bluuebunny/crossref_metadata_2025 huggingface dataset:
Starch metabolism in tubers of transgenic potato (Solanum tuberosum) with increased ADPglucose pyrophosphorylase
Authors: SWEETLOVE Lee J. , BURRELL Michael M. , REES Tom ap
Published: August 2015

The aim of this work was to use tubers from transgenic lines of potato (Solanum tuberosum) containing increased amounts of ADPglucose pyrophosphorylase to study the role of this enzyme in the control of starch synthesis. A 4–5-fold increase in activity of the enzyme, achieved by transformation with the Escherichia coli ADPglucose pyrophosphorylase gene glgC-16, had no detectable effect on the starch content of developing or mature tubers. No significant effects were found on the contents of ADPglucose, UDPglucose, glucose 1-phosphate, glucose 6-phosphate, PPi, ATP and ADP. Flux from [U-14C]sucrose, supplied to tubers still attached to the plant, to starch increased roughly in proportion to the increase in ADPglucose pyrophosphorylase activity. These measurements of flux gave a response coefficient close to 1 for the activity of the pyrophosphorylase in respect of starch synthesis. Pulse–chase experiments with [U-14C]sucrose showed that the increased flux into starch in the transformed tubers was accompanied by an increased rate of starch turnover. Further experiments suggested that the increased turnover was associated with an increase in the capacity of the tubers to degrade starch.
A novel trans-spliced mRNA from Onchocerca volvulus encodes a functional S-adenosylmethionine decarboxylase
Authors: DA'DARA Akram A. , HENKLE-DÜHRSEN Kimberly , WALTER Rolf D.
Published: August 2015

Complete cDNA and genomic sequences encoding the Onchocerca volvulus S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, have been isolated and characterized. The deduced amino acid sequence encodes a 42 kDa proenzyme with a moderate level of sequence homology to eukaryotic SAMDCs. Enzymically active O. volvulusSAMDC was expressed at a high level in an Escherichia colimutant strain lacking endogenous SAMDC. The recombinant enzyme was purified to homogeneity using DEAE-cellulose, methylglyoxal bis(guanylhydrazone)–Sepharose and Superdex S-200 chromatography. It was determined that the recombinant proenzyme is cleaved to produce 32 and 10 kDa subunits. The sequence of the N-terminal portion of the large subunit was determined and comparison with the sequence of the proenzyme revealed that the precise cleavage site lies between Glu86 and Ser87. Gel-filtration experiments demonstrated that these two subunits combine to form an active heterotetramer. Comparison of the cDNA and genomic sequences revealed that the SAMDC mRNA undergoes both cis- and trans-splicing in its 5´-untranslated region (UTR). Anchored PCR on O. volvulusmRNA confirmed the cDNA sequence and identified two distinct trans-spliced products, a 22-nucleotide spliced-leader sequence and a 138 bp sequence containing the 22 nucleotide spliced-leader sequence. Genomic Southern-blot analysis suggests that the O. volvulusSAMDC is encoded by a single-copy gene. This gene spans 5.3 kb and is comprised of nine exons and eight introns. The first intron is located in the 5´-UTR and processing of this intron has a potential regulatory function. The 5´-flanking region of the gene contains potential transcriptional regulatory elements such as a TATA box, two CAAT boxes and AP-1-, C/EBP-, ELP-, H-APF-1-, HNF-5- and PEA3-binding sites.
F1F0-ATPase, early target of the radical initiator 2,2′-azobis-(2-amidinopropane) dihydrochloride in rat liver mitochondria in vitro
Authors: BEAUSEIGNEUR Frédéric , GOUBERN Marc , CHAPEY Marie-France , GRESTI Joseph , VERGELY Catherine , TSOKO Marcelline , DEMARQUOY Jean , ROCHETTE Luc , CLOUET Pierre
Published: August 2015

This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2′-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1FO-ATPase, comparable with dicyclohexylcarbodi-imide, which alters FO proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1FO-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1FO-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.
Investigations into the molecular size and shape of tomato extensin
Authors: BROWNLEADER Michael D. , BYRON Olwyn , ROWE Arthur , TREVAN Michael , WELHAM Kevin , DEY Prakash M.
Published: August 2015

The molecular characteristics of soluble extensin from tomato have been investigated. An apparent molecular mass greater than 240 kDa has been previously observed with the shape-dependent method of gel-filtration chromatography [Brownleader and Dey (1993) Planta (Berlin) 191, 457–469]. Tomato extensin is a heavily glycosylated protein that does not migrate into SDS/polyacrylamide gels. This shape-dependent behaviour raises doubts about agreement between the observed apparent mass and the absolute value. The molecular mass measured with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS) was 72.3 kDa, with no evidence of any other species except a doubly charged ion. The sample was therefore considered to be monodisperse under the conditions used. Electron microscopy of soluble extensin showed the presence of particles 40–50 nm in length and 2.0–2.5 nm in width. A minority of these particles showed a central ‘kink’. A number of smaller and generally wider particles (20 nm×2–4 nm) were considered to be folded monomers and larger particles were thought to be dimers. Sedimentation analysis showed that extensin exists in a rapid monomer–dimer equilibrium in the concentration range and buffer used. Sedimentation equilibrium data gave a Kd of 8.5 µM and sedimentation velocity data generated a Kd between 1 and 10 µM. The concentration dependence of the measured sedimentation coefficient was used, together with hydrodynamic bead modelling, to define plausible shapes for monomer and dimer. This suggests that monomeric extensin is an elongated rod of length 40 nm and width 2 nm, which forms staggered dimers of average length 50 nm and width 3 nm. Extensin is an integral component of the primary cell wall. The physical characteristics (size, shape and form) of the rod-like extensin have been evaluated in this paper so that the role that extensin plays in primary cell wall architecture and during plant disease resistance can be more fully understood.
p30, a novel protein target of mouse calcyclin (S100A6)
Authors: FILIPEK Anna , WOJDA Urszula
Published: August 2015

A novel protein target of mouse calcyclin (S100A6) was detected by a gel overlay method with 125I-labelled calcyclin. Interaction of calcyclin with its 30 kDa target protein (p30) present in Ehrlich ascites tumour (EAT) cells depended on the presence of Ca2+ ions. The binding of p30, evidenced by the reaction with 125I-labelled calcyclin, was found to be of higher affinity than the binding between mouse calcyclin and annexin II or glyceraldehyde-3-phosphate dehydrogenase. Examination of tissue extracts by the gel overlay method has shown that p30 is present not only in the EAT cells but also in mouse brain and spleen. This novel target protein of mouse calcyclin was purified to homogeneity from EAT cells by means of Phenyl-Sepharose chromatography, affinity chromatography and CM-cellulose chromatography. Purified p30 was digested with α-chymotrypsin and a partial amino acid sequence of one of the resulting peptides was established. A database search analysis revealed that the sequence is unique, with a similarity of less than 55% to any other known protein sequence.
Inhibition of collagen-induced platelet aggregation as the result of cleavage of α2β1-integrin by the snake venom metalloproteinase jararhagin
Authors: KAMIGUTI Aura S. , HAY Charles R. M. , ZUZEL Mirko
Published: August 2015

Jararhagin is a high-molecular-mass (52 kDa) haemorrhagic metalloproteinase from Bothrops jararacavenom and a member of the metalloproteinase/disintegrin/cysteine-rich protein family. The disintegrin domain of jararhagin has been implicated in the inhibition of platelet responses to collagen by a mechanism that is not entirely known. The present investigation demonstrates that both active and 1,10-phenanthroline-inactivated jararhagin inhibit platelet aggregation by collagen with an IC50 of 40 and 140 nM respectively. The apparently higher inhibitory effect of the active enzyme clearly indicates that, in addition to the disintegrin region, the metalloproteinase domain of jararhagin also participates in this inhibition. As collagen interacts with platelets via α2β1-integrin, we investigated the effects of jararhagin on this integrin using selected function-blocking monoclonal antibodies against both of its subunits. Flow cytometry of platelets treated with native jararhagin and immunoprecipitation of platelet surface glycoproteins from lysates after jararhagin treatment showed an apparently selective reduction of α2β1-integrin immunoreactivity with both anti-α2 and anti-β1 monoclonal antibodies. The loss of immunoreactivity was not due to integrin internalization, since it also took place in cytochalasin D-treated platelets. Here we show that jararhagin cleaved isolated α2β1-integrin resulting in the generation of a 115 kDa β1 fragment. We therefore propose that the inhibition by jararhagin of platelet response to collagen is mediated through the binding of jararhagin to platelet α2-subunit via the disintegrin domain, followed by proteolysis of the β1-subunit with loss of the integrin structure (conformation) necessary for the binding of macromolecular ligands.
Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid
Authors: WIGGINS David , GIBBONS Geoffrey F.
Published: August 2015

When rat hepatocytes were cultured for 24 h in the absence of exogenous fatty acid, the amount of very-low-density lipoprotein (VLDL) triacylglycerol (TAG) secreted (114±14 µg/mg of cell protein) could not be accounted for by the mass of TAG lost from the cells (29±6.1 µg/mg of cell protein) during this period (n = 12). Of the balance (85±14 µg/mg; 94±15 nmol/mg), a maximum of only 37 nmol/mg of cell protein of TAG could be accounted for by fatty acids synthesized de novo. When labelled exogenous oleate (initial concentration, 0.75 mM) was present in the culture medium, the net gain in cellular plus VLDL TAG (253±38 µg/mg of cell protein per 24 h) was greater than that contributed by the exogenous fatty acid (155±18.2 µg/mg of cell protein, n = 5). Again, the balance (98.8±18.2 µg/mg of cell protein per 24 h) was too great to be accounted for by fatty acid synthesis de novo. In experiments in which cellular glycerolipids were prelabelled with [9,10(n)-3H]oleic acid, following removal of the labelled fatty acid, there was a net increase in labelled cellular plus VLDL TAG over the next 24 h. That cellular phospholipids are the source of a substantial part of the excess TAG synthesized is supported by the following evidence. (1) The loss of prelabelled cellular phospholipid during culture was greater than could be accounted for by secretion into the medium. (2) During culture of cells prelabelled with 1,2-di-[1-14C]palmitoyl phosphatidylcholine, a substantial amount of label was secreted as VLDL TAG. (3) In pulse–chase experiments, the kinetics of labelled phospholipid turnover were consistent with conversion into a non-phospholipid pool. The enzymology involved in the transfer of phospholipid fatty acids into TAG is probably complex, but the present results suggest that this pathway may represent an important route by which extracellular fatty acids are channelled into VLDL TAG.
Structural elucidation of XR586, a peptaibol-like antibiotic from Acremonium persicinum
Authors: SHARMAN Gary J. , TRY Andrew C. , WILLIAMS Dudley H. , AINSWORTH A. Martyn , BENEYTO Richard , GIBSON Trevor M. , McNICHOLAS Carole , RENNO Didier V. , ROBINSON Neil , WOOD Keith A. , WRIGLEY Stephen K.
Published: August 2015

A novel peptide, XR586, has been isolated from fermentations of Acremonium persicinum (Xenova culture collection number X21488). The structure of XR586 has been elucidated by means of NMR spectroscopy, electrospray and fast-atom bombardment MS, derivatization and enzymic digestion. It has been shown to be helical by CD measurements. XR586 shows many structural and conformational features in common with peptaibols, particularly the zervamicins. Peptaibol antibiotics are peptides, typically of 15–20 residues, containing a large proportion of α-aminoisobutyric acid (Aib) residues. These peptides adopt a helical conformation in solution and display anti-bacterial and toxic properties due to their ability to form pores in membranes. However, while XR586 contains several Aib residues, it lacks a terminal phenylalaninol and terminates in the sequence Phe-Gly. The lack of reduction of the penultimate residue at the C-terminus may indicate that this step is normally at the end of the biosynthetic pathway of peptaibols and occurs with cleavage of Gly. The 1H chemical shift assignments of XR586 are reported in Supplementary Publication SUP 50179 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1996) 313, 9 (‘Deposition of data’).
Feedback repression of ornithine decarboxylase synthesis mediated by antizyme
Authors: MITCHELL John L. A. , CHOE Chung-youl , JUDD Gary G.
Published: August 2015

The induction of antizyme by spermidine and the resulting enhancement of ornithine decarboxylase (ODC) degradation have been well studied; however, little is known about the mechanism whereby elevated spermidine levels decrease synthesis of the polyamine biosynthetic enzyme. To evaluate the relative contribution of inhibited synthesis, as distinct from enhanced degradation of ODC, spermidine levels were manipulated in a variant cell line that overproduces a stable form of ODC. Spermidine did not selectively inhibit ODC synthesis in these variant cells, supporting the concept that spermidine diminishes ODC synthesis in normal cells owing to enhanced degradation of the protein in the presence of elevated antizyme levels. This model was further investigated in vitro by use of rabbit reticulocyte lysate, which catalyses simultaneous ODC mRNA translation and antizyme-stimulated degradation of ODC protein. Antizyme strongly repressed the incorporation of labelled amino acids into normal rat ODC. Unexpectedly it also diminished the apparent translation of ODC mRNA species coding for enzyme forms that are not destabilized by the post-translational addition of antizyme. The effect of antizyme on ODC translation was not observed in wheatgerm extract, in which there is no antizyme-induced degradation. Further, deletion of a short segment of antizyme necessary for the destabilization of ODC (amino acid residues 113–118) resulted in a form that bound ODC but did not diminish its apparent translation. These results suggest that the co-translational addition of antizyme to ODC results in a complex that is different from, and innately less stable than, that formed when antizyme is added post-translationally.
Post-translational peptide bond formation during concanavalin A processing in vitro
Authors: SHELDON Philip S. , KEEN Jeffrey N. , BOWLES Dianna J.
Published: August 2015

Post-translational processing of concanavalin A (Con A) is complex, involving deglycosylation, proteolytic cleavage on the carboxy group side of asparagine residues and formation of a peptide bond de novo. This has been studied with the 125I-labelled Con A glycoprotein precursor as a substrate for processing in vitro. Extracts of immature jackbean cotyledons and the commercially available purified preparation of asparaginylendopeptidase were able to catalyse the above processes. The processing resulted in the conversion of the 33.5 kDa inactive glycoprotein precursor into an active lectin. Processing activity was maximal at approx. pH 5.5. Evidence to support processing at authentic sites was obtained by observation of the release of 125I at positions in the sequence where tyrosine residues were present.
The cytochrome c3 superfamily: amino acid sequence of a dimeric octahaem cytochrome c3 (Mr 26,000) isolated from Desulfovibrio gigas
Authors: BRUSCHI Mireille , LEROY Gisèle , BONICEL Jacques , CAMPESE Daniel , DOLLA Alain
Published: August 2015

Cytochrome c3 (Mr 26000) isolated from Desulfovibrio gigas is a dimeric cytochrome consisting of two identical subunits of 109 amino acids, each of which contains four haem groups. On the basis of its amino acid sequence, this cytochrome clearly belongs to the cytochrome c3 superfamily, and will be classified in class III of the c-type cytochromes as defined by Ambler [(1980) in From Cyclotrons to Cytochromes (Robinson, A. B. and Kaplan, N. O., eds.), pp. 263–279, Academic Press, London]. It contains ten cysteine and nine histidine residues in each subunit, and eight cysteines and eight histidines linked to the four haem groups were found to be invariant on alignment of all known cytochrome c3 sequences. Two intermolecular disulphide bridges have been determined between cysteine residues 5 and 46 of the two monomers. Cytochrome c3 (Mr 26000) from D. gigas is clearly different from cytochrome c3 (Mr 13000) from the same strain, with which it shows only 27% sequence identity. Compared with cytochrome c3 (Mr 26000) from D. desulfuricans Norway, the three-dimensional structure of which has been determined, 26.95% of the residues have been conserved. In the enzyme from D. desulfuricans Norway, hydrophobic interactions have been described across the dimer interface. Residues involved in similar interactions seem to be well conserved in the equivalent D. gigas cytochrome. This sequence provides structural data to allow specification of this new subclass of polyhaem cytochromes. Furthermore, D. gigas cytochrome c3 (Mr 26000) is the first polyhaem cytochrome shown to contain two disulphide bridges linking two identical subunits, which could induce more rigid folding. The folding and the evolution of this family of polyhaem cytochromes are discussed.
The characterization of endosomal insulin degradation intermediates and their sequence of production
Authors: SEABRIGHT Paul J. , SMITH Geoffrey D.
Published: August 2015

Insulin degradation within isolated rat liver endosomes was studied in vitro with the aid of three 125I-insulin isomers specifically labelled at tyrosine (A14, B16 and B26). Chloroquine and 1,10-phenanthroline were used to minimize insulin proteolysis during endosome preparation, whereas the manipulation of endosomal processing of insulin in vitro by Co2+ ions (to activate) and 1,10-phenanthroline (to inhibit) permitted the study of degradation intermediates and their time-dependent production. Structural and kinetic analysis of intermediates isolated from both intra- and extra-endosomal compartments allowed the determination of major cleavage sites and the probable sequence of proteolytic events. It was found that 125I-tyrosine is the ultimate labelled degradation product of all iodo-insulin isomers, suggesting that endosomal proteases are able to degrade insulin to the level of its constituent amino acids. 125I-tyrosine was also the only radiolabelled product able to cross the endosomal membrane. Intra-endosomal insulin degradation proceeds via two inter-related cleavage routes after metalloendoprotease cleavage of the B-chain. One pathway results from an initial cleavage in the centre region of the B-chain (B7–19), probably at B14-15, whereas the major route results from a cleavage at B24-25. B24-25 cleavage removes the B-chain C-terminal hexapeptide (B25–30), which is subsequently cleaved by an aminopeptidase activity to produce first the pentapeptide B26–30 and then 125I-tyrosine. The isolation of intact radiolabelled A-chain from the degradation of 125I-[A14]-insulin suggests that further degradation of proteolytic intermediates containing cleaved B-chain proceeds via interchain disulphide reduction. The A-chain is then processed by several cleavages, one of which occurs at A13-14.
Isolation and characterization of the androgen-dependent mouse cysteine-rich secretory protein-1 (CRISP-1) gene
Authors: SCHWIDETZKY Uta , SCHLEUNING Wolf-Dieter , HAENDLER Bernard
Published: August 2015

In mice, cysteine-rich secretory protein-1 (CRISP-1) is mainly found in the epididymis and also, to a lesser extent, in the salivary gland of males, where androgens control its expression. We have now isolated and characterized overlapping phage clones covering the entire length of the CRISP-1gene. DNA sequencing revealed that the gene is organized into eight exons, ranging between 55 and 748 bp in size, and seven introns. All exonŐintron junctions conformed to the GT/AG rule established for eukaryotic genes. The intron length, as determined by PCR, varied between 1.05 and 4.0 kb so that the CRISP-1gene spans over 20 kb of the mouse genome. The transcription-initiation site was determined by primer extension and localized at the expected distance downstream of a consensus TATA box. Approximately 3.7 kb of the CRISP-1promoter region were isolated and sequenced, and several stretches fitting the androgen-responsive element consensus were found. Those that most resembled the consensus were analysed by electrophoretic mobility-shift assay and found to form specific complexes with the liganded androgen receptor in vitro, but with different affinities. Putative binding elements for the transcription factors Oct, GATA, PEA3, CF1, AP-1 and AP-3 were also found in the promoter region.
Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes
Authors: DASGUPTA Shashiprabha , DASGUPTA Dipak , CHATTERJEE Aruna , BISWAS Susweta , BISWAS Birendra B.
Published: August 2015

The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3 and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro.Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 ŶC), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 ƁM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores, because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500ŷ200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting SternŐVolmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205Ő250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (ΔH), entropy (ΔS) and free energy (ΔG), for the formation of the two InsP3Őreceptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.
Reduced very-low-density lipoprotein fractional catabolic rate in apolipoprotein C1-deficient mice
Authors: JONG Miek C. , REE Janine H. van , DAHLMANS Vivian E. H. , FRANTS Rune R. , HOFKER Marten H. , HAVEKES Louis M.
Published: August 2015

The function of apolipoprotein (apo) C1 in vivo is not clearly defined. Because transgenic mice overexpressing human apoC1 show elevated triacylglycerol (TG) levels [Simonet, Bucay, Pitas, Lauer and Taylor (1991) J. Biol. Chem. 266, 8651Ő8654], an as yet unknown role for apoC1 in TG metabolism has been suggested. Here we investigated directly the effect of the complete absence of apoC1 on very-low-density lipoprotein (VLDL)-TG lipolysis, clearance and production, by performing studies with the previously generated apoC1-deficient mice. On a sucrose-rich, low fat/low cholesterol (LFC) diet, apoC1-deficient mice accumulate in their circulation VLDL particles, which contain relatively lower amounts of lipids when compared with VLDL isolated from control mice. Lipolysis assays in vitro on VLDL from apoC1-deficient and control mice showed no differences in apparent Km and Vmax values (0.27ŷ0.06 versus 0.24ŷ0.03 mmol of TG/litre and 0.40ŷ0.03 versus 0.36ŷ0.03 mmol of non-esterified fatty acid (NEFA)/min per litre respectively). To correct for potential differences in the size of the VLDL particles, the resulting Km values were also expressed relative to apoB concentration. Under these conditions apoC1-deficient VLDL displayed a lower, but not significant, Km value when compared with control VLDL (3.44ŷ0.71 versus 4.44ŷ0.52 mmol of TG2/g apoB per litre). VLDL turnover studies with autologous injections of [3H]TG-VLDL in vivo showed that the VLDL fractional catabolic rate (FCR) was decreased by up to 50% in the apoC1-deficient mice when compared with control mice (10.5ŷ3.4 versus 21.0ŷ1.2/h of pool TG). No significant differences between apoC1-deficient and control mice were observed in the hepatic VLDL production estimated by Triton WR139 injections (0.19ŷ0.02 versus 0.21ŷ0.05 mmol/h of TG per kg) and in the extra-hepatic lipolysis of VLDL-TG (4.99ŷ1.62 versus 3.46ŷ1.52/h of pool TG) in vivo. Furthermore, [125I]VLDLŐapoB turnover experiments in vivo also showed a 50% decrease in the FCR of VLDL in apoC1-deficient mice when compared with control mice on the LFC diet (1.1ŷ0.3 versus 2.1ŷ0.1/h of pool apoB). When mice were fed a very high fat/high cholesterol (HFC) diet, the VLDLŐapoB FCR was further decreased in apoC1-deficient mice (0.4ŷ0.1 versus 1.4ŷ0.4/h of pool apoB). We conclude that, in apoC1-deficient mice, the FCR of VLDL is reduced because of impaired uptake of VLDL remnants by hepatic receptors, whereas the production and lipolysis of VLDL-TG is not affected.
Effect of pH on kinetic parameters of NAD+-dependent formate dehydrogenase
Authors: MESENTSEV Alexander V. , LAMZIN Victor S. , TISHKOV Vladimir I. , USTINNIKOVA Tatyana B. , POPOV Vladimir O.
Published: August 2015

To define in detail the molecular mechanism of NAD+-dependent formate dehydrogenase, the pH dependences of various kinetic and spectroscopic parameters have been studied: Vmax, Km (NAD+), Km (formate), inhibition constants for structural analogues of substrate (NO3-) and product (CNS-, CNO-, N3-), CD and fluorescence properties. The value of Vmax, rate-limiting hydride transfer, is nearly constant throughout the entire pH range of enzyme stability (6.0Ő11.2) but decreases below 6. The Km values for both substrates remain constant within the pH range 6Ő10. At pH values below 6 (for the coenzyme) and above 10 (for both substrate and coenzyme) the Km values increase. In the acidic range this change is attributed to the ionization of two carboxy groups (pK approx. 5.5Ő6.0) located at the NAD+-binding site of the enzyme active centre. The pH transition in the basic region (pK 10.5ŷ0.2) has a conformational origin and affects the enzyme's affinity for substrates and anion inhibitors. A similar transition has been observed for formate dehydrogenases from yeast Candida boidinii and Hansenula polymorpha. The results complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme.
Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis
Authors: BROEK Peter J. A. van den , GOMPEL Angeline E. van , LUTTIK Marijke A. H. , PRONK Jack T. , LEEUWEN Carla C. M. van
Published: August 2015

Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome coxidase as a proton-motive-force-generating system. Addition of reduced cytochrome cgenerated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40Ő50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+Őglucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+Őmaltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilisthe transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.
Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide
Authors: KETTLE Anthony J. , GEDYE Craig A. , WINTERBOURN Christine C.
Published: August 2015

Hypochlorous acid is the most powerful oxidant generated by neutrophils and is likely to contribute to the damage mediated by these inflammatory cells. The haem enzyme myeloperoxidase catalyses its production from hydrogen peroxide and chloride. 4-Aminobenzoic acid hydrazide (ABAH) is a potent inhibitor of hypochlorous acid production. In this investigation we show that, in the presence of hydrogen peroxide, ABAH irreversibly inactivates myeloperoxidase. ABAH was oxidized by myeloperoxidase, and kinetic analysis of the inactivation conformed to that for a mechanism-based inhibitor. Inactivation was exacerbated by concentrations of hydrogen peroxide greater than 50 ƁM and by the absence of oxygen. Hydrogen peroxide alone caused minimal inactivation. Reduced glutathione inhibited the oxidation of ABAH as well as the irreversible inhibition of myeloperoxidase. In the presence of oxygen, ABAH and hydrogen peroxide initially converted myeloperoxidase into compound III, which susequently lost haem absorbance. In the absence of oxygen, the enzyme was converted into ferrous myeloperoxidase and its haem groups were rapidly destroyed. We propose that myeloperoxidase oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous myeloperoxidase reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation.
Polymorphism and structure of the gene coding for the α1 subunit of the Artemia franciscana Na/K-ATPase
Authors: GARCÍA-SÁEZ Alberto , PERONA Rosario , SASTRE Leandro
Published: August 2015

Genomic clones coding for one of the two identified Artemia franciscana Na/K-ATPase α subunits, the α1 subunit, have been isolated. Several overlapping clones were obtained, although their restriction maps showed a large heterogeneity. Sequencing of their exons showed that they differ in up to 3.46% of their nucleotides in translated regions and 8.18% in untranslated regions. Southern blot analysis of DNA purified from different lots of A. franciscana cysts and from isolated individuals suggests that the variation is due to the existence of multiple Na/K-ATPase α1 subunit alleles in A. franciscana. The Na/K-ATPase α1 subunit gene is divided into 15 exons. Ten of the 14 introns are located in identical positions in this gene as in the human Na/K-ATPase α3 subunit gene. Analysis of the 5ƀ flanking region of the gene has allowed identification of the transcription-initiation sites. The adjacent upstream region has been shown to have functional promoter activity in cultured mammalian cells, suggesting the evolutionary conservation of some of the promoter regulatory sequences.
Partition of the organochlorine insecticide lindane into the human sperm surface induces membrane depolarization and Ca2+ influx
Authors: SILVESTRONI Leopoldo , FIORINI Roberto , PALLESCHI Simonetta
Published: August 2015

The effects of the insecticide lindane (the γ-isomer of 1,2,3,4,5,6-hexachlorocyclohexane) on membrane potential, cytosolic free Ca2+ concentration ([Ca2+]i) and surface biophysical properties were studied in human spermatozoa. The insecticide induces rapid, transient and reproducible membrane depolarization and opening of voltage-dependent Ca2+ channels leading to an increase in [Ca2+]i. In contrast with the effect in somatic cells, lindane did not affect γ-aminobutyric acid receptor-linked Cl- currents. Ca2+ and K+ currents were found to drive lindane-induced membrane depolarization and repolarization respectively, whereas Na+ and Cl- fluxes appear not to have a role in the phenomenon. The insecticide was still able to produce membrane depolarization both in the combined absence of extracellular Ca2+ and Na+ and in high-K+ buffer, suggesting that lindane alters the membrane dipole potential. In agreement with this, Laurodan and Prodan fluorescence spectroscopy revealed that lindane partition into the sperm plasma membrane lowers water molecular dynamics in the uppermost region of the membrane external leaflet, probably as the result of reordering of water dipoles. We propose that the first effect of lindane partitioning into the sperm plasma membrane is a change in the membrane dipole potential, which results in the activation of membrane-located Ca2+-influx pathways.
Interaction of human CYP17 (P-45017α, 17α-hydroxylase-17,20-lyase) with cytochrome b5: importance of the orientation of the hydrophobic domain of cytochrome b5
Authors: LEE-ROBICHAUD Peter , KADERBHAI Mustak A. , KADERBHAI Naheed , WRIGHT J. Neville , AKHTAR Muhammad
Published: August 2015

Human CYP17 (P-45017α, 17α-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17α-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b5. The native form of cytochrome b5 is composed of a globular core, residues 1Ő98, followed by a membrane insertable C-terminal tail, residues 99Ő133. In the present study the abilities of five different forms of cytochrome b5 to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b5 (native pig), its genetically engineered rat counterpart (coreŐtail), the soluble core form of the latter (core), the core with the secretory signal sequence of alkaline phosphatase appended to its N-terminal (signalŐcore) and the latter containing the C-terminal tail of the native rat protein (signalŐcoreŐtail). When examined by Edman degradation and MS, the engineered proteins were shown to have the expected N-terminal amino acid sequences and molecular masses. The native pig was found to be acetylated at the N-terminal. The native pig and coreŐtail enzymes were equally efficient at enhancing the side-chain cleavage activity of human CYP17 and the signalŐcoreŐtail was 55% as efficient. The core and signalŐcore constructs were completely inactive in the aforementioned reaction. All the five derivatives were reduced to varying degrees by NADPH:cytochrome P-450 (NADPH-P450) reductase and the relative efficiencies of this reduction were reminiscent of the behaviour of these derivatives in supporting the side-chain cleavage reaction. In the side-chain cleavage assay, however, NADPH-P450 reductase was used in large excess so that the reduction of cytochrome b5 derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b5 and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b5 but also by its defined spatial orientation at the C-terminal.
Electrospray mass-spectrometric, spectrophotometric and electrochemical methods do not provide evidence for the binding of nitric oxide by pyocyanine at pH7
Authors: VUKOMANOVIC Dragic V. , ZOUTMAN Dick E. , STONE John A. , MARKS Gerald S. , BRIEN James F. , NAKATSU Kanji
Published: August 2015

In several recent publications on pyocyanine, its mechanism of action has been attributed to an ability to react with nitric oxide (NO), resulting in the formation of an adduct. We examined the chemical interaction of pyocyanine and NO using electrospray (ES) MS, spectrophotometry and voltammetry at neutral pH and with 10Ő100 ƁM pyocyanine. No binding of NO to pyocyanine was observed. Alternative mechanisms for the inhibition of NO-induced vasorelaxation by pyocyanine should be sought.
Structural instability of mutant β-cell glucokinase: implications for the molecular pathogenesis of maturity-onset diabetes of the young (type-2)
Authors: KESAVAN Prabakaran , WANG Liqun , DAVIS Elizabeth , CUESTA Antonio , SWEET Ian , NISWENDER Kevin , MAGNUSON Mark A. , MATSCHINSKY Franz M.
Published: August 2015

The catalytic function and thermal stability of wild-type and mutant recombinant human pancreatic β-cell glucokinase was investigated. The mutants E70K and E300K, which are thought to be the cause of impaired insulin production by the pancreatic β-cell and decreased glucose uptake by the liver of patients with maturity-onset diabetes of the young, were found to be functionally indistinguishable from the wild-type, i.e. their kcat,S0.5, inflection point and hwere normal. However, these two mutants showed markedly reduced stability under a variety of test conditions. Glucokinase instability, not low enzyme catalytic activity, may be the cause of diabetes mellitus with E70K and E300K mutants.
cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1
Authors: CSAR Xavier F. , WARD Alister C. , HOFFMANN Brigitte W. , GUY Graeme G. , HAMILTON John A.
Published: August 2015

The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [γ-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF-treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization.
Involvement of two basic residues (Lys-270 and Arg-276) of human liver 3α-hydroxysteroid dehydrogenase in NADP(H) binding and activation by sulphobromophthalein: site-directed mutagenesis and kinetic analysis
Authors: MATSUURA Kazuya , TAMADA Yoshiyuki , SATO Kumiko , IWASA Harunori , MIWA Gunpei , DEYASHIKI Yoshihiro , HARA Akira
Published: August 2015

A human liver 3α-hydroxysteroid dehydrogenase isoenzyme, a member of the aldoŐketo reductase family, shows a marked preference for NADP(H) over NAD(H), and is activated by sulphobromophthalein, which increases the Km values for both NADP(H) and substrates. Here we report kinetic alterations in binding of the coenzymes and the activator to the enzyme caused by site-directed mutagenesis of Lys-270 and Arg-276, which are strictly conserved among the aldoŐketo reductase family of enzymes. The mutated enzymes, K270M and R276M, showed increases in the Km for NADP+ of 22- and 290-fold respectively; the Km for alcohol substrate and the kcat of the NADP+-linked reaction were also elevated, by 9- and 5-fold respectively. No kinetic constant of the NAD+-linked reaction was altered by more than 3-fold. Calculation of the free-energy changes showed that the 2ƀ-phosphate group of NADP+ contributes 16.3 kJ/mol (3.9 kcal/mol) of binding energy to its interaction with the wild-type enzyme, and the mutagenesis to K270M and R276M destabilized the binding energy of NADP+ by 6.3 and 13.0 kJ/mol (1.5 and 3.1 kcal/mol) respectively. In addition, the mutations attenuated enzyme activation by sulphobromophthalein, which bound to the mutant enzymes as an inhibitor. The inhibition for the R276M mutant was competitive with respect to NADP+ and non-competitive with respect to the substrate, whereas that for the K270M mutant was mixed-type, showing activation at coenzyme concentrations greater than 20ȕKm. These results suggest that the two basic residues in the 3α-hydroxysteroid dehydrogenase isoenzyme play crucial roles in binding both the negatively charged 2ƀ-phosphate group of NADP+ and the sulphonic groups of sulphobromophthalein.
Analysis of mitogen-activated protein kinase activation by naturally occurring splice variants of TrkC, the receptor for neurotrophin-3
Authors: GUNN-MOORE Frank J. , WILLIAMS Alan G. , TAVARÉ Jeremy M.
Published: August 2015

TrkC is a receptor tyrosine kinase that binds neurotrophin-3(NT-3) with high affinity. A number of naturally occurring splice variants of TrkC exist, including one (TrkC.ki14) with a 14 amino acid insertion between subdomains VII and VIII of the tyrosine kinase domain. This kinase insert blocks the ability of NT-3 to stimulate neurite outgrowth in PC12 cells and proliferation in fibroblasts. The inserts also block the ability of TrkC to form a high-affinity complex with Shc and phospholipase Cγ(PLCγ) and the activation of PtdIns 3-kinase, and attenuates the sustained activation of mitogen-activated protein kinase (MAPK). In the current study we set out to determine whether the attenuation of the activation of MAPK by the insert was the result of the inability of TrkC to activate the ShcŐRas pathway, PtdIns 3-kinase activation, PLCγ activation, or a combination thereof. Experiments with the use of cell-permeant inhibitors argue against a major role for PLCγ and PtdIns 3-kinase in the activation of MAPK by TrkC. The introduction of the 14 amino acid kinase insert appeared to slow the kinetics of NT-3-stimulated Shc phosphorylation and ShcŐGrb2 association and reduce their magnitude; an effect which was associated with a delayed, and only transient, activation of MAPK. Taken together, our data suggest that the apparent defect in MAPK activation caused by the kinase insert may result predominantly from an inhibition of high-affinity Shc binding, although a role for PLCγ and PtdIns 3-kinase cannot be completely excluded.
mRNA encoding the β-subunit of the mitochondrial F1-ATPase complex is a localized mRNA in rat hepatocytes
Authors: EGEA Gustavo , IZQUIERDO José M. , RICART Javier , MARTÍN Carmen SAN , CUEZVA José M.
Published: August 2015

Subcellular mRNA localization has emerged as a mechanism for regulation of gene expression and protein-sorting pathways. Here we describe the different cytoplasmic presentation in rat hepatocytes of two nuclear mRNA species encoding subunits α and β of the mitochondrial F1-ATPase complex. α-F1-ATPase mRNA is dispersed and scattered in the cytoplasm. In contrast, β-F1-ATPase mRNA appears in rounded electron-dense clusters, often in close proximity to mitochondria. Hybridization experiments with β2-microglobulin and β-actin cDNA species reveal an expected subcellular distribution pattern of the mRNA species and a non-clustered appearance. Development does not alter the presentation of β-F1-ATPase mRNA hybrids, although it affects the relative abundance of β-F1-ATPase mRNA clusters in the cytoplasm of the hepatocyte. These findings illustrate in vivo the existence of two different sorting pathways for the nuclear-encoded mRNA species of mitochondrial proteins. High-resolution immunocytochemistry and immunoprecipitation experiments allowed the identification of the β-subunit precursor in the cytoplasm of the hepatocyte, also suggesting a post-translational import pathway for this precursor protein. It is suggested that the localization of β-F1-ATPase mRNA in a subcellular structure of the hepatocyte might have implications for the control of gene expression at post-transcriptional levels during mitochondrial biogenesis in mammals.