Induction of glyoxylate cycle enzymes in rat liver upon food starvation

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K_m(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K_m(acetyl-CoA) of 0.2 μM, a K_m(glyoxylate) of 3 mM and a pH optimum of 7.6.     

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.     

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.     

Kinetic study of sphingomyelin hydrolysis for ceramide production

Kinetic study of sphingomyelin hydrolysis catalyzed by Clostridium perfringens phospholipase C was, at the first time, conducted for ceramide production. Ceramide has the major role in maintaining the water-retaining properties of the epidermis. Hence, it is of great commercial potential in cosmetic and pharmaceutical industries such as in hair and skin care products. The enzymatic hydrolysis of sphingomyelin has been proved to be a feasible method to produce ceramide. The kinetic performance of sphingomyelin hydrolysis in the optimal two-phase (water:organic solvent) reaction system was investigated to elucidate the possible reaction mechanism and also to further improve the hydrolysis performance. Enzyme in solution had less thermal stability than the enzyme powder and the immobilized enzyme. The thermal inactivation of phospholipase C in all the three forms did not follow the first order reaction at 65 °C. The reactions for both the soluble and immobilized enzymes followed Michaelis–Menten kinetics. K_m's for the soluble and immobilized enzymes were 1.07 ± 0.32 and 1.26 ± 0.19 mM, respectively. The value of V_max was markedly decreased by the immobilization without much change in K_m, as if the immobilization functioned as the non-competitive inhibition. Ceramide as product activated the hydrolysis reaction, however, and its addition mainly caused the increase in the affinity of the enzyme–substrate complex.     

A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems

Bacterial β-ketoacyl-ACP reductase (FabG) and the β-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its β-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The K_m and k_cat values of FabG obtained for EAA were 127 mM and 0.30 s^− 1, while those of this enzyme for NADPH were 10.0 μM and 0.59 s^− 1, respectively. The corresponding K_m and k_cat values of FAS were 126 mM and 4.63 s^− 1 for EAA; 8.7 μM and 4.09 s^− 1 for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.     

P, P-bis(5′-adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl-tRNA synthetase

Bovine tryptophanyl-tRNA synthetase (TrpRS, E.C. 6.1.1.2) is unable to catalyze in vitro formation of Ap₄A in contrast to some other aminoacyl-tRNA synthetases. However, in the presence of -tryptophan, ATP-Mg²⁺ and ADP the enzyme catalyzes the Ap₃A synthesis via adenylate intermediate. Ap₃A (not Ap₄A) may serve as a substrate for TrpRS in the reaction of E·(Trp AMP) formation and in the tRNA^Trp charging. The K_m value for Ap₃A was higher than the K_m for ATP (approx. 1.00 vs. 0.22 mM) and V_max was 3 times lower than for ATP. The Zn²⁺-deficient enzyme catalyzes Ap₃A synthesis in the absence of exogenous ADP due to ATPase activity of Zn²⁺-deprived TrpRS. The inability of mammalian TrpRS to synthesize Ap₄A, might be considered as a molecular tool preventing the removal of Zn²⁺ due to chelation by Ap₄A and therefore preserving the enzyme activity.     

The Mechanism of Salt Activation of Thermolysin: Relation with Pressure Activation and Implications of Hydration Change Coupled with Rate Process

Thermolysin (E.C. 3.4.24.4) shows a remarkable increase in catalytic activity at elevated salt concentrations or hydrostatic pressures. Salt effected K_cat, only, whilst the effect of pressure was related to both K_cat, and K_m. The turnover, derived from k_cat/K_m(V), of the hydrolysis of an N-acyldipeptide amide substrate was scarcely affected by addition of salt. These results were interpreted in terms of the stabilization of increased (or exposed) charges at the transition state of the reaction.     

Partial purification and characterization of fructokinase from developing taproots of sugar beet (Beta vulgaris)

Fructokinase (FK) has been purified from developing sugar beet (Beta vulgaris L.) taproots by ion exchange chromatography and gel filtration. One major isoform was identified. The protein appears to be a dimer (M_r 38 000). Kinetically, the purified sugar beet fructokinase has a pH optimum of 8.5 and a high specificity for fructose (K_m = 0.068 mM). The enzyme can utilise a range of nucleotide triphosphates, although ATP is the most effective. Sugar beet fructokinase is inhibited by fructose concentrations in excess of 0.6 mM. Fructose-6-phosphate and Mg ADP are also inhibitory, but at relatively high concentrations. K⁺ at 10 mM stimulates activity by 30%. Fructokinase activity and the level of FK protein remain high throughout taproot development. Tissue-blots showed that high levels of FK protein were associated with conducting tissues.     

Bioconversion of phospholipids by immobilized phospholipase A2

Phospholipase A₂ selectively hydrolyses the ester linkage at the sn-2 position of phospholipids forming lysocompounds. This bioconversion has importance in biotechnology since lysophospholipids are strong bioemulsifiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A₂ from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex^® support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A₂ was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A₂ was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V_max 883.4 μmol mg⁻¹ min⁻¹ and a K_m 12.9 mM for soluble form and V_max = 306 μmol mg⁻¹ min⁻¹ and a K_m = 3.9 for immobilized phospholipase A₂) were in agreement with the immobilization effect. The results obtained with CM-Sephadex^®-phospholipase A₂ system give a good framework for the development of a continuous phospholipid bioconversion process.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase

Myo-inositol may be incorporated in the formation of phosphatidylinositol by two mechanisms. One reaction utilizes CDP-diacylglycerol and is catalyzed by phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11). The second reaction is the phosphatidylinositol: myo-inositol exchange reaction, in which a free inositol is exchanged for an existing inositol headgroup. This characterization of inositol incorporation into phosphatidylinositol in the green alga Chlamydomonas reinhardtii provides evidence for the presence of both reactions. The transferase reaction required a divalent cation and exhibited its maximum activity at 2.0 mM Mn²⁺. The optimal pH for this reaction was 8.5–9.0. The best substrate concentrations were 0.5 mM CDP-diacylglycerol and 1.2 mM myo-inositol, with an estimated K_m for myo-inositol of 0.2 mM. The exchange reaction also required Mn²⁺ for activity, but became saturated at 0.5 mM Mn²⁺. The optimal pH of the exchange reaction was 8.0, the optimal myo-inositol concentration was 0.3 mM, and the estimated K_m for myo-inositol in this reaction was 0.015 mM. Measurement of the transferase reaction in cell fractions of C. reinhardtii indicated that the activity occurred primarily in the microsomal fraction, with little or no activity in the plastids.     

Nonionic detergent-induced activation of an esterase from Bacillus megaterium 20-1

An esterase-producing Bacillus megaterium strain (20-1) was isolated from a soil sample collected in South Korea. The cloned gene showed that the esterase 20-1 composed of 310 amino acids corresponding to a molecular mass (M_r) of 34,638. Based on the M_r and the protein sequence, the esterase 20-1 belonged to the H lipase/esterase group. The optimum temperature and pH of the purified His-tagged enzyme were 20–35 °C and 8.0, respectively. The esterase 20-1 showed a ‘nonionic detergent-induced activation’ phenomenon, which was a detergent type- and concentration-dependent process. In comparison with the native enzyme, the Tween 80-treated enzyme had relatively a similar k_cat value of 274 s⁻¹ but a very low K_m value of 0.037 mM for PNPC (C₆), therefore, it showed a 14-fold increase in k_cat/K_m value.     

Phosphatidylinositol synthase of Tetrahymena: inositol isomers as substrates in phosphatidylinositol biosynthesis and headgroup exchange reactions

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 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees 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.     

Different responses of rat cerebral mitochondrial 2-oxoglutarate dehydrogenase activity to ammonia and hepatic encephalopathy in synaptic and nonsynaptic mitochondria

The effects of in vitro treatment with ammonium chloride and acute hepatic encephalopathy (HE) induced by thioacetamide treatment (TAA), on the 2-oxoglutarate dehydrogenase (OGDH) activity in synaptic and nonsynaptic mitochondria from rat brain were examined. In control conditions, V_max and K_m for 2-oxoglutaric acid (2-OG) were higher in the synaptic than in nonsynaptic mitochondria by about 45 and 55%, respectively. A particularly high sensitivity of OGDH to ammonium ions in vitro was observed in nonsynaptic mitochondria, as manifested by a 30% decrease of V_max and a 60% decrease of K_m for 2-OG. Synaptic mitochondria showed a slight response to HE which was manifested by a 12% increase of V_max. In nonsynaptic mitochondria a 19% decrease of K_m for 2-OG was observed, but V_max was unaffected. Nonsynaptic mitochondria from HE rats reacted to the addition of ammonium ions in vitro with a 30% inhibition of V_max but with no alteration of K_m for 2-OG. In synaptic mitochondria from HE rats there was a slight inhibition of V_max, but an about 15% decrease of K_m for 2-OG. Based on these results, the different responses of OGDH in two mitochondrial populations to HE and ammonium ions in vitro would appear to be due to intrinsic differences between the properties of the enzyme in the synaptic and nonsynaptic brain compartments.     

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。     

黄土高原不同植被带人工刺槐林土壤氮磷转化酶动力学参数及其温度敏感性

土壤酶是有机质降解的催化剂,其动力学特征是表征酶催化性能的重要指标,对评价土壤健康质量有重要作用。本研究选择黄土高原3种植被带下人工刺槐林土壤为对象,探讨了土壤酶动力学参数对温度变化的响应及其温度敏感性(Q₁₀)的变化特征。结果表明: 随着培养温度的升高,土壤丙氨酸转氨酶、亮氨酸氨基肽酶和碱性磷酸酶的潜在最大反应速率(V_max)和半饱和常数(K_m)均呈线性增加,且V_max呈现出森林带>森林草原带>草原带的地带性规律。V_max的温度敏感性(Q10(V_max))为1.14～1.62,K_m的温度敏感性(Q_10(Km))为1.05～1.47,且两者在森林草原带的值均低于其他植被带。在低、高温区,不同土壤酶的Q₁₀在各植被带间的变化也不尽相同。冗余分析显示,Q₁₀与环境变量尤其是土壤养分有显著的相关关系,这表明Q₁₀可能还受到除温度以外其他环境因子的影响。     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Use of 3-D computer modelling and kinetic studies to analyse grapefruit pyrophosphate-dependent phosphofructokinase

The glycolytic reaction of grapefruit PPi-dependent phosphofructokinase (PFP) depends on the presence of Fru-2,6-P₂ (K_a=6.7 nM). This molecule was further demonstrated in grapefruit juice sac cells. Citrate, -ketoglutarate and isocitrate competitively inhibited the binding of Fru-2,6-P₂ to PFP. The affinity for Fru-6-P (K_m=159 μM) and PPi (K_m=33 μM) were not affected by the addition of these molecules. In the gluconeogenic reaction, the presence of Fru-2,6-P₂ did not affect the K_m of Fru-1,6-P₂ (61 μM) in contrast to orange fruit PFP. These results led to the building of a computer model of PFP, based on the known structure of Bacillus stearothermophilus ATP-dependent phosphofructokinase (ATP-PFK). The results show that catalysis of Fru-6-P in the chain is most unlikely, due to amino-acid substitutions and that Fru-2,6-P₂ can bind between the and β subunits.     

The effect of assay composition, detergent solubilization and reconstitution on red beet (Beta vulgaris L.) plasma membrane H-ATPase kinetic properties

Modification of the salt concentration, composition and/or buffer type in the assay of plasma membrane ATPase activity caused substantial changes in the K_m and slight changes in the temperature dependence of this enzyme. The K_m and temperature dependence were also affected by detergent solubilization of the ATPase and its subsequent reconstitution into liposomes. Modulation of kinetic properties by assay composition and hydrophobic state reflect the sensitivity of the plasma membrane H⁺-ATPase to its immediate environment. This may indicate a possible regulatory mechanism for this important plant enzyme.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.