bluuebunny/crossref_metadata_2025 huggingface dataset:
Flux control exerted by overt carnitine palmitoyltransferase over palmitoyl-CoA oxidation and ketogenesis is lower in suckling than in adult rats
Authors: KRAUSS Stefan , LASCELLES Carol V. , ZAMMIT Victor A. , QUANT Patti A.
Published: August 2015

We examined the potential of overt carnitine palmitoyltransferase (CPT I) to control the hepatic catabolism of palmitoyl-CoA in suckling and adult rats, using a conceptually simplified model of fatty acid oxidation and ketogenesis. By applying top-down control analysis, we quantified the control exerted by CPT I over total carbon flux from palmitoyl-CoA to ketone bodies and carbon dioxide. Our results show that in both suckling and adult rat, CPT I exerts very significant control over the pathways under investigation. However, under the sets of conditions we studied, less control is exerted by CPT I over total carbon flux in mitochondria isolated from suckling rats than in those isolated from adult rats. Furthermore the flux control coefficient of CPT I changes with malonyl-CoA concentration and ATP turnover rate.
Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts
Authors: TETLOW Ian J , BOWSHER Caroline G , EMES Michael J
Published: August 2015

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl β-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent Km for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.
Molecular cloning and further characterization of rat peroxisomal trihydroxycoprostanoyl-CoA oxidase
Authors: BAUMGART Eveline , VANHOOREN Johannes C. T. , FRANSEN Mark , LEUVEN Fred VAN , FAHIMI H. Dariush , VAN VELDHOVEN Paul P. , MANNAERTS Guy P
Published: August 2015

The composite trihydroxycoprostanoyl-CoA oxidase cDNA sequence, derived from overlapping clones isolated via screening of two different rat liver expression libraries, consisted of 2509 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76711 Da. The reading frame and identity of the trihydroxycoprostanoyl-CoA oxidase cDNA were confirmed by the location of various tryptic peptides, obtained from the purified enzyme, in the deduced amino acid sequence. The C-terminus (His-Lys-Met) of trihydroxycoprostanoyl-CoA oxidase did not seem to interact with the C-terminal peroxisomal targeting signal 1 (PTS1) import receptor, although the tripeptide fits the rule of conserved PTS1 variants for targeting of proteins to glycosomes of Trypanosomatidae. At the protein level, trihydroxycoprostanoyl-CoA oxidase showed 45% identical amino acids with rat palmitoyl-CoA oxidase, whereas the identity with pristanoyl-CoA oxidase was much lower (22%). Northern analysis of multiple rat tissues revealed a signal (approx. 2.6 kb) only in liver and (although much weaker) in kidney. Dot-blot analysis of total liver RNA revealed that the mRNA for trihydroxycoprostanoyl-CoA oxidase is not induced after treatment of rats with structurally unrelated peroxisome proliferators and indicates that highly similar mRNAs are present in other mammals, including man. Immunocytochemistry showed a decrease in trihydroxycoprostanoyl-CoA oxidase protein in individual liver peroxisomes (‘diluting-out effect’) after treatment of rats with bezafibrate, whereas the palmitoyl-CoA oxidase labelling was significantly increased.
Anionic tobacco peroxidase is active at extremely low pH: veratryl alcohol oxidation with a pH optimum of 1.8
Authors: G. GAZARIAN Irina , LAGRIMINI L. Mark , GEORGE Simon J. , THORNELEY Roger N. F.
Published: August 2015

Tobacco peroxidase (36 kDa, pI 3.5) exhibits unique catalytic and spectral properties that are modulated by pH, calcium and magnesium ions. It catalyses the oxidation of veratryl alcohol by hydrogen peroxide over a wide pH range (1.5–5.0) in the presence of these metal ions with a pH optimum of 1.8. This is the only example of a holoperoxidase described so far that is active and comparatively stable at such a low pH. The enhancement of tobacco peroxidase activity by magnesium ions is to our knowledge the first example of a magnesium-induced peroxidase activation. UV/visible spectra of tobacco peroxidase showed that the Soret band shifted and its absorption coefficient increased upon the addition of calcium or magnesium ions and on lowering the pH. The tobacco peroxidase spectrum at pH 1.85, in the presence of calcium chloride (> 50 mM), is similar to that of lignin peroxidase at pH 6.0, with the Soret band shifting from 403 to 409 nm and the molar absorption coefficient increasing from 108000 to 148000±2000 M-1·cm-1 (results given ±S.E.M.; n = 3). The data provide evidence for a low-affinity site for bivalent metal ion binding in addition to the two constitutive calcium sites that are present in all plant peroxidases. The presence of a glutamic acid residue (Glu-141) at the entrance to the haem-binding pocket, analogous to Glu-146 in lignin peroxidase and not present in other plant peroxidases, may account for these novel properties.
Evidence for the presence of a large keratan sulphate proteoglycan in the human uterine cervix
Authors: FISCHER Dagmar-Christiane , HENNING Astrid , WINKLER Matthias , RATH Werner , HAUBECK Hans-Dieter , GREILING Helmut
Published: August 2015

Profound changes occur in the uterine cervix during pregnancy. In particular, the extracellular matrix of the connective tissue is remodelled extensively. To elucidate the mechanisms involved in this process, we have analysed the proteoglycan pattern in the human cervix from pregnant and non-pregnant women. Proteoglycans of the cervix tissue specimen were extracted with 4 M guanidine hydrochloride and precipitated with 80% ethanol. Purification of proteoglycans was performed by several chromatographic steps. Characterization of proteoglycans was done by SDS/PAGE before and after digestion with glycosaminoglycan-specific enzymes. Proteoglycans were detected by combined Alcian Blue/silver staining or, after blotting of biotin-labelled proteoglycans on to poly(vinylidene difluoride) membrane, with peroxidase-conjugated avidin or by the use of keratan sulphate- or decorin-specific monoclonal antibodies. In contrast with previous reports, where only chondroitin/dermatan sulphate proteoglycans have been found in the uterine cervix, we have shown in the present study the existence of a large keratan sulphate proteoglycan with an Mr > 220000 in cervix samples from non-pregnant and pregnant women. This proteoglycan showed a strong reaction with the keratan sulphate-specific monoclonal antibody 5D4 and could be degraded by keratanases. The size of the core protein of this keratan sulphate proteoglycan was estimated to be about Mr 220000.
Higher-plant medium- and short-chain acyl-CoA oxidases: identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal β-oxidation
Authors: HOOKS Mark A. , BODE Kornelia , COUÉE Ivan
Published: August 2015

Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10–C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4–C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 µM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3–8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570–8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal β-oxidation are discussed.
Peroxisomal multifunctional enzyme of β-oxidation metabolizing d-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization
Authors: QIN Yong-Mei , POUTANEN Matti H. , HELANDER Heli M. , KVIST Ari-Pekka , SIIVARI Kirsi M. , SCHMITZ Werner , CONZELMANN Ernst , HELLMAN Ulf , HILTUNEN J. Kalervo
Published: August 2015

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an Mr of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578–21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted Mr of 79331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17β-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal β-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase–hydratase–dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of β-oxidation but through reciprocal stereochemical courses.
The MAL proteolipid is a component of the detergent-insoluble membrane subdomains of human T-lymphocytes
Authors: MILLÁN Jaime , PUERTOLLANO Rosa , FAN Li , RANCAÑO Carmen , ALONSO Miguel A.
Published: August 2015

The human mal gene, identified during a search for cDNAs selectively expressed during T-cell development, encodes a highly hydrophobic protein belonging to a group of proteins, termed proteolipids, characterized by their unusual property of being soluble in organic solvents used to extract cell lipids. To study the localization of the MAL protein we have prepared stable transfectants expressing the MAL protein tagged with a c-myc epitope (MAL/c-myc) using human epithelial A-498 cells. Immunofluorescence analysis suggested that MAL/c-myc is localized mainly to cholesterol-enriched structures with a post-Golgi location and, at low levels, in early endosomes. Moreover, extraction of A-498 cell membranes with Triton X-100 (TX100) and fractionation by centrifugation to equilibrium in sucrose gradients demostrated the presence of MAL/c-myc in the detergent-insoluble buoyant fraction, known to be enriched in glycolipids and cholesterol. To compare the behaviour of MAL in T-cells with that in epithelial A-498 cells, we prepared stably transfected cells expressing MAL/c-myc using human Jurkat T-cells. When TX100 extracts from Jurkat cells were subjected to centrifugation to equilibrium in sucrose gradients we found MAL exclusively in the floating fractions, together with molecules characteristic of the T-cell insoluble complexes, such as the tyrosine kinase p56lck, the glycosylphosphatidylinositol-anchored protein CD59 and the ganglioside GM1. These results, taken together, indicate that the MAL proteolipid is a component of the detergent-resistant membrane microdomains present in T-lymphocytes, and suggest that MAL might play a role in modulating the function of these microdomains during T-cell differentiation.
The β-d-xylosidase of Trichoderma reesei is a multifunctional β-d-xylan xylohydrolase
Authors: HERRMANN Michael C. , VRSANSKA Maria , JURICKOVA Milada , HIRSCH Jan , BIELY Peter , KUBICEK Christian P.
Published: August 2015

An extracellular multifunctional α-d-xylan xylohydrolase, previously described as α-xylosidase, was purified fromTrichoderma reesei RUT C-30 to physical homogeneity. The active enzyme was a 100 (ŷ5) kDa glycosylated monomer that exhibited a pI of 4.7. Its activity was optimal at pH 4 and it was stable between pH 3 and 6. Its temperature-stability was moderate (70% of activity remaining after 60 min at 50 ŶC) and optimal activity was observed at 60 ŶC. It is capable of hydrolysing α-1,4-xylo-oligosaccharides [degree of polymerization (DP) 2Ő7], the apparent Vmax increasing with increasing chain length. The enzyme also attacked debranched beech-wood (Lenzing) xylan and 4-O-methylglucuronoxylan, forming xylose as the only end product. The Km for xylan was 0.7 g/l. For this reason we consider the enzyme to be a α-d-xylan xylohydrolase. The enzyme also exhibits α-l-arabinofuranosidase activity on 4-nitrophenyl α-l-arabinofuranoside, and evidence is presented that this is not caused by an impurity in the enzyme preparation. The α-d-xylan xylohydrolase exhibits glycosyltransferase activity with xylo-oligosaccharides and at high concentrations of 4-nitrophenyl α-d-xylopyranoside (4-Nph-α-Xyl). The enzyme hydrolyses α-1, 4-linkages preferentially to α-1,3-linkages, and α-1,2-linked xylo-oligosaccharides are not hydrolysed at all. The enzyme liberates terminal α-1,4-xylopyranose residues linked to a 2-O-substituted xylopyranose residue, but not that linked to a 3-O-substituted xylopyranose residue. The enzyme does not attack methyl, methyl 1-thio-, benzyl or butyl 1-thio-α-d-xylopyranosides and 4-naphthyl, 2-naphthyl and phenyl α-d-xylopyranosides.
Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase
Authors: BERTRAND Luc , VERTOMMEN Didier , DEPIEREUX Eric , HUE Louis , RIDER Mark H. , FEYTMANS Ernest
Published: August 2015

Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642Ő9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted.
Effects of pH on phosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum by inorganic phosphate
Authors: KHAN Yamin M. , Malcolm EAST J. , LEE Anthony G.
Published: August 2015

The fluorescence intensity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin has been shown to decrease on phosphorylation of the ATPase with Pi, this providing a convenient measure of the level of phosphorylation. Comparison of the fluorescence decrease observed with ATP and with high concentrations of Pi fix the value of the equilibrium constant for the phosphorylation reaction E2PMg ⇌ E2PiMg at pH 6.0 at about 2. Studies of the pH-dependence of phosphorylation show that H2PO4- and HPO42- bind to the ATPase with equal affinity, but that only binding of H2PO4- leads to phosphorylation, described by an equilibrium constant of 2.3. Luminal Ca2+ can bind to a pair of sites on the ATPase, with affinities of 1.3ȕ103 and 1.7ȕ103 M-1 for the unphosphorylated and phosphorylated forms of the ATPase respectively, with stronger binding of Ca2+ to the phosphorylated form resulting in an increase in the effective equilibrium constant for phosphorylation.
The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein
Authors: PAPANASTASIOU Philemon , McCONVILLE Malcolm J. , RALTON Julie , KÖHLER Peter
Published: August 2015

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis(syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardiaisolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is post-translationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of [3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.
Mitochondrial creatine kinase isoform expression does not correlate with its mode of action
Authors: ANFLOUS Keltoum , VEKSLER Vladimir , MATEO Philippe , SAMSON Françoise , SAKS Valdour , VENTURA-CLAPIER Renée
Published: August 2015

In adult mammalian ventricular tissue, mitochondrial creatine kinase (mi-CK), which is bound to the outer surface of the mitochondrial inner membrane, is functionally coupled to oxidative phosphorylation. This is shown, in saponin-permeabilized rat ventricular fibres, by a shift in the apparent Km of mitochondrial respiration for ADP from 300ŷ56 ƁM to 111ŷ40 ƁM (P < 0.001) on the addition of 25 mM creatine, due to a local accumulation of ADP close to the ATP/ADP translocator. We have found that, in atrial fibres, the apparent Km for ADP is high, but is not decreased by creatine, suggesting an absence of coupling in this tissue, as has previously been observed in smooth muscle. mi-CK is encoded by two different genes, which are expressed in a tissue-specific manner: the sarcomeric isoform is expressed in ventricular and skeletal muscles, while the ubiquitous isoform is expressed in smooth muscle, brain and other tissues. In order to determine whether a specific function can be attributed to the expression of a specific isoform, we investigated mi-CK mRNA expression by Northern blot analysis. Hybridization with synthetic oligonucleotides specific for each mi-CK isoform showed the expression of only the sarcomeric isoform in rat atria. This result was confirmed by PCR using primers specific for each isoform. In addition, electrophoretic analysis of CK isoforms showed no difference in the octamer/dimer ratio of mi-CK in the atria and ventricles. In atria, unlike the soleus or ventricles, the maximum potential rate of mitochondrial phosphocreatine synthesis was lower than the maximal rate of ATP production by the mitochondria. The total CK/adenylate kinase ratio was also lower in atria than in the other tissues, suggesting a greater contribution of adenylate kinase to adenine nucleotide compartmentation in this tissue. The functional differences between mi-CK in the two cardiac tissues seem to imply a specific arrangement of the proteins in the intermembrane space rather than the expression of specific isoforms.
The CD46 transmembrane domain is required for efficient formation of measles-virus-mediated syncytium
Authors: SEYA Tsukasa , KURITA Mitsue , IWATA Kazunori , YANAGI Yusuke , TANAKA Kazuhiko , SHIDA Kyoko , HATANAKA Michiyo , MATSUMOTO Misako , JUN Sheng , HIRANO Akiko , UEDA Sigeharu , NAGASAWA Sigeharu
Published: August 2015

Two phosphatidylinositol (PI)-anchored versions of a measles virus (MV) receptor membrane cofactor protein (MCP; CD46) were generated by fusing the extracellular domain of MCP to the decay-accelerating factor (DAF; CD55) or its PI anchor. The PI-anchored forms of MCP expressed on Chinese hamster ovary cells, otherwise non-permissive to MV, conferred a smaller MV cytopathic effect than a wild-type MCP, a Ser/Thr-rich domain-deletion mutant and a cytoplasmic tail-deletion mutant of MCP. Therefore the differences in MV receptor properties between the two PI-anchored and three transmembrane forms were investigated. The PI-anchored forms were predominantly expressed on microvilli as in DAF, whereas the other transmembrane forms were found on intracellular membranes. The PI-anchored forms conferred high MV-binding capacity compared with the transmembrane versions. MV replication was, however, severely suppressed in cells expressing the PI-anchored forms, resulting in ineffective syncytium formation. In contrast, cell-to-cell fusion occurred efficiently after co-transfection of cDNA species encoding MV-H, MV-F and any version of MCP. Thus the PI-anchored forms, despite showing sufficient MV binding and cell-to-cell fusion competence together with MV-H and MV-F, mediate inefficient MV entry or replication, which causes severe suppression of the MV cytopathic effect. A biased receptor distribution on microvilli might participate in the selection of a low MV uptake pathway in the PI-anchored forms of MCP. Taken together, the transmembrane portion of MCP is a critical factor for effective virusŐcell fusion and the subsequent MV replication.
Transient inactivation of phosphatidylethanolamine N-methyltransferase-2 and activation of cytidine triphosphate:phosphocholine cytidylyltransferase during non-neoplastic liver growth
Authors: TESSITORE Luciana , CUI Zheng , VANCE Dennis E.
Published: August 2015

Phosphatidylethanolamine N-methyltransferase-2 (PEMT2) may contribute to the control of hepatocyte cell division, since its inactivation is associated with several types of liver proliferation including tumorigenesis [Cui, Houweling and Vance (1994) J. Biol. Chem. 269, 24531Ő24533]. To determine if the inactivation of PEMT2 was involved in non-neoplastic proliferation of hepatocytes, we studied the expression of this enzyme in a model of lead nitrate-induced liver proliferation in vivo in rats. A maximal decrease in PEMT activity (60%) and loss of PEMT2 protein (95%) coincided with maximal DNA synthesis and maximal cytidine triphosphate:phosphocholine cytidylyltransferase activity 36 h and 48 h after lead nitrate stimulation in male and female livers respectively. The decrease in expression of PEMT2 corresponded to a decrease in its mRNA. Compared with males, female rats exhibited a 12 h delay in the peak of DNA synthesis, in cytidylyltransferase activity and in the minimum of PEMT2 expression. Supplementation of the rats with dietary choline shifted the female pattern of PEMT2 inactivation, DNA synthesis and activation of cytidylyltransferase to 12 h earlier so that it was similar to the time frame of the expression of these activities in males. These results are consistent with the proposal that the inactivation of PEMT2 may have a role in the regulation of non-neoplastic growth of liver.
Effects of charged lipids on the interaction of cholesteryl ester transfer protein with lipid microemulsions
Authors: RAJARAM Otthakal V. , SAWYER William H.
Published: August 2015

This study reports the effects of charged lipids on the transfer of cholesteryl-1-pyrene decanoate (Py-CE) between apolipoprotein-free microemulsion particles mediated by cholesteryl ester transfer protein (CETP). The surface charge characteristics of microemulsion particles composed of cholesteryl oleate and egg yolk phosphatidylcholine were altered by incorporating phosphatidylserine, oleate or stearylamine into the phosphatidylcholine that forms the surface monolayer of the particle. The transfer of Py-CE was measured continuously by following the decrease in excimer fluorescence that accompanies the transfer of the probe from donor to acceptor particles [Rajaram, Chan and Sawyer (1994) Biochem. J. 304, 423Ő430]. The inclusion of 20 mol% phosphatidylserine relative to the phospholipid in the surface monolayer of the emulsion caused a 64% decrease in the first-order rate constant describing the transfer. An increase in ionic strength caused a partial reversal of this effect, indicating that electrostatic factors are only partially responsible for the interaction with lipid. Complete inhibition of transfer was observed when 10 mol% sodium oleate was incorporated into the surface monolayer. The incorporation of stearylamine into the emulsion caused a 32% increase in the transfer rate. The binding of CETP to the different emulsion surfaces was also examined using a surface plasmon resonance biosensor. The presence of negatively charged lipid (phosphatidylserine or oleic acid) decreased the rate of association of CETP with the emulsion without a significant change in the dissociation rate constant. The presence of the positively charged lipid stearylamine increased the rate of association of CETP with the lipid surface. It is concluded that a negative surface charge on the monolayer decreases the rate of transfer by decreasing the affinity of CETP for these particles.
Highly conserved residue arginine-15 is required for the Ca2+-dependent properties of the γ-carboxyglutamic acid domain of human anticoagulation Protein C and activated Protein C
Authors: THARIATH Abraham , CASTELLINO Francis J.
Published: August 2015

The function of the rigidly conserved amino acid residue R15 in the Ca2+/phospholipid-dependent properties of the γ-carboxyglutamic acid (Gla)-containing domain (GD) of human Protein C (PC) were investigated through site-directed mutagenesis strategies. A series of recombinant (r) mutants, namely r-[R15K]PC, r-[R15H]PC, r-[R15L]PC, and r-[R15W]PC, were constructed, expressed and purified, and their relevant properties investigated. As revealed by intrinsic fluorescence analysis, all of the variant proteins underwent Ca2+-dependent structural transitions. Nonetheless, they displayed altered binding properties to acidic phospholipid vesicles, and also did not interact with a monoclonal antibody specific for the type of Ca2+-dependent conformation of the GD that characterizes the wild-type protein. On conversion into their activated forms, these variant enzymes possessed less than 10% of the ex vivo plasma anticoagulant activity of wild-type r-PC. Similar activities were found when the r-active PC mutants were assayed directly for inactivation of factor Va and factor VIII, in the complete prothrombinase and tenase complexes respectively. We conclude that R15 is a critical residue in allowing the GD of PC, and probably of other proteins of this class, to adopt a Ca2+-dependent conformation that allows functional phospholipid binding, thus explaining the strict conservation of this amino acid residue in GD modules of various proteins. As a result of an analysis of structural models of the Ca2+ŐGD complex of PC, it is postulated that hydrogen bonds between the side chain of R15 and the functionally important Gla16 residue, as well as between the side chain of R15 and the carbonyl oxygen in the peptide bond of H10, are critical for adoption of a Ca2+-dependent conformation of the GD that allows functional phospholipid binding.
Effect of membrane environment on the activity and inhibitability by malonyl-CoA of the carnitine acyltransferase of hepatic microsomal membranes
Authors: BROADWAY Neil M. , SAGGERSON E. David
Published: August 2015

We have investigated the extent to which membrane environment affects the catalytic properties of the malonyl-CoA-sensitive carnitine acyltransferase of liver microsomal membranes. Arrhenius-type plots of activity were linear in the absence and presence of malonyl-CoA (2.5 μM). Sensitivity to malonyl-CoA increased with decreasing assay temperature. Partly purified enzyme displayed an increased K0.5 (substrate concentration supporting half the maximal reaction rate) for myristoyl-CoA and a reduced sensitivity to malonyl-CoA compared with the enzyme in situ in membranes. Reconstitution with liposomes of a range of compositions restored the K0.5 for myristoyl-CoA to values similar to that seen in native membranes. The lipid requirements for restoration of sensitivity to malonyl-CoA were more stringent. When animals were starved for 24 h the specific activity of carnitine acyltransferase in microsomal membrane residues was increased 3.3-fold, whereas sensitivity to malonyl-CoA was decreased to 1/2.8. When enzymes partly purified from fed and starved animals were reconstituted into crude soybean phosphatidylcholine liposomes there was no difference in sensitivity to malonyl-CoA. When partly purified enzyme from fed rats was reconstituted into liposomes prepared from microsomal membrane lipids from fed animals it was 2.2-fold more sensitive to malonyl-CoA than when reconstituted with liposomes prepared from microsomal membrane lipids from starved animals. This suggests that the physiological changes in sensitivity to malonyl-CoA are mediated via changes in membrane lipid composition rather than via modification of the enzyme protein itself. The increased specific actvity of acyltransferase observed on starvation could not be attributed to changes in membrane lipid composition.
Identification and partial characterization of a novel membrane glycoprotein induced by amino acid deprivation in renal epithelial cells
Authors: BURSTON Judy , McGIVAN John
Published: August 2015

We have identified a protein of 110 kDa in the renal epithelial cell line NBL-1, which is induced on incubation of the cells in an amino-acid-free medium. The protein was purified on conA–Sepharose and subjected to N-terminal sequencing. The sequence obtained, VDRINFKT, does not correspond to any protein in the databases. Antipeptide antibodies made to this sequence recognised a single protein of 110 kDa in whole cell membranes and in a conconavalin A protein extract. Using the antibody on Western blots, the protein was induced 2.5–3 fold in 10–15 h and the induction was inhibited by cycloheximide and tunicamycin. The protein was found also in rat liver plasma membranes. A procedure for the partial purification of this protein from rat liver is described, and some internal sequence is reported. The possible relationship of the induction of this novel protein to the induction of amino acid transport in these cells by amino acid deprivation is discussed.
Functional interaction of the carboxylic acid group of agonists and the arginine residue of the seventh transmembrane domain of prostaglandin E receptor EP3 subtype
Authors: CHANG Chang-sheng , NEGISHI Manabu , NISHIGAKI Nobuhiro , ICHIKAWA Atsushi
Published: August 2015

Prostaglandin (PG) E2 binds to PGE receptor EP3 subtype and induces Gi activity. To assess the role of the interaction of the carboxylic acid group of agonists and its putative binding site, Arg-309 in the seventh transmembrane domain of EP3α receptor, in receptor activation, we have mutated the positively charged Arg-309 to the polar but uncharged Gln (EP3α-R309Q) and Asn (EP3α-R309N), and to the non-polar Leu (EP3α-R309L). Wild-type, EP3α-R309Q and EP3α-R309N receptors showed high-affinity binding for PGE2, but the EP3α-R309L receptor showed very-low-affinity binding. Guanosine 5´-[γ-thio]triphosphate increased the PGE2 binding to the wild-type receptor, decreased the binding to EP3α-R309Q and EP3α-R309N receptors, but did not affect that to the EP3α-R309L receptor. Furthermore we examined the Gi activities of two types of EP3 agonist, TEI-3356 with a negatively charged carboxylic acid, and TEI-4343, a methyl ester of TEI-3356 with an uncharged but polar group, towards those receptors. Both agonists inhibited the forskolin-stimulated cAMP formation in wild-type, EP3α-R309Q and EP3α-R309N receptors in the same concentration-dependent manner, but the agonists showed a very low inhibition of EP3α-R309L receptor. These findings demonstrate that the hydrogen-bonding interaction of EP3 agonists and residue 309 in the seventh transmembrane domain of the EP3α receptor is sufficient for the functional activation of the EP3α receptor.
Regulation of platelet-activating factor receptor gene expression in vivo by endotoxin, platelet-activating factor and endogenous tumour necrosis factor
Authors: WANG Hao , TAN Xiao-di , CHANG Hong , GONZALEZ-CRUSSI Frank , REMICK Daniel G. , HSUEH Wei
Published: August 2015

A competitive PCR assay was developed to quantify platelet-activating factor (PAF) receptor (PAF-R) transcripts in rat tissues using a synthetic RNA as a competitor. We found PAF-R mRNA constitutively expressed in the eight organs tested, with the ileum containing the highest concentration [(3.49±0.15)×107 molecules/μg of RNA]. Significant but lower levels were also detected in the jejunum, spleen, lungs, kidneys, heart, stomach and liver. Furthermore we defined the regulatory role of inflammatory mediators in ileal PAF-R gene expression using a rat model of intestinal injury induced by PAF or lipopolysaccharide (LPS). Injection of LPS or low-dose PAF resulted in a marked increase in ileal PAF-R mRNA within 30 min. The up-regulation on PAF-R elicited by PAF was biphasic, peaking first at 90 min, then again at 6 h. In contrast, LPS elicited a weak monophasic response. The second phase of PAF-R mRNA increase after PAF administration was completely abolished by WEB 2170, a PAF antagonist, and partially inhibited by anti-tumour necrosis factor (TNF) antibody. These observations indicate the involvement of endogenous PAF and TNF in this event. In conclusion, we found: (a) preferential PAF-R expression in the ileum, suggesting a role for PAF in intestinal inflammation; (b) induction of PAF-R expression in vivo by its own agonist; (c) a complex regulation of PAR-R gene expression in vivoinvolving a network of various pro-inflammatory mediators.
Selective inhibition of mitochondrial respiration and glycolysis in human leukaemic leucocytes by methylglyoxal
Authors: BISWAS Swati , RAY Manju , MISRA Sanjoy , DUTTA D. P. , RAY Subhankar
Published: August 2015

The effect of methylglyoxal on the oxygen consumption of mitochondria of both normal and leukaemic leucocytes was tested by using different respiratory substrates and complex specific artificial electron donors and inhibitors. The results indicate that methylglyoxal strongly inhibits mitochondrial respiration in leukaemic leucocytes, whereas, at a much higher concentration, methylglyoxal fails to inhibit mitochondrial respiration in normal leucocytes. Methylglyoxal strongly inhibits ADP-stimulated α-oxoglutarate and malate plus NAD+-dependent respiration, whereas, at a higher concentration, methylglyoxal fails to inhibit succinate and α-glycerophosphate-dependent respiration. Methylglyoxal also fails to inhibit respiration which is initiated by duroquinone and cannot inhibit oxygen consumption when the N,N,N´,N´-tetramethyl-p-phenylenediamine by-pass is used. NADH oxidation by sub-mitochondrial particles of leukaemic leucocytes is also inhibited by methylglyoxal. Lactaldehyde, a catabolite of methylglyoxal, can exert a protective effect on the inhibition of leukaemic leucocyte mitochondrial respiration by methylglyoxal. Methylglyoxal also inhibits l-lactic acid formation by intact leukaemic leucocytes and critically reduces the ATP level of these cells, whereas methylglyoxal has no effect on normal leucocytes. We conclude that methylglyoxal inhibits glycolysis and the electron flow through mitochondrial complex I of leukaemic leucocytes. This is strikingly similar to our previous studies on mitochondrial respiration, glycolysis and ATP levels in Ehrlich ascites carcinoma cells [Ray, Dutta, Halder and Ray (1994) Biochem. J. 303, 69–72; Halder, Ray and Ray (1993) Int. J. Cancer 54, 443–449], which strongly suggests that the inhibition of electron flow through complex I of the mitochondrial respiratory chain and inhibition of glycolysis by methylglyoxal may be common characteristics of all malignant cells.
Transgenic mice overexpressing ornithine and S-adenosylmethionine decarboxylases maintain a physiological polyamine homoeostasis in their tissues
Authors: HELJASVAARA Ritva , VERESS Ildiko , HALMEKYTÖ Maria , ALHONEN Leena , JÄNNE Juhani , LAAJALA Pasi , PAJUNEN Antti
Published: August 2015

Recent work has shown that transgenic mice overexpressing human ornithine decarboxylase display no marked changes in the tissue concentrations of spermidine or spermine in spite of a dramatic increase in putrescine levels. In the tissues of transgenic mice carrying the human spermidine synthase gene and in those of hybrid mice overexpressing both ornithine decarboxylase and spermidine synthase, spermidine and spermine levels remain within normal limits. To test whether the amount of the propylamine group donor, decarboxylated S-adenosylmethionine, limits the conversion of putrescine into the higher polyamines, we have produced transgenic mouse lines harbouring the rat S-adenosylmethionine decarboxylase gene in their genome. However, neither these mice nor the hybrid mice overexpressing both ornithine decarboxylase and S-adenosylmethionine decarboxylase displayed significant changes in their spermidine and spermine tissue levels. To study the mechanism by which cells maintain the constancy of the polyamine concentrations, we have determined the metabolic flux of polyamines in transgenic primary fibroblasts using pulse labelling. The results indicate that the polyamine flow is faster in transgenic primary fibroblasts than in non-transgenic fibroblasts and that the intracellular homoeostasis of higher polyamines is maintained at least partly by the acetylation of spermidine and spermine and their secretion into the medium.
ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope
Authors: MÖHLMANN Torsten , TJADEN Joachim , HENRICHS Gundrun , QUICK Paul W. , HÄUSLER Rainer , NEUHAUS Ekkehard H.
Published: August 2015

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, 395–401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport [14C]Glc6P or [32P]Pi in counter exchange with Pi, Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied [14C]ADPGlc, reaching a maximum at 2 mM. Ultrasonication of isolated amyloplasts greatly reduces the rate of ADPGlc-dependent starch synthesis, indicating that the process is dependent on the intactness of the organelles. The plastid ATP/ADP transporter is not responsible for ADPGlc uptake. Data are presented that indicate that ADPGlc is transported by another translocator in counter exchange with AMP. To analyse the physiology of starch synthesis in more detail, we examined how Glc6P- and ADPGlc-dependent starch synthesis in isolated maize endosperm amyloplasts interact. Glc6P-dependent starch synthesis is not inhibited by increasing concentrations of ADPGlc. In contrast, the rate of ADPGlc-dependent starch synthesis is reduced by increasing concentrations of ATP necessary for Glc6P-dependent starch synthesis. The possible modes of inhibition of ADPGlc-dependent starch synthesis by ATP are discussed with respect to the stromal generation of AMP required for ADPGlc uptake.
Isolation of peptides from phage-displayed random peptide libraries that interact with the talin-binding domain of vinculin
Authors: ADEY Nils. B , KAY Brian. K
Published: August 2015

Peptides isolated from combinatorial libraries typically interact with, and thus help to characterize, biologically relevant binding domains of target proteins. To characterize the binding domains of the focal adhesion protein vinculin, vinculin-binding peptides were isolated from two phage-displayed random peptide libraries. Altogether, five non-similar vinculin-binding peptides were identified. Despite the lack of obvious sequence similarity between the peptides, binding and competition studies indicated that all five interact with the talin-binding domain of vinculin and do not disrupt the binding of α-actinin or paxillin to vinculin. The identified peptides and talin bind to vinculin in a comparable manner; both bind to immobilized vinculin, but neither binds to soluble vinculin unless the C-terminus of vinculin has been deleted. An analysis of amino acid variants of one of the peptides has revealed three non-contiguous motifs that also occur in the region of talin previously demonstrated to bind vinculin. Amino acid substitutions within a 127-residue segment of talin capable of binding vinculin confirmed the importance of two of the motifs and suggest that residues critical for binding are within a 16-residue region. This study demonstrates that the vinculin-binding peptides interact with vinculin in a biologically relevant manner and represent an excellent tool for further study of the biochemistry of vinculin.
1-O-Octadecyl-2-O-methylglycerophosphocholine inhibits protein kinase C- dependent phosphorylation of endogenous proteins in MCF-7 cells
Authors: ZHOU Xi , ARTHUR Gilbert
Published: August 2015

Studies with leukaemic cells, based primarily on in vitro assays, have suggested that antitumour ether lipids have only a moderate effect on protein kinase C (PKC) activity, and, furthermore, inhibition of PKC is unlikely to be involved in the mechanism of inhibition of cell proliferation by these compounds. To determine if this is also the case for epithelial cancer cells, we examined the effect of 1-O-octadecyl-2-O-methylglycerophosphocholine (ET18-OCH3) on PKC-induced phosphorylation of endogenous proteins in MCF-7 cells under incubation conditions where the drug inhibited cell proliferation. As expected, stimulation of quiescent 32P-labelled MCF-7 cells with 1 μM PMA resulted in the phosphorylation of a number of proteins. The PMA-induced phosphorylation of the proteins was abolished by preincubation of the cells with Ro 31-8220 (5 μM) for 20 min, or 10 μg/ml ET18-OCH3 for 3 h before stimulation with PMA. Thus under incubation conditions where ET18-OCH3 inhibited the proliferation of MCF-7 cells, the ether lipid potently inhibited the activity of PKC in intact cells. This inhibition was unlikely to be due to the effect of the compound on PKC translocation since there was little effect of ET18-OCH3 on the translocation of the α, γ and ϵ species of PKC. These results suggest that a role for the inhibition of PKC activity by ET18-OCH3 in the mechanism of inhibition of cell proliferation by ET18-OCH3 cannot yet be discounted in epithelial cancer cells. In addition, we also observed that ET18-OCH3 enhanced the phosphorylation of selected proteins under basal unstimulated conditions. Although some of these proteins were also observed to be phosphorylated in response to PMA stimulation, the phosphorylation induced by ET18-OCH3 was not inhibited by Ro 31-8220, indicating that this was not mediated by PKC.
Internalization and down-regulation of the prostacyclin receptor in human platelets
Authors: GIOVANAZZI Serenella , ACCOMAZZO Maria R. , LETARI Ornella , OLIVA Daniela , NICOSIA Simonetta
Published: August 2015

The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 °C, addition of unlabelled ligand at either 37 °C or 4 °C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 °C or 4 °C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 °C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 °C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 °C, two classes of binding sites were present, whereas after preincubation at 37 °C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.