Purified Enzyme Preparation Research Articles

Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida

BackgroundAromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction.ResultsThe reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL− 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L− 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g− 1 DCW h− 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)–polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product.ConclusionsBioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.Graphical abstract

DNA Probes for Analysis the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells

The important role of DNA damage in the occurrence of various diseases, including cancer, stimulates studies of the mechanisms of genetic information stability, carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and an increased risk of diseases developing still remains relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and AP endonuclease APE1. The sensitivity of DNA probes was determined on purified enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed a significant variability in the level of enzyme activity in these cell lines. Obtained results can become the test system platform for analyzing the activity of the base excision DNA repair system in the human body.

DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells

The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.

Valorization of rice straw, sugarcane bagasse and sweet sorghum bagasse for the production of bioethanol and phenylacetylcarbinol

Open burning of agricultural residues causes numerous complications including particulate matter pollution in the air, soil degradation, global warming and many more. Since they possess bio-conversion potential, agro-industrial residues including sugarcane bagasse (SCB), rice straw (RS), corncob (CC) and sweet sorghum bagasse (SSB) were chosen for the study. Yeast strains, Candida tropicalis, C. shehatae, Saccharomyces cerevisiae, and Kluyveromyces marxianus var. marxianus were compared for their production potential of bioethanol and phenylacetylcarbinol (PAC), an intermediate in the manufacture of crucial pharmaceuticals, namely, ephedrine, and pseudoephedrine. Among the substrates and yeasts evaluated, RS cultivated with C. tropicalis produced significantly (p ≤ 0.05) higher ethanol concentration at 15.3 g L−1 after 24 h cultivation. The product per substrate yield (Yeth/s) was 0.38 g g-1 with the volumetric productivity (Qp) of 0.64 g L−1 h−1 and fermentation efficiency of 73.6% based on a theoretical yield of 0.51 g ethanol/g glucose. C. tropicalis grown in RS medium produced 0.303 U mL−1 pyruvate decarboxylase (PDC), a key enzyme that catalyzes the production of PAC, with a specific activity of 0.400 U mg−1 protein after 24 h cultivation. This present study also compared the whole cells biomass of C. tropicalis with its partially purified PDC preparation for PAC biotransformation. The whole cells C. tropicalis PDC at 1.29 U mL−1 produced an overall concentration of 62.3 mM PAC, which was 68.4% higher when compared to partially purified enzyme preparation. The results suggest that the valorization of lignocellulosic residues into bioethanol and PAC will not only aid in mitigating the environmental challenge posed by their surroundings but also has the potential to improve the bioeconomy.

The glycine N-acyltransferases, GLYAT and GLYATL1, contribute to the detoxification of isovaleryl-CoA - an in-silico and in vitro validation

Isovaleric acidemia (IVA), due to isovaleryl-CoA dehydrogenase (IVD) deficiency, results in the accumulation of isovaleryl-CoA, isovaleric acid and secondary metabolites. The increase in these metabolites decreases mitochondrial energy production and increases oxidative stress. This contributes to the neuropathological features of IVA. A general assumption in the literature exists that glycine N-acyltransferase (GLYAT) plays a role in alleviating the symptoms experienced by IVA patients through the formation of N-isovalerylglycine. GLYAT forms part of the phase II glycine conjugation pathway in the liver and detoxifies excess acyl-CoA’s namely benzoyl-CoA. However, very few studies support GLYAT as the enzyme that conjugates isovaleryl-CoA to glycine. Furthermore, GLYATL1, a paralogue of GLYAT, conjugates phenylacetyl-CoA to glutamine. Therefore, GLYATL1 might also be a candidate for the formation of N-isovalerylglycine. Based on the findings from the literature review, we proposed that GLYAT or GLYATL1 can form N-isovalerylglycine in IVA patients. To test this hypothesis, we performed an in-silico analysis to determine which enzyme is more likely to conjugate isovaleryl-CoA with glycine using AutoDock Vina. Thereafter, we performed in vitro validation using purified enzyme preparations. The in-silico and in vitro findings suggested that both enzymes could form N-isovaleryglycine albeit at lower affinities than their preferred substrates. Furthermore, an increase in glycine concentration does not result in an increase in N-isovalerylglycine formation. The results from the critical literature appraisal, in-silico, and in vitro validation, suggest the importance of further investigating the reaction kinetics and binding behaviors between these substrates and enzymes in understanding the pathophysiology of IVA.

Inter-Species Redox Coupling by Flavin Reductases and FMN-Dependent Two-Component Monooxygenases Undertaking Nucleophilic Baeyer-Villiger Biooxygenations.

Using highly purified enzyme preparations throughout, initial kinetic studies demonstrated that the isoenzymic 2,5- and 3,6-diketocamphane mono-oxygenases from Pseudomonas putida ATCC 17453 and the LuxAB luciferase from Vibrio fischeri ATCC 7744 exhibit commonality in being FMN-dependent two-component monooxygenases that promote redox coupling by the transfer of flavin reductase-generated FMNH2 by rapid free diffusion. Subsequent studies confirmed the comprehensive inter-species compatibility of both native and non-native flavin reductases with each of the tested monooxygenases. For all three monooxygenases, non-native flavin reductases from Escherichia coli ATCC 11105 and Aminobacter aminovorans ATCC 29600 were confirmed to be more efficient donators of FMNH2 than the corresponding tested native flavin reductases. Some potential practical implications of these outcomes are considered for optimising FMNH2-dependent biooxygenations of recognised practical and commercial value.

The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids

Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK β1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKβ1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKβ1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.

A comparative study on fibrinolytic enzymes extracted from six Bacillus spp. isolated from fruit-vegetable waste biomass

Fibrinolytic enzymes are considered as a significant therapeutic agent for thrombosis. Keeping the quest for identification of effective fibrinolytic enzyme(s) from microbial source, a comparative study was done on six Bacillus spp. isolated from fruit and vegetable waste. The fibrinolytic activities of the isolates were confirmed by a fibrin plate assay. Six potential isolates were identified by 16s rRNA sequencing as Bacillus altitudinus HEM05, Bacillus halotolerance ARKH1, Bacillus safensis CO05, Bacillus subtilis subsp. inaquorum BE1, Bacillus tequilensis SAS23 and Bacillus siamensis BHO27. Extracellular fibrinolytic enzymes were purified to homogeneity from all six isolates by acetone precipitation, anion exchange chromatography and gel filtration chromatography. Purified fibrinolytic enzymes showed fibrinolytic specific activity ranging from 60.92 U mg-1 to 220.19 U mg-1. Molecular weight (SDS PAGE) of purified enzymes ranged from 30,200 kDa to 69,500 kDa. Enzymes were effective over wide range of pH from 5 to 10, with optimum pH between 7 and 10. The enzymes were active between 37 °C to 60 °C. Effect of inhibitory activity of metal ions, protease inhibitors and amidolytic activity on purified enzymes indicated fibrinolytic enzymes from Bacillus altitudinus HEM05, Bacillus halotolerance ARKH1, Bacillus safensis CO05 as plasmin serine protease, from Bacillus subtilis subsp. inaquorum BE1, Bacillus tequilensis SAS23 as subtilin chymotrypsin like metalloprotease and from Bacillus siamensis BHO27 as subtilin chymotrypsin. In vitro assays for the purified enzyme preparations from all six isolates confirmed enzymes could activate plasminogen and significantly degraded the fibrin net of blood clot.

The effect of ethanol on protein-ligand interactions

The relevance of studying the regulation of protein-ligand interactions is due to the emergence of new views on the role of metabolites and their key importance in vital processes. To study the protein-ligand interaction, the AB0 antigen-antibody blood system and the enzyme-substrate system of dehydrogenases were used as a test system, and ethanol was used as an influencing factor. In experiments performed with A and B blood erythrocyte antigens, natural AB0 system antibodies and monoclonal antibodies under the influence of ethanol performed change of the degree of agglutination and the time to onset of erythrocyte agglutination. It was found that ethanol can regulate the enzyme-substrate interactions of dehydrogenases: lactate dehydrogenase (EC 1.1.1.27), glyceraldehyde phosphate dehydrogenase (EC 1.1.1.12), and α-glycerol phosphate dehydrogenase (EC 1.1.1.8). The increase in the activity of studied enzymes under the influence of ethanol in the whole blood hemolysate was 2.5 - 3 times higher than in the isolated medium (with pure enzyme preparations).

Partial purification and characterization of fatty acid esterase from pearl millet

Aim: The present study aimed to identify a negative modulator of lipolytic enzyme fatty acid esterase (FAE) for exploring possibilities of increasing shelf life of pearl millet flour through arresting in-situ hydrolysis of lipids. Methodology: FAE was partially purified from flour of pearl millet hybrid HHB 234 by (NH4)2SO4 fractionation (30-60% saturation) and gel filtration chromatography using Sephadex G-75. The enzyme was characterized for physico-chemical properties viz., molecular mass, optimum pH, optimum temperature and thermal stability and kinetic properties viz., Km value and effect of modulators. Results: Crude extract contained 1008 units of activity and 421 mg proteins resulting in to specific activity of 2.4 units mg-1 protein. The enzyme was purified 10.7 fold with a recovery of 21.52% and specific activity of 25.7 units mg-1 protein by ammonium sulphate fractionation followed by gel filtration. The molecular weight of purified enzyme preparation was 60 kDa, as determined by gel filtration through Sephadex G-75. The enzyme exhibited maximum activity at pH 8.2 and 45°C. The enzyme was stable up to 60°C for 20 min and showed Km value of 0.65 µM for p-NPB. At 10 mM concentration, Mg2+ and Zn2+ altered the activity positively by 54% and negatively by 42% whereas EDTA, DTT, PMSF, ascorbic acid inhibited the activity by 75, 68, 50 and 48%, respectively. Interpretation: Partially purified lipolytic enzyme FAE from pearl millet flour was strongly inhibited by ascorbic acid. This novel information might be useful in developing processing technologies for improving shelf life of flour. Key words: Characterization, Fatty acid esterase, Pearl millet, Purification, Shelf life

D-Aspartate N-methyltransferase catalyzes biosynthesis of N-methyl-d-aspartate (NMDA), a well-known selective agonist of the NMDA receptor, in mice

N-Methyl-d-aspartate (NMDA), which is a selective agonist for the NMDA receptor, has recently been shown to be present in various biological tissues. In mammals, the activity of d-aspartate N-methyltransferase (DDNMT), which produces NMDA from d-aspartate, has been detected only in homogenates prepared from rat tissues. Moreover, the enzymatic properties of DDNMT have been poorly studied and its molecular entity has not yet been identified. In this report, we show for the first time that the activity of DDNMT is present in mouse tissues and succeed in obtaining a partially purified enzyme preparation from a mouse tissue homogenate with a purification fold of 1900 or more, and have characterized the enzymatic activity of this preparation. The results indicate that DDNMT, which is highly specific for d-aspartate and is S-adenosyl-l-methionine-dependent, is a novel enzyme that clearly differs from the known methylamine-glutamate N-methyltransferase (EC 2.1.1.21) and glycine N-methyltransferase (EC 2.1.1.20).

Humic Acid Transformation by the Fungus Cerrena unicolor Growing on Cellulose and Glucose

Microbial degradation of lignocellulose and soil organic matter is an important process controlling CO2 flow into the atmosphere, which is mostly carried out by fungi. The alkali-soluble fraction, which is represented mostly by the so-called humic acids (HA), dark-colored compounds of phenolic nature, constitute ~30‒70% of soil Corg. This is the first report on efficient degradation of soil HA under cellulolytic conditions; cellobiose dehydrogenase (CDH), an oxidative enzyme of the cellulolytic complex, was shown to play the key role in this process. Growth of a wood-decomposing fungus Cerrena unicolor on cellulose (CDH and laccase production) and glucose (laccase production) resulted in HA decolorization by 60 and 20%, respectively. HA depolymerization in the presence of CDH and its polymerization in the presence of laccase were shown by gel filtration experiments with the culture liquid and pure enzyme preparations. HA degradation in the presence of CDH was inhibited by radical scavengers, indicating the radical nature of this process (probably the Fenton’s reaction). HA depolymerization by CDH decreased in the presence of laccase, while HA polymerization by laccase decreased in the presence of CDH. Thus, the interaction of these enzymes affects HA transformation under cellulolytic conditions. These results improve our understanding of the mechanisms of microbial degradation of the aromatic components of soil humus and of the ecological functions of wood-decomposing fungi grown under cellulolytic conditions.

Molecular Imaging of Inflammation in Osteoarthritis Using a Water-Soluble Fluorocoxib.

Clinical imaging approaches to detect inflammatory biomarkers, such as cyclooxygenase-2 (COX-2), may facilitate the diagnosis and therapy of inflammatory diseases. To this end, we report the discovery of N-[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide chloride salt (fluorocoxib D), a hydrophilic analog of fluorocoxib A. Fluorocoxib D inhibits COX-2 selectively in purified enzyme preparations and cells. It exhibits adequate photophysical properties to enable detection of COX-2 in intact cells, in a mouse model of carrageenan-induced acute footpad inflammation and inflammation in a mouse model of osteoarthritis. COX-2-selectivity was verified either by blocking the enzyme's active site with celecoxib or by molecular imaging with nontargeted 5-carboxy-X-rhodamine dye. These data indicate that fluorocoxib D is an ideal candidate for early detection of inflammatory or neoplastic lesions expressing elevated levels of COX-2.

Biochemical and molecular characterization of lactase producing bacterium isolated from dairy effluent

In the present study, the microbial source for potent lactase producers was explored to supplement the lactase intolerant individuals. Dairy effluent was screened for lactase producing bacteria by conventional microbiological methods. Among the positive isolates, one isolate VUVD001 was found to be a strong producer of lactase enzyme and this strain identified by 16S rDNA analysis. The lactase producing bacterium is identified as Bacillus subtilis by biochemical and 16S rDNA analysis. The VUVD001 strain found to survive in a temperature range of 20–55 °C, pH range of 5–8 and a salt concentration up to 8%. Further, the partially purified enzyme preparation was tested by zymogram analysis for activity testing using x-gal and the enzyme activity of cell free supernatant was estimated to be 15.10 U/ml at 37 °C, 4% NaCl and pH of 7.0. Our finding reports a new isolate Bacillus subtilis VUVD001 strain which can be used as potential strain for commercial production of lactase.

A Simple In Vitro Assay to Measure the Activity of Geranylgeranyl Diphosphate Synthase and Other Short-Chain Prenyltransferases.

Most carotenoids are C40 metabolites produced from C20 geranylgeranyl diphosphate (GGPP). The enzymes that produce this precursor, GGPP synthases (GGPPS), are members of the short-chain prenyltransferase (SC-PT) family. SC-PTs are enzymes that catalyze the sequential head-to-tail addition of one or more C5 molecules of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) with the concomitant release of pyrophosphate (PPi). SC-PTs produce linear isoprenyl diphosphates of up to C20 (GGPP) that serve as precursors for many groups of isoprenoids with a wide range of essential biological functions in Eucarya, Bacteria, and Archaea. Enzymatic analysis of SC-PT activity normally requires complex, laborious and expensive methods such as radioactivity-based assays or liquid chromatography-mass spectrometry (LC-MS). Here we describe a fast and inexpensive spectrophotometric protocol for determining the kinetic parameters of SC-PTs in purified enzyme preparations, using an adapted assay for PPi quantification. We developed the method using the Arabidopsis thaliana GGPPS11 enzyme, which produces geranylgeranyl diphosphate for the synthesis of carotenoids in the chloroplast.

Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations.

Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3-1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51-120%. In circular dichroism measurement, the melting temperatures of variants were 50-53°C, lower than that of WT (56°C). These results suggested that A-G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2.

Construction of a strain-producer of the chimeric protein consisting of RNA polymerase and a DNA-affinity domain

One of the recent perspective trends of molecular biotechnology is cell-free synthesis of protein. The procedure of cell-free synthesis of protein is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes. Procreation of the transcription stage requires participation of specific RNA polymerase which initiates process of mRNA synthesis from the particular sites of recognition. Often the DNA-dependent RNA polymerase of a bacteriophage of T7 (T7 RNA polymerase) is for this purpose applied. For improvement of qualitative characteristics of the T7 RNA polymerase in the real work the new strain of Escherichia coli producing this enzyme fused with the DNA-affine Sso7d domain of a thermophilic bacterium Sulfolobus solfataricus is created. The producing ability of the received recombinant strain concerning synthesized chimera protein reaches 625 un/l of cultural liquid, and the specific activity of the purified enzyme preparation was 80 un/ μg of protein. The received enzyme is intended for use as tools at synthesis of proteins in cell-free system.

Dual character of GABA action on Cl–-transport by the reconstituted Cl–/-ATPase from rat brain

The Cl–/-ATPase from the plasma membranes of animal brains is an ATP-consuming Cl–-transporting ATPase P-type that is structurally coupled to GABAA receptors. The aim of work was to study the GABA effect on Cl– transport across liposomal membranes by the reconstituted ATPase under various conditions (i.e. in the absence and in the presence of ATP in the incubation medium). We reconstituted the affinity-purified enzyme in the liposomes with a fluorescent dye for Cl–. The Cl–-transport in proteoliposomes was evaluated from variations in fluorescence. Native-PAGE of the purified enzyme preparation, as well as western blot analysis with an antibody against the GABAAR β3 subunit, showed one band with a molecular mass of 300 kDa. The addition of GABA (100 μM) quickly resulted (1–3 s) in an increase in the Cl– influx into proteoliposomes. The application of ATP (3 mM) resulted in a gradual increase in the Cl– inflow into the proteoliposomes at 0.5–4 min. Vanadate (10 μM) did not change the GABA-induced Cl– inflow into the liposomes, but completely inhibited the ATP-dependent Cl– input. Under conditions where Cl– was previously loaded into proteoliposomes by ATP-dependent Cl– input, the addition of GABA to the incubation medium caused a short-lasting Cl– efflux from the proteoliposomes. However, an additional long-time incubation of these proteoliposomes resulted in the restoration of Cl– accumulation. Pentobarbital, propofol, or diazepam elevate and picrotoxin or furosemide eliminate the effect of GABA on the Cl–-transport processes. These findings indicate a dual nature of GABA action on the Cl–/-ATPase.

Effect of phenol on the GABAAR-coupled Cl−/HCO3−-ATPase from fish brain: An in vitro approach on the enzyme function

Phenol (C6H5OH) has a toxic effect on the central nervous system of animals and humans. The Cl−/HCO3−-ATPase from the plasma membranes of animal brains is the primary active P-type Cl−-transporting system that is coupled to GABAA receptor (GABAAR). In this paper, we used an in vitro approach to assess the effects of phenol (1–500μM) on the functional parameters of the Cl−/HCO3−-ATPase isolated from the fish brain. The enzyme is insensitive to phenol in the presence of Cl− or HCO3− in the incubation medium. By contrast, in the presence of Cl−/HCO3−, phenol inhibits (I50=27μM) both the enzyme activity and its participation in ATP-dependent Cl− transport through the membranes of artificial liposomes. Enriched plasma membranes and purified enzyme preparations were separated using hrCNE-PAGE. The ATPase activity in native gels was detected in the presence of phenol (100μM). Detection of ATPase activity in a purified preparation, showed a native protein of 300kDa, in agreement with western blot analysis with antibodies against GABAAR β3 subunits. SDS-PAGE showed that one subunit with a molecular weight of 56kDa was directly phosphorylated by γ-32P–ATP and dephosphorylated in the presence of phenol. The in vitro approach described in this work allowed the first demonstration that GABAAR-coupled Cl−/HCO3−-ATPase can be a protein marker for assessment of the toxicity of phenolics on the central nervous system.

Characterization of the functional interactions of plastidial starch phosphorylase and starch branching enzymes from rice endosperm during reserve starch biosynthesis

Functional interactions of plastidial phosphorylase (Pho1) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Pho1 and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Pho1. Based on our in vitro data, in the developing rice endosperm, Pho1 appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Pho1. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Pho1 performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Pho1 is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis.