BBA - General Subjects (v.1570, #2)

Reduction of streptavidin RYDS-mediated renal adhesion by site-directed mutagenesis by Samuel Murray; Anthony Maraveyas; Tammy Dougan; Anthony C Chu (81-88).
Naturally occurring core-Streptavidin (c-Strep) would serve as a more useful agent in vivo if not for its high kidney retention. This retention is mediated by an integrin-binding motif—RYDS—that shares homology to the more common RGDS. We generated a c-Strep molecule constituting amino acids 13–139 of streptavidin and by site-directed mutagenesis altered the RYDS motif to RYES. RYDS-c-Streptavidin and RYES-c-Streptavidin were expressed in E. coli and purified on a 2-imminobiotin matrix. Each demonstrated an affinity for biotin similar to that of native post-secretory streptavidin while maintaining their ability to form dimers and tetramers. The mutant RYES-c-Streptavidin was no longer able to mediate normal rat kidney cell attachment in an in vitro assay. RYDS-c-Streptavidin-mediated kidney cell attachment was inhibited by competition with c-Streptavidin, RYDS-c-Streptavidin and RGDS-containing peptides but not with an irrelevant peptide or RYES-c-Streptavidin. Therefore, the point mutation D49E generates a molecule, which may not display the in vivo kidney retention observed for RYDS-c-Streptavidin, potentially finding more widespread clinical application.
Keywords: Streptavidin; Integrin; Kidney retention; Biotin; Core-Streptavidin; RGDS;

Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu,Zn-superoxide dismutase modification by Jung Hoon Kang; Kyung Sik Kim; Soo Young Choi; Hyeok Yil Kwon; Moo Ho Won; Tae-Cheon Kang (89-96).
Carnosine (β-alanyl-l-histidine), homocarnosine (γ-amino-butyryl-l-histidine) and anserine (β-alanyl-1-methyl-l-histidine) have been proposed to act as anti-oxidants in vivo. The protective effects of carnosine and related compounds against the oxidative damage of human Cu,Zn-superoxide dismutase (SOD) by peroxyl radicals generated from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation, which is associated with the inactivation of enzyme. Carnosine, homocarnosine and anserine significantly inhibited the fragmentation and inactivation of Cu,Zn-SOD by AAPH. All three compounds also inhibited the release of copper ions from the enzyme and the formation of carbonyl compounds in AAPH-treated Cu,Zn-SOD. These compounds inhibited the fragmentation of other protein without copper ion. The results suggest that carnosine and related compounds act as the copper chelator and peroxyl radical scavenger to protect the protein fragmentation. Oxidation of amino acid residues in Cu,Zn-SOD induced by AAPH were significantly inhibited by carnosine and related compounds. It is proposed that carnosine and related dipeptides might be explored as potential therapeutic agents for pathologies that involve Cu,Zn-SOD modification mediated by peroxyl radicals.
Keywords: Cu,Zn-superoxide dismutase; Fragmentation; Peroxyl radical; Carnosine;

7-Chloro-4-hydroxyquinoline (CQ) is an antitumor drug but its efficiency is not very satisfactory. This fact motivates us to study the relationship between the structure of 4-hydroxyquinoline with various substituent and its antioxidant effect against free-radical-initiated peroxidation: the hemolysis of human erythrocyte initiated thermally by water-soluble initiator, 2,2′-azobis (2-amidinopropane hydrochloride) (AAPH), acts as an experimental system. 7-Fluoro-4-hydroxyquinoline (FQ) and CQ can be synthesized by decarboxylation of 7-fluoro-4-hydroxyquinoline-3-carboxylic acid (FQCA) and 7-chloro-4-hydroxyquinoline-3-carboxylic acid (CQCA), respectively, and FQCA and CQCA are prepared by hydrolysis of ethyl 7-fluoro-4-hydroxyquinoline-3-carboxylate (FQCE) and ethyl 7-chloro-4-hydroxyquinoline-3-carboxylate (CQCE), respectively. The inhibitory concentration of 50% inhibition (IC50) of AAPH-induced hemolysis of the erythrocyte has been studied and found that all these chemicals dissolved in dimethyl sulfoxide (DMSO) can inhibit the free-radical-induced peroxidation. To clarify the relationship between the distributive status of the chemicals and their antioxidant effect, the chemical has been dissolved in the vesicle of dipalmitoyl phosphatidylcholine (DPPC) by sonication and suspended in the reaction system. It is found that FQCE, CQCE, FQCA and CQCA act as prooxidants either used alone or used in combination with α-tocopherol (TOH), demonstrating that FQCE, CQCE, FQCA and CQCA play a prooxidative role when they are packaged in the DPPC vesicle. This can be understood that the electron-attracting group, i.e. -COOC2H5, -COOH, at the ortho position to the hydroxy group of quinoline makes the phenoxy radical of quinoline derivatives active by attracting negative charge from the electron-deficient radical site. These unstable free radicals preserved in DPPC vesicle can initiate additional propagation of lipid peroxidation and cause hemolysis. However, FQ and CQ without electron-attracting group are antioxidants even in DPPC vesicle either used alone, or mixed with TOH. Moreover, the antioxidative activity of FQ is much better than CQ either used alone or in combination with TOH, indicating that FQ has the potential to replace CQ to be an antioxidant drug. Therefore, the antioxidant/prooxidant effect is not only correlated with the molecular structure but also the distributive status in the reaction system.
Keywords: Antioxidant; Prooxidant; Erythrocyte; Free radical; 4-Hydroxyquinoline; Hemolysis;

Time-resolved 1H and 13C NMR spectroscopy for detailed analyses of the Azotobacter vinelandii mannuronan C-5 epimerase reaction by Martin Hartmann; Anne Sissel Duun; Sidsel Markussen; Hans Grasdalen; Svein Valla; Gudmund Skjåk-Bræk (104-112).
AlgE2, AlgE4, and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyze the post-polymerization conversion of β-d-mannuronic acid residues into α-l-guluronic acid residues. To study the kinetics and mode of action of these enzymes, homopolymeric mannuronan and other alginate samples with various composition were epimerized by letting the enzymatic reaction take place in an NMR tube. Series of 1H NMR spectra were recorded to obtain a time-resolved picture of the epimerization progress and the formation of specific monomer sequences. Starting from mannuronan, guluronic acid contents of up to 82% were introduced by the enzymes, and the product specificity, substrate selectivity, and reaction rates have been investigated. To obtain direct information of the GulA-block formation, similar experiments were performed using a 13C-1-enriched mannuronan as substrate. The NMR results were found to be in good agreement with data obtained by a radioisotope assay based on 3H-5-labeled substrates.
Keywords: Azotobacter vinelandii; Mannuronan; C-5 epimerase; AlgE2; AlgE4; AlgE6;

PGE2, PGF and the thromboxane agonist U-46619 bind to bovine aortic endothelial cells and compete on the same binding site with similar affinity. In addition, binding remains unaffected by prolonged exposure to the ligand. These characteristics differ significantly from those of any known G-coupled prostaglandin receptor. Binding of PGE2 to the cells is reduced in the presence of the cyclic nucleotides cGMP and cAMP, and is unaffected by protein kinase inhibitors. Removal of permeable cyclic nucleotides from the cell medium results in a fast and complete restoration of PGE2 binding to the cells, suggesting that both cyclic nucleotides reduce PGE2 binding by a reversible interaction with the prostaglandin-binding site, without the involvement of second messenger-activated protein kinases. Our data further show that binding of prostaglandins to bovine aortic endothelial cells is sensitive to heavy metals and to activators and blockers of calcium, ATP-sensitive K(+) and chloride channels. Nickel, a specific cyclic nucleotide-gated (CNG) channel activator, decreases PGE2 binding and so do the CNG channel activators Rp-8-Br-PET-cGMPS and Sp-8-Br-PET-cGMPS. On the other hand, the calcium channel blockers pimozide, diltiazem as well as LY-83,583, a guanylate cyclase inhibitor, which were reported to block CNG channels, enhance PGE2 binding. The sensitivity of PGE2 binding to selective CNG channel modifying agents, as well as the rapid and reversible interaction with cyclic nucleotides, may suggest that the common low-affinity prostanoid-binding site on bovine aortic endothelial cells is associated with a molecular entity, which possess several properties of a CNG channel.
Keywords: Channel modulator; Endothelial cell; Bovine; PGE2; CNG channel;

Calpains are neutral Ca2+-dependent cysteine proteases. In this study, we utilized casein zymography to detect such a proteolytic activity in Drosophila melanogaster extracts throughout the life of this organism. One calpain-like activity that was sensitive to the general cysteine protease inhibitors, E64 and calpain inhibitor I, but insensitive to the human calpain-specific inhibitor, calpastatin, is demonstrated. The relevance of this finding is discussed with respect to the absence of a corresponding Drosophila gene, homologous to the vertebrate calpastatin genes, as concluded from our unsuccessful attempts to clone such a gene and our Blast searches using the FlyBase. The mechanisms of Drosophila calpain regulation require further investigation. However, we suggest that single chain, non-heterodimeric calpains may be insensitive to calpastatin and that Drosophila cystatin-like molecules may play a role in negatively regulating Drosophila calpain.
Keywords: Calpain; Ca2+ activation; Cysteine protease; Calpastatin; Cystatin; Drosophila;

In aqueous methyl linoleate emulsions (pH 7.4, 25 °C, air-saturated), nitrosylmyoglobin and saturated fatty acid anions (palmitate and stearate investigated) each showed antioxidant effect on metmyoglobin-induced peroxidation as measured by oxygen depletion rate. For equimolar concentration of nitrosylmyoglobin and metmyoglobin and for metmyoglobin in moderate excess, a reduction in oxygen consumption rate of ∼70% was observed. Fatty acid anions reduced oxygen consumption rate most significantly for palmitate (up to 60% for a fatty acid:heme protein ratio of 90:1). No further antioxidative effect was seen for fatty acid anions in the presence of nitrosylmyoglobin, whereas nitrosylmyoglobin showed a further antioxidant effect in presence of fatty acid anions in the metmyoglobin-catalyzed process. The antioxidative mechanism of nitrosylmyoglobin and fatty acid anions is different, and while the fatty acid anions seem active in inhibiting initiation of oxidation through protection against metmyoglobin activation into perferrylmyoglobin, as shown by freeze-quench Electron Spin Resonance (ESR) spectroscopy, nitrosylmyoglobin is rather active in the oxygen consuming (propagation) phase.
Keywords: Nitrosylmyoglobin; Metmyoglobin; Free fatty acid; Perferrylmyoglobin; Lipid peroxidation; Antioxidative mechanism;

Ethanol perfusion increases the yield of oxidative phosphorylation in isolated liver of fed rats by Marie-Christine Beauvieux; Pierre Tissier; Patrice Couzigou; Henri Gin; Paul Canioni; Jean-Louis Gallis (135-140).
The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria.The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using 31P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O2 Clark electrodes.The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86±0.02 μmol/min g wet weight (min g ww)] than in control [1.44±0.18 μmol/min g ww]. However, mitochondrial respiration remained unchanged [2.20±0.13 μmol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33±0.035 (control) to 0.42±0.015 (ethanol).The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.
Keywords: Ethanol; Liver; Nuclear magnetic resonance; Oxidative phosphorylation; ATP/O;

Functional investigation of a gene encoding pteridine glycosyltransferase for cyanopterin synthesis in Synechocystis sp. PCC 6803 by Yoon Kyung Hwang; Ji Youn Kang; Hyun Joo Woo; Yong Kee Choi; Young Shik Park (141-144).
A gene (slr1166) putatively encoding pteridine glycosyltransferase was disrupted with a kanamycin resistance cassette in Synechocystis sp. PCC 6803, which produces cyanopterin. The deduced polypeptide from slr1166 consisted of 354 amino acid residues sharing 45% sequence identity with UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) isolated previously from Synechococcus sp. PCC 7942. The knockout mutant was unable to produce cyanopterin but only 6-hydroxymethylpterin-β-galactoside, verifying that slr1166 encodes a pteridine glycosyltransferase, which is responsible for transfer of the second sugar glucuronic acid in cyanopterin synthesis. The mutant was affected in its intracellular pteridine content and growth rate, which were 74% and 80%, respectively, of wild type, demonstrating that the second sugar residue is still required for quantitative maintenance of cyanopterin. This supports the previous suggestion that glycosylation may contribute to high cellular concentration of pteridine compounds.
Keywords: Cyanopterin; Pteridine glycoside; Pteridine glycosyltransferase; Gene disruption; Synechocystis sp. PCC 6803;