Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Effects of Ischemia and Reperfusion on P2X(2) Receptor Expressing Neurons of the Rat Ileum Enteric Nervous System

Purpose We investigated the effects of ischemia/reperfusion in the intestine (I/R-i) on purine receptor P2X(2)-immunoreactive (IR) neurons of the rat ileum. Methods The superior mesenteric artery was occluded for 45 min with an atraumatic vascular clamp and animals were sacrificed 4 h later. Neurons of the myenteric and submucosal plexuses were evaluated for immunoreactivity against the P2X(2) receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT), calbindin, and calretinin. Results Following I/R-i, we observed a decrease in P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of neurons of the myenteric and submucosal plexuses. These studies also revealed an absence of calbindin-positive neurons in the I/R-i group. In addition, the colocalization of the P2X(2) receptor with NOS, ChAT, and calretinin immunoreactivity in the myenteric plexus was decreased following I/R-i. Likewise, the colocalization between P2X(2) and calretinin in neurons of the submucosal plexus was also reduced. In the I/R-i group, there was a 55.8% decrease in the density of neurons immunoreactive (IR) for the P2X(2) receptor, a 26.4% reduction in NOS-IR neuron, a 25% reduction in ChAT-IR neuron, and a 47% reduction in calretinin-IR neuron. The density of P2X(2) receptor and calretinin-IR neurons also decreased in the submucosal plexus of the I/R-i group. In the myenteric plexus, P2X(2)-IR, NOS-IR, ChAT-IR and calretinin-IR neurons were reduced in size by 50%, 49.7%, 42%, and 33%, respectively, in the I/R-i group; in the submucosal plexus, P2X(2)-IR and calretinin-IR neurons were reduced in size by 56% and 72.6%, respectively. Conclusions These data demonstrate that ischemia/reperfusion of the intestine affects the expression of the P2X(2) receptor in neurons of the myenteric and submucosal plexus, as well as density and size of neurons in this population. Our findings indicate that I/R-i induces changes in P2X(2)-IR enteric neurons that could result in alterations in intestinal motility.

NAD(P)H Oxidase Participates in the Palmitate-Induced Superoxide Production and Insulin Secretion by Rat Pancreatic Islets

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose-stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free-albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI-diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase-I (CPT-I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47(PHOX) translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up-regulated the protein content of p47(PHOX) and the mRNA levels of p22(PHOX), gp91(PHOX), p47(PHOX), proinsulin and the G protein-coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate. J. Cell. Physiol. 226: 1110-1117, 2011. (C) 2010 Wiley-Liss, Inc.

O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway

Aims Glycosylation with beta-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. Methods and results Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 mu M) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan. Conclusion We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.

Peripheral Oxidative Stress Biomarkers in Mild Cognitive Impairment and Alzheimer`s Disease

Oxidative stress has been associated with normal aging and Alzheimer`s disease (AD). However, little is known about oxidative stress in mild cognitive impairment (MCI) patients who present a high risk for developing AD. The aim of this study was to investigate plasma production of the lipid peroxidation marker, malonaldehyde (MDA) and to determine, in erythrocytes, the enzymatic antioxidant activity of catalase, glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) in 33 individuals with MCI, 29 with mild probable AD and 26 healthy aged subjects. GR/GPx activity ratio was calculated to better assess antioxidant defenses. The relationship between oxidative stress and cognitive performance was also evaluated by the Mini Mental State Examination (MMSE). AD patients showed higher MDA levels than both MCI and healthy elderly subjects. MCI subjects also exhibited higher MDA levels compared to controls. Catalase and GPx activity were similar in MCI and healthy individuals but higher in AD. GR activity was lower in MCI and AD patients than in healthy aged subjects. Additionally, GR/GPx ratio was higher in healthy aged subjects, intermediate in MCI and lower in AD patients. No differences in GST activity were detected among the groups. MMSE was negatively associated with MDA levels (r = -0.31, p = 0.028) and positively correlated with GR/GPx ratio in AD patients (r = 0.68, p < 0.001). MDA levels were also negatively correlated to GR/GPx ratio (r = -0.31, p = 0.029) in the AD group. These results suggest that high lipid peroxidation and decreased antioxidant defenses may be present early in cognitive disorders.

O-GlcNAcylation Contributes to Augmented Vascular Reactivity Induced by Endothelin 1

O-GlcNAcylation augments vascular contractile responses, and O-GlcNAc-proteins are increased in the vasculature of deoxycorticosterone-acetate salt rats. Because endothelin 1 (ET-1) plays a major role in vascular dysfunction associated with salt-sensitive forms of hypertension, we hypothesized that ET-1-induced changes in vascular contractile responses are mediated by O-GlcNAc modification of proteins. Incubation of rat aortas with ET-1 (0.1 mu mol/L) produced a time-dependent increase in O-GlcNAc levels and decreased expression of O-GlcNAc transferase and beta-N-acetylglucosaminidase, key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine vasoconstriction (maximal effect [in moles]: 19 +/- 5 versus 11 +/- 2 vehicle). ET-1 effects were not observed when vessels were previously instilled with anti-O-GlcNAc transferase antibody or after incubation with an O-GlcNAc transferase inhibitor (3-[2-adamantanylethyl]-2-[{4-chlorophenyl}azamethylene]-4-oxo-1,3-thiazaperhyd roine-6-carboxylic acid; 100 mu mol/L). Aortas from deoxycorticosterone-acetate salt rats, which exhibit increased prepro-ET-1, displayed increased contractions to phenylephrine and augmented levels of O-GlcNAc proteins. Treatment of deoxycorticosterone-acetate salt rats with an endothelin A antagonist abrogated augmented vascular levels of O-GlcNAc and prevented increased phenylephrine vasoconstriction. Aortas from rats chronically infused with low doses of ET-1 (2 pmol/kg per minute) exhibited increased O-GlcNAc proteins and enhanced phenylephrine responses (maximal effect [in moles]: 18 +/- 2 versus 10 +/- 3 control). These changes are similar to those induced by O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate, an inhibitor of beta-N-acetylglucosaminidase. Systolic blood pressure (in millimeters of mercury) was similar between control and ET-1-infused rats (117 +/- 3 versus 123 +/- 4 mm Hg; respectively). We conclude that ET-1 indeed augments O-GlcNAc levels and that this modification contributes to the vascular changes induced by this peptide. Increased vascular O-GlcNAcylation by ET-1 may represent a mechanism for hypertension-associated vascular dysfunction or other pathological conditions associated with increased levels of ET-1. (Hypertension. 2010; 55: 180-188.)

Impaired Vasodilator Activity in Deoxycorticosterone Acetate-Salt Hypertension Is Associated With Increased Protein O-GlcNAcylation

Hyperglycemia, which increases O-linked beta-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E(max) [mN] = 17.6 +/- 4 versus 10.7 +/- 2 control; n = 6) and decreased relaxation to acetylcholine (47.6 +/- 6% versus 73.2 +/- 10% control; n = 8) versus arteries from uninephrectomized rats. O- GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units = 3.8 +/- 0.3 versus 2.3 +/- 0.3 control; n = 5). PugNAc (O- GlcNAcase inhibitor; 100 mu mol/L; 24 hours) increased vascular O- GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2 +/- 2 versus 10.7 +/- 3 vehicle; n = 6), and decreased acetylcholine dilation in uninephrectomized (41.4 +/- 6 versus 73.2 +/- 3 vehicle; n = 6) but not in DOCA rats (phenylephrine, 16.5 +/- 3 versus 18.6 +/- 3 vehicle, n = 6; acetylcholine, 44.7 +/- 8 versus 47.6 +/- 7 vehicle, n = 6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) phosphorylation (P < 0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) (P < 0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc-modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O- GlcNAc transferase, glutamine: fructose-6-phosphate amidotransferase, and O- GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P < 0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O- GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension. (Hypertension. 2009; 53: 166-174.)

TICK INNATE IMMUNITY

Ticks are blood feeding parasites transmitting a wide variety of pathogens to their vertebrate hosts. The vector competence of ticks is tightly linked with their immune system. Despite its importance, our knowledge of tick innate immunity is still inadequate and the limited number of sufficiently characterized immune molecules and cellular reactions are dispersed across numerous tick species. The phagocytosis of microbes by tick hemocytes seems to be coupled with a primitive complement-like system, which possibly involves self/nonself recognition by fibrinogen-related lectins and the action of thioester-containing proteins. Ticks do not seem to possess a pro-phenoloxidase system leading to melanization and also coagulation of tick hemolymph has not been experimentally proven. They are capable of defending themselves against microbial infection with a variety of antimicrobial peptides comprising lysozymes, defensins and molecules not found in other invertebrates. Virtually nothing is known about the signaling cascades involved in the regulation of tick antimicrobial immune responses. Midgut immunity is apparently the decisive factor of tick vector competence. The gut content is a hostile environment for ingested microbes, which is mainly due to the antimicrobial activity of hemoglobin fragments generated by the digestion of the host blood as well as other antimicrobial peptides. Reactive oxygen species possibly also play an important role in the tick-pathogen interaction. The recent release of the Ixodes scapularis genome and the feasibility of RNA interference in ticks promise imminent and substantial progress in tick innate immunity research.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Notch signaling controls the balance of ciliated and secretory cell fates in developing airways

Although there is accumulated evidence of a role for Notch in the developing lung, it is still unclear how disruption of Notch signaling affects lung progenitor cell fate and differentiation events in the airway epithelium. To address this issue, we inactivated Notch signaling conditionally in the endoderm using a Shh-Cre deleter mouse line and mice carrying floxed alleles of the Pofut1 gene, which encodes an O-fucosyltransferase essential for Notch-ligand binding. We also took the same conditional approach to inactivate expression of Rbpjk, which encodes the transcriptional effector of canonical Notch signaling. Strikingly, these mutants showed an almost identical lung phenotype characterized by an absence of secretory Clara cells without evidence of cell death, and showed airways populated essentially by ciliated cells, with an increase in neuroendocrine cells. This phenotype could be further replicated in cultured wild-type lungs by disrupting Notch signaling with a gamma-secretase inhibitor. Our data suggest that Notch acts when commitment to a ciliated or non-ciliated cell fate occurs in proximal progenitors, silencing the ciliated program in the cells that will continue to expand and differentiate into secretory cells. This mechanism may be crucial to define the balance of differentiated cell profiles in different generations of the developing airways. It might also be relevant to mediate the metaplastic changes in the respiratory epithelium that occur in pathological conditions, such as asthma and chronic obstructive pulmonary disease.

Occupational airborne contamination in South Brazil: 2. Oxidative stress detected in the blood of workers of incineration of hospital residues

One of the most useful methods for elimination of solid residues of health services (SRHS) is incineration. However, it also provokes the emission of several hazardous air pollutants such as heavy metals, furans and dioxins, which produce reactive oxygen species and oxidative stress. The present study, which is parallel to an accompanied paper (Avila Jr. et al., this issue), investigated several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of vitamin E, lipoperoxidation = TBARS, reduced glutathione = GSH, oxidized glutathione = GSSG, and activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in three different groups (n = 20 each) exposed to airborne contamination associated with incineration of SRHS: workers directly (ca. 100 m from the incinerator) and indirectly exposed (residents living ca. 5 km the incineration site), and controls (non-exposed subjects). TBARS and GSSG levels were increased whilst GSH, TG and alpha-tocopherol contents were decreased in workers and residents compared to controls. Increased GST and CAT activities and decreased GPx activities were detected in exposed subjects compared to controls, while GR did not show any difference among the groups. In conclusion, subjects directly or indirectly exposed to SRHS are facing an oxidative insult and health risk regarding fly ashes contamination from SRHS incineration.

Antioxidant therapy attenuates oxidative insult caused by benzonidazole in chronic Chagas` heart disease

Chronic chagasic cardiac patients are exposed to oxidative stress that apparently contributes to disease progression. Benznidazole (BZN) is the main drug used for the treatment of chagasic patients and its action involves the generation of reactive species. 41 patients with Chagas` heart disease were selected and biomarkers of oxidative stress were measured before and after 2 months of BZN treatment (5 mg/kg/day) and the subsequent antioxidant supplementation with vitamin E (800 UI/day) and C (500 mg/day) during 6 months. Patients were classified according to the modified Los Andes clinical hemodynamic classification in groups IA, IB, II and III, and the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR), as well as the contents of reduced glutathione (GSH), thiobarbituric acid reactive species (TBARS), protein carbonyl (PC), vitamin E and C and nitric oxide (NO), myeloperoxidase (MPO) and adenosine deaminase (ADA) activities were measured in their blood. Excepting in group III, after BZN treatment SOD, CAT, GPx and GST activities as well as PC levels were enhanced while vitamin E levels were decreased in these groups. After antioxidant supplementation the activities of SOD, GPx and GR were decreased whereas PC, TBARS, NO, and GSH levels were decreased. In conclusion, BZN treatment promoted an oxidative insult in such patients while the antioxidant supplementation was able to attenuate this effect by increasing vitamin E levels, decreasing PC and TBARS levels, inhibiting SOD, GPx and GR activities as well as inflammatory markers, mainly in stages with less cardiac involvement. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Antioxidant therapy attenuates oxidative stress in the blood of subjects exposed to occupational airborne contamination from coal mining extraction and incineration of hospital residues

Coal mining and incineration of solid residues of health services (SRHS) generate several contaminants that are delivered into the environment, such as heavy metals and dioxins. These xenobiotics can lead to oxidative stress overgeneration in organisms and cause different kinds of pathologies, including cancer. In the present study the concentrations of heavy metals such as lead, copper, iron, manganese and zinc in the urine, as well as several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of lipoperoxidation = TBARS, protein carbonyls = PC, protein thiols = PT, alpha-tocopherol = AT, reduced glutathione = GSH, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of six different groups (n = 20 each) of subjects exposed to airborne contamination related to coal mining as well as incineration of solid residues of health services (SRHS) after vitamin E (800 mg/day) and vitamin C (500 mg/day) supplementation during 6 months, which were compared to the situation before the antioxidant intervention (Avila et al., Ecotoxicology 18:1150-1157, 2009; Possamai et al., Ecotoxicology 18:1158-1164, 2009). Except for the decreased manganese contents, heavy metal concentrations were elevated in all groups exposed to both sources of airborne contamination when compared to controls. TBARS and PC concentrations, which were elevated before the antioxidant intervention decreased after the antioxidant supplementation. Similarly, the contents of PC, AT and GSH, which were decreased before the antioxidant intervention, reached values near those found in controls, GPx activity was reestablished in underground miners, and SOD, CAT and GST activities were reestablished in all groups. The results showed that the oxidative stress condition detected previously to the antioxidant supplementation in both directly and indirectly subjects exposed to the airborne contamination from coal dusts and SRHS incineration, was attenuated after the antioxidant intervention.

Occupational airborne contamination in south Brazil: 1. Oxidative stress detected in the blood of coal miners

Reactive oxygen species and nitrogen species have been implicated in the pathogenesis of coal dust-induced toxicity. The present study investigated several oxidative stress biomarkers (Contents of lipoperoxidation = TBARS, reduced = GSH, oxidized = GSSG and total glutathione = TG, alpha-tocopherol, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of three different groups (n = 20 each) exposed to airborne contamination associated with coal mining activities: underground workers directly exposed, surface workers indirectly exposed, residents indirectly exposed (subjects living near the mines), and controls (non-exposed subjects). Plasma TBARS were increased and whole blood TG and GSH levels were decreased in all groups compared to controls. Plasma alpha-tocopherol contents showed approximately half the values in underground workers compared to controls. GST activity was induced in workers and also in residents at the vicinity of the mining plant, whilst CAT activity was induced only in mine workers. SOD activity was decreased in all groups examined, while GPx activity showed decreased values only in underground miners, and GR did not show any differences among the groups. The results showed that subjects directly and indirectly exposed to coal dusts face an oxidative stress condition. They also indicate that people living in the vicinity of the mine plant are in health risk regarding coal mining-related diseases.

Overexpression and Purification of the Oat (Avena Fatua) Protein AFN1 and Construction of a pMAL/AFN1 Expression Plasmid

Abscisic acid (ABA) is an important phytohormone with regulatory roles in many physiological processes. ABA expression is induced by environmental stresses such as drought and it is known to be an inhibitor of seed germination. A wild oat (Avena fatua) called AFN1 has been hypothesized to initiate the early stages of germination as its mRNA accumulates in nondormant seed embryos during imbibition. The polypeptide sequence of AFN1 suggests that it is an ABA glucosyl transferase. Glucosylation by AFN1 and thereby inactivation of ABA could lead to seed germination. In order to understand the role of AFN1 in germination, an ample quantity of AFN1 polypeptide is needed to test for enzymatic ABA glucosylase activity. My work has been to overexpress recombinant AFN1containing a (His)6 tag using a pRSETC E.coli expression system followed by Purification of the AFN1 protein by means of a nickel-affinity column that bind to the (His)6 tag. Due to the insufficient yield of AFN1 fusion protein obtained with this procedure, another method using a pMAL-c2x vector is now being employed. The pMAL expression system provides a method for expressing and purifying protein by tagging proteins with maltose-binding protein (MBP). It is anticipated that MBP tag will be advantageous as it can make the fusion protein more soluble and thereby yield a larger quantity of protein. Currently, work is underway on the construction of pMAL/AFN1 plasmid.