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1.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1814-1818
The effect of the oxidation of linoleic acid on the interfacial adsorptivity of lysophosphatidylcholine (LPC)/free fatty acid (FFA)/ovalbumin (OA) complexes was investigated by 31P-NMR. The interfacial adsorptivity of the complexes was evaluated by the mean droplet size, phosphorus signal and relaxation time of an emulsion composed of each complex. The interfacial adsorptivity of the LPC/FFA/OA complexes became lower with the oxidation of linoleic acid, which formed a complex with LPC and OA. Reduction of the T2 relaxation time and peak broadening of Ser-P68 for OA correlated well with the formation of fine emulsions from an LPC/linoleic acid/OA complex. The bilayer vesicles composed of LPC and linoleic acid with a low POV value were destroyed by coupling with protein and show high interfacial adsorptivity. On the other hand, the vesicles composed of LPC and linoleic acid with a high POV value remained in liposome and show low interfacial adsorptivity. These results suggest that the affinity of bilayer vesicles composed of LPC and FFA mainly promoted the interfacial adsorption of an LPC/FFA/OA complex, and that the region of Ser-P68 in OA was adsorbed at the interface when the complex formed a fine emulsion. 相似文献
2.
Saunders LP Cao W Chang WC Albright RA Braddock DT De La Cruz EM 《The Journal of biological chemistry》2011,286(34):30130-30141
Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k(1) ≥500 μm(-1) s(-1)) and is hydrolyzed slowly (k(2) = 0.024 s(-1)). Release of the first hydrolysis product is random and rapid (≥1 s(-1)), whereas release of the second is slow and rate-limiting (0.005-0.007 s(-1)). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics. 相似文献
3.
The binding of theophylline (Th, 11-840 microM) to bovine serum albumin (BSA, 10 microM) using microdialysis technique in the presence of fatty acids (2.5-50.0 microM) and cholesterol (20-500 nM) indicates that fatty acids and cholesterol inhibit the binding of Th to BSA. The maximum inhibition (90.5%) occurs in presence of acetic acid (AA) followed by lauric acid (LA, 83.3%), palmitic acid (PA, 72.2%), oleic acid (OA, 44.4%) and cholesterol (22.2%). Fatty acids and cholesterol also decrease the number of binding sites and the affinity for the binding of Th to BSA. Such a decrease is maximum in the presence of AA followed by LA, PA, OA and cholesterol. Complete abolition of the low affinity binding site in the presence of AA indicates that the low affinity binding is predominantly ionic in nature while the high affinity binding involves ionic and other type(s) of unidentified force(s). This makes high affinity binding stronger than low affinity binding. 相似文献
4.
Haehner T Massing U Diesinger T Müller-Enoch D 《Zeitschrift für Naturforschung. C, Journal of biosciences》2004,59(7-8):599-605
The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1-O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2-5.0 microM and 2.8-3.5 microM, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 microM) and rac-2-O-acetyl (Ki = 1.33 microM) differs slightly from those of rac-2-NH2 (Ki = 2.2 microM) and rac-2-N-acetyl (Ki = 2.2 microM), but with the reconstituted CYP2B1: NADPH-P450-reductase system all Ki values are in the small range of 1.8 - 2.6 microM, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity. 相似文献
5.
Okazaki N Tamada T Feese MD Kato M Miura Y Komeda T Kobayashi K Kondo K Blaber M Kuroki R 《Protein science : a publication of the Protein Society》2012,21(4):539-552
Glycosyltrehalose trehalohydrolase (GTHase) is an α-amylase that cleaves the α-1,4 bond adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild-type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3-Tre), was determined to 2.6-Å resolution. The structure with G3-Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3-Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose. 相似文献
6.
The role of a prominent surface-exposed loop (residues 88-97) in the alpha domain of streptokinase (SK), in human plasminogen (HPG) activation was explored through its selective mutagenesis and deletion studies. We first made a conformationally constrained derivative of the loop by the substitution of sequences known to possess a strong propensity for beta-turn formation. The mutant so formed (termed SK(88-97-Beta Turn)), when tested for co-factor activity against substrate HPG, after first forming a 1:1 molar complex with human plasmin (HPN), showed a nearly 6-fold decreased co-factor activity compared to the wild-type, native SK. The major catalytic change was observed to be at the k(cat) level, with relatively minor changes in K(m) values against HPG. Real-time binary interaction (i.e. the 1:1 complexation between SK, or its mutant/s, with HPG), and ternary complexation studies (i.e. the docking of a substrate HPG molecule into the preformed SK-HPG complex) using Surface Plasmon Resonance were done. These studies revealed minor alterations in binary complex formation but the ternary interactions of the substitution and/or deletion mutants were found to be decreased for full-length HPG compared to that for native SK.HPG. In contrast, their ternary interactions with the isolated five-kringle domain unit of plasminogen (K1-5) showed K(d) values comparable to that seen with the native SK.HPG complex. Taking into consideration the overall alterations observed in catalytic levels after site-specific mutagenesis and complete loop deletion of the 88-97 loop, on the one hand, and its known position at the SK-HPG interface in the binary complex, suggests the importance of this loop. The present results suggest that the 88-97 loop of the alpha domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme-substrate affinity per se is minimal. 相似文献
7.
Parrill AL Echols U Nguyen T Pham TC Hoeglund A Baker DL 《Bioorganic & medicinal chemistry》2008,16(4):1784-1795
Autotaxin (ATX, NPP-2) catalyzes the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a mitogenic cell survival factor that stimulates cell motility. The high expression of both ATX and receptors for LPA in numerous tumor cell types has produced substantial interest in exploring ATX as an anticancer chemotherapeutic target. ATX inhibitors reported to date are analogs of LPA, a phospholipid, and are more hydrophobic than is typical of orally bioavailable drugs. This study applied both structure-based and ligand-based virtual screening techniques with hit rates of 20% and 37%, respectively, to identify a promising set of non-lipid, drug-like ATX inhibitors. Structure-based virtual screening necessitated development of a homology model of the ATX catalytic domain due to the lack of structural information on any mammalian NPP family member. This model provided insight into the interactions necessary for ATX inhibition, and produced a suitably diverse training set for the development and application of binary QSAR models for virtual screening. The most efficacious compound identified in this study was able to completely inhibit ATX-catalyzed hydrolysis of 1 microM FS-3 (a synthetic, fluorescent LPC analog) at a 10 microM concentration. 相似文献
8.
The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure. 相似文献
9.
In the present study, the effect of increasing concentrations of palmitic (PA, C16:0), stearic (SA, C18:0), oleic (OA, C18:1, n-9), linoleic (LA, C18:2n-6), docosahexaenoic (DHA, C22:6 n-3) and eicosapentaenoic (EPA, C20:5 n-3) acids on lymphocyte proliferation was investigated. The maximal non-toxic concentrations of these fatty acids for human lymphocytes in vitro were determined. It was also evaluated whether these fatty acids at non-toxic concentrations affect IL-2 induced lymphocyte proliferation and cell cycle progression. OA and LA at 25 microM increased lymphocyte proliferation and at higher concentrations (75 microM and 100 microM) inhibited it. Both fatty acids promoted cell death at 200 microM concentration. PA and SA decreased lymphocyte proliferation at 50 microM and promoted cell death at concentrations of 100 microM and above. EPA and DHA decreased lymphocyte proliferation at 25 and 50 microM being toxic at 50 and 100 microM, respectively. PA, SA, DHA and EPA decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases. OA and LA caused an even greater pronounced effect. The treatment with all fatty acids increased neutral lipid accumulation in the cells but the effect was more pronounced with PA and DHA. In conclusion, PA, SA, DHA and EPA decreased lymphocyte proliferation, whereas OA and LA stimulated it at non-toxic concentrations. 相似文献
10.
Vilkaitis G Merkiene E Serva S Weinhold E Klimasauskas S 《The Journal of biological chemistry》2001,276(24):20924-20934
Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-l-methionine (AdoMet) and product S-adenosyl-l-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI.AdoMet complex (k(off) = 22 s(-)1, K(D) = 6 microm) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI.(Me)DNA.AdoHcy (k(chem) = 0.26 s(-)1) is followed by a slower decay step (k(off) = 0.045 s(-)1), which is the rate-limiting step in the catalytic cycle (k(cat) = 0.04 s(-)1). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K(D)(DNA(ternary)), k(off)(DNA(ternary)), K(M)(AdoMet(ternary)) values but small changes in K(D)(DNA(binary)), K(D)(AdoMet(binary)), k(chem), and k(cat). When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI.DNA complex is inhibited 400-fold (k(chem)(5FC) = 0.7 x 10(-)3 s(-)1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k(chem). We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k(chem)) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI. 相似文献
11.
M. Gey 《Engineering in Life Science》1988,8(2):197-205
Suitable and optimized chromatographic separation systems for HPLC analyses of mono-, di- and oligomeric carbohydrates, organic acids (e.g. gluconic acid, α-ketoglutaric acid), phospholipids (PE, LPE, LPC, PC, SPH) and neutral lipids (squalene, cholesterol) are demonstrated. Applications of HPLC technique are separation examples of sugars from hydrolyzed starches which were isolated from potatos, calculations of organic acids in fermentation mediums and determinations of neutral lipids and phospholipids which were isolated from microbial biomass. The liquid chromatographic separations are based on self-packed highly efficient (approximately 80 000 theoretical plates per meter, N/m) glass columns. 相似文献
12.
Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC50 = 0.8 microM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20. OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 microM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin-induced platelet activation. 相似文献
13.
E. Jeffrey North Daniel A. Osborne Peter K. Bridson Daniel L. Baker Abby L. Parrill 《Bioorganic & medicinal chemistry》2009,17(9):3433-3442
Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to form the bioactive lipid lysophosphatidic acid (LPA). LPA stimulates cell proliferation, cell survival, and cell migration and is involved in obesity, rheumatoid arthritis, neuropathic pain, atherosclerosis and various cancers, suggesting that ATX inhibitors have broad therapeutic potential. Product feedback inhibition of ATX by LPA has stimulated structure–activity studies focused on LPA analogs. However, LPA displays mixed mode inhibition, indicating that it can bind to both the enzyme and the enzyme–substrate complex. This suggests that LPA may not interact solely with the catalytic site. In this report we have prepared LPC analogs to help map out substrate structure–activity relationships. The structural variances include length and unsaturation of the fatty tail, choline and polar linker presence, acyl versus ether linkage of the hydrocarbon chain, and methylene and nitrogen replacement of the choline oxygen. All LPC analogs were assayed in competition with the synthetic substrate, FS-3, to show the preference ATX has for each alteration. Choline presence and methylene replacement of the choline oxygen were detrimental to ATX recognition. These findings provide insights into the structure of the enzyme in the vicinity of the catalytic site as well as suggesting that ATX produces rate enhancement, at least in part, by substrate destabilization. 相似文献
14.
A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (k(cat)), the Michaelis constant (K(M)), and the specificity constant (k(cat)/K(M)) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K(M), and k(cat)/K(M) values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K(M) values (0.93, 0.37, 0.027, and 0.00015 microM(-1) s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K(M) value for Xyl(5) (136 microM) was roughly twice as high as that for Xyl(6) (73 microM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed. 相似文献
15.
In vivo investigation of the placental transfer of (13)C-labeled fatty acids in humans 总被引:1,自引:0,他引:1
Placental fatty acid transfer in humans in vivo was studied using stable isotopes. Four pregnant women undergoing cesarean section received 4 h before delivery an oral dose of [(13)C]palmitic acid (PA), [(13)C]oleic acid (OA), [(13)C]linoleic acid (LA), and [(13)C]docosahexaenoic acid (DHA). Maternal blood samples were collected at -4 h (basal), -3 h, -2 h, -1 h, 0 h, and +1 h relative to time of cesarean section. At the time of birth, venous cord blood and placental tissue were collected. Fatty acid composition was determined by gas-liquid chromatography and isotopic enrichment by gas chromatography-combustion-isotope ratio mass spectrometry. (13)C-enrichment of fatty acids in the nonesterified fatty acids (NEFA) of cord plasma tended to be higher than in NEFA of placenta, with statistically significant differences for the nonesterified OA and DHA ([(13)C]PA, 0.024 +/- 0.011 vs. 0.001 +/- 0.001; [(13)C]OA, 0.042 +/- 0.008 vs. 0.005 +/- 0.003; [(13)C]LA, 0.038 +/- 0.010 vs. 0.008 +/- 0.002; [(13)C]DHA, 0.059 +/- 0.009 vs. 0.010 +/- 0.003). The ratio of tracer fatty acid concentrations of placenta to maternal plasma was significantly higher for [(13)C]DHA than for the other fatty acids ([(13)C]PA, 7.1 +/- 1%; [(13)C]OA, 3.8 +/- 0.4%; [(13)C]LA, 9.2 +/- 1.3%; [(13)C]DHA, 25.9 +/- 3.4%). These results suggest that only a part of the placental NEFA participated in fatty acid transfer, and that the placenta showed a preferential accretion of DHA relative to the other fatty acids. 相似文献
16.
Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.
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Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger. 相似文献
17.
Dietary hydroperoxides are being discussed as potential health hazards contributing to oxidative stress-related diseases. However, how food-born hydroperoxides could exert systemic effects remains elusive in view of the limited chances to be absorbed. Therefore, the metabolic fate of 13-HPODE (13-hydroperoxy octadecadienoic acid), 13-HODE (13-hydroxy octadecadienoic acid) and linoleic acid (LA) was investigated in a CaCo-2 cell monolayer as a model of the intestinal epithelium. [1-14C]-13-HPODE, up to a non-cytotoxic concentration of 100 microM, did not cross the CaCo-2 cell monolayer unreduced if applied to the luminal side. The [1 -14C]-HPODE-derived radioactivity was preferentially recovered from intracellular and released diacylglycerols (DG), phospholipids (PL) and cholesterol esterified with oxidized fatty acids (oxCE). A similar distribution pattern was obtained with 13-HODE. In contrast, LA is preferentially incorporated into triacylglycerols (TG), cholesteryl esters (CE) and PL (but mainly released as TG). 13-HPODE dose-dependently decreased the incorporation of LA into released TG, while LA accumulated in cellular and released DGs, effects similarily exerted by 13-HODE. We concluded that food-born hydroperoxy fatty acids are instantly reduced by the gastrointestinal glutathione peroxidase, which was previously shown to persist in selenium deficiency. Accordingly, modulation of the glutathione peroxidases by selenium deprivation/repletion did not modify the disturbance of the lipid metabolism by 13-HPODE. Thus, hydroperoxy fatty acids disturb intestinal lipid metabolism by being esterified as hydroxy fatty acids into complex lipids, and may render lipoproteins synthesized thereof susceptible to further oxidative modifications. 相似文献
18.
Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme 总被引:4,自引:0,他引:4
G P Mullen P Shenbagamurthi A S Mildvan 《The Journal of biological chemistry》1989,264(33):19637-19647
The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme. 相似文献
19.
Christopher M. Anderson Richard J. Bridges † A. Richard Chamberlin ‡ Keiko Shimamoto § Yoshimi Yasuda-Kamatani§ Raymond A. Swanson 《Journal of neurochemistry》2001,79(6):1207-1216
Na(+)-dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. L-threo-beta-hydroxyaspartate (L-TBHA), DL-threo-beta-benzyloxyaspartate (DL-TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC) (+/-)-cis-4-methy-trans-pyrrolidine-2,4-dicarboxylic acid (cis-4-methy-trans-2,4-PDC) and L-antiendo-3,4-methanopyrrolidine-2,4-dicarboxylic acid (L-antiendo-3,4-MPDC) inhibited L-[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 microM (DL-TBOA and L-TBHA) - 43 microM (cis-4-methy-trans-2,4-PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While L-TBHA, L-trans-2,4-PDC, cis-4-methy-trans-2,4-PDC and L-antiendo-3,4-MPDC displayed significant transporter substrate activities in neurons and astrocytes, DL-TBOA was a substrate only in astrocytes. This effect of DL-TBOA was concentration-dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal DL-TBOA uptake velocity (< or = 10 microM), DL-TBOA blocked uptake reversal in ATP-depleted astrocytes; this blockade was negated at concentrations that drove substantial DL-TBOA uptake (> 10 microM). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions. 相似文献
20.
The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (=0.5 microM) is significantly lower than that of the measured K(m(MBP)) (4.2 +/- 0.8 microM). Furthermore, MBP binds to the ERK2 x ATP complex at least 1500-fold more tightly than does ERKtide (K(d(ERKtide)) >/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (=1.2 s(-1)) versus that of the synthetic peptide (>/=56 s(-1)), from the ERK2 active site. 相似文献