Biomedical subjects

S N Sarkar

Publications and source records attributed to S N Sarkar.

At least 19 recordsLinked to original sources

PubMed2006

Induction of oxidative stress in erythrocytes of male rats subchronically exposed to a mixture of eight metals found as groundwater contaminants in different parts of India.

Exposure of animals and humans to different metal components through contaminated drinking water can result in a wide range of adverse clinical conditions. Toxicological consequences arising from the concurrent repeated exposure to multiple metal contaminants are not known. The purpose of the present study was to evaluate the oxidative stress-inducing potential of a mixture of eight metals (arsenic, cadmium, lead, mercury, chromium, nickel, manganese, iron), representative of groundwater contamination in different areas of India, in erythrocytes of male rats subchronically exposed to environmentally relevant doses via drinking water. The selection of these metals, as determined by literature survey of groundwater contamination in India, was primarily based on the frequency of their occurrence and contamination level above World Health Organization maximum permissible limit (MPL) in drinking water. Male albino Wistar rats were exposed to the metal mixture at 0, 1, 10, and 100 times the mode concentrations (the most frequently occurring concentration) of the individual metals in drinking water for 90 days. In addition, one group of rats was also exposed to the mixture at a concentration equal to the MPL of individual components. The oxidative stress in erythrocytes was evaluated by assessing the magnitude of malondialdehyde production and reduced glutathione (GSH) content and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) after 30, 60, and 90 days of exposure. MPL and 1x dose levels did not cause any changes. The mixture at 10x and 100x doses caused dose- and time-dependent effects. After 30 days, the 10x dose did not cause any changes except increase in SOD activity. The 100x dose increased the activities of SOD, catalase and GR and the GSH level, but caused no alterations in lipid peroxidation (LPO) and GPx activity. After 60 days, the 10x dose did not cause any changes. The 100x dose increased LPO and decreased all the antioxidant parameters, except GSH. After 90 days, both 10x and 100x levels elevated LPO. The 10x dose decreased GSH level and activities of SOD and catalase, but not of GPx and GR, whereas the 100x dose decreased all the antioxidative systems. Overall, the present study demonstrates that the subchronic exposure of male rats to the mixture of metals via drinking water results in induction of oxidative stress and concomitant reduction in antioxidative defense system in erythrocytes at 10 and 100 times the mode concentrations of the individual metals in contaminated groundwater.

Administration, Oral↗

PubMed2006

Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine.

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.

Animals↗

PubMed2006

Cytogenetic effects of a mixture of selected metals following subchronic exposure through drinking water in male rats.

The current study examines the genotoxic effects of subchronic exposure via drinking water to a mixture of eight metals (arsenic, cadmium, lead, mercury, chromium, nickel, manganese and iron) found as contaminants of water sources in different parts of India and its possible association with oxidative stress. Male rats were exposed to the mixture at 0, 1, 10 and 100 times the mode concentration of each metal daily for 90 days. Another dose group at concentration equivalent to maximum permissible limit (MPL) for each metal and a reference group given ip cyclophosphamide were incorporated. The mixture at 100x level significantly increased chromosomal aberrations and micronuclei induction (2.4 folds) in bone marrow cells and reduced the ratio of polychromatic to normochromatic erythrocytes by 25%. The mixture significantly increased sister chromatid exchange in bone marrow (1.67 and 2.3 folds) and spleen (1.57 and 1.98 folds) cells with both 10x and 100x doses. Cyclophosphamide was more potent than the mixture in causing cytogenetic damage in these parameters. In rat spleen, the mixture at 10x and 100x doses caused dose-dependent increase in lipid peroxidation (25.95 and 52.71%) and decrease in the activities of superoxide dismutase (20.36 and 40.62%), catalase (18.24 and 35.50%), glutathione peroxidase (22.33 and 36.12%) and glutathione reductase (19.22 and 31.35%) and in the level of GSH (19.76 and 35.15%). The results suggest that the mixture induced genotoxicity in rat bone marrow and spleen cells at concentrations relatively higher than that found in groundwater sources and the genotoxic effect could relate to induction of oxidative stress. However, observations with lower doses indicate that additive or synergistic interactions following exposure to metal components at MPL levels or at mode concentrations of contemporary groundwater levels in India may not result in clastogenicity in male rats.

Animals↗

PubMed2005

Effects of calcium channel blocker, mibefradil, and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium.

The present study was undertaken to investigate the in vitro influence of mibefradil, a calcium channel blocker, and pinacidil, a potassium channel opener, on pregnant goat myometrial spontaneous rhythmic contractility and contractions induced with the agonist, oxytocin. Longitudinal strips from the distal region of uterus, collected from goats at midgestation, were mounted in an organ bath for recording isometric contractions. Mibefradil (10(-8)-10(-4) M) or pinacidil (10(-10)-10(-4) M), added cumulatively to the bath at an increment of 1 log unit, caused concentration-dependent inhibition of the spontaneous rhythmic contractions of isolated uterine strips. The rhythmic contraction was, respectively, abolished at 100 and 10 microM concentrations of mibefradil and pinacidil. In a concentration-dependent manner, mibefradil (1 and 10 microM) antagonized the contractions elicited with oxytocin (10(-5)-10(-2) IU). Pretreatment of uterine strips with glibenclamide (10 microM), a selective KATP channel blocker, caused a rightward shift of the concentration-response curve of pinacidil with a concomitant decrease in its pD2 value. Pinacidil (0.3, 1 and 3 microM), in a concentration-related manner, antagonized the oxytocin (10(-5)-10(-2) IU)-induced contractile response. The inhibition of spontaneous rhythmic contractions and antagonism of oxytocin-induced contraction by mibefradil in the pregnant goat myometrium may be related to the antagonism of voltage-dependent Ca2+ channels, while by pinacidil suggests that KATP channel could be a therapeutic target for tocolysis.

Adenosine Triphosphate↗

PubMed2003

Influence of malathion pretreatment on the toxicity of anilofos in male rats: a biochemical interaction study.

Toxicity of organophosphates stems mainly from the accumulation of acetylcholine due to inhibition of acetylcholinesterase (AChE). The consequences of excess acetylcholine depend on the events initiated by the interaction of acetylcholine with cholinergic receptors. Lipid peroxidation (LPO) induced by organophosphates also seems to be mediated via cholinergic receptors. Anilofos is a widely used thionoorganophosphate herbicide, while malathion is a thionoorganophosphate insecticide. Thionoorganophosphates undergo mixed function oxidase (MFO)-catalyzed bioactivation to oxons and can induce cholinergic crisis in mammals. Thus, factors (e.g. exposure to certain xenobiotics) which alter the MFO activity, can be assumed to affect the toxicity of these organophosphates. It was investigated in rats if malathion as an inhibitor of MFO can alter the toxicity of anilofos, examining certain biochemical traits in blood, brain and liver. Malathion or anilofos and their combination did not produce any obvious signs of toxicity. Malathion did not alter the anticholinesterase action of anilofos in blood, brain and liver. LPO was increased in erythrocytes, brain and liver with anilofos or malathion and their combination. Production of lipid peroxide in brain of malathion-pretreated rats given anilofos was significantly greater than in rats given anilofos alone. Malathion decreased glutathione (GSH) contents of liver and blood. Glutathione-S-transferase (GST) activity was decreased in the liver with malathion and its combination with anilofos. Total adenosine triphosphatase (ATPase) activity was not affected. Activities of Mg(2+)-ATPase and Na(+)-K(+)-ATPase were increased in the liver and erythrocytes, respectively, with the pesticide combination. Protein level in plasma was decreased with malathion and its combination with anilofos, but only with the combination in the liver. Results of the study indicate that malathion pretreament may not essentially alter the anticholinesterase action of anilofos, but may enhance anilofos-mediated oxidative damage to rat brain.

Adenosine Triphosphatases↗

PubMed2001

Effect of isoproturon pretreatment on the biochemical toxicodynamics of anilofos in male rats.

Anilofos and isoproturon are important herbicides of organophosphorus and substituted phenylurea groups, respectively. Isoproturon is an inducer of hepatic drug-metabolizing enzymes. Animals and humans have the potential to be exposed to the mixture of these intentionally introduced environmental xenobiotics, but toxicological interactions between these herbicides are not known. Effects of isoproturon pretreatment (675 mg/kg/day for 3 consecutive days) on the toxic actions of anilofos administered orally as a single dose (850 mg/kg) were evaluated by determining some biochemical attributes in blood (erythrocyte/plasma), brain and liver of rats. Anilofos or isoproturon alone or in combination failed to produce any noticeable signs of cholinergic hyperactivity and behavioural alterations. Isoproturon did not potentiate the anticholinesterase action of anilofos in blood and liver. Inhibition of brain acetylcholinesterase was significantly protected. No significant alteration in anilofos-mediated production of lipid peroxidation was observed in erythrocyte and brain of isoproturon-pretreated rats, but it was significantly increased in liver. Anilofos did not affect GSH and GST. The isoproturon-mediated increase in GSH levels of brain (threefold) and liver (3.6-fold) was also not affected following combined administration. GST activity was increased in liver of rats given isoproturon alone (fourfold) or in combination with anilofos (2.8-fold). Activities of total ATPase, Mg2+-ATPase and Na+-K+-ATPase were not affected in rats given either anilofos alone or herbicides in sequence. With these treatments, there were no alterations in the protein content of plasma, brain and liver. Overall findings of the study indicate that isoproturon pretreatment does not alter the toxicity of anilofos, the GSH-GST metabolic pathway may not have a significant implication in the detoxification of anilofos and the production of a reactive oxygen species may be a factor in mediating anilofos toxicity.

Adenosine Triphosphatases↗

PubMed2001

A specific isozyme of 2'-5' oligoadenylate synthetase is a dual function proapoptotic protein of the Bcl-2 family.

2-5(A) synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5' linked oligoadenylates that activate RNase L and cause mRNA degradation. Because they all can synthesize 2-5(A), the reason for the existence of so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes apoptosis in mammalian cells. The proapoptotic activity of 9-2 was isozyme-specific and enzyme activity-independent. The 9-2-expressing cells exhibited many properties of cells undergoing apoptosis, such as DNA fragmentation, caspase activation, and poly ADP-ribose polymerase and lamin B cleavage. The isozyme-specific carboxyl-terminal tail of the 9-2 protein was shown, by molecular modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that it may be able to interact with members of the Bcl-2 family that contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal microscopy showed that 9-2 can indeed interact with the anti-apoptotic proteins Bcl-2 and Bclx(L) in vivo and in vitro. Mutations in the BH3 domain that eliminated the 9-2-Bcl-2 amd 9-2-Bclx(L) interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an interferon-induced dual function protein of the Bcl-2 family that can synthesize 2-5(A) and promote cellular apoptosis independently. Moreover, the cellular abundance of this protein is regulated by alternative splicing; the other isozymes encoded by the same gene are not proapoptotic.

2',5'-Oligoadenylate Synthetase↗

PubMed2001

Subacute toxicity of anilofos, a new organophosphorus herbicide, in male rats: effect on some physical attributes and acetylcholinesterase activity.

In acute toxicity study, rats showed dose-dependent signs of cholinergic hyperactivity and behavioural alterations. Maximum intensity of symptoms was not associated with mortality. Oral LD50 was 1681 mg/kg. In subacute toxicity study, rats were orally administered 50, 100 or 200 mg/kg of anilofos once daily for 28 days. Signs and symptoms were observed mainly with 200mg/kg. At this dose, anilofos induced hypothermia and progressive weight loss. None of the anilofos-treated rats died. Weight of brain, lung, testis was not altered, while of liver, heart, spleen and kidney increased. Anilofos inhibited cholinesterase (ChE) activities of erythrocyte (41-67%), plasma (36%), blood (37-64%), brain (63-73%) and liver (28-48%). Total protein was decreased in plasma and liver. Results indicate moderate toxic potential of anilofos in mammals, substantial contribution of CNS-mediated effects in causing anilofos toxicity and no direct relationship between hypothermia and level of ChE inhibition.

Acetylcholinesterase↗

PubMed2001

Subacute toxicity of anilofos, a new organophosphorus herbicide in male rats: effect on lipid peroxidation and ATPase activity.

Effects of anilofos on lipid peroxidation--an index of oxidative stress, ATPase activity--an integral part of active transport mechanisms for cations, GSH level and GST activity were evaluated in blood (erythrocyte/plasma), brain and liver of male rats after daily oral exposure to 50, 100 or 200 mg/kg for 28 days. None of the doses increased lipid peroxidation. The lowest dose, rather, produced marginally significant decrease in peroxidation in liver. Different doses of anilofos decreased GSH content and activities of GST and ATPases. Inhibition of total ATPase (34-44%) and Na+-K+-ATPase (45-52%) activities was maximum in liver, while that of Mg2+-ATPase (46-56%) was more in erythrocyte. Results indicate that anilofos may not cause oxidative damage to cell membrane in repeatedly exposed animals and may cause neuronal/cellular dysfunction by affecting ionic transport across cell membrane.

Adenosine Triphosphatases↗

PubMed2000

Cell growth regulatory and antiviral effects of the P69 isozyme of 2-5 (A) synthetase.

Among the many interferon-induced proteins that carry out multiple cellular functions of interferons is the family of enzymes called 2'-5' oligoadenylate synthetases. We examined the anticellular and antiviral activities of a specific member of that family, the P69 isozyme. P69 was expressed in human cells by transfection and shown to be localized primarily in the endoplasmic reticulum. For further studies, permanent cell lines expressing different levels of P69 or an enzymatically inactive mutant were isolated. Constitutive P69 expression caused inhibition of replication of encephalomyocarditis virus but not of vesicular stomatitis virus, Sendai virus, or reovirus. Increasing levels of P69 expression also caused increasing perturbations in cell growth properties. There was increasing accumulations of the P69-expressing cells in the G1 phase of the cell cycle; cell-doubling time was increased by P69 expression; and there were many multinucleated cells in the P69-expressing line, indicating a defect in cytokinesis.

2',5'-Oligoadenylate Synthetase↗

PubMed1999

The nature of the catalytic domain of 2'-5'-oligoadenylate synthetases.

2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol beta, P69 can assume an alphabetabetaalphabetabetabeta structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes. Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.

2',5'-Oligoadenylate Synthetase↗

PubMed1999

Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase.

P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2'-5'-linked oligoadenylate products containing up to 30 residues. 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.

2',5'-Oligoadenylate Synthetase↗

PubMed1998

Excess nucleoside triphosphates (or zinc) allow recovery of alkaline phosphatase activity following refolding under reducing conditions.

The contribution of ATP and other nucleotides to the stabilization of non-native structures has been described for some proteins. We report here the effect of GTP, ATP, and their nonhydrolyzable analogues on the denaturation and renaturation of the enzyme Escherichia coli alkaline phosphatase. We show that GTP, ATP, and their nonhydrolyzable analogues considerably stimulate renaturation of AP in the presence of 2-mercaptoethanol where spontaneous renaturation is completely arrested due to reduction of S-S bonds. GTP is the most efficient inducer of reconstitution of the active site and appears to play a specific role besides being a substrate. The reconstituted protein was found to be in the reduced form despite having near-normal activity. The self-refolded oxidized form and the GTP-refolded reduced form had the same KM/kcat values and showed similar structural properties. We conclude that GTP can not only induce reconstitution of dimerization-competent monomers because of its substrate nature but also act as a modulator of the activity of AP. We also report here on the Zn2+-assisted reconstitution of E. coli AP under reducing condition. The prior formation of a disulfide bond for positioning the active site residues in the proper geometry is unnecessary under this condition.

Adenosine Triphosphate↗

PubMed1998

Production and purification of recombinant 2'-5' oligoadenylate synthetase and its mutants using the baculovirus system.

Investigation of the structure-function relationship of the 2'-5' oligoadenylate [2-5 (A)] synthetases has been hampered by the lack of an efficient expression system for a recombinant enzyme. Here, we report that the 9-2 isozyme of murine 2-5 (A) synthetase can be efficiently expressed in insect cells using the baculovirus system. The recombinant protein was purified to apparent homogeneity, and its enzymatic activity was characterized. It had a high specific activity, required double-stranded RNA as a cofactor, and synthesized dimers to hexamers of 2-5 (A). The utility of our expression system was demonstrated by studying the properties of two previously reported mutant proteins. Both of these mutants, when produced in bacteria, are enzymatically inactive, although similarly produced wild-type protein is active. Unexpectedly, when expressed in insect cells, both mutant proteins were enzymatically as active as the wild-type protein. These results suggest that in the eukaryotic expression system described here, the mutant proteins can undergo appropriate modifications or folding that is required for attaining an enzymatically active conformation.

2',5'-Oligoadenylate Synthetase↗

PubMed1998

Production, purification, and characterization of recombinant 2', 5'-oligoadenylate synthetases.

2',5'-Oligoadenylate [2-5(A)] synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5'-linked oligoadenylates in the presence of double-stranded RNA (dsRNA), their cofactor. The 2-5(A) molecules, in turn, activate the latent ribonuclease RNase L by promoting its dimerization. The 2-5(A) synthetase pathway has been implicated in interferon's antiviral and anticellular activities. In addition to their interesting cellular properties, these enzymes are also enzymologically interesting because they are the only known template and primer independent nucleotide (DNA or RNA)polymerases that synthesize 2'-5'-linked oligonucleotides. Moreover, their mode of activation by dsRNA remains unknown. In the past, biochemical and structure-function studies have been hampered by the lack of a convenient system for expressing recombinant 2-5(A) synthetases. These proteins are toxic to mammalian cells, probably because of RNase L activation, and proteins produced in bacteria do not have full enzymatic activity. To circumvent these problems, we have developed a baculovirus-insect cell system for high-yield expression of the small and medium isozymes. Here, methods are described for the production, purification, and characterization of the mouse small (9-2) (S. K. Ghosh, J. Kusari, S. K. Bandyopadhyay, H. Samanta, R. Kumar, and G. C. Sen, 1991, J. Biol. Chem. 266, 15293-15299) and human medium (P69) (I. Marie and A. G. Hovanessian, 1992, J. Biol. Chem. 267, 9933-9939) 2-5(A) synthetase isozymes and their mutants using the insect cell system. We also report methods for studying 2-5(A) synthetase-dsRNA interactions and protein-protein interactions among the subunits of the two isozymes.

2',5'-Oligoadenylate Synthetase↗

PubMed1998

Spectroscopic studies of the interactions of the pyrethroid insecticide fenvalerate with gramicidin.

Fenvalerate is a pyrethroid insecticide which interacts with ionic channels. Using circular dichroism technique we have studied the interaction of fenvalerate with gramicidin, a model channel peptide which transports ions. In most organic solvents, gramicidin exists as a double helix except in trifluoroethanol where it exists as a channel forming single stranded beta6.3 helical monomer. In model lipid membranes, under certain experimental conditions, gramicidin exists as a channel forming single stranded beta6.3 helical dimer. Our results show that fenvalerate interacts more with the single stranded beta6.3 helical monomer or dimer than with the double helical form of gramicidin. This was further confirmed by an increase in the rate of gramicidin mediated proton transport in liposomes by fenvalerate, using the pH sensitive fluorophore, pyranine.

Anti-Bacterial Agents↗

PubMed1997

Enzymatic activity of 2'-5'-oligoadenylate synthetase is impaired by specific mutations that affect oligomerization of the protein.

Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2'-5'-oligoadenylate (2-5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293-15299). Sequence comparison of this region among the different isozymes of 2-5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia ni insect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses of in vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed by in vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2-5(A) synthetases.

2',5'-Oligoadenylate Synthetase↗

PubMed1997

Effects of mutating specific residues present near the amino terminus of 2'-5'-oligoadenylate synthetase.

In this study, we investigated the role of specific amino acid residues present near the amino terminus of the 9-2 isozyme of 2'-5'-oligoadenylate synthetase. In vitro expression of deletion mutants showed that residues 1-9 are required for enzyme activity. Within this region, residues 3, 7, and 8 were found to be conserved among all known isozymes of 2'-5'-oligoadenylate synthetase. Mutation of these residues singly or in combination resulted in partial or total loss of enzyme activity. Substitution of the proline residue at position 7 by different residues caused a partial or complete loss of activity. The properties of the inactive P7Q mutant were further explored by expressing the protein in bacteria. The bacterially expressed protein was also enzymatically inactive. The mutant protein could bind the substrate ATP and the activator double-stranded RNA normally. Oligomerization properties of the protein were examined by an affinity-based interaction assay and by glycerol gradient centrifugation; there was no detectable difference between the wild type and the P7Q mutant. These results demonstrated the importance of the proline residue at position 7 in conferring enzyme activity to the protein without affecting its other properties.

2',5'-Oligoadenylate Synthetase↗