.
Published: March 2005

The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose‐l,7‐bisphosphatase [Sed(l,7)P2ase], was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72–507 bp) and seven introns (85–626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose‐1,6‐bisphosphatase[Fru(1,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(l,6)P2ase are highly conserved in the Sed(1,7)P2ase molecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1,6)P2ase is not found in the Sed(l,7)P2ase enzyme.
Calponin and SM 22 isoforms in avian and mammalian smooth muscle
Authors: GIMONA Mario , SPARROW Malcolm P. , STRASSER Peter , HERZOG Monika , SMALL J. Victor
Published: March 2005

Calponin is a basic smooth‐muscle‐specific protein capable of binding to F‐actin, tropomyosin and calmodulin in vitro. Using two‐dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a PI identical to the most basic adult a variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees‐Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985–5991], which has sequence motifs related to calponin, displays a similar isoform pattern during development; one isoform (a) is present in the embryo and three in the adult.

In living smooth‐muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32P04, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two‐dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. These results support the claim [Barany, M., Rokolya, A. & Barany, K. (1991) FEBS Lett. 279, 65–68] that calponin phosphorylation is not involved in smooth muscle regulation in vivo, as has been suggested from in vitro studies [Winder, S. J. & Walsh, M. J. (1990) J. Biol. Chem. 265, 10 148–10 1551]. In vitro translation of porcine and chicken smooth‐muscle mRNA produced only a single (α) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the α isoform and a second more basic spot, not observed in tissue samples.

Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down‐regulated like calponin, metavinculin and heavy‐caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the α SM 22 isoform during smooth‐muscle phenotype modulation in vitro.
Structure of neuropeptide Y dimer in solution
Authors: COWLEY David J. , HOFLACK Jan M. , PELTON John T. , SAUDEK Vladimir
Published: March 2005

The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D2O was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11 – 36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N‐terminus (residues 1–9) appears as an unstructured mobile segment.

Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 ± 0.6 μM at pH 2–8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.
Stopped‐flow studies of calcium dissociation from calcium‐binding‐site mutants of Drosophila melanogaster calmodulin
Authors: MARTIN Stephen R. , MAUNE John F. , BECKINGHAM Kathy , BAYLEY Peter M.
Published: March 2005

The kinetics of calcium dissociation from two groups of site‐specific mutants of calmodulin from Drosophila melanogaster have been studied by stopped‐flow kinetic methods, using the fluorescent calcium chelator 8‐amino‐2‐[(2‐amino‐5‐methylphenoxy)methyl]‐6‐methoxyquinoline‐N,N,N′,N′‐tetraacetic acid (Quin 2). The BQ series of mutants consists of four proteins in which one of the four bidentate glutamate residues (Glu 12 of each of the four calcium binding loops) has been replaced by glutamine. In the BK series of mutants, the corresponding glutamate has been replaced by lysine. Calcium‐dissociation kinetics of proteins with a mutation in site I or II (N‐terminal domain) are consistent with a model in which the mutation weakens binding at the non‐mutated N‐terminal partner site and has a small, but significant, effect on the kinetic properties of sites III and IV (C‐terminal domain). The proteins with a mutation in site III or IV show a large effect, with decreased Ca2+ dissociation rate from the unmodified N‐terminal Ca2+‐binding sites I and II. A structural interpretation is proposed, based on enhanced interactions between the domains when the affinity of individual sites have been dramatically reduced by mutation. This effect is greatest for the mutations in the C‐terminal domain, which appear to destroy the co‐operativity of Ca2+ binding at sites III and IV. The results show that site‐specific mutation can have surprisingly far‐ranging effects on kinetic properties of calmodulin. The kinetic analysis also shows that studies of specifically engineered mutants may in principle help to unmask the values of intrinsic rate constants for the wild‐type protein which are not normally observable in the process of Ca2+ dissociation.
Swainsonine is a useful tool to monitor the intracellular traffic of N‐linked glycoproteins as a function of the state of enterocytic differentiation of HT‐29 cells
Authors: HOURI Jean‐Jacques , OGIER‐DENIS Eric , BAUVY Chantal , AUBERY Michèle , SAPIN Catherine , TRUGNAN Germain , CODOGNO Patrice
Published: March 2005

After treatment with swainsonine, an inhbitor of both lysosomal a‐mannosidase and Golgi α‐mannosidase‐I1 activities, analysis of [3H]mannose‐labeled glycans showed that HT‐29 cells, derived from a human colonic adenocarcinoma, displayed distinct patterns of N‐glycan expression, depending upon their state of enterocytic differentiation. In differentiated HT‐29 cells hybrid‐type chains were detected, whereas undifferentiated HT‐29 cells accumulated high‐mannose‐type oligosaccharide, de‐ spite our demonstration of Golgi α‐mannosidase‐II activity in both cell populations. Pulse/chase experiments carried out in the presence of swainsonine revealed that the persistence of high‐mannose‐ type chains in undifferentiated HT‐29 cells was the result of the stabilization of glycoproteins substi‐ tuted with these glycans. These data suggest that in undifferentiated HT‐29 cells, glycoproteins with high‐mannose‐type oligosaccharides are delivered to a degradative compartment containing swainsonine‐sensitive α‐mannosidase(s), whereas in differentiated HT‐29 cells glycoproteins enter a compartment in which α‐mannosidase II (Golgi apparatus) is present. Thus, this apparent dual effect of swainsonine on N‐glycan trimming may reflect differences in the intracellular traffic of glycoproteins as a function of the state of enterocytic differentiation of HT‐29 cells.
A novel 24‐kDa microtubule‐associated protein purified from sea urchin eggs
Authors: MAEKAWA Shohei , TORIYAMA Masaru , SAKAI Hikoichi
Published: March 2005

Chromatographic fractionation of a crude extract of sea urchin eggs on a hydrophobic column enabled us to find a new 24‐kDa microtubule‐associated protein (SU‐MAP24) that bound tightly to the column and was eluted under alkaline conditions. Biochemical studies using the purified protein showed its direct binding to microtubules reconstituted from tubulin purified from starfish sperm outer fibers. SU‐MAP24 promoted tubulin polymerization in a dose‐dependent manner. Immunoblotting analysis showed that SU‐MAP24 is present in a microtubule protein fraction obtained from a crude extract using taxol, and immunostaining of paraffin‐sectioned metaphase eggs showed its localization in the mitotic apparatus. These results show that SU‐MAP24 is a newly identified microtubule‐ associated protein.
Role of GDP in formyl‐peptide‐receptor‐induced activation of guanine‐nucleotide‐binding proteins in membranes of HL 60 cells
Authors: WIELAND Thomas , KREISS Janine , GIERSCHIK Peter , JAKOBS Karl H.
Published: March 2005

Membranes of myeloid differentiated human leukemia (HL 60) cells contain receptors for the chemotactic peptide, fMet‐Leu‐Phe (Met, N‐formylmethionine), interacting with pertussis‐toxin‐sensitive guanine‐nucleotide‐binding proteins (G proteins). Agonist activation of the receptors in‐ creases binding of the GTP analog, guanosine 5′‐[γ‐thioltriphosphate (GTP[S]), to membrane G proteins, at 30°C only in the presence of exogenous GDP. In contrast, at 0°C fMet‐Leu‐Phe stimulated binding of GTP[S] to G proteins maximally without addition of GDP. Under conditions resulting in marked degradation of membrane‐bound GDP, control binding of GTP[S] measured at 0°C was significantly increased, whereas the extent of agonist‐stimulated binding was reduced. Furthermore, there was a rapid spontaneous release of membrane‐bound GDP at 30°C, but not at 0°C. The data suggest that in intact membranes of HL 60 cells G proteins are initially in a GDP‐liganded form, which state allows the receptor‐induced exchange of bound GDP for GTP[S] at low temperature. In contrast, at or near physiological temperature. bound GDP is rapidly released (and degraded), resulting in unligated G proteins to which GTP[S] will bind independently of agonist‐activated receptors.
Conformational analysis of the melanin‐concentrating hormone core by circular dichroic spectroscopy
Authors: SILIGARDI Giuliano , CAMPBELL Malcolm M. , GIBBONS William A. , DRAKE Alex F.
Published: March 2005

A detailed circular dichroic (CD) study of the conformational flexibility of the melanin‐concentrating hormone core [MCH(5 – 14)] is reported. Variable pH (2 – 10) and temperature (−80° to + 80°C) in aqueous media reveal that CD contributions from tyrosine, disulphide bridge and the amide backbone can be discriminated. Only below −10°C does a preferred ‐S‐S‐conformation (P chirality, dihedral angle φ= 90 ± 10°) dominate. The amide backbone CD contribution varies over all temperatures (−80° to +80°C) providing evidence for a type‐II β‐turn at low temperatures, with the emergence of a type‐I β‐turn at higher temperatures. Tyrosine exhibits a special behaviour at pH 7. These conclusions are in broad agreement with published NMR studies. Nevertheless, the MCH(5–14) core is seen to be conformationally flexible in aqueous solution at ambient temperatures. Conformation differences are observed in a non‐aqueous environment.
Isolation and partial characterization of membrane‐associated cyclophilin and a related 22‐kDa glycoprotein
Authors: THALHAMMER Theresia , KIEFFER Lynda J. , JIANG Tian‐rong , HANDSCHUMACHER Robert E.
Published: March 2005

The presence of membrane‐associated proteins which stereospecifically bind cyclosporin A and react with anti‐cyclophilin antibodies has been documented in rat tissue. Extraction of membranes with 6 M urea or 0.5% Chaps releases cyclosporin‐binding activity that is 5–12% of that found in cytosol. Cyclosporin‐A‐binding proteins are present in most subcellular organelles of liver, but microsomes contain the greatest activity. These proteins can be purified by adsorption onto a cyclosporin‐A affinity column and elution with cyclosporin A. Two major fractions are resolved on SDS/PAGE: an 18‐kDa fraction is comprised of two isoforms that are similar if not identical to the two major cytosolic isoforms of cyclophilin. In addition, in microsomes an approximately equal quantity of a 22‐kDa glycoprotein was detecte. Based on partial sequencing (five peptides, 89 amino acids) this protein is similar but not identical to human cyclophilin B. This 22‐kDa isoform is poorly recognized by affinity‐purified anti‐cyclophilin antibodies and comprises several predominant isoforms (pI∼ 9.3–9.6). Selective binding of membrane 22‐kDa cyclophilin to peanut lectin suggests the oligosaccharides contain a terminal galctosyl‐N‐galactosamine residue.
Tissue distribution of constitutive and induced soluble peroxidase in rat
Authors: DE Prabir K.
Published: March 2005

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen‐induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane‐bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases.

As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane‐bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5–3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane‐bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross‐react with lactoperoxidase it did interact with lactoperoxidase on Western blot.

The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane‐bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue‐specific regulation of gene expression at the molecular level.
The quinone‐reactive Ni/Fe‐hydrogenase of Wolinella succinogenes
Authors: DROSS Felix , GEISLER Viktor , LENGER Ruth , THEIS Friedbert , KRAFFT Torsten , FAHRENHOLZ Falk , KOJRO Elzbieta , DUCHÊNE Andrea , TRIPIER Dominique , JUVENAL Karin , KRÖGER Achim
Published: March 2005

The hydrogenase (Hyd) isolated from the cytoplasmic membrane of Wolinella succinogenes consists of three polypeptides (HydA, HydB and HydC) and contains cytochrome b (6.4 μmol/g protein), which was reduced upon the addition of H2. The enzyme catalyzed the reduction of 2,3‐dimethyl‐1,4‐naphthoquinone with H2, in contrast to an earlier preparation which was made up of HydA and HydB only and did not contain cytochrome b (Unden, G., Böcher, R., Knecht, J. & Kröger, A. (1982) FEBS Lett. 145, 230–234). This suggests that HydC is a cytochrome b which serves as a mediator in the electron transfer from H2 to the quinone.

The hydrogenase genes were cloned, sequenced and identified by sequence comparison with the N‐termini of the three subunits. The three genes were arranged in the order hydA, hydB, hydC, with the transcription start site in front of hydA, and were present only once on the genome. Separated by an intergene region of 69 nucleotides, hydC was followed by at least two more open reading frames of unknown function. The amino acid sequences derived from hydA, hydB and hydC were similar to those of the membrane Ni‐hydrogenases of seven other bacteria. HydA and HydB also showed similarity to the small and the large subunits of periplasmic Ni‐hydrogenases. HydC was predicted to contain four hydrophobic segments which might span the bacterial membrane. Two histidine residues located in hydrophobic segments are conserved in the corresponding sequences of the other membrane hydrogenases and might ligate the haem B.
Quantitative expression patterns of multidrug‐resistance P‐glycoprotein (MDR1) and differentially spliced cystic‐fibrosis transmembrane‐conductance regulator mRNA transcripts in human epithelia
Authors: BREMER Silvia , HOOF Torsten , WILKE Martina , BUSCHE Roger , SCHOLTE Bob , RIORDAN John R. , MAASS Günter , TÜMMLER Burkhard
Published: March 2005

P‐glycoprotein (MDR1), that confers multidrug resistance in cancer, and the cystic‐fibrosis transmembrane‐conductance regulator (CFTR), that is causative defective in cystic fibrosis, belong to the family of ATP‐binding transport proteins. The expression of MDR1 and CFTR in human epithelial tissues and the cell lines T84 and HT29 was estimated by primer‐directed reverse transcription (RT) and subsequent monitoring of the kinetics of cDNA product formation during the polymerase chain reaction (PCR), MDR1 mRNA was found in high levels, 15–50 amol mRNA/μg RNA, in the intestine, kidney, liver and placenta, and in low levels, 0.2 amol/μg RNA, in respiratory epithelium. Large amounts of CFTR mRNA were measured in the gastrointestinal tract, whereas the kidney, as the phenotypically normal organ, and the lung, as the most severely affected organ in cystic fibrosis, both contained low amounts, 3 amol CFTR/μg RNA. CFTR transcript levels of 1–5 amol/μg RNA were determined in lymphocytes and lymphoblast cell lines, suggesting that lymphoblasts are an accessible source for the study of the molecular pathogenesis of cystic fibrosis. When transcripts were scanned by overlapping RT/PCR analyses, only transcript of expected size was detected for MDR1 mRNA, whereas variable in‐frame deletions of either exon 4, 9 or 12 were observed in CFTR mRNA. The complete loss of single exons was seen at proportions of 1–40% in all investigated tissues and cell lines with large donor‐to‐donor variation. Exons 9 and 12 of the CFTR gene encode parts of the evolutionarily well‐conserved first nucleotide‐binding fold including the two Walker motifs. Alternative splicing may give rise to various CFTR forms of different function and localization.