Decrease In Km Values Research Articles

Anthracene detoxification by Laccases from indigenous fungal strains Trichoderma lixii FLU1 and Talaromyces pinophilus FLU12

The persistence and ubiquity of polycyclic aromatic hydrocarbons (PAHs) in the environment necessitate effective remediation strategies. Hence, this study investigated the potential of purified Laccases, TlFLU1L and TpFLU12L, from two indigenous fungi Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12), respectively for the oxidation and detoxification of anthracene. Anthracene was degraded with vmax values of 3.51 ± 0.06 mg/L/h and 3.44 ± 0.06 mg/L/h, and Km values of 173.2 ± 0.06 mg/L and 73.3 ± 0.07 mg/L by TlFLU1L and TpFLU12L, respectively. The addition of a mediator compound 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to the reaction system significantly increased the degradation of anthracene, with up to a 2.9-fold increase in vmax value and up to threefold decrease in Km values of TlFLU1L and TpFLU12L. The GC–MS analysis of the metabolites suggests that anthracene degradation follows one new pathway unique to the ABTS system—hydroxylation and carboxylation of C-1 and C-2 position of anthracene to form 3-hydroxy-2-naphthoic acid, before undergoing dioxygenation and side chain removal to form chromone which was later converted into benzoic acid and CO2. This pathway contrasts with the common dioxygenation route observed in the free Laccase system, which is observed in the second degradation pathways. Furthermore, toxicity tests using V. parahaemolyticus and HT-22 cells, respectively, demonstrated the non-toxic nature of Laccase-ABTS-mediated metabolites. Intriguingly, analysis of the expression level of Alzheimer’s related genes in HT-22 cells exposed to degradation products revealed no induction of neurotoxicity unlike untreated cells. These findings propose a paradigm shift for bioremediation by highlighting the Laccase-ABTS system as a promising green technology due to its efficiency with the discovery of a potentially less harmful degradation pathway, and the production of non-toxic metabolites.

Immobilization of Xylanase on ZnO nanoparticles obtained by green synthesis from Eupatorium cannabinum L. and its application in enrichment of fruit juices

Juice production is one of the main components of the nutritional food industry. Therefore, improving fruit juice quality and preventing the formation of negative by-products is important for human health. The role of environmentally friendly, safe and economically more affordable green nanocarriers in improving the quality of fruit juice is increasing day by day. For this purpose, zinc oxide nanoparticles (ZnONPs) were prepared from the aqueous extract of the Eupatorium cannabinum L. using the green synthesis method. After immobilization, structural and morphological characterizations of the prepared ZnONPs and xylanase (Xyl)@ZnONPs were carried out, which confirmed that both the synthesis of ZnONPs and the immobilization of Xyl enzyme on ZnONPs were successfully carried out. The biochemical parameters of Xyl@ZnONPs such as optimum pH and temperature, kinetic parameters, thermal stability, reusability, and storage stability were investigated and compared with free Xyl. After immobilization, the optimum pH of free Xyl shifted from 5.0 to an alkaline pH of 8.0, while no change in the optimum temperature (70 °C) was observed for both enzymes. While the activation energy of free Xyl was 5.56 kJ/mol, it was found to be 7.27 kJ/mol for Xyl@ZnONPs. Xyl@ZnONPs outperformed free Xyl due to its long-term storage stability and reusability. Moreover, Xyl@ZnONPs retained 50 % of its activity after 5 reuses and 87 % of its activity after 4 weeks of storage at room temperature. In addition, a decrease in Km value and an increase in Vmax value were observed after immobilization, which means the increased catalytic activity of the Xyl@ZnONPs. As a result of re-using Xyl@ZnONPs in orange juice for 6 cycles, clarification yield of 66.43 % was obtained under optimized conditions. Overall, this study prepared a suitable, environmentally friendly, and robust carrier support for Xyl immobilization and developed a biocatalyst that will provide high performance in the industrial field.

Bioactivities and Mode of Actions of Dibutyl Phthalates and Nocardamine from Streptomyces sp. H11809.

Dibutyl phthalate (DBP) produced by Streptomyces sp. H11809 exerted inhibitory activity against human GSK-3β (Hs GSK-3β) and Plasmodium falciparum 3D7 (Pf 3D7) malaria parasites. The current study aimed to determine DBP’s plausible mode of action against Hs GSK-3β and Pf 3D7. Molecular docking analysis indicated that DBP has a higher binding affinity to the substrate-binding site (pocket 2; −6.9 kcal/mol) than the ATP-binding site (pocket 1; −6.1 kcal/mol) of Hs GSK-3β. It was suggested that the esters of DBP play a pivotal role in the inhibition of Hs GSK-3β through the formation of hydrogen bonds with Arg96/Glu97 amino acid residues in pocket 2. Subsequently, an in vitro Hs GSK-3β enzymatic assay revealed that DBP inhibits the activity of Hs GSK-3β via mixed inhibition inhibitory mechanisms, with a moderate IC50 of 2.0 µM. Furthermore, the decrease in Km value with an increasing DBP concentration suggested that DBP favors binding on free Hs GSK-3β over its substrate-bound state. However, the antimalarial mode of action of DBP remains unknown since the generation of a Pf 3D7 DBP-resistant clone was not successful. Thus, the molecular target of DBP might be indispensable for Pf survival. We also identified nocardamine as another active compound from Streptomyces sp. H11809 chloroform extract. It showed potent antimalarial activity with an IC50 of 1.5 μM, which is ~10-fold more potent than DBP, but with no effect on Hs GSK-3β. The addition of ≥12.5 µM ferric ions into the Pf culture reduced nocardamine antimalarial activity by 90% under in vitro settings. Hence, the iron-chelating ability of nocardamine was shown to starve the parasites from their iron source, eventually inhibiting their growth.

β-Galactosidase mediated synthesized nanosupport for the immobilization of same enzyme: Its stability and application in the hydrolysis of lactose

β-Galactosidase was immobilized on modified nanosilver reduced graphene oxide (Ag@rGO) nanocomposite prepared by in vitro synthesis using same enzyme. The effectiveness factor, η value of the immobilized enzyme was calculated to be 0.968, suggesting enhancement in enzyme activity after immobilization. The morphological structure of the crosslinked biopolymer was analyzed using electron microscopy and other characterization techniques. The kinetics displayed a decrease in Km value from 0.50 to 0.44 mmol L−1 while there was an increase in Vmax values from 0.031 to 0.039 μmol min−1 mL−1. The immobilized enzyme retained 85% activity after its 10th repeated use. Inhibition constant (Ki) value suggests galactose to be a more potent inhibitor of the enzyme. Despite the inhibitory potential of these hydrolysis products, the immobilized enzyme preparation retained 44.2% activity in the presence of both inhibitory sugars. The as-synthesized nanobiocatalyst was found quite effective in hydrolyzing 89% of lactose from whey. Hence, this nanobiocatalyst can be used in removing lactose from dairy waste, whey before releasing it into the water bodies. Also, the cytotoxicity and genotoxicity of Ag@rGO NC was assessed on human blood lymphocytes using flow cytometry and comet assay, respectively.

Structural and Functional Insights into Aldosterone Biosynthesis

The steroid hormone aldosterone synthesized by cytochrome P450 11B2 (CYP11B2, aldosterone synthase) regulates blood pressure and mineral homeostasis. For aldosterone synthesis, CYP11B2 requires the small iron/sulfur cluster protein adrenodoxin (Adx) which provides electrons for the reaction. Overproduction of aldosterone leads to primary aldosteronism, the most common form of secondary hypertension with very limited treatment options. Herein a structure/function approach was used to examine the role of adrenodoxin as allosteric modulator of aldosterone biosynthesis and to define the structural basis for CYP11B2 interaction with adrenodoxin. Studies revealed that adrenodoxin binding on the CYP11B2 surface increases the affinity to its physiological substrate 11-deoxycorticosterone in the buried active site up to 5.5-fold. Kinetic studies showed an increase of CYP11B2 turnover and a decrease in Km values at higher adrenodoxin concentrations. When performing inhibition studies, higher concentrations of adrenodoxin enhance the inhibitory effect of the antihypertensive drug osilodrostat. To elucidate the structural basis for aldosterone biosynthesis, an adrenodoxin/CYP11B2 fusion protein was generated to examine the protein/protein interaction. An X-ray structure of this complex with the breast cancer drug fadrozole was obtained in high resolution and identified the adrenodoxin binding site on the proximal face of CYP11B2 and specific electrostatic interactions. Overall, the studies presented here improve our understanding of the aldosterone biosynthetic system and the impact of adrenodoxin on CYP11B2 interaction with substrates and drugs. This information can be exploited for an orthogonal approach to treat primary aldosteronism and associated cardiac disease.

Application of poly(vinyl alcohol)-assisted silver nanoparticles immobilized β-keratinase composite as topical antibacterial and dehairing agent

For developing an environmentally friendly, antibacterial, dehairing topical formulation, a β-keratinase enzyme (20.1 kDa) purified from Bacillus subtilis strain RM-01 was coupled to silver nanoparticles(AgNPs) of 8.0 ± 2.0 nm dimensions, prepared using poly(vinyl alcohol) matrix. The Plackett–Burman factorial design demonstrated that the pH of the buffer, amount of silver nanoparticles (Ag NPs), and concentration of β-keratinase enzyme play the most significant role in the Ag NP-enzyme-binding process. Biophysical techniques using scanning electron microscope, transmission electron microscope, X-ray diffraction, and Fourier-transform infrared spectroscopy (FT-IR) were used to characterize the free Ag NPs as well as AgNPs immobilized by β-keratinase. Binding of β-keratinase to Ag NPs resulted in a 5.6-fold increase in specific activity, a considerable increase in Vmax with a corresponding decrease in Km values towards keratin, and a significant improvement of the thermal and storage stabilities of immobilized enzyme compared to free enzyme. The enhanced antibacterial potency, dehairing activity, and organoleptic analysis of AgNPs immobilized with β-keratinase supports the industrial application of β-keratinase-immobilized Ag NPs in the pharmaceutical and cosmetic industries.

Comparison of steroid hormone hydroxylation mediated by cytochrome P450 3A subfamilies

Hydroxylation activity at the 6β-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6β-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6β-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6β-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6β-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6β-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6β-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.

Molecular Aspects of the Interaction of Organic Solvents and Proteinase K: Kinetics and Docking Studies

Recently, the number of studies dealing with nonaqueous enzymology has increased. Proteases are enzymes that catalyze the hydrolysis of peptide bonds in other proteins. The 279-residue serine protease proteinase K (EC 3.4.21.14) from the fungus Tritirachium album limber belongs to the subtilisin family of enzymes. The enzyme is extensively applied for protein-related studies due to its stability at high temperature and ability to remain active in a wide range of pH (3.0–7.0). The effect of organic solvents (1,4-butanediol and butanol) on the activity of native proteinase K was studied by ultraviolet–visible (UV–Vis) spectroscopy and molecular docking. The results of spectroscopy show that 1,4-butanediol and butanol lead to increase in Vmax and decrease in Km values at low concentrations. Furthermore, they indicated that butanol is a slightly better enzyme activator in comparison with 1,4-butanediol. Molecular docking data illustrate interaction between organic solvents and PK, where hydrogen bonds and hydrophobic interactions play a major role in the PK-organic solvents complex formation. Therefore, we could conclude that these interactions lead to change in the native conformation of PK and increased enzyme activity.

Comparative Results in a Combined Procedure of Intrastromal Corneal Rings Implantation and Cross-linking in Patients with Keratoconus: A Retrospective Study.

IntroductionCorneal thinning disorders caused by keratoconus often lead to protrusion, irregular astigmatism, and even perforation. Keratoconus, like other corneal ectasia, does not have a known cause. Severe cases of keratoconus may require correction or restoration of tectonic integrity of the cornea by surgical means. Intracorneal ring segments are a new modality in the treatment of corneal ectactic disorders. A new technique of stopping the evolution of keratoconus and strengthening the cornea is combining techniques of intrastromal corneal ring implantation and corneal collagen cross-linking. The objective of the study is to compare the effectiveness of combined procedures: intrastromal corneal ring implantation followed by cross-linking, with cross-linking followed by intrastromal corneal ring implantation.MethodsThe study comprised two groups of patients with different evolutionary stages of keratoconus, which met the eligibility criteria for intrastromal corneal ring segment implantation and corneal collagen cross-linking. Group 1 included patients (41 eyes) who underwent intrastromal corneal ring implantation followed by cross-linking and group 2 (30 eyes) included patients who underwent cross-linking first followed by intrastromal corneal ring implantation.ResultsA decrease in Km values of about 1.5 D and refraction was observed in group 1, compared to a decrease in Km values of about 1 D and refraction in group 2. Recovery of visual acuity was higher in group 1 than group 2.ConclusionsThe sequence of intrastromal corneal ring implantation followed by cross-linking proved to be more effective in reducing Km values, spherical equivalent and cylinder compared with cross-linking followed by intrastromal corneal ring implantation.

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-Hydroxyalkanoates.

Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids; interestingly the enantiopreference toward methyl 3-hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of Vmax and a 9.6 fold decrease of Km values toward methyl 3-hydroxyhexanoate (Vmax : 2.48 U mg- and Km : 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30-fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S-enantiomer ≥99 % activity decrease and R-enantiomer >20-fold activity improvement) toward methyl 3-hydroxybutyrate.

Improvement of chitinase Pachi with nematicidal activities by random mutagenesis

Chitinase, an enzyme that can degrade the main compositions of insect intestine and cuticle, has been used in the bio-control field. Our previous work has reported the chitinase Pachi with nematicidal activity (Caenorhabditis elegans). In the present study, to improve the chitinolytic and nematicidal activities of Pachi, a random mutant library was constructed by error-prone PCR and screened by bacteriophage T7-based high-throughput screening system. One mutant, PachiN35D was obtained from about 10, 000 clones. The kinetics analysis revealed that PachiN35D exhibited a 63% decrease in Km value against chitosan, a 2.1-fold enhancement in kcat/Km value and a 1.2-fold increase in specific activity over the wild-type Pachi. Moreover, the mortality analysis against Caenorhabditis elegans showed that the 50% lethal concentration (LC50) of PachiN35D is 309.6±1.1μg/ml and a 20% increase in nematicidal activity over the wild-type Pachi (with a LC50 value of 387.3±31.7μg/ml). The structure modeling and superimposition indicated that the substitution N35D reduced the distance between substrate and substrate-binding site Asp141, finally resulting in an increase in substrate affinity, catalytic efficiency and specific activity. These results provide useful information for the study of structure-function relationship of Pachi and lay a foundation for its potential applications in agro-biotechnology.

Allosteric activation of midazolam CYP3A5 hydroxylase activity by icotinib – Enhancement by ketoconazole

Icotinib (ICO), a novel small molecule and a tyrosine kinase inhibitor, was developed and approved recently in China for non-small cell lung cancer. During screening for CYP inhibition potential in human liver microsomes (HLM), heterotropic activation toward CYP3A5 was revealed. Activation by icotinib was observed with CYP3A-mediated midazolam hydroxylase activity in HLM (∼40% over the baseline) or recombinant human CYP3A5 (rhCYP3A5) (∼70% over the baseline), but not in the other major CYPs including rhCYP3A4. When co-incubated with selective CYP3A4 inhibitor CYP3cide or monoclonal human CYP3A4 inhibitory antibody in HLM, the activation was extended to ∼60%, suggesting CYP3A5 might be the isozyme involved. Further, the relative activation was enhanced to ∼270% in rhCYP3A5 in the presence of ketoconazole. The activation was substrate and pathway dependent and observed only in the formation of 1′-OH-midazolam, and not 4-OH-midazolam, 6β-OH-testosterone, or oxidized nifedipine. The activation requires the presence of cytochrome b5 and it is only observed in the liver microsomes of dogs, monkeys, and humans, but not in rats and mice. Kinetic analyses of 1′-OH-midazolam formation showed that ICO increased the Vmax values in HLM and rhCYP3A5 with no significant changes in Km values. By adding CYP3cide with ICO to the incubation, the Vmax values increased 2-fold over the CYP3cide control. Addition of ketoconazole with ICO alone or ICO plus CYP3cide resulted in an increase in Vmax values and decrease in Km values compared to their controls. This phenomenon may be attributed to a new mechanism of CYP3A5 heterotropic activation, which warrants further investigation.

Syntheses, characterizations, crystal structures, and biological activities of two new mixed ligand Ni(II) and Cu(II) Schiff base complexes

Two mixed-ligand complexes, [Cu(L)(2imi)] (1) and [Ni(L)(2imi)]·MeOH (2) [L = 2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato) and 2imi = 2-methyl imidazole], have been prepared by the reaction of appropriate metal salts with H2L and 2-methyl imidazole. Their structures were characterized by microanalysis, FT-IR, UV–vis, molar conductivity, and 1H NMR for [Ni(L)(2imi)]·MeOH. The structures were determined using single crystal X-ray diffraction. Each four-coordinate metal center, Cu(II) in 1 and Ni(II) in 2, is surrounded by donors of Schiff base (L2−) and N of 2-methyl imidazole in square planar geometries. α-Amylase activities of these compounds have also been investigated. The experimental data showed that α-amylase was inhibited by Ni(II) complex while the Cu(II) complex causes a 1.3-fold decrease in Km value. Antimicrobial results show that these compounds, especially the Cu(II) complex, have potential for antibacterial activity against Gram negative and Gram positive bacteria and antifungal activity against Aspergillus fumigatus.

Characterization of immobilized β-galactosidase from Aspergillus niger

β-galactosidase enzyme was isolated from Aspergillus niger, and immobilized in sodiumalginate gel. The maximum activity of the free enzyme was obtained at 65oC, pH 3.5 and its notaffected by immobilization. The free enzyme had pH stability range from 3.5 to 6.5 and it wasincreased by immobilization process especially at acid pH values. The free enzyme retained90.28, 85.09, 45.49, and 19.2 % of its initial activity after incubation at 30, 40, 50, and 60oC, for60 min respectively. Thermal stability was enhanced by immobilization process. The kineticparameters for soluble and immobilized enzyme were also determined, and immobilization ledto decrease in Km value (5.12 mM for free form to 1.48 mM for immobilized form), indicatingdecreased affinity by the enzyme for its substrate. Vmax was also decreased by immobilizationprocess, and it was reached from 86.66 μmol ONP.min-1 for free enzyme to .38.02 μMONP.min-1 for immobilized form.

Mechanistic, Mutational, and Structural Evaluation of a Taxus Phenylalanine Aminomutase

The structure of a phenylalanine aminomutase (TcPAM) from Taxus canadensis has been determined at 2.4 Å resolution. The active site of the TcPAM contains the signature 4-methylidene-1H-imidazol-5(4H)-one prosthesis, observed in all catalysts of the class I lyase-like family. This catalyst isomerizes (S)-α-phenylalanine to the (R)-β-isomer by exchange of the NH2/H pair. The stereochemistry of the TcPAM reaction product is opposite of the (S)-β-tyrosine made by the mechanistically related tyrosine aminomutase (SgTAM) from Streptomyces globisporus. Since TcPAM and SgTAM share similar tertiary- and quaternary-structures and have several highly conserved aliphatic residues positioned analogously in their active sites for substrate recognition, the divergent product stereochemistries of these catalysts likely cannot be explained by differences in active site architecture. The active site of the TcPAM structure also is in complex with (E)-cinnamate; the latter functions as both a substrate and an intermediate. To account for the distinct (3R)-β-amino acid stereochemistry catalyzed by TcPAM, the cinnamate skeleton must rotate the C1-Cα and Cipso-Cβ bonds 180° in the active site prior to exchange and rebinding of the NH2/H pair to the cinnamate, an event that is not required for the corresponding acrylate intermediate in the SgTAM reaction. Moreover, the aromatic ring of the intermediate makes only one direct hydrophobic interaction with Leu-104. A L104A mutant of TcPAM demonstrated an ∼1.5-fold increase in kcat and a decrease in KM values for sterically demanding 3'-methyl-α-phenylalanine and styryl-α-alanine substrates, compared to the kinetic parameters for TcPAM. These parameters did not change significantly for the mutant with 4'-methyl-α-phenylalanine compared to those for TcPAM.

Mutant APH(2″)-IIa Enzymes with Increased Activity against Amikacin and Isepamicin

Directed evolution by random PCR mutagenesis of the gene for the aminoglycoside 2''-IIa phosphotransferase generated R92H/D268N and N196D/D268N mutant enzymes, resulting in elevated levels of resistance to amikacin and isepamicin but not to other aminoglycoside antibiotics. Increases in the activities of the mutant phosphotransferases for isepamicin are the result of decreases in K(m) values, while improved catalytic efficiency for amikacin is the result of both a decrease in K(m) values and an increase in turnover of the antibiotic. Enzymes with R92H, D268N, and D268N single amino acid substitutions did not result in elevated MICs for aminoglycosides.

THE EFFECTS OF PHOSPHITE ON PHOSPHATE STARVATION RESPONSES OF ULVA LACTUCA (ULVALES, CHLOROPHYTA)1

Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH2PO4) or Pi‐sufficient (100 μM NaH2PO4) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH2PO4 decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The Km value of Pi uptake rate was decreased by incubation with 1 μM NaH2PO4 and the decrease in Km value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions.

The comparative interaction of human and bovine serum albumins with CYP2C9 in human liver microsomes

The effect of human serum albumin (HSA), in its endogenous, free fatty acid free (FAF) and globulin free (GF) form, on the activity of CYP2C9 was studied in human liver microsomes using tolbutamide as the substrate. The widely used BSA was included to assess the differential effect of BSA and HSA. CYP2C9 activity was expressed as CL int (Vmax/Km). HSA(FAF) and BSA showed a concentration-dependent and biphasic (activation and inhibition) interaction with CYP2C9 activity. HSA(GF) and HSA exhibited an inhibitory effect, with an inhibition constant, Ki, of 19.9 μM (0.13% albumin) and 42.2 μM (0.35% albumin), respectively. Enzyme-kinetics revealed that the activation is accompanied by a decrease in Km values, while with inhibition Km values increased. A simplified method to calculate clearance, utilizing a single slope (V/S) determination based on V over the lowest linear range of [S] (designated as CL one) was assessed. Virtually identical values were obtained for CL int and CL one. The free-drug hypothesis was tested by comparing ratios of relative CL int/unbound fraction (FDH Test ratio). The FDH Test ratio for HSA was about 1, indicating that HSA binding of tolbutamide reduced the CYP2C9 activity in accord with the free-drug hypothesis. The FDH Test ratios for BSA and HSA(FAF) were 3.7 and 3.0, revealing a monophasic activation of CYP2C9. For 2%HSA(GF) the ratio of 0.3 confirmed inhibition. As revealed by their removal, free fatty acids and globulins, significantly alter the interaction of HSA with CYP2C9. In addition, HSA and BSA showed different effects on the oxidation of tolbutamide by CYP2C9.

Ascorbate peroxidase from rice seedlings: properties of enzyme isoforms, effects of stresses and protective roles of osmolytes

Two isoforms of ascorbate peroxidase, namely, APX 1 and APX 2 with subunit molecular weight of 28.5 and 34 kDa, respectively, were purified from leaves of 15-day grown rice ( Oryza sativa L.) plants. The molecular and enzymatic properties of the two isoforms differed significantly but both belonged to the class of ascorbate specific heme peroxidases and were inhibited by thiol reagents. The two isozymes were separated from each other at the DEAE-Sephacel chromatography step of purification and were named APX 1 and APX 2 in the order of elution in the chromatography. APX 2 accounted for 74% of the total activity. The Km values of APX 1 and APX 2 for ascorbate were 400 and 200 μM and those for hydrogen peroxide were 33 and 76 μM, respectively. Both isoforms had very high preference for ascorbate as electron donor. pH optima for APX 1 and APX 2 were 6.5 and 7.0, respectively. Activity of both isoforms was completely lost by heat treatment at 80 °C for 20 min or in presence of 150 mM NaCl in the assay medium. A 40% polyethylene glycol (PEG-6000) in the assay medium led to about 50% inhibition in the activity of APX 1 and 65% inhibition of APX 2 with an increase in enzyme Km values. Incorporation of 1 M proline, glycine betaine or sucrose in presence of PEG in the assay medium led to a pronounced restoration in enzyme activity with decrease in Km values. Glycine betaine appeared to be an effective protectant for both isoforms under in vitro water stressed conditions. APX 2 appeared to be more sensitive to the metals Ni, Al and As when compared to APX 1. When rice seedlings were raised in sand cultures in nutrient solution containing 1 mM Al 2(SO 4) 3, during a 5–20 day growth period about 0.5–1.3 times higher activity of APX was observed compared to the activity in controls. Similarly, rice seedlings subjected to polyethylene glycol induced water deficit for 24 h showed elevated APX activity compared to unstressed seedlings. Ascorbate peroxidase in rice seedlings appears to have an important role in detoxification of H 2O 2 under abiotic stressful conditions.

Regulation of Na+/H+ exchanger by urogastrone, a potent activator of cell proliferation

We tested the effects of epidermal growth factor (EGF) on Na+/H+ exchanger (NHE) activity using urogastrone for treatment of Wistar rats and rat kidney tissue slices. NHE activity was monitored in isolated kidney brush border membrane vesicles by following fluorescence quenching of acridine orange. A significant increase of NHE activity was detected as early as 5 min after addition of urogastrone to rat kidney slices in vitro. In Wistar rats treated with urogastrone we also found increased NHE activity (by about 12 %). Both changes of NHE activity were the result of a significant rise of Vmax value and an apparent decrease in Km value in in vitro experiments. The rise of NHE activity caused by urogastrone was sensitive to the inhibitors of transcription and translation. The presence of phosphatase inhibitor, NaF, elevated NHE activity of non-stimulated as well as of urogastrone-stimulated exchanger, suggesting that phosphorylation plays an important role in Na+/H+ exchange. Osmolarity of the medium seems to regulate NHE activity in such a manner that both hyper- and hypoosmolar conditions inhibited NHE activity. The absence of Ca2+ions produced a 60 % decrease of NHE activity. The chemical modification of histidine residues with diethyl pyrocarbonate or SH groups with N-ethylmaleimide inhibited NHE activity.