Kinetic characterization of sulphur-containing excitatory amino acid uptake in primary cultures of neurons and astrocytes

The uptake of the neuroactive sulphur amino acids -cysteine sulphinate, -cysteate, -homocysteine sulphinate and -homocysteate was investigated in astrocytes cultured from the prefrontal cortex; in neurons, cultured from cerebral cortex; and, in granule cells, cultured from cerebellum. It was shown that each amino acid acted as a substrate for a plasma membrane transporter in both neurons and astrocytes. Astrocytes and neurons exhibited a high-affinity uptake for -cysteine sulphinate and -cysteate with K_m values ranging from 14–100 μM, and a low-affinity uptake for -homocysteine sulphinate and -homocysteate, with K_m values ranging from 225–1210 μM. The uptake of all transmitter candidates studied was partially sodium-dependent. This sodium-dependency was most evident at low (< 100 μM) concentrations of each substrate. The apparent uptake measured in the absence of sodium was included as a component in corrections made for non-saturable influx. With the exception of -cysteine sulphinate, uptake of each sulphur amino acid was greatest in astrocytes, with V_max values ranging between 15–32 nmol min⁻¹ mg⁻¹ cell protein. Moreover, the uptake of each sulphur amino acid in cerebellar granule cells (V_max values ranging between 10–25 nmol min⁻¹ mg⁻¹ cell protein) was consistently greater than that in cerebral cortex neurons (V_max values ranging between 1.5–6 nmol min⁻¹ mg⁻¹ cell protein).     

A kinetic study of almond-β-glucosidase catalysed synthesis of hexyl-glycosides in low aqueous media: Influence of glycosyl donor and water activity

A variety of alkyl and aryl glycosides were investigated as substrates for almond β-glucosidase catalysed synthesis of hexyl-β- -glycosides in low aqueous hexanol media. The rate-limiting step in the organic media was determined to be the glycosylation of the enzyme. The kinetic constants V_max, K_m (glycosyl donor) and V_max/K_m were all influenced by the water activity and they all increased in value with increasing water activity. The increase in V_max/K_m was mainly determined by the increase in V_max and a plot of log(V_max/K_m) versus water activity resulted in a straight line with similar slopes for all glycosides but with different absolute values and thus the most reactive substrate p-nitrophenyl glucoside was the best one in the entire water activity range studied (0.53–0.96). The preference for the two competing acceptors, hexanol and water, was not affected by the aglycon part of the glucoside. Surprisingly, the ratio between trans glycosylation and hydrolysis increased with increasing water activity. A decrease in water activity caused an increase in equilibrium yield of hexyl glycoside, as expected, but was not beneficial for the kinetically controlled yield.     

Enzymic properties of β-mannanase from Polyporus versicolor

Substrate specificities and the kinetic parameters, K_m and V_max, of the four multiple enzyme forms of extracellular β-mannanase activity purified from Polyporus versicolor were determined. Although K_m values were significantly greater than those encountered in other β-mannanase systems V_max values were equivalent or much greater, rendering the physiological efficiencies of the β-mannanase comparable to those of other β-mannanases. All enzymes preferred glucomannan as substrate, were highly refractory at low concentrations to n-octylglucopyranoside, sodium deoxylcholate, and sodium dodecylsulfate, and were largely insensitive to methanol, ethanol, acetonitrile, and dimethylsulfoxide.     

Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution

A newly isolated enantioselective esterase from Pseudomonas fluorescens KCTC 1767, which is currently considered as a biocatalyst for the production of a commercially valuable (S)-ketoprofen, has revealed a low structural and thermal stability. In order to enhance the stability, directed evolution was attempted on this enantioselective esterase by successive steps of an error prone and staggered extension PCR. After the second round of evolution, the best mutant 6–52 with enhanced thermal stability was selected and analyzed. DNA sequence analyses of 6–52 revealed that the three amino acid residues (L120P, I208V, and T249A) were changed and the mutation L120P was presumed as a structurally important residue due to its presence in all positive variants. The purified mutant 6–52, when incubated at 50 and 55 °C for 2 h, remained its activity over 30 and 10%, respectively, whereas there were no detectable activities in wild-type enzyme. The analysis of 6–52 in the presence of 15% ethanol showed 1.8-fold increase in the activity, compared to that of wild-type enzyme. The K_m and V_max values of 6–52 were estimated to be slightly increased, leading to 1.2-fold-higher the catalytic efficacy k_cat/K_m than that of wild-type enzyme. Additionally, the mutant 6–52 was more resistant to high substrate concentrations than that of wild-type enzyme.     

Renal transport and disposition of Na-2-mercaptoethane sulfonate disulfide (Dimesna) in the rat

The transport and reduction of dimesna (NA-2-mercaptoethane sulfonate disulfide) was studied in vitro using isolated, perfused rat kidney, and isolated renal epithelial cells. Cellular uptake of dimesna was found to be dependent on an active transport mechanism working across the luminal brush border, with an app. K_m of 22 μM and V_max 1.4 nmol. 10⁶ cells⁻¹.min⁻¹. Among other low molecular thiols or disulfides reduced glutathione was the only one to exert competitive inhibition. γ-GT-activity or cellular GSH status had no influence on renal uptake of dimesna, but the intracellular reduction rate was dependent on access to reduced glutathione as a cofactor.     

Over-expression of human type I (placental) 3β-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus

Human type I placental 3β-hydroxy-5-ene-steroid dehydrogenase/steroid 5→4-ene-isomerase (3β-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3β-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3β-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3β-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3β-HSD substrate, 5-androstan-3β-o1-17-one, in the Sf-9 cell homogenate (K_m = 17.9 μM) and placental microsomes (K_m = 16.7 μM). The 3β-HSD activity (V_max = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (V_max = 9.1 nmol/min/mg). The K_m values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (K_m = 14.7 μM) and placental microsomes (K_m = 14.4 μM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (V_max = 25.7 nmol/min/mg) relative to placenta (V_max = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3β-HSD/isomerase in human placenta.     

基于FvCB模型分析盐分胁迫对棉花叶片光合作用的影响

为深入理解叶片光合特性对盐胁迫的响应机理,以棉花为试验材料,设置5个盐分(NaCl)浓度处理:0(CK)、50、100、150和200 mmol·L^-1,利用FvCB模型分析盐胁迫对棉花幼苗叶片光合特性的影响。结果表明:与CK相比,50和100 mmol·L^-1盐分处理增加了棉花叶片的最大羧化速率(V_{c max})和最大电子传递速率(J_max),但150和200 mmol·L^-1盐分处理显著降低了V_{c max}和J_max。叶片净光合速率(P_n)、叶肉导度(g_m)和暗呼吸速率(R_d)随盐分浓度升高而下降;与CK相比,50和100 mmol·L^-1盐分处理对g_m无显著影响,但P_n和R_d显著降低。150和200 mmol·L^-1盐分处理明显降低了P_n、g_m和R_d,且与0、50和100 mmol·L^-1盐分处理间存在显著差异;利用FvCB模型模拟了不同盐分胁迫下叶片净光合速率。与不考虑g_m的模拟结果相比,考虑g_m提高模拟值和实测值间的决定系数,并降低了平均绝对误差。棉花幼苗耐盐阈值为100～150 mmol·L^-1,随盐分浓度的增加,光合限制因素由叶肉因素转变为光合机构受损;引入g_m可以提高FvCB模型的模拟精度。     

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360–410 mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores.

The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C₃₄₀) which has an oxidation-reduction midpoint potential close to 340 mV (n = 1) at pH 6.0–7.6. The E_m of this component varies by 60 mV/pH unit between pH 7.6 and 9.2.

The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C₃₄₀.     

DNA replication fidelity: kinetics and thermodynamics

Mechanisms that control the fidelity of DNA replication are discussed. Data are reviewed for 3 steps in a fidelity pathway: nucleotide insertion, exonucleolytic proofreading, and extension from matched and mismatched 3′-primer termini. Fidelity mechanisms that involve predominately K_m discrimination, V_max discrimination, or a combination of the two are analyzed in the context of a simple model for fidelity. Each fidelity step is divided into 2 components, thermodynamics and kinetic. The thermodynamic component, which relates to free-energy differences between right and wrong base pair, is associated with a K_m discrimination mechanism for polymerase. The kinetic component, which represents the enzyme's ability to select bases for insertion and excision to achieve fidelity greater than that availablek from base pairing free-energy differences, is associated with a V_max discrimination mechanism for polymerase. Currently available fidelity data for nucleotide insertion and primer extension in the absence of proofreading appears to have relatively large K_m and small V_max components. An important complication can arise when analyzing data from polymerases containing an associated 3′-exonuclease activity. In the presence of proofreading, a V_max discrimination mechanisms is likely to occur, but this may be the result of two K_m discrimination mechanisms acting serially, one for nucleotide insertion and other for excision. Possible relationships between base pairing free energy differences measured in aqueous solution and those defined within the polymerase active cleft are considered in the context of the enzyme's ability to exclude water, at least partially, from the vicinity of its active site.     

Stimulation of platelet serotonin and Rb uptake by N-ethylmaleimide

The effects of N-ethylmaleimide (NEM) on mouse platelet serotonin (5-HT) and ⁸⁶Rb⁺ uptake were studied. The 5-HT transport system showed a biphasic response to increasing concentrations of NEM, with low concentrations (25–50 μM) stimulating and high concentrations (200–400 μM) inhibiting 5-HT transport. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked NEM-induced stimulation of 5-HT transport. The kinetics of 5-HT uptake indicated that NEM (50 μM) markedly increased the maximal rate of 5-HT transport (V_max control = 28.4±1.4 pmol/10⁸ platelets/4 min vs V_max NEM = 64.5±9.5 pmol/10⁸ platelets/4 min but had no significant effect on the K_m value. Platelet Na⁺ K⁺ ATPase activity was determined by measuring ⁸⁶Rb⁺ uptake. Platelet ⁸⁶Rb⁺ uptake showed a biphasic response to NEM, with low concentrations (25–100 μM) significantly stimulating and high concentrations (400 μM) inhibiting uptake. These changes in platelet ⁸⁶Rb⁺ uptake paralleled the biphasic changes in 5-HT transport. In the presence of fluoxetine, 5-HT transport was markedly inhibited but no change in the ability of NEM to stimulate ⁸⁶Rb⁺ uptake was observed. These data suggest that low concentrations of NEM activate plasma membrane Na⁺ K⁺ ATPase which results in a marked stimulation of platelet 5-HT transport.     

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10⁴. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential (E_m) of that carrier should be a function of the solution ionic strength (I). of that carrier should be a function of the solution ionic strength (I).

The E_m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E_m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases.

The E_m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E_m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane.

The E_m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.     

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF₄ than in other media tested (i.e. E=100, V ₀=0.21 mM min^-1 in BMIM·BF₄, E=2, V ₀=0.43 mM min^-1 in phosphate buffer, E=14-92, V ₀=0.22-0.25 mM min^-1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF₄ on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.     

Potential activities of androgen metabolizing enzymes in human prostate

The entire androgen metabolism of the human prostate is an integral part of the DHT mediated cellular processes, which eventually give rise to the androgen responsiveness of the prostate. Therefore, the potential activities of various androgen metabolizing enzymes were studied. Moreover, the impact of aging on the androgen metabolism and the inhibition of 5-reductase by finasteride were studied. In epithelium (E) and stroma (S) of normal (NPR) and hyperplastic human prostate (BPH), for each enzyme being involved in the conversion either of testosterone via DHT, 3- and 3β-diol to the C₁₉O₃-triols or from testosterone to androstenedione and vice versa, the amount (V_max) and Michaelis constant (K_m) were determined by Lineweaver-Burk plots. Furthermore, V_max/K_m quotients were calculated, which served as an index for the potential enzyme activity. 17 enzymes showed a mean V_max/K_m ≥ 0.10. The top four were the 5-reductases in E and S of NPR and BPH. Among those, the highest activity was found in E of NPR (1.6 ± 0.2). Moreover, in E a significant age-dependent decrease of 5-reductase activity occurred, whereas in stroma rather constant activities were found over the whole age range. Similar age-dependent alterations were found for the cellular DHT levels. Finally, the finasteride inhibition of 5-reductase (IC₅₀;nM) was stronger in E (35 ± 17) than in S (126 ± 15). In conclusion, 5-reductase is: (a) the outstanding androgen metabolizing enzyme in NPR and BPH; (b) dictating the DHT enrichment in the prostate; (c) under the impact of aging; and (d) preferentially inhibited by finasteride in E.     

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H

3β-hydroxysteroid dehydrogenase 5-ene isomerase (3βHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3βHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K_m=0.17±0.03 and 1.02 μM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V_max while K_m was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K_i was calculated from replot of Dixon's slope for each substrate concentration. K_i from the intercept and the slope of this replot were similar (0.276±0.01 and 0.263±0.02 μM) and higher than the K_m for DHEA. The K_m and K_i values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V_max while K_m values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.     

Ionic dependence of adenosine uptake by isolated nerve endings from Torpedo electric organ

Pure cholinergic synaptosomes from the electric organ of Torpedo marmorata have a saturable adenosine uptake mechanism (calculated K_m, 3 μM; V_max, 24 pmol/min/mg prot.). In this preparation, high intracellular calcium elicited by increasing external calcium concentration, potassium depolarization, sodium-calcium exchange inhibition or divalent cation ionophore A23187 action, inhibits adenosine uptake into synaptosomes. The data presented in this paper are consistent with the interpretation that high intracellular calcium participates in the regulation of the high-affinity adenosine uptake system.     

Characterization of cocaine-sensitive dopamine uptake in PC12 cells

Dopamine uptake in rat pheochromocytoma (PC12) cells is a carrier-mediated process which follows Michaelis Menten kinetics. Uptake was saturable with an apparent K_m of 0.71 μM for dopamine and a V_max of 3.2 pmol/2 × 10⁵ cells/min. The rank order of potency for various amines was norepinephrine copamine > epinephrine. Uptake increased with increasing temperature and showed a sharp break in the Arrhenius plot at 27.5 C. The Q₁₀ was 1.39 above and 2.95 below 27.5 C. Cocaine inhibited uptake in a dose-dependent manner with a K₁ of 0.97 μM. The presence of cocaine lowered the apparent K_m but did not affect the V_max, indicating competitive inhibition. Tunicamycin inhibited [³H]dopamine accumulation in a dose- and time-dependent fashion suggesting the dopamine uptake site in PC12 cells is an asparagine-linked glycoprotein. Kinetic analysis showed a decrease in V_max but not in the apparent K_m after tunicamycin treatment, consistent with the notion that tunicamycin treatment results in the loss of a substantial amount of active carrier molecules.     

氮添加对毛竹林土壤酸性磷酸单酯酶动力学参数的影响

土壤磷酸酶在有机磷矿化和磷循环过程中发挥着重要作用,然而,土壤磷酸酶响应氮(N)沉降的动力学机制仍不清楚。本研究在亚热带毛竹林中设置对照(0)、20(低氮)、40(中氮)和80 g N·hm^-2·a^-1(高氮)4种不同氮添加处理,在氮添加满3年、5年和7年时采集0～15 cm土层土壤样本,测定了土壤化学性质、微生物生物量,并分析了酸性磷酸单酯酶(ACP)的最大反应速率(V_m)、半饱和常数(K_m)和催化效率(K_a)。结果表明: 氮添加显著降低了土壤可溶性有机碳、有效磷和有机磷含量,显著增加了土壤铵态氮、硝态氮含量和V_m,且V_m与有效磷、有机磷和可溶性有机碳含量存在显著相关关系;总体上,氮添加显著提高了K_a;除了在氮添加满5年时高氮处理下K_m显著高于对照外,氮添加对K_m无显著影响,且K_m与有效磷和有机磷含量有显著负相关关系。中、高氮处理对ACP动力学参数的影响大于低氮处理。方差分解分析表明,土壤化学性质的变化而非微生物学性质的变化主导了V_m(47%)和K_m(33%)的变化。总之,氮添加显著影响了毛竹林土壤的基质有效性,通过调控ACP动力学参数(尤其是V_m)进而影响了土壤磷循环。本研究有助于了解氮素富集下土壤微生物调节土壤磷循环的潜在机制,并为全球变化下土壤磷循环模型优化提供重要参数。     

Properties of endogenous β-glucosidase of a Saccharomyces cerevisiae strain isolated from Sicilian musts and wines

Three hundred sixty-one yeast strains (80 of which ascribable to Saccharomyces cerevisiae) were isolated from Sicilian musts and wines with the purpose of looking for β-glucosidase (βG, EC 3.2.1.21) activity. Of these, the AL 41 strain had highest endogenous βG activity and was identified as belonging to the species S. cerevisiae by biochemical and molecular methods. This enzyme was subsequently characterized. It had optimum effect at pH 3.5–4.0, whilst optimum temperature was 20 °C, compatible with typical wine-cellar conditions; it was not inhibited by ethanol, at concentrations of 12–14%, or fructose and glucose. The βG was also characterised in terms of the kinetic parameters K_m (2.55 mM) and V_max (1.71 U mg⁻¹ of protein). Finally, it remained stable for at least 35 days in model solutions of must and wine.     

A model for metalloprotein-catalysed ADP, ATP transport in mitochondria

We propose a hypothetical model for the transmembrane exchange reaction catalysed by the mitochondrial adenine nucleotide carrier protein, which basically consists of an alternating reorientation of a transitory carrier—metal—nucleotide complex. The key features of the model are: the participation of an intrinsic divalent metal ion in the course of transport catalysis; the different stability constants of protonated and deprotonated nucleotide—metal complexes; the exposure and retraction of strategic arginyl residues; the alternating reorientation of the active center involving a change from the cytosolic conformation (C_c) to the matrix conformation (C_m).