Biomedical subjects

V Anantharam

Publications and source records attributed to V Anantharam.

At least 19 recordsLinked to original sources

PubMed2006

Methamphetamine induces autophagy and apoptosis in a mesencephalic dopaminergic neuronal culture model: role of cathepsin-D in methamphetamine-induced apoptotic cell death.

Autophagy is a phylogenetically conserved process that plays a critical role in the degradation of oxidatively damaged proteins and organelle turnover. The role of oxidative stress and apoptosis in methamphetamine (METH)-induced neurotoxicity is well known; however, the potential contribution of autophagy to METH-induced oxidative damage in dopaminergic neuronal systems remains unclear. The goals of the present article were twofold: (a) to develop an in vitro dopaminergic cell culture model to study cellular and molecular mechanisms underlying METH-induced autophagy and apoptosis, and (b) to determine whether lysosomal protease cathepsin-D activation, resulting from the loss of lysosomal membrane integrity, contributes to METH-induced apoptosis. To accomplish these goals, we characterized morphological and biochemical changes in an immortalized mesencephalic dopaminergic neuronal cell line (N27 cells) following treatment with METH. Exposure of METH (2 mM) to N27 cells resulted in the appearance of cytoplasmic vacuolar structures reminiscent of autophagic vacuoles within 3 h. In order to ascertain the identity of the vacuolar structures that are formed following METH exposure, immunohistochemical staining for markers of autophagy were performed. LAMP 2, a classical marker of autophagolysosomes, revealed an extensive punctuate pattern of distribution on the vacuolar membrane surface, with exclusive localization in the cytoplasm. Additionally, using DNA fragmentation analysis we showed a dose-dependent increase in fragmented DNA in METH treated N27 cells. Since METH-induced autophagy preceded DNA fragmentation, we tested whether dysfunction of the autophagolysosomal system contributes to nuclear damage. Immunofluorescence studies with cathepsin-d demonstrated a granular pattern of staining in untreated cells, whereas an increased cathepsin- D immunoreactivity with a globular pattern of staining was observed in METH-treated cells. Nevertheless, blockade of cathepsin-D activation by pepstatin-A, cathepsin-D inhibitor, failed to alter METH-induced DNA fragmentation. Collectively, these results demonstrate that N27 dopaminergic neuronal cell model may serve as an excellent in vitro model to study the mechanisms of METH-induced autophagy and apoptosis. Furthermore, it is less likely that cathepsin-D may serve as a trigger for the induction of apoptosis subsequent to exposure of N27 dopaminergic neuronal cells to METH.

Animals↗

PubMed2004

Blockade of PKCdelta proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methylcyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death.

The use of methylcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline additive has raised health concerns and increased interest in understanding the neurotoxic effects of manganese. Chronic exposure to inorganic manganese causes Manganism, a neurological disorder somewhat similar to Parkinson's disease. However, the cellular mechanism by which MMT, an organic manganese compound, induces neurotoxicity in dopaminergic neuronal cells remains unclear. Therefore, we systematically investigated apoptotic cell-signaling events following exposure to 3-200 microM MMT in mesencephalic dopaminergic neuronal (N27) cells. MMT treatment resulted in a time- and dose-dependent increase in reactive oxygen species generation and cell death in N27 cells. The cell death was preceded by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers MMT-induced apoptotic cell death. Importantly, MMT induced proteolytic cleavage of protein kinase Cdelta (PKCdelta), resulting in persistently increased kinase activity. The proteolytic activation of PKCdelta was suppressed by treatment with 100 microM Z-VAD-FMK and 100 microM Z-DEVD-FMK, suggesting that caspase-3 mediates the proteolytic activation of PKCdelta. Pretreatment with 100 microM Z-DEVD-FMK and 5 microM rottlerin (a PKCdelta inhibitor) also significantly attenuated MMT-induced DNA fragmentation. Furthermore, overexpression of either the kinase inactive dominant negative PKCdelta(K376R) mutant or the caspase cleavage resistant PKCdelta(D327A) mutant rescued N27 cells from MMT-induced DNA fragmentation. Collectively, these results demonstrate that the mitochondrial-dependent apoptotic cascade mediates apoptosis via proteolytic activation of PKCdelta in MMT-induced dopaminergic degeneration and suggest that PKCdelta may serve as an attractive therapeutic target in Parkinson-related neurological diseases.

Animals↗

PubMed2003

Dieldrin induces apoptosis by promoting caspase-3-dependent proteolytic cleavage of protein kinase Cdelta in dopaminergic cells: relevance to oxidative stress and dopaminergic degeneration.

We previously reported that dieldrin, one of the potential environmental risk factors for development of Parkinson's disease, induces apoptosis in dopaminergic cells by generating oxidative stress. Here, we demonstrate that the caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) mediates as well as regulates the dieldrin-induced apoptotic cascade in dopaminergic cells. Exposure of PC12 cells to dieldrin (100-300 microM) results in the rapid release of cytochrome C, followed by the activation of caspase-9 and caspase-3 in a time- and dose-dependent manner. The superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride significantly attenuates dieldrin-induced cytochrome C release, indicating that reactive oxygen species may contribute to the activation of pro-apoptotic factors. Interestingly, dieldrin proteolytically cleaves native PKCdelta into a 41 kDa catalytic subunit and a 38 kDa regulatory subunit to activate the kinase. The dieldrin-induced proteolytic cleavage of PKCdelta and induction of kinase activity are completely inhibited by pretreatment with 50-100 microM concentrations of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), indicating that the proteolytic activation of PKCdelta is caspase-3-dependent. Additionally, Z-VAD-FMK, Z-DEVD-FMK or the PKCdelta specific inhibitor rottlerin almost completely block dieldrin-induced DNA fragmentation. Because dieldrin dramatically increases (40-80-fold) caspase-3 activity, we examined whether proteolytically activated PKCdelta amplifies caspase-3 via positive feedback activation. The PKCdelta inhibitor rottlerin (3-20 microM) dose-dependently attenuates dieldrin-induced caspase-3 activity, suggesting positive feedback activation of caspase-3 by PKCdelta. Indeed, delivery of catalytically active recombinant PKCdelta via a protein delivery system significantly activates caspase-3 in PC12 cells. Finally, overexpression of the kinase-inactive PKCdelta(K376R) mutant in rat mesencephalic dopaminergic neuronal cells attenuates dieldrin-induced caspase-3 activity and DNA fragmentation, further confirming the pro-apoptotic function of PKCdelta in dopaminergic cells. Together, we conclude that caspase-3-dependent proteolytic activation of PKCdelta is a critical event in dieldrin-induced apoptotic cell death in dopaminergic cells.

Animals↗

PubMed2003

Proteolytic activation of proapoptotic kinase PKCdelta is regulated by overexpression of Bcl-2: implications for oxidative stress and environmental factors in Parkinson's disease.

We previously demonstrated that the organochlorine pesticide dieldrin, a potential chemical risk factor for development of Parkinson's disease (PD), impairs mitochondrial function and promotes apoptosis in dopaminergic PC12 cells. We further demonstrated that caspase-3-dependent proteolytic activation of a member of the novel PKC family, protein kinase Cdelta (PKCdelta), contributes to apoptotic cell death in dopaminergic cells. In the present study, we report that the proapoptotic function of PKCdelta can be regulated by overexpression of the mitochondrial anti-apoptotic protein Bcl2 in dieldrin-treated dopaminergic cells. Exposure to dieldrin (30 or 100 micro M) for 3 h produced a dose-dependent increase in caspase-3 activation and DNA fragmentation in vector-transfected PC12 cells. Overexpression of human Bcl-2 in PC12 cells completely suppressed dieldrin-induced caspase-3 activation and DNA fragmentation. Furthermore, dieldrin-induced proteolytic activation of PKCdelta was also remarkably reduced in Bcl-2-overexpressed cells. Together, these results suggest that the proapoptotic function of PKCdelta can be regulated by mitochondrial redox modulators during neurodegenerative processes.

Animals↗

PubMed2001

Dieldrin-induced oxidative stress and neurochemical changes contribute to apoptopic cell death in dopaminergic cells.

We examined the acute toxicity of dieldrin, a possible environmental risk factor of Parkinson's disease, in a dopaminergic cell model, PC12 cells, to determine early cellular events underlying the pesticide-induced degenerative processes. EC(50) for 1 h dieldrin exposure was 143 microM for PC12 cells, whereas EC(50) for non-dopaminergic cells was 292-351 microM, indicating that dieldrin is more toxic to dopaminergic cells. Dieldrin also induced rapid, dose-dependent releases of dopamine and its metabolite, DOPAC, resulting in depletion of intracellular dopamine. Additionally, dieldrin exposure caused depolarization of mitochondrial membrane potential in a dose-dependent manner. Flow cytometric analysis showed generation of reactive oxygen species (ROS) within 5 min of dieldrin treatment, and significant increases in lipid peroxidation were also detected following 1 h exposure. ROS generation was remarkably inhibited in the presence of SOD. Dieldrin-induced apoptosis was significantly attenuated by both SOD and MnTBAP (SOD mimetic), suggesting that dieldrin-induced superoxide radicals serve as important signals in initiation of apoptosis. Furthermore, pretreatment with deprenyl (MAO-inhibitor) or alpha-methyl-L-p-tyrosine (TH-inhibitor) also suppressed dieldrin-induced ROS generation and DNA fragmentation. Taken together, these results suggest that rapid release of dopamine and generation of ROS are early cellular events that may account for dieldrin-induced apoptotic cell death in dopaminergic cells.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2000

Conditionally-immortalized astrocytic cell line expresses GAD and secretes GABA under tetracycline regulation.

We have engineered conditionally-immortalized mouse astrocytes to express beta-galactosidase or GAD(65) in a tetracycline-controlled fashion. The engineered cell lines, BASlinbetagal and BASlin65, divide at 33 degrees C but cease division at 39 degrees C. We carried out morphological and biochemical analyses to further understand GABA production and release, and to determine the suitability of these cells for transplantation. Using the BASlinbetagal cell line, we showed a dramatic regulation of beta-galactosidase expression by tetracycline. The BASlin65 cell line showed functional GAD(65) enzymatic activity and GABA production, both of which were suppressed by growth in the presence of tetracycline. When cultured in the absence of tetracycline, BASlin65 cells have a total GABA content equal to or greater than other GABA-ergic cell lines. Immunofluorescence microscopy revealed that GAD(65) had a distinct perinuclear localization and punctate staining pattern. GABA, on the other hand, showed diffuse staining throughout the cytoplasm. BASlin65 cells not only synthesize GABA, they also release it into the extracellular environment. Their ability to produce and release significant amounts of GABA in a tetracycline-regulated manner makes BASlin65 cells a useful cellular model for the study of GABA production and release. Furthermore, their non-tumorigenicity makes them excellent candidates for transplantation into specific regions of the brain to provide a localized and regulatable source of GABA to the local neuronal circuitry.

Animals↗

PubMed2000

Conditionally immortalized cell lines, engineered to produce and release GABA, modulate the development of behavioral seizures.

Transplantation of genetically engineered cells can provide sustained focal delivery of naturally occurring molecules, including neurotransmitters and growth factors. We have engineered immortalized mouse cortical neurons and glia to deliver GABA by driving GAD(65) expression. Engineered cell lines showed GAD(65) mRNA expression, enzymatic activity, and GABA release. In vitro, basal flux of GABA was approximately 20% of total cellular GABA. We transplanted these GABA-producing cells bilaterally into either the anterior or the posterior substantia nigra of 43 rats. The rats were subsequently kindled through an electrode placed in the entorhinal cortex. GABA-producing cells, but not beta-galactosidase-producing cells, affected kindling rates. The number of stimulations needed to reach the first stage-5 seizure and to achieve full kindling differed significantly between the anterior and posterior transplantation sites when GAD(65)-producing cells were transplanted but not when beta-galactosidase-producing cells were transplanted. Our data show that transplanted engineered cells can make and release GABA at physiologically meaningful concentrations.

Animals↗

PubMed1998

Ethanol increases the activity of Ca(++)-dependent K+ (mslo) channels: functional interaction with cytosolic Ca++.

Ethanol (EtOH) reversibly activates large conductance, Ca(++)-activated K+ (BK) channels in rat neurohypophysial terminals, an effect that probably contributes to the inhibition of vasopressin release by this drug. Heterogeneity in the terminal channel population makes it difficult to determine the mechanisms underlying this activation. Here, we report the effects of EtOH on the steady-state activity of BK channels cloned from mouse brain (mslo, alpha subunit) and expressed in Xenopus oocytes. EtOH reversibly increased mslo channel activity in excised patches, showing a potency (EC50 = 24 mM) similar to that reported using native channels. EtOH activation was observed under conditions that make it highly improbable that it is mediated by freely diffusible second messengers, or secondary to G-protein modulation. Rather, it probably results from a functional interaction between the drug and the channel alpha subunit. Activation occurred without increase in the number of functional channels present in the patch and resulted from actions that were a function of EtOH concentration: at < or = 10 mM, activation was due to a decrease in the channel mean closed time, whereas between 25 and 100 mM, activation was due to both a decrease in the mean closed time and an increase in the mean open time. The characteristic high unitary conductance and ionic selectivity of BK channels were unaltered by the drug. Whereas the voltage dependence of channel gating remained unchanged, channel activation mediated by the response of the Ca(++)-sensing site(s) to increases in the concentration of intracellular calcium, [Ca++]ic, was reduced by EtOH. This finding is consistent with EtOH and [Ca++]ic behaving functionally as partial and full agonists of mslo channels, respectively. Because the potentiation of mslo activity by the drug decreased as Ca++ levels were increased, EtOH-activation of BK channels would be most evident when [Ca++]ic is near resting levels, rather than during periods of high activity and Ca++ influx.

Animals↗

PubMed1996

Alternative splicing of the NMDAR1 subunit affects modulation by calcium.

Four splice variants of the NR1 receptor subunit, characterized by the presence or absence of cassettes encoding inserts of 21 (Insert 1) and 37 (Insert 2) amino acids were expressed in Xenopus oocytes and studied using voltage-clamp techniques. In 1.8 mM Ca2+, a slow inward current (Islow), which peaked 20 s after exposure to NMDA was evident when Insert I was present, but not when absent. However, in elevated external Ca2+ medium a similar Islow was observed in variants missing Insert I. The Ca2+ dependency of Islow reflected a requirement for intracellular accumulation of Ca2+. The divalent ion permeability of Insert I containing and Insert 1 lacking receptor channels expressed alone, as well as in heteromeric assemblies with NR2A and NR2B, was similar for all combinations tested. Thus, the lower Ca2+ dependency for Islow in oocytes expressing Insert I was not due to higher calcium entry. Islow was less sensitive to blockers of ICl(Ca) than were endogenous calcium-activated chloride currents (ICl(Ca)). Also, Islow was not abolished in Cl(-)-free external medium, when voltage was manipulated such that Islow was outward-going. Thus, Islow, while containing a component due to activation of endogenous ICl(Ca), is primarily due to current flowing through the receptor ion channel. Development of Islow was unaffected by PKC or PKA inhibitors. The modulation of the Ca2+ dependency of Islow by Insert I occurs in a range of Ca2+ concentrations which are physiologically relevant, and may provide an important means of modulation of glutamate transmission under normal and pathological conditions.

Alternative Splicing↗

PubMed1996

Inhibitory effects of ketamine and halothane on recombinant potassium channels from mammalian brain.

BACKGROUND: There is increasing evidence that direct interactions between volatile anesthetics and channel proteins may result in general anesthesia. Using voltage-clamp techniques, the authors examined the effect of two general anesthetics (ketamine and halothane) on a rat brain potassium channel of known amino acid sequence, and further assessed whether the inhibition of the channel is altered by a partial deletion of the C-terminal sequence of this channel. METHODS: Xenopus laevis oocytes were microinjected with either Kv2.1 or delta C318 (a mutated channel in which the last 318 amino acids of the C-terminus have been deleted) cRNA, and channel function in translated channels was observed before, during, and after exposure to graded concentrations of ketamine (25, 50, and 75 micrometers) and halothane (1%, 2%, and 4%). RESULTS: Ketamine and halothane reduced Kv2.1 and delta C318 peak current amplitude in a dose-dependent and reversible fashion. The inhibition of current was voltage dependent for halothane but not for ketamine. Halothane accelerated the time constant of current inactivation, whereas ketamine affected this parameter minimally in both channel types. Use dependence of ketamine and halothane action was observed in both Kv2.1 and the mutant channel, attributable to augmentation of C-type inactivation. CONCLUSIONS: Although both ketamine and halothane inhibit potassium currents through the Kv2.1 channel, their mechanisms of action at this potential target may be different. Deletion of the C-terminal sequence resulted in decreased sensitivity to both anesthetics. Although it is not clear whether anesthetics interact directly with the C-terminus, which is thought to reside intracellularly, this portion of the channel protein clearly influences the actions of both anesthetics.

Anesthetics, Dissociative↗

PubMed1995

Abolition of G protein inhibition of alpha 1A and alpha 1B calcium channels by co-expression of the beta 3 subunit.

Three different classes of alpha 1 Ca2+ channel (alpha 1A, alpha 1B, alpha 1C) were expressed in Xenopus oocytes to determine whether G protein-mediated inhibition is an inherent property of the alpha 1 subunit itself, and if so, whether co-expression of auxiliary subunits modulates the inhibition seen. From our data it is apparent that either alpha 1A or alpha 1B Ca2+ channels expressed alone are sufficient for voltage-dependent G protein inhibition. alpha 1C Ca2+ channels expressed alone do not exhibit the G protein inhibition seen in alpha 1A and alpha 1B channels. Additionally, co-expression of the beta 3 subunit abolishes the ability of G proteins to inhibit currents through alpha 1A and alpha 1B Ca2+ channels. Differential sensitivity of alpha 1 as well as modulation of properties by beta 3 provide a potential mechanism for the regulation of G protein-mediated inhibition in neurons.

Animals↗

PubMed1995

Block of the N-methyl-D-aspartate receptor by phencyclidine-like drugs is influenced by alternative splicing.

N-Methyl-D-aspartate (NMDA) receptors were expressed in Xenopus oocytes from injected mRNA. The presence of an alternatively-spliced insertion encoding 21 amino acids at the N-terminus of the NMDAR1 (NR1(111)) subunit, made homomeric assemblies of the receptor more sensitive to ketamine and MK-801 than receptors assembled from NMDAR1 subunits lacking this insert (NR1(011) and NR1(001)). The influence of this insert was maintained when NR1 subunits were co-expressed in heteromeric combinations with NR2B. The increased sensitivity of the receptors containing the insert (NR1(111)) was accompanied by a faster on-rate for drug action than was observed for receptors lacking the insert (NR1(011) and NR1(001)). Our results suggest that the action of phencyclidine-like drugs is influenced by the presence of Insertion I in the NMDA isoforms, generated by alternative splicing.

Alternative Splicing↗

PubMed1995

Ethanol inhibition of recombinant heteromeric NMDA channels in the presence and absence of modulators.

The NMDA receptor/channel has been shown to be inhibited by ethanol in the clinically relevant range 25-100 mM. We studied heteromeric assemblies (NR1b/NR2) of the NMDA receptor, expressed in oocytes, to address three questions regarding this inhibition, and discovered the following: (1) The inhibition was nearly equivalent when ethanol was coapplied with the agonist, and when ethanol was introduced after steady-state current was established, suggesting that ethanol does not act by interfering with the activation process of the NMDA receptor. (2) The degree of inhibition was controlled by the NR2 subunit, with both NR2A and NR2B significantly more sensitive to ethanol than NR2C and NR2D. (3) Manipulation of the NMDA receptor with a number of agents that normally modulate it did not alter the degree of inhibition produced by ethanol. The presence of Mg2+ (3 and 12.5 microM), Zn2+ (1 and 10 microM), or the glycine antagonist 7-chlorokynurenic acid (1.25 or 5 microM), did not alter the ethanol sensitivity of heteromeric (NR1b/NR2A, NR1b/NR2B, NR1b/NR2C) NMDA receptors. Redox modulation of the NMDA receptor by dithiothreitol (2 mM) or 5,5'-dithiobis(2-nitrobenzoic acid) (1 mM) also did not alter the degree to which ethanol inhibits NMDA receptors. Taken together, these results indicate that the ethanol sensitivity of native NMDA receptors, which likely exist in heteromeric form, results from actions at a site different from those of known modulators of the receptor.

Animals↗

PubMed1993

Halothane acts on many potassium channels, including a minimal potassium channel.

There has been considerable controversy over whether general anesthetics act directly on membrane proteins, and if so, whether there are uniquely sensitive protein targets upon which they act. Here, we examine the actions of halothane on a diverse collection of voltage-gated potassium channels expressed in Xenopus oocytes, and find that they are all sensitive at clinically relevant concentrations. To investigate further the molecular basis of this commonality, human and rat minimal potassium (minK) channels, which have exceedingly short amino acid sequences, were examined. Current through these channels is reversibly reduced to 68% of control values by 0.5% (0.34 mM) halothane. A double deletion mutant of the 130-amino acid minK protein, in which 30 amino acids of the N-terminus, thought to be extracellular, and 37 amino acids of the putative intracellular C-terminus are deleted (resulting in a protein in which more than half of both the extracellular and intracellular domains have been removed) responds to low halothane concentrations similarly to the parent channel. While alternative explanations are possible, this result is consistent with a model whereby halothane interacts with the channel protein from within the lipid bilayer.

Animals↗

PubMed1993

Homomeric assemblies of NMDAR1 splice variants are sensitive to ethanol.

Studies have shown that the neuronal NMDA receptor is a target for the actions of ethanol. Recently, a number of subunits of the NMDA receptor have been cloned and functionally expressed in various combinations. We have expressed four splice variants of the NMDAR1 subunit in Xenopus oocytes, and find that homomeric assemblies of this subunit, in the absence of other subunits, exhibit ethanol sensitivity comparable to that seen in neurons. In the presence of calcium, the reduction of total current was greatest in the NMDAR1-LL splice variant, and was significantly less in the NMDAR1-SS variant. The increased sensitivity of NMDAR1-LL may be attributed to a particularly sensitive slow current 'hump' which is more pronounced in NMDAR1-LL than in NMDAR1-SS. The reduction of NMDA-evoked current by ethanol was significantly different when calcium was replaced by barium in the external medium. In this case, the slow current hump was significantly reduced, current through NMDAR1-LL was less reduced by ethanol, and the percent reduction of NMDAR1-LL and NMDAR1-SS currents was similar. NMDA-evoked currents in heteromeric receptors formed by coinjection of the mouse epsilon-1 subunit with the NMDAR1 splice variants responded to ethanol similarly to homomeric assemblies.

Animals↗

PubMed1992

Combinatorial RNA splicing alters the surface charge on the NMDA receptor.

Transcripts encoding four NMDA receptor subunits, generated from the NMDAR1 gene by alternative RNA splicing, have been demonstrated in adult rat brain. RNA transcripts derived from cDNAs encoding each form direct the formation of functional NMDA receptors in Xenopus oocytes. The two amino acid cassettes of 21 and 37 amino acids found in the splice variants increase the positive extracellular surface charge on the subunits and may thereby modulate the functional properties of the receptor.

Amino Acid Sequence↗

PubMed1992

Measurement of the substrate dissociation constant of a solubilized membrane carrier. Substrate stabilization of OxlT, the anion exchange protein of Oxalobacter formigenes.

OxlT, a secondary carrier found in Oxalobacter formigenes, mediates the exchange of divalent oxalate and monovalent formate. Because OxlT has an unusually high turnover number (greater than or equal to 1000/s), and because formate, one its substrates, shows high passive membrane permeability as formic acid, it has been difficult to obtain information on protein-substrate interactions using traditional methods in membrane biology. For this reason, we devised a new way to measure substrate dissociation constants. Detergent-solubilized material was exposed to inactivating temperatures in the absence or presence of OxlT substrates, and periodic reconstitution was used to monitor the kinetics of thermal decay. The data were consistent with a simple scheme in which only unliganded OxlT was temperature-sensitive; this premise, along with the assumption of equilibrium between liganded and unliganded species, allowed calculation of substrate dissociation constants for oxalate (18 +/- 3 microM), malonate (1.2 +/- 0.2 mM), and formate (3.1 +/- 0.6 mM). Further analysis revealed that substrate binding energy contributed at least 3.5 kcal/mol to stabilization of solubilized OxlT. Accordingly, we suggest that substrate binding energy is directly involved in driving protein structure reorganization during membrane transport. This new approach to analyzing protein-substrate interactions may have wider application in the study of membrane carriers.

Anions↗

PubMed1992

Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of Oxalobacter formigenes.

We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis.

Bacterial Proteins↗