A multistep mechanism of nucleophilic substitution of vitamin B1 in methanol

Thiamin (1) and an analog (2) having a pyridine ring in place of the thiazole portion react with amine nucleophiles in methanol at 71.5°C to give substitution products 3. The nucleophiles replace the thiazole and pyridine rings. The course of the substitution reactions which were followed by PMR are first order in substrate and zero order in amine. Proposed is a multistep mechanism analogous to that observed for second-order reactions between sulfite ion, for example, and thiamin, its derivatives, and analogs. The conjugate acid of the substrate forms a sigma adduct with methoxide ion. Following fragmentation to a resonance-stabilized cation the amine nucleophile becomes incorporated into this charged intermediate. Loss of methoxide ion generates the aromatic substitution product. Substrate protonation and methoxide ion addition are kinetically equivalent to the addition of solvent, a process that cannot be detected kinetically. Addition of the amine occurs in a fast step. Overall then, the scheme is only first order in substrate as required by the experimental observations.     

Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom

The L-21 ScaI ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a guanosine-dependent endonuclease reaction that is analogous to the first step in self-splicing of this intervening sequence. We now describe pre-steady-state kinetic experiments, with sulfur substituting for the pro-RP (nonbridging) phosphoryl oxygen atom at the site of cleavage, that test aspects of a kinetic model proposed for the ribozyme reaction (Herschlag, D., & Cech, T. R. (1990) Biochemistry 29, 10159-10171). Thio substitution does not affect the reaction with subsaturating oligonucleotide substrate and saturating guanosine ((kcat/Km)S), consistent with the previous finding that binding of the oligonucleotide substrate limits this rate constant. In contrast, there is a significant decrease in the rate of single-turnover reactions of ribozyme-bound (i.e., saturating) oligonucleotide substrate upon thio substitution, with decreases of 2.3-fold for the reaction with guanosine ((kcat/Km)G) and 7-fold for hydrolysis [i.e., with solvent replacing guanosine; kc(-G)]. These "thio effects" are consistent with rate-limiting chemistry, as shown by comparison with model reactions. Nonenzymatic nucleophilic substitution reactions of the phosphate diester, methyl 2,4-dinitrophenyl phosphate monoanion, are slowed 4-11-fold by thio substitution for reactions with hydroxide ion, formate ion, fluoride ion, pyridine, and nicotinamide. In addition, we have confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA (Burgers, P. M. J., & Eckstein, F. (1979) Biochemistry 18, 592-596). Considering the strong preference of Mg2+ for binding to oxygen rather than sulfur, the modest thio effect on the chemical step of the ribozyme-catalyzed reaction and the absence of a thio effect on the equilibrium constant for binding of the oligonucleotide substrate suggest that the pro-RP oxygen atom is not coordinated to Mg2+ in the E.S complex or in the transition state. General implications of thio effects in enzymatic reactions of phosphate diesters are discussed.     

Pentacopper(II) complexes of alpha-aminohydroxamic acids: uranyl-induced conversion of a 12-metallacrown-4 to a 15-metallacrown-5

Effects of uranyl on the pentacopper(II) complexes of alpha-leucinehydroxamic acid and alpha-tyrosinehydroxamic acid were studied in water and methanol by means of electrospray ionisation mass spectrometry (ES-MS), absorption spectrophotometry, circular dichroism spectroscopy and proton NMR spectroscopy. All the measurements were consistent with the complete conversion of a 12-metallacrown-4 to a 15-metallacrown-5 upon addition of one equivalent of the uranyl ion. The uranyl ion is accommodated in the cavity formed by five copper(II) ions and five alpha-aminohydroxamate ligands. The 15-metallacrown-5 inclusion complexes have a high affinity for the uranyl ion. Competition studies showed that even in the presence of a large excess of calcium(II), the 15-metallacrown-5 remained stable, and no exchange reactions between calcium(II) and uranyl were observed. Extraction of uranyl from the 15-metallacrown-5 was also not detected in the presence of a large excess of 18-crown-6. Trivalent lanthanide ions can be partially sequestered by the 15-metallacrown-5, however, even these trivalent ions are displaced by uranyl.     

The monensin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR

Addition of monesin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine (EPC) in sodium or potassium chloride solution and from dioleoylphosphatidylcholine (DOPC) in sodium chloride solutions gives rise to dynamic 23Na- and 39K-NMR spectra. The dynamic spectra arise from the monensin-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of monensin and metal ion concentrations and are compatible with a model in which one monensin molecule transports one metal ion. Rate constants for the association and dissociation of the monensin-metal complex in the membrane/water interface are extracted and the stability constants for complex formation are evaluated. The rate constants in DOPC are similar to those in EPC, confirming that diffusion is not rate-limiting in the transport process and that dissociation of the complex is the rate-limiting step. Although potassium on its own is transported more rapidly, sodium forms the more stable complex and is therefore transported preferentially in competition with potassium.     

Leaving group effects on ligand substitution reactions of pentacyanoferrate(II) complexes: rate constant and activation volume correlations

The kinetics of the reaction of cyanide ions with pentacyanoferrate(II) complexes have been studied spectrophotometrically at pressures of 1 bar and up to 1 kbar, at 298.2 K. An excess of cyanide ions was employed and first-order kinetics were observed both in aqueous solution and in aqueous-mono-ol mixtures. For several pyridine derivative leaving groups, neutral or mono-positively charged, the rate constant variation in aqueous medium is only over one half-order of magnitude, although thiourea and quinoxaline are much more labile, dissociating with rate constants about ten and three hundred times greater than this range, respectively. Very modest changes in rate constant are observed upon addition of 40% methanol, and in a few examples studied, kinetic differences become significant only in cosolvent-rich mixtures. Volumes of activation, Δ V^*, are all positive, for reaction in water, confirming the expected bond extension of the leaving group in a D mechanism. Solvation changes and ligand differences do not wholly explain the variation in Δ V^* values, or the changes in this parameter found when cosolvents are added. Reasonably good correlations are found for the logarithms of rate constants both with the pK_a of the ligand and with Δ V^*. Other potential correlations of the leaving group property and kinetic parameter are discussed.     

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.     

Methanol biosynthesis by covalently immobilized cells of Methylosinus trichosporium: batch and continuous studies

The DEAE-cellulose linked cells of Methylosinus trichosporium displaying high specific methane mono-oxygenase activity (66 mumol methane oxidized/h mg cells) were used for methanol biosynthesis from biogas derived methane in a batch and a continuous cell reactor. The optimum cell-to-carrier ratio was determined to be 0.5 g cells/g dry weight cellulose. Batch experiments indicated that 100 mM phosphate ion concentration was necessary to inhibit further oxidation of methanol; excess oxygen supply favored methanol accumulation with an increase in methane conversion efficiency to 27%. A pulse of 40 mM sodium formate at the end of 6 h resulted in restoration of methanol accumulation by regenerating NADH(2) required for the sustained activity of methane mono-oxygenase. Maximum methanol level of 50 mumol/mg cells was obtained in the batch reactor. In a standard 50-mL ultrafiltration continuous reactor, the covalently linked cells produced methanol at a continuous rate of 100 mumol/h for the first 10 h, after which the methanol accumulation rate fell low due to the depletion of NADH(2). The methanol accumulation could be stimulated by supplying sodium formate (40 mM) in either 20 or 100 mM phosphate buffer. Maximum methanol accumulation rate of 267 mumol/h was obtained when 20 mM formate was supplied in the feed stream containing 100 mM phosphate ions, and this level of biosynthesis was maintained for over 72 h. The stoichiometric balance made at various points of formate addition indicated that the molar amount of methanol generated at steady state is dependent on the equimolar addition of sodium formate to the feed. The half-life t(1/2) and thermal denaturation rate constant K(d) were computed to be 108 h and 6.42 x 10(-3) h(-1), respectively.     

Effect of hydraulic residence time on microbial sulfide production in an upflow sludge blanket denitrification reactor fed with methanol

Summary In the combined ion exchange/biological denitrification process for nitrate removal from ground water anion exchange resins are regenerated in a closed circuit by way of an upflow sludge blanket denitrification reactor. The regenerant (a concentrated sodium bicarbonate solution) is recirculated through the ion exchanger in the r generation mode and the denitrification reactor. In the closed system sulfate accumulates to very high concentrations. For that reason it was examined under what process conditions sulfate reduction occurs in an upflow sludge blanket denitrification reactor, when the influent contains high sulfate concentrations (5.45 g SO ₄ ^2- /l) and high sodium bicarbonate concentrations (19.8 g NaHCO₃/l) in addition to nitrate and methanol. It appeared that at a hydraulic residence time of 5 h sulfide production started, when the nitrate loading rate was 20% of the denitrification reactor capacity and methanol was added in excess. The excess of methanol was converted into acetate after nitrate was depleted. Conversion of methanol into acetate was a function of the hydraulic residence time. At hydraulic residence times above 8 h this conversion was complete. Also in batch experiments it was observed that excess of methanol was converted into acetate, and that sulfate reduction started when nitrate was depleted. From all experiments it is clear that, provided that methanol is added in good relation to the quantity of nitrate that has to be denitrified, acetate will not be produced and sulfate reduction will not occur in the denitrification reactor, even in the presence of very high sulfate concentrations.     

The mechanism of mitochondrial swelling. V. Permeability of mitochondria to alkali metal salts of strong acid anions

Mitochondria do not swell appreciably when suspended in media containing the chlorides or bromides of alkali metal or ammonium ions. On the other hand, extensive swelling takes place when mitochondria are suspended in ammonium or sodium acetate. These findings have been widely interpreted to mean that the mitochondrial membrane is impermeable to chloride and bromide ions. However, the resistance of the mitochondria to volume changes is not necessarily a valid criteria of impermeability to a given ion pair. Such a conclusion presumes the as yet untested assumptions that (1) permeability to the ion pair is pair is always the rate-limiting step in swelling, and (2) permeability to the ion pair is equivalent to the driving force for water influx. We have conducted experiments addressed to the question of mitochondrial permeability by methods (tracer exchange diffusion) which are independent of volume changes. Our findings indicate that the mitochondrial membrane is very readily penetrated by alkali metal chloride and bromide salts. Further, we have concluded that the resistance to swelling in such media is associated with a lack of driving force.     

Quantitative analysis of polychlorinated biphenyls, organochlorine insecticides, polycyclic aromatic hydrocarbons, polychlorinated hydrocarbons and polynitrohydrocarbons in spiked samples of soil, water and plasma by selected-ion monitoring gas chromatography–mass spectrometry

A broad range of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated hydrocarbons (PCHs), polynitrohydrocarbons (PNHs), polychlorinated biphenyls (PCBs) and organochlorine (OCs) insecticides were simultaneously analyzed in spiked soil, water or plasma samples by using gas chromatography–mass spectrometry (GC–MS). Water and plasma samples containing the pollutants were extracted by a solid-phase extraction (SPE) method using florisil columns. The soil samples, fortified with the toxicants, were extracted with water, methanol or dichloromethane (DCM). The water extract was processed by the SPE method. The methanol and DCM samples were dried, dissolved in acetonitrile and subjected to the SPE extraction. The extracted samples were analyzed by GC–MS programmed to monitor selected ions. The deuterium labelled compounds were used as the internal standards. The chromatographic profile of total ions indicated complete separation of some compounds such as isophorone, naphthalene, all PCBs, most OC insecticides and PNHs; high M_r PAHs and some PCHs were partially or incompletely separated. The chromatographic profile of individual ion indicated good separation of each ion. The minimum detection limit ranged from 1 to 4 pg injected when 1 or 2 ions were monitored or from 20 to 200 pg injected when 20 ions were monitored. The SPE method that provided 60–105% recovery of pollutants from water samples, provided only 2–60% recovery from plasma samples. This may be due to the binding of pollutants to plasma proteins. Water recovered 1–30%, while methanol or DCM recovered 65–100% of the pollutants added to the soil samples. The use of internal standards corrected for the loss of pollutants from plasma or soil.     

A laser-temperature-jump method for the study of the rate of transfer of hydrophobic ions and carriers across the interface of thin lipid membranes

The first application of a laser-temperature-jump apparatus for the study of ion transport through planar (artificial) lipid membranes is described. The relaxation of the electric current is detected, either continuously at a constant applied voltage or discontinuously by a series of short voltage pulses. The second technique, a combined voltage- and temperature-jump method, is especially appropriate to investigate the kinetics of the adsorption/desorption process of hydrophobic ions and neutral carriers of cations at the membrane interface and to separate this phenomenon from the diffusion process through the unstirred aqueous layers adjacent to the membrane. The aim is to determine the rate-limiting step of transport. The permeation rate of the hydrophobic anion 2,4,6-trinitrophenolate is limited by the inner membrane barrier. For tetraphenylberate the rate constant of translocation across the inner barrier and that of desorption from the membrane into water are found to be of comparable magnitude. The membrane permeability of the neutral macrocyclic ion carrier enniatin B is strongly interface limited by its comparatively small rate of desorption into water. These results show that the frequently used a priori assumption of partition equilibrium at the membrane interfaces during transport is not justified.     

The effect of halides on the equilibrium and reactivity of aspartate aminotransferase.

For the pork heart, extramitochondrial aspartate aminotransferase (EC 2.6.1.1), the “half-reaction” equilibrium, amino acid + phosphopyridoxal enzyme ? keto acid + phosphopyridoxamine enzyme, is displaced in favor of the phosphopyridoxamine enzyme by the addition of halide ions. The order of effectiveness is I^? > Br^? > Cl^? > F^?. A kinetic analysis of this equilibrium with alanine and pyruvate as substrates showed that halide ions (0.01–0.1 m) both increase the rate of the forward reaction and decrease the rate of the reverse reaction. Chloride ions decrease the rate of the reverse reaction by competitively inhibiting the formation of an intermediate enzyme-pyruvate complex. The rate of the forward reaction is proportional to the alanine concentration up to 0.5 m alanine, indicating that the initial combination of alanine with the enzyme is the rate-limiting step in this direction. The activation by anions must therefore involve the initial binding of the substrates to the enzyme. Chloride ions also cause a marked activation of the enzyme in the presence of glutarate by displacing the inhibitory glutarate from the enzyme. These results indicate that some enzyme activations may be due to relieving a preexisting inhibition by ligand substitution reactions. The finding that aspartate aminotransferase has an anion-sensitive “half-reaction” equilibrium, or redox potential, suggests that transaminases may function in both active and passive transport of anions across membranes.     

Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.     

Further studies on the catalytic mechanism of human liver alpha-L-fucosidase

Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) with [1-3H]conduritol C trans-epoxide revealed that there are four active sites per tetrameric enzyme complex. Solvent isotope effect experiments give evidence for a proton transfer at the rate-limiting step in catalysis. Transglycosylase activity was observed using methanol as an alternative glycone acceptor to produce methyl alpha-L-fucoside, suggesting that alpha-L-fucose is formed when water is the acceptor. Initial burst kinetics experiments suggest that a glycosyl-enzyme intermediate is formed, although the magnitude of the burst is not stoichiometric with the number of active sites. These data, along with previous results, suggest a general acid-general base catalytic mechanism involving double inversion of stereochemistry at C-1 of fucose, as well as the formation of either a covalent glycosyl-enzyme intermediate or a tight ion pair between a charged active-site residue and a hypothetical fucosyl oxocarbonium ion intermediate.     

Studies on lung tumours. III. Oxidative metabolism of dimethylnitrosamine by rodent and human lung tissue.

The oxidative metabolism of the carcinogen dimethylnitrosamine (DMN) was studied in mouse, rat, hamster and human respiratory tissue. [14C]DMN was purified by Dowex-1-bisulfite column chromatography to remove a contaminant (probably [14C]formaldehyde) interfering with the enzyme assay. Since formaldehyde and methyl carbonium ions - yielding methanol with water - are considered to be the primary products of DMN metabolism, tissue slices were assayed for the production of [14C]CO2 from 14C-labelled methanol, formaldehyde, formate, and DMN. Oxidation of formaldehyde to formate was not, but oxidation of formate to CO2 was very much rate-limiting. This rate-limiting step was circumvented by introducing quantitative chemical oxidation of formate to CO2 by mercury(II)chloride following the enzymic reaction. Since oxidation of methanol to CO2 proved to be insignificant, production of CO2 from DMN by lung tissue enzymes and HgCl2 may serve as a parameter for N-demethylating activity and the production of the suspected carcinogenically active methyl carbonium ions. The DMN-N-demethylating activities of lung tissue slices of two mouse strains with widely different susceptibilities to formation of lung adenomas by DMN differed significantly, but the difference seemed too small to explain the divergence in tumourigenic response. The enzymatic activities decreased in hamster bronchus, hamster trachea, hamster lung, GRS/A mouse lung, C3Hf/A mouse lung, human lung, Sprague-Dawley rat lung, in that order. The reported resistance of the hamster respiratory system to tumour induction by DMN may therefore not be due to poor DMN-N-demethylating capacity.     

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.     

Environmental relevance of heavy metal-substituted chlorophylls using the example of water plants

Following experiments which studied the substitution of thecentral ion of isolated chlorophylls by heavy metal ions invitro, in vivo experiments with submersed water plants werecarried out. It was discovered that the substitution of thecentral atom of chlorophyll, magnesium, by heavy metals (mercury,copper, cadmium, nickel, zinc, lead) in vivo is an importantdamage mechanism in stressed plants. This substitution preventsphotosynthetic light-harvesting in the affected chlorophyllmolecules, resulting in a breakdown of photosynthesis. The reactionvaries with light intensity. In low light irradiance all thecentral atoms of the chlorophylls are accessible to heavy metals,with heavy metal chlorophylls being formed, some of which aremuch more stable towards irradiance than Mg-chlorophyll. Consequently,plants remain green even when they are dead. In high light,however, almost all chlorophyll decays, showing that under suchconditions most of the chlorophylls are inaccessible to heavymetal ions. Key words: Heavy metal chlorophylls, submersed water plants, antenna pigments, copper, zinc     

Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate. The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively. For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates. The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used. The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting. The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent. The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate. Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate. Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair. Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant. These results are discussed in terms of a kinetic and chemical mechanism for the enzyme.     

两歧双歧杆菌胞外多糖对胃癌细胞及hTERT的影响

【目的】研究从双歧杆菌属两歧双歧杆菌(Bifidobacterium bifidum)提取的细胞表面成分胞外多糖(Exopolysaccharide, B.EPS)对人胃癌细胞BGC-823的生长抑制作用及对端粒酶限速因子hTERT活性的影响。【方法】将三种不同浓度B.EPS体外作用于胃癌细胞BGC823,MTT法检测细胞生长抑制率并辅以形态学观察;异硫氰酸盐(FITC)联合PI染色,通过流式细胞术检测肿瘤细胞初期调亡情况;肿瘤细胞端粒酶限速因子hTERT mRNA经RT-PCR检测B.EPS对端粒酶活性抑制作用;通过荧光分光光度计显示B.EPS 对胃癌细胞作用后胞内Ca2+含量变化。【结果】经过检测发现,B.EPS对人胃癌细胞BGC823的生长显著抑制(P＜0.05)呈剂量时间反应关系;细胞中hTERT mRNA在B.EPS的作用下表达降低(P＜0.05),有一定剂量效应关系;随着B.EPS对肿瘤细胞的抑制,细胞内Ca2+含量显著增加(P＜0.05)。【结论】B.EPS诱导人胃癌细胞BGC823调亡的机制可能与改变肿瘤细胞端粒酶限速因子hTERT mRNA表达量和细胞内钙离子浓度有关。