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mM MgCl2 (mm + mgcl2)
Selected AbstractsDevelopment of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substratesELECTROPHORESIS, Issue 12 2006Jamshed Iqbal Abstract Fast and convenient CE assays were developed for the screening of adenosine kinase,(AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260,nm. An MEKC method using borate buffer (pH,9.5) containing 100,mM SDS (method,A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH,7.5 or 8.5) was used and a constant current (95,,A) was applied (method,B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10,min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method,C). After hydrodynamic injection of a plug of reaction buffer (20,mM Tris-HCl, 0.2,mM MgCl2, pH,7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1,mM,ATP, 100,,M adenosine, and 20,,M,UMP as an internal standard,(I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5,kV separation voltage (negative polarity) for 0.20,min to let the plugs interpenetrate. The voltage was turned off for 5,min (zero-potential amplification) and again turned on at a constant current of ,60,,A to elute the products within 7,min. The method employing a polyacrylamide-coated capillary of 20,cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose,response curves and calculated Ki values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay. [source] MYELINATION DEFICIT IN NERVE OF SUCKLING RATS DUE TO CYCLOLEUCINE -INDUCED DEFICIENCY OF METHYL DONORSJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2000R. Bianchi We used cycloleucine (CL) , which prevents methionine conversion to S-adenosyl-methionine (SAMe) by inhibiting ATP-L-methionine-adenosyl-transferase (MAT) , to characterize the lipid and protein changes induced by methyl donors deficit in peripheral nerve and brain myelin in rats during development. We have previously shown that CL (400 mg/kg ip) given to suckling rats at days 7, 8, 12, and 13 after birth reduced brain and sciatic nerve weight gain, brain myelin content, protein, phospholipid (PL), and galactolipid concentration in comparison to control. Among PLs, only sphingomyelin (SPH) significantly increased by 35,50%. SAMe p-toluensulphonate (SAMe-SD4) (100 mg/kg, ip) given daily from day 7, as with exogenous SAMe, partially prevented some lipid alterations induced by CL, particularly galactolipid and SPH. To test the ability of CL to affect PL metabolism we have measured de novo PL biosynthesis, ex vivo in nerve homogenates (in comparison with brain homogenates) from control and CL-treated animals killed at day 18 after birth, starting from labelled substrates ([3H]-choline, specific activity 20 mCi/mmol) in a Tris/HCl buffer, containing 5 mM MgCl2, 0.2 mM EDTA, 0.1 mM ATP, and 0.5 mM of the labelled substrates. After 60 min incubation, lipids were extracted, PL separated by TLC, and corresponding silica gel fractions scraped and counted in a liquid scintillator. Phosphatidylcholine enrichment in labelled choline resulted in slight increases in brain and sciatic nerve of CL-treated rats, suggesting an increased synthesis rate via the Kennedy pathway, possibly due to the reduced availability of methyl donors. Interestingly, choline incorporation into SPH in brain and nerve myelin resulted in significant increases of 30,40%. In agreement with the observed decrease of galactolipid content and the relative increase in SPH, these data suggest an alteration in sphingolipid metabolism after CL. Among proteins, in sciatic nerves of CL-treated pups the relative content of a number of polypeptides, namely the 116, 90, 66, 58, and 56 kDa bands, decreased, whereas others increased; the most abundant PNS protein, protein zero, remained unchanged. The analyses of myelin basic protein isoforms revealed a dramatic increase in the 14.0 and 18.5 forms, indicating early active myelination. SAMe-SD4 treatment counteracted, and in some cases normalized, these changes. In summary, methyl donor deficiency induced by MAT inhibition produces myelin lipid and protein alterations, partly counteracted by SAMe-SD4 administration. The financial support of Telethon-Italy (grant No. D 51) is gratefully acknowledged. [source] Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: Off-pathway aggregation propensity does not determine the co-chaperonin requirementPROTEIN SCIENCE, Issue 12 2000Paul A. Voziyan Abstract One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions. [source] Phosphatidylinositol Synthase of Tetrahymena: Inositol Isomers as Substrates in Phosphatidylinositol Biosynthesis and Headgroup Exchange ReactionsTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2007BRIDGET M. RIGGS ABSTRACT. Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30°C. The enzyme retained approximately 80% of its activity after incubation at 70°C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo -inositol and scyllo -inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non- myo -inositols in Tetrahymena and the potential for the production of novel, non- myo -inositol-containing second messengers. [source] Crystallization and preliminary X-ray crystallographic studies of the thermoactive pullulanase type I, hydrolyzing ,-1,6 glycosidic linkages, from Fervidobacterium pennivorans Ven5ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2000Joyce H. G. Lebbink Crystals of the thermoactive recombinant F. pennivorans type I pullulanase, purified from the supernatant of a Bacillus subtilis culture, have been obtained by the vapour-diffusion method in the presence of the inhibitor ,-cyclodextrin (2,mM) by mixing protein (15,mg,ml,1) with an equal volume of crystallization solution containing 0.1,M bis,tris propane pH 6.5, 50,mM MgCl2 and 15% polyethylene glycol 3350. Crystals diffracted to 3.0,Å using conventional Cu,K, radiation and belong to space group P212121, with unit-cell parameters a = 76.8, b = 96.2, c = 98.5,Å. The asymmetric unit contains one monomer. A preliminary 26% complete data set has been collected at 2.2,Å resolution using synchrotron radiation. [source] Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breveACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Ryuichiro Suzuki The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293,K using 0.05,mM thiamine diphosphate, 0.25,mM MgCl2, 24%(w/v) PEG 6000 and 0.1,M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8,Å, and diffracted to beyond 1.7,Å resolution. [source] | |||||||||||||