The interaction of the Chrna5 D398N variant with developmental nicotine exposure.

A single nucleotide polymorphism (SNP) in CHRNA5 (rs16969968, change from an aspartic acid [D] to asparagine [N] at position 398 of the human α5 nicotinic acetylcholine receptor subunit) has been associated with increased risk for nicotine dependence. Consequently, carriers of the risk variant may be at elevated risk for in utero nicotine exposure. To assess whether this gene-environment interaction might impact nicotine intake in developmental nicotine-exposed offspring, we utilized a mouse expressing this human SNP. D and N dams drank nicotine (100 µg/mL) in 0.2% saccharin water or 0.2% saccharin water alone (vehicle) as their sole source of fluid from 30 days prior to breeding until weaning of offspring. The nicotine (D Nic, N Nic) or vehicle (D Veh, N Veh) exposed offspring underwent a 2-bottle choice test between postnatal ages of 30 to 46 days. N Nic offspring consumed the most nicotine at the highest concentration (400 µg/mL) compared with all other groups. In contrast, D Nic offspring drank the least amount of nicotine at all concentrations tested. Nicotine-stimulated dopamine (DA) release measured from striatal synaptosomes was increased in D Nic offspring, while decreased in N Nic offspring relative to their genotype-matched controls. These data suggest that the α5 variant influences the effect of developmental nicotine exposure on nicotine intake of exposed offspring. This gene-environment interaction on striatal DA release may provide motivation for increased nicotine seeking in N Nic offspring and reduced consumption in D Nic offspring.

Nicotinic cholinergic mechanisms causing elevated dopamine release and abnormal locomotor behavior.

Firing rates of dopamine (DA) neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) control DA release in target structures such as striatum and prefrontal cortex. DA neuron firing in the soma and release probability at axon terminals are tightly regulated by cholinergic transmission and nicotinic acetylcholine receptors (nAChRs). To understand the role of α6* nAChRs in DA transmission, we studied several strains of mice expressing differing levels of mutant, hypersensitive (leucine 9' to serine [L9'S]) α6 subunits. α6 L9'S mice harboring six or more copies of the hypersensitive α6 gene exhibited spontaneous home-cage hyperactivity and novelty-induced locomotor activity, whereas mice with an equal number of WT and L9'S α6 genes had locomotor activity resembling that of control mice. α6-dependent, nicotine-stimulated locomotor activation was also more robust in high-copy α6 L9'S mice versus low-copy mice. In wheel-running experiments, results were also bi-modal; high-copy α6 L9'S animals exhibited blunted total wheel rotations during each day of a 9-day experiment, but low-copy α6 L9'S mice ran normally on the wheel. Reduced wheel running in hyperactive strains of α6 L9'S mice was attributable to a reduction in both overall running time and velocity. ACh and nicotine-stimulated DA release from striatal synaptosomes in α6 L9'S mice was well-correlated with behavioral phenotypes, supporting the hypothesis that augmented DA release mediates the altered behavior of α6 L9'S mice. This study highlights the precise control that the nicotinic cholinergic system exerts on DA transmission and provides further insights into the mechanisms and consequences of enhanced DA release.

Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.

Compensation in pre-synaptic dopaminergic function following nigrostriatal damage in primates.

Clinical symptoms of Parkinson's disease only become evident after 70-80% reductions in striatal dopamine. To investigate the importance of pre-synaptic dopaminergic mechanisms in this compensation, we determined the effect of nigrostriatal damage on dopaminergic markers and function in primates. MPTP treatment resulted in a graded dopamine loss with moderate to severe declines in ventromedial striatum (approximately 60-95%) and the greatest reductions (approximately 95-99%) in dorsolateral striatum. A somewhat less severe pattern of loss was observed for striatal nicotinic receptor, tyrosine hydroxylase and vesicular monoamine transporter expression. Declines in striatal dopamine uptake and transporter sites were also less severe than the reduction in dopamine levels, with enhanced dopamine turnover in the dorsolateral striatum after lesioning. The greatest degree of adaptation occurred for nicotine-evoked [(3)H]dopamine release from striatal synaptosomes, which was relatively intact in ventromedial striatum after lesioning, despite > 50% declines in dopamine. This maintenance of evoked release was not due to compensatory alterations in nicotinic receptor characteristics. Rather, there appeared to be a generalized preservation of release processes in ventromedial striatum, with K(+)-evoked release also near control levels after lesioning. These combined compensatory mechanisms help explain the finding that Parkinson's disease symptomatology develops only with major losses of striatal dopamine.

Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum.

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [(3)H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. alpha-Conotoxin MII did not inhibit this release, while alpha-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [(3)H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the beta 2 subunit null mutation. The deletion of the beta 2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the beta 2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC(50) value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.

Nicotinic-agonist stimulated (86)Rb(+) efflux and [(3)H]epibatidine binding of mice differing in beta2 genotype.

Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in beta2 subunit expression. Function was measured by on-line detection of (86)Rb(+) efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 microM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-beta-erythroidine (DHbetaE); and (2) stimulation with 10 microM epibatidine in the presence of 2 microM DHbetaE, a response that is relatively resistant to inhibition by DHbetaE. Deletion of the beta2 subunit profoundly reduced both DHbetaE-sensitive and -resistant (86)Rb(+) efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [(3)H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the beta2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in beta2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [(3)H]epibatidine binding was heterogeneous in that a fraction of the sites required the beta2 subunit. Many remaining sites were sensitive to inhibition by alpha-bungarotoxin indicating that a subset of the low affinity [(3)H]epibatidine binding are of the alpha7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for beta2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.

Pharmacological and null mutation approaches reveal nicotinic receptor diversity.

We have developed an array of assays for nicotinic acetylcholine receptor binding and function. [125I]alpha-Bungarotoxin-, (-)-[3H]nicotine-, and [3H]epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic acetylcholine receptor function was quantified using synaptosomal [3H]dopamine, [3H]gamma-aminobutyric acid ([3H]GABA), and 86Rb(+) efflux techniques. Additionally, the effects of beta2 subunit deletion on each of the measures were assessed. Detailed pharmacological comparison revealed minimally six nicotinic binding subtypes: [125I]alpha-bungarotoxin-binding nicotinic acetylcholine receptors; beta2-subunit-dependent and -independent high-affinity (-)-[3H]nicotine-binding sites; beta2-dependent and -independent cytisine-resistant [3H]epibatidine-binding sites; and a beta2-dependent low-affinity [3H]epibatidine binding site. Comparative pharmacology suggested that [3H]GABA and dihydro-beta-erythroidine (DHbetaE)-sensitive 86Rb(+) efflux are mediated by the same (probably alpha4beta2) nicotinic acetylcholine receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke [3H]dopamine and DHbetaE-resistant 86Rb(+) efflux. In whole-brain preparations, each measure of nicotinic acetylcholine receptor function was beta2 dependent. The majority of beta2-independent [3H]epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification of novel, native nicotinic acetylcholine receptors.

Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit.

Nicotinic agonist-stimulated efflux of 86Rb+ from mouse brain synaptosomes was monitored continuously by on-line radioactivity detection. The concentration-effect curve following a 5-s stimulation with acetylcholine was biphasic (EC50 = 7.2 and 550 microM). alpha-Bungarotoxin (100 nM) did not inhibit the response, but dihydro-beta-erythroidine (DHbetaE) blocked both phases with differing potency (average IC50 =.22 and 8.9 microM for responses activated by low and high acetylcholine concentrations, respectively). Differential sensitivity DHbetaE inhibition was used to measure stimulation of 86Rb+ efflux by 17 nicotinic agonists, which differed markedly in potency and efficacy. All agonists were more potent at the DHbetaE-sensitive site. Both components were inhibited by the six antagonists tested. Methyllycaconitine and DHbetaE were more potent for the DHbetaE-sensitive component, whereas hexamethonium was more potent at the DHbetaE-resistant component. Both DHbetaE-sensitive and DHbetaE-resistant responses were reduced more than 95% in beta2-null mutant mice, establishing the requirement for the beta2 subunit for both components. Both components were widely, but not identically, distributed throughout the brain. The DHbetaE-sensitive component appears to be identical with agonist-stimulated 86Rb+ efflux described previously and is likely to be mediated by alpha4beta2 receptors. The DHbetaE-resistant component is a novel, active, and widely distributed response mediated by nicotinic receptor(s) that also require the beta2 subunit.

Pharmacological comparison of transient and persistent [3H]dopamine release from mouse striatal synaptosomes and response to chronic L-nicotine treatment.

L-Nicotine stimulates a biphasic release of [3H]dopamine from mouse striatal synaptosomes which does not persist after agonist is removed. Approximately 80% of the initial release is transient and disappears with a half-time of less than 1 min; the other 20% persists for several minutes (t(1/2), 5-10 min). Both the transient and persistent phases were investigated by 10-min exposures to agonists with an in vitro perfusion technique. A series of nicotinic agonists and antagonists were used to determine the pharmacological relationship of the two phases. Parameters measured included EC50 and Vmax values and desensitization rates for both phases for agonists, Ki values for antagonists and Ki values for low concentrations of agonists. The results are consistent with both phases being mediated by a single type of receptor. In addition, the effects of chronic nicotine treatment on transient and persistent [3H]DA release were measured. For both phases, release was decreased approximately 15% by chronic infusion of 4.0 mg/kg/hr L-nicotine. Correlation of the results with inactivation of a portion of the receptors rather than a reversible desensitization is discussed.

Neurosteroids modulate nicotinic receptor function in mouse striatal and thalamic synaptosomes.

Progesterone and its A-ring reduced metabolites are allosteric activators of GABA(A) receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an 86Rb+ efflux assay that likely measures the function of alpha4beta2-type nicotinic receptors and [3H]dopamine release, which may be modulated by an alpha3-containing nicotinic receptor. Both of the A-ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the 86Rb+ efflux assay. The 86Rb+ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays (t1/2 = 0.4 min) and was reversed even more slowly (t1/2 = 10-15 min). Steroid addition did not alter either the rate of association of [3H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.

Desensitization of nicotine-stimulated 86Rb+ efflux from mouse brain synaptosomes.

The desensitization of nicotine-stimulated 86Rb+ efflux from synaptosomes prepared from C57BL/6 mouse brain was investigated. Nicotine stimulated a saturable, concentration-dependent efflux of 86Rb+ from synaptosomes (EC50 = 0.60 microM), but the response decreased with time of exposure to nicotine. The rate of decrease of the response (desensitization) increased as the nicotine concentration was increased (EC50 = 0.35 microM; maximal rate of desensitization = 1.1 min-1). Desensitization of nicotine-stimulated 86Rb+ efflux was also observed when synaptosomes were exposed to low (1-200 nM) concentrations of nicotine that caused little or no stimulation of efflux (EC50 = 13 nM). The rate of desensitization observed with low nicotine concentrations (0.30 min-1) was less than that measured at stimulating concentrations. Desensitization was not fully reversible for synaptosomes exposed to nicotine concentrations between 10 nM and 10 microM: Only 60-40% of the control response was regained after a 10-min washout period. The kinetics of functional desensitization were compared with the kinetics of [3H]nicotine binding. [3H]Nicotine binding to midbrain particulate fractions displayed both a fast and a slow phase. The EC50 values for these two phases were 2.6 and 14 nM, respectively. Data obtained from functional desensitization and ligand binding experiments were analyzed using a two-state model. The kinetic constants obtained from the analyses of these two processes were very similar. Overall, the results suggest that nicotinic receptor function measured with ion flux desensitizes when exposed to either stimulating or nonstimulating concentrations of nicotine. In addition, the kinetic properties calculated for the functional desensitization are comparable to those for [3H]nicotine binding.

Desensitization of nicotine-stimulated [3H]dopamine release from mouse striatal synaptosomes.

Potential desensitization of brain nicotinic receptors was studied using a [3H]dopamine release assay. Nicotine-stimulated [3H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC50 of 0.33 +/- 0.13 microM and a Hill coefficient of 1.44 +/- 0.18. Desensitization by activating concentrations of nicotine had a similar EC50 and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC50 = 6.9 +/- 3.6 nM, t1/2 = 1.6-2.0 min, nH = 1.02 +/- 0.01). Both types of desensitization produced a maximum 75% decrease in [3H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [3H]nicotine to striatal membrane at 22 degrees C included a KD of 3.7 +/- 0.5 nM, Bmax of 67.5 +/- 2.2 fmol/mg, and Hill coefficient of 1.07 +/- 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [3H]-nicotine binding site could be the presynaptic receptor involved in [3H]dopamine release in mouse striatal synaptosomes.

Downregulation of nicotinic receptor function after chronic nicotine infusion.

Chronic nicotine treatment generally results in tolerance to several actions of nicotine and a paradoxical increase in brain nicotinic receptor numbers. Receptor upregulation, it has been argued, arises as a consequence of functional desensitization. In the studies reported here, mice were chronically infused with saline (control) or one of five doses of nicotine (0.25-4.0 mg/kg/hr) for 10 days. This treatment resulted in a dose-dependent tolerance to nicotine-induced decreases in body temperature as well as decreases in locomotor and rearing activities in a Y-maze. The anticipated increase in [3H]nicotine binding was also observed. To assess functional status of the nicotinic receptors, nicotine-stimulated release of [3H]dopamine from striatal synaptosomes and 86Rb+ efflux from cortical and midbrain synaptosomes were also measured. Chronic nicotine infusion resulted in an infusion dose-dependent decrease in [3H]dopamine release from striatum and 86Rb+ efflux from midbrain; cortical 86Rb+ efflux was not affected by chronic nicotine treatment. Dose-response analyses of the release and efflux assays demonstrated that chronic nicotine infusion evoked decreases in the maximal effects of nicotine on the functional assays; potency was not altered by chronic drug treatment. These results are consistent with the hypothesis that behavioral tolerance to nicotine is a consequence of down-regulation of brain nicotinic receptor function.

Nicotinic receptor function determined by stimulation of rubidium efflux from mouse brain synaptosomes.

The ability of nicotinic agonists to activate ion channels resulting in Na+ and K+ fluxes has been used to develop a functional assay by using mouse brain synaptosomes. Synaptosomes prepared using Percoll gradients were enriched in binding sites for [3H]nicotine and were capable of accumulating the K+ analog, 86Rb+. The efflux of 86Rb+ from the synaptosomes was subsequently monitored using continuous superfusion at 21 degrees C. Ion flux was stimulated in a concentration-dependent manner by several nicotinic agonists, including L-nicotine, acetylcholine, N-methylcarbamylcholine and dimethylphenylpiperazinium. The process was stereoselective: L-nicotine was 30-fold more potent than D-nicotine. Cytisine stimulated ion flux at low concentrations, but this drug was less efficacious than most other agonists tested. Anabasine was also less efficacious than the other agonists. The EC50 values for agonist-stimulated efflux correlated closely to the IC50 values for inhibition of [3H]nicotine binding, but concentrations required to inhibit binding were lower than those required to stimulate ion flux. Nicotine-induced 86Rb+ efflux was blocked by several nicotinic antagonists including mecamylamine, D-tubocurarine, hexamethonium and decamethonium. Mecamylamine was approximately 50 times as potent as hexamethonium. Neither alpha-bungarotoxin nor atropine were effective antagonists and neuronal-bungarotoxin was a relatively ineffective inhibitor. The amount of nicotine-induced efflux varied among brain regions with midbrain (thalamus and mesencephalon) having the largest response and cerebellum the smallest. The magnitude of the ion flux correlated closely with the amount of [3H] nicotine binding in each brain region. The results indicate that a nicotinic-receptor-mediated ion flux can be measured in brain tissue and that the ion flux may serve as a useful functional assay for nicotinic receptors in the central nervous system. Furthermore, it is postulated that the nicotinic-agonist stimulated ion flux may be mediated by receptors measured by high affinity [3H]nicotine binding.

Involvement of cell-cell adhesion molecules in liver colonization by metastatic murine lymphoma/lymphosarcoma variants.

Metastatic variant sublines of the murine RAW117 large cell lymphoma or lymphosarcoma have been established in vitro by sequential cycles of harvesting of liver tumor nodules after intravenous inoculation of tumor cell suspensions into syngeneic BALB/c mice. After five and ten in vivo selections for liver colonization, variant sublines RAW117-H5 and -H10, respectively, were established, and these formed significantly more surface liver tumors than the parental RAW117-P line. RAW117 sublines were tested for their abilities to adhere to embryonic mouse liver or brain cells in an in vitro cell-cell adhesion assay. Liver colonizing RAW117-H10 cells adhered with greater selectivity to liver cells than to brain cells. Parental RAW117-P cells were more homotypically adhesive, but they were nonselective in their organ cell adhesion properties. We examined RAW117 cells for the presence of liver cross-reactive antigens using polyclonal xenoantibody preparations directed against embryonic murine liver cells. These antibody preparations block organ-specific homotypic adhesion of embryonic murine liver cells in vitro. The amount of fetal liver antigen(s) expressed on RAW117 sublines correlated with liver colonization potentials (H10 greater than H5 greater than P) in quantitative absorption assays. Treatment of the highly metastatic RAW117-H10 subline with polyclonal anti-embryonic murine liver F(ab')2 or Fab' antibody fragments had no effect on RAW117-H10 cell viability or growth in vitro or in vivo, but inhibited liver colonization (median liver tumor colonies reduced from greater than 200 to 0) and prolonged life expectancy. In contrast, pretreatment of RAW117-H10 cells with polyclonal anti-H-2 did not modify the in vivo biologic properties of these metastatic cells.

Organ and class specificity of cell adhesion blocking antisera.

Rabbit antisera prepared against embryonic chick liver cells have been characterized using cell cytotoxicity tests, antibody absorption tests, inhibition of cell-cell reaggregation and cell-aggregate adhesion assays. The sera contain antibodies which specifically block embryonic chick liver cell-cell aggregation with no effect on other cell types or embryonic liver cells of other classes tested. Assays with sera absorbed with live cells, neural retina cells, or erythrocytes as well as with sera raised against these cells, indicate that this anti-aggregation effect is not merely the result of coating the cell surface with non-specific antibody. The anti-aggregation specificities of the sera have been checked by absorption experiments with a variety of membrane preparations. These results suggest significant cross-reactivity only between chick liver and kidney, perhaps reflecting the relatedness of the epithelial elements of these tissues. The immunoglobulin G fraction, F(ab')2 and monomeric fragments Fab' retain activity suggesting that the antisera inhibit cell-cell adhesion via blockage of cell surface ligands and not by cell surface modulation effects or cytotoxicity. The organ and class specificities of the antisera suggest that the inhibition of cell-cell aggregation is mediated by the direct binding of antibody to the embryonic chick liver cell adhesion ligand system.

Steady-state kinetics and inhibition studies of the aldol condensation reaction catalyzed by bovine liver and Escherichia coli 2-keto-4-hydroxyglutarate aldolase.

Two sensitive assays, one which fluorometrically measures only the L isomer of 2-keto-4-hydroxyglutarate after decarboxylation to L-malate and the other which spectrophotometrically determines both enantiomers by reductive amination with glutamate dehydrogenase, are described. By use of these assays, the steady-state kinetics of the aldol condensation of pyruvate with glyoxylate, as catalyzed by 2-keto-4-hydroxyglutarate aldolase from either bovine liver or Escherichia coli, were studied as was the inhibition of this reaction by glyoxylate and other anions. For the E. coli aldolase, double-reciprocal plots are linear except at high (above 5 mM) glyoxylate concentrations; apparent Km values increase with increasing concentrations of the fixed substrate. The data are consistent with an ordered reaction sequence. Inhibition by halides follows the lyotropic or Hofmeister series. Esters are not good inhibitors; mono-, di-, and tricarboxylic acids are increasingly inhibitory. Of the substrate analogues tested, hydroxypyruvate is the most potent inhibitor. Inhibition studies with citrate, acetaldehyde, and glyoxylate (all competitive inhibitors) suggest there are two domains at the active site-the Schiff base forming lysyl residue which interacts with carbonyl analogues (like acetaldehyde) and a center of positive charge which binds anions (like citrate). In contrast to the bacterial enzyme, liver 2-keto-4-hydroxyglutarate aldolase is inhibited in a competitive manner by much lower concentrations (0.1 mM or even lower) of glyoxylate. Many salts and some carboxylic acids activate the liver enzyme. Similarly, substrate analogues like 2-ketobutyrate and fluoropyruvate are mild activators; no effect is seen with acetaldehyde. Besides glyoxylate, only glyoxal, 2-ketoglutarate, and hydroxypyruvate inhibit the aldol condensation reaction. A uniform value of 1 is found for the number of inhibitor molecules bound per active site of either liver or E. coli 2-keto-4-hydroxyglutarate aldolase.

Tritium exchange reactions catalyzed by 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli K-12.

Tritiated water and tritiated substrates have been used to study exchange reactions catalyzed by Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16, 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate). With pyruvate, the enzyme catalyzes a rapid first-order exchange of all three methyl hydrogens in the absence of added acceptor aldehyde (i.e. glyoxylate). This reaction is not rate limiting for aldol condensation or cleavage; quite different pH-activity profiles for the exchange reaction versus aldol cleavage and also comparative effects that pH changes have on Km and V values for the two processes favor this conclusion. The exchange reaction with 2-oxobutyrate, a substrate analog, is stereoselective; one methylene hydrogen is removed at a 6-fold faster rate than the other but eventually both are exchanged. No tritium exchange occurs with glyoxylate.