Pre- and postsynaptic localization of central muscarinic receptors.

3H-Quinuclidinyl benzylate and dimethyl 14C-d-tubocurarine were used to localize muscarinic and nicotinic receptors in synaptosomal membranes of the rat cerebral cortex. The results obtained, after the action of different concentrations of Triton X-100, suggest that, while nicotinic receptors are postsynaptic, the muscarinic ones are both pre- and postsynaptic.     

Improvement of 5-HT3 receptor binding assay: enhancement of specific [3H]quipazine binding with Triton X-100-treated membranes from rat cortex.

The 5-hydroxytryptamine (5-HT)3 receptor binding assay using [3H]quipazine was examined. It was impossible to obtain specific [3H]quipazine binding with the membrane fractions from rat cortex prepared by the usual procedure. When the membranes were pretreated with detergent Triton X-100, the ratio of specific [3H]quipazine binding markedly increased, depending upon the concentration of Triton X-100 in the range of 0.01-0.1% (w/v). At a concentration of more than 0.05%, the specific binding reached a maximum of 55 to 60% of the total binding. The specific [3H]quipazine binding to the Triton X-100-treated membranes was reversible and was potently inhibited by several 5-HT3 antagonists, while 5-HT1, 5-HT2 receptor antagonists and other receptor-specific ligands had no effect on the binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.62 nM and Bmax of 97 fmol/mg protein. Thus, the Triton X-100-treated membranes retained the characteristics of 5-HT3 binding sites, making it possible to use [3H]quipazine for a 5-HT3 receptor binding assay with a high ratio of specific binding.     

Strychnine-insensitive binding of [3H]glycine to synaptic membranes in rat brain, treated with Triton X-100

Binding of radiolabelled glycine, a putative inhibitory neurotransmitter in mammalian lower central structures, was examined by using the synaptic membranes of the brain of rat, treated with Triton X-100. This treatment with Triton markedly potentiated the binding of [3H]glycine detected at 2 degrees C and 30 degrees C. However, this binding was not affected by three different convulsants, strychnine, picrotoxin and bicuculline. The binding was saturable at 2 degrees C, with increasing concentrations of [3H]glycine up to 1 microM. Scatchard analysis revealed that the binding sites consisted of a single component with a Kd of 202 nM and a Bmax of 1.74 pmol/mg protein. The binding was inhibited, not only by various amino acids structurally related to glycine, including D- and L-serine and D-, L- and beta-alanine, but was also eliminated by some peptides containing glycine, such as gamma-D- and gamma-L-glutamylglycine, glycine methylester and N-methyl-glycine. In addition, the strychnine-insensitive binding of [3H]glycine was significantly abolished by numerous quinoxaline antagonists for excitatory amino acid receptors in the brain. These results suggest that synaptic membranes of brain, treated with Triton X-100, are useful to detect the strychnine-insensitive binding of [3H]glycine and superior to untreated membranes in terms of the freedom from the confounding effects of some endogenous amino acids.     

Protection by atropine of the inhibition caused by triton X-100 on central muscarinic receptors

Synaptosomal membranes from cat cerebral cortex were labelled with [³H]-quinuclidinyl benzylate ([³H]-QNB). There was shown to be a single type of binding site with Kd = 0.34 nM, Bmax = 2.2 nmol/g protein and Hill No. = 1.01. Triton X-100 at 5 × 10^?4% inhibited the specific binding of [³H]QNB and the inhibition was almost complete at 10^?2%. Treatment with 2.5 × 10^?6M atropine, followed by centrifugation washings protected the receptor site from the inhibitory action of the detergent. The protection afforded by other cholinergic drugs was less effective. The use of this technique has confirmed the results of our previous work on the possible pre- and postsynaptic location of central muscarinic receptors. These findings open the possibility for protection of other detergent-sensitive receptors and for their isolation and purification as well-defined macromolecules.     

GABA and benzodiazepine receptors in the offspring of dams receiving diazepam: ontogenetic studies

Ontogenesis of the regulation of ³H-GABA and ³H-diazepam binding to rat brain plasma membranes treated with 0.05% Triton X-100 has been studied. The density of ³H-diazepam and ³H-GABA binding in cortex, cerebellum and corpus striatum at birth was approximately one third of the adult values. They increased at the same rate and reached the adult values between 14–21 days after birth. Study of the binding characteristics showed that the K_D for high and low affinity for ³H-GABA, and for ³H-diazepam did not change during ontogenesis and the increase reflects only an increase of Bmax. The number of Triton X-100 treatments of crude synaptic membrane (CSM) required to maximize ³H-GABA for the high affinity component were different at various postnatal days: only one treatment was required in 1-day old rats, two in 7- and 14-day old rats and three in adult animals. In addition, the capability of muscimol to stimulate ³H-diazepam binding in both frozen-thawed and Triton X-100 treated membrane preparations decreased with increasing age. Binding of ³H-GABA and ³H-diazepam to brain of newborn rats whose dams received diazepam throughout pregnancy (100 mg/kg, × os, bid) was also studied. No significant differences were observed in the ontogenetic development of both bindings. However, in the cortex of these newborn rats the capability of muscimol to stimulate ³H-diazepam binding was greatly reduced in Triton X-100-treated membranes.     

Agonist potency at P2X7 receptors is modulated by structurally diverse lipids

BACKGROUND AND PURPOSE: The P2X(7) receptor exhibits a high degree of plasticity with agonist potency increasing after prolonged receptor activation. In this study we investigated the ability of lipids to modulate agonist potency at P2X(7) receptors. EXPERIMENTAL APPROACH: A variety of lipids, including lysophosphatidylcholine, sphingosylphosphorylcholine and hexadecylphosphorylcholine were studied for their effect on P2X(7) receptor-stimulated ethidium bromide accumulation in cells expressing human recombinant P2X(7) receptors and on P2X(7) receptor-stimulated interleukin-1 beta (IL1 beta) release from THP-1 cells. The effects of the lipids were also assessed in radioligand binding studies on human P2X(7) receptors. KEY RESULTS: At concentrations (3-30 microM) below the threshold to cause cell lysis, the lipids increased agonist potency and/or maximal effects at P2X(7) receptors in both ethidium accumulation and IL1 beta release studies. There was little structure activity relationship (SAR) for this effect and sub-lytic concentrations of Triton X-100 partially mimicked the effects of the lipids. The lipids caused cell lysis and increased intracellular calcium at higher concentrations (30-100 microM) which complicated interpretation of their effects in functional studies. However, the lipids (3-100 microM) also increased agonist potency 30-100 fold in radioligand binding studies. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that a diverse range of lipids increase agonist potency at the P2X(7) receptor in functional and binding studies. The broad SAR, including the effect of Triton X-100, suggests this may reflect changes in membrane properties rather than a direct effect on the P2X(7) receptor. Since many of the lipids studied accumulate in disease states they may enhance P2X(7) receptor function under pathophysiological conditions.     

Differential effects of triton X-100 on benzodiazepine and GABA binding in a frozen-thawed synaptosomal fraction of rat brain

The effect of Triton X-100 on 3H-GABA and 3H-diazepam binding was measured in a frozen-thawed synaptosomal fraction of rat brain. Specific binding activity (amount bound per mg protein) of both ligands was increased by the treatment. Diazepam binding capacity in the pellet was progressively decreased, while GABA binding was increased, then decreased by increasing Triton X-100. Diazepam binding affinity was unchanged, while GABA binding affinity increased. Triton X-100 appears to preferentially solubilize benzodiazepine binding sites, indicating GABA and benzodiazepine binding sites are on separate macromolecules.     

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.     

DIDS, an anion transport blocker, modulates ivermectin-induced enhancement of benzodiazepine receptor binding in rat brain

Ivermectin enhancement of [3H]diazepam binding was abolished by pretreatment of membranes with 0.05% Triton X-100, whereas GABA-induced enhancement was not changed after this treatment. Ivermectin enhancement was neither affected by picrotoxinin nor dependent on the chloride ion. 4,4'-Diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) dose-dependently reduced [3H]diazepam binding enhanced by 10(-6) M ivermectin, without affecting the basal specific binding. The effects of DIDS were derived from reduction of increased binding affinity of benzodiazepine receptors by ivermectin, but were not dependent on chloride ion in the assay medium. DIDS inhibited GABA- and pentobarbital- but not chloride ion-induced enhancement of [3H]diazepam binding.     

Characterization of Glutamate Receptor by Spider Toxin

Abstract

Recent study has shown that the venom of some orb-web spiders contain potent blockers of the glutamate receptors. Joro spider toxin (JSTX) derived from Nephila clavata has been found to block excitatory postsynaptic potentials and glutamate-evoked responses in the neuromuscular synapse of crustacea, the squid giant synapse and the mammalian brain synapse. Structures of the toxins (JSTXs, NSTXs) of spiders belonging to the genus Nephila were determined and it was found that a unique 2,4-dihydroxyphenylacetyl asparaginyl cadaverine part was conserved between all toxins, indicating that this part is intimately involved in the blocking activity.

Labeling of synthesized JSTX-3 was used for histological investigation of glutamate receptors. Using autoradiography ¹²⁵I-JSTX-3 was found to bind at the lobster neuromuscular synapse. A histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat hippocampus and cerebellum. JSTX-3 was used for isolation of glutamate receptors from brain. A crude synaptic membrane fraction from rat hippocampus and cerebellum was solubilized by Triton X-100. SDS-PAGE of the affinity purified JSTX-3 binding proteins showed at least 4 bands around 70 K daltons.     

Sigma-2 receptors are specifically localized to lipid rafts in rat liver membranes

We have previously shown that sigma-2 receptors are relatively difficult to solubilize (Eur. J. Pharmacol. 304 (1996) 201), suggesting possible localization in detergent-resistant lipid raft domains. Rat liver membranes were treated on ice with 1% Triton X-100 or 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and the extract subjected to centrifugation on a discontinuous gradient of 5%, 38%, and 40% sucrose. Gradient fractions were analyzed for sigma-1 receptors using [3H]+-pentazocine and for sigma-2 receptors using [3H]1,3-di-o-tolylguanidine ([3H]DTG), in the presence of dextrallorphan. Flotillin-2 was assessed by immunoblotting as a marker for lipid rafts. Sigma-2 receptors were found to discretely co-localize with flotillin-2 in lipid raft fractions. However, sigma-1 receptors were found throughout the gradient. Rafts prepared in CHAPS had sigma-2 receptors with normal pharmacological characteristics, whereas those in Triton X-100-prepared rafts had about seven-fold lower affinity for [3H]DTG and other ligands. Thus, sigma-2 receptors are resident in membrane lipid rafts, whereas sigma-1 receptors appear in both raft and non-raft membrane domains. Lipid rafts may play an important role in the mechanism of sigma-2 receptor-induced apoptosis.     

Effects of phosphorylation and cytoskeleton-affecting reagents on GABA(A) receptor recruitment into synaptosomes following acute stress

The [3H]-flunitrazepam receptor density, measured ex vivo in synaptosomes at 4 degrees C, increased by about 30% because of acute stress in chicks. This increase was first reported to be a receptor recruitment due to the fact that the increase induced by subsolubilizing concentrations of Triton X-100 was not additive to the receptor increase induced by acute stress [J Neural Transm 87 (1992) 97]. In synaptosomal membranes from stressed chicks, the incorporation of alkaline phosphatase or ATP into the lumen abolished or increased, respectively, the receptor unmasking after incubation at 4 and 37 degrees C, suggesting that phosphorylation plays a role in the recruitment mechanism. Moreover, both colchicine and vinblastine, but not taxol, abolished the recruitment induced by stress at 37 degrees C only in synaptosomes, suggesting that micrutubule depolymerization plays a role in the masking of receptors. Furthermore, both cytochalasins C and D induced an increase of the receptor density, abolished by N-ethylmaleimide, in both the stressed and nonstressed conditions, suggesting that microfilament depolymerization induced the exposure to the radioligand of a cytosolic vesicular receptor pool, which had not fused yet with the postsynaptic membrane.     

Morphological and physiological changes in Tetrahymena pyriformis for the in vitro cytotoxicity assessment of Triton X-100.

Non-ionic surfactants such as Triton X-100 have been widely used in industrial processing and in cleaning products for almost 50 years, being effective and economic emulsifying, wetting agents, dispersants and solubilizers. Cleaning products containing these surfactants are disposed of mainly by discharge into wastewater, which receives biological treatment in wastewater treatment systems. However, surface-active agents interact with eukaryotic cell membranes leading to biological damage at high concentrations. Tetrahymena pyriformis was used here as model organism to assess the effects of Triton X-100 through a series of in vitro cytotoxicity tests. Growth rates and morphological changes were, by their simplicity and reproducibility, the simplest toxicological assays. Cytoskeleton analysis seemed to be related with phagocytosis rate. Viability was evaluated by two different tests. Calcein AM/EthD-1 was used to assess T. pyriformis membrane damage during the 48-h experiment. The colorimetric MTT assay proved to be highly sensitive even at very short periods of Triton X-100 exposure. Tests performed in this study included simple and fast bioassays that provide overall information on the morphological and physiological state of cells exposed to different non-lytic and lytic concentrations of Triton X-100.     

Effects of substance P on the binding of ligands to nicotinic acetylcholine receptors

The effect of substance P on the binding of many ligands that interact with the nicotinic acetylcholine receptor was examined in membrane preparations of Torpedo electroplaque and BC3H-1 cells and in solubilized membranes of rat, chick, and goldfish brain. In the absence of carbamylcholine, the affinity of [3H]phencyclidine for the high affinity local anesthetic binding site on Torpedo membranes was increased by substance P with an EC50 of approximately 5 microM. In the presence of carbamylcholine, which itself increases [3H]phencyclidine binding affinity, substance P caused a decrease in the affinity of [3H]phencyclidine. The concentration dependence of the inhibition, however, was inconsistent with a competitive interaction, since the apparent Hill coefficient was significantly less than one. We conclude from these results that substance P does not directly interact with the high affinity local anesthetic binding site on the nicotinic receptor of Torpedo membranes. Substance P also does not appear to interact directly with the agonist binding site since the peptide had no significant effect on [3H]acetylcholine binding to Torpedo membranes. Substance P inhibited [125I]alpha-bungarotoxin binding to both native and Triton X-100 solubilized Torpedo membranes, although the IC50 was 8-fold higher for the solubilized preparation (12 versus 93 microM). We interpret this inhibition in solubilized membranes as evidence that the peptide may interact directly with a binding site on the nicotinic acetylcholine receptor. Substance P also decreased the initial rate of [125I]alpha-bungarotoxin to membranes prepared from BC3H-1 cells (IC50 = 108 microM) and to solubilized membranes from rat, chick, and goldfish brain. In the brain membranes, however, the peptide did not completely inhibit binding; at the highest concentration examined (100 microM), the maximum inhibition observed was 60%. Consistent with the results for [3H]acetylcholine binding to Torpedo membranes, the peptide had no effect on the binding of the cholinergic agonist [3H](-)nicotine to these tissue preparations. These data suggest that substance P may have a general modulatory action on a subclass of nicotinic receptors that include muscle-type, ganglionic-type, and a putative subpopulation of central nervous system receptors.     

Developmental changes in ANP-stimulated guanylyl cyclase activity enhanced by ATP in rat lung membrane fractions.

1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system.     

Demonstration of alpha-adrenoceptors in the rabbit bladder base and urethra with 3H-dihydroergocryptine ligand binding

The purpose of this work was to demonstrate the presence of alpha-adrenoceptors in a crude membrane preparation made from rabbit bladder base and urethra. This was achieved by radioligand binding studies, using 3H-dihydro-alpha-ergocryptine (3H-DHE) as the radioligand. The specific binding, i.e. the binding that could be inhibited by 10(-5) M phentolamine, was saturable with 73 fmol 3H-DHE bound per mg membrane protein. Binding was at steady state after 60 min., and reversible. Rate constants for association and dissociation were 3 X 10(7) M-1 min.-1, and 2 X 10(-2) min.-1, respectively. A number of compounds were tested for their abilities to compete with 3H-DHE for the binding sites. The relative affinity of some adrenoceptor agonists was: (-)-adrenaline greater than (-)-noradrenaline much greater than (+/-)-isoprenaline. Stereoselectivity was shown, since (-)-noradrenaline had 42 times higher affinity than (+)-noradrenaline. Adrenoceptor antagonists inhibited 3H-DHE binding in the following order of potency: DHE greater than phentolamine much greater than (+/-)-propranolol. The dissociation constant, KD, for DHE to the binding sites was estimated in three different ways. The constants were derived from saturation, competition, and kinetic studies, and gave KD values of 1.1, 1.4 and 0.7 nM, respectively. The results suggest that alpha-adrenoceptors were labelled by 3H-DHE in the tissue homogenates.     

Characterization of a domoic acid binding site from Pacific razor clam

The Pacific razor clam, Siliqua patula, is known to retain domoic acid, a water-soluble glutamate receptor agonist produced by diatoms of the genus Pseudo-nitzschia. The mechanism by which razor clams tolerate high levels of the toxin, domoic acid, in their tissues while still retaining normal nerve function is unknown. In our study, a domoic acid binding site was solubilized from razor clam siphon using a combination of Triton X-100 and digitonin. In a Scatchard analysis using [3H]kainic acid, the partially-purified membrane showed two distinct receptor sites, a high affinity, low capacity site with a KD (mean +/- S.E.) of 28 +/- 9.4 nM and a maximal binding capacity of 12 +/- 3.8 pmol/mg protein and a low affinity, high capacity site with a mM affinity for radiolabeled kainic acid, the latter site which was lost upon solubilization. Competition experiments showed that the rank order potency for competitive ligands in displacing [3H]kainate binding from the membrane-bound receptors was quisqualate > ibotenate > iodowillardiine = AMPA = fluorowillardiine > domoate > kainate > L-glutamate. At high micromolar concentrations, NBQX, NMDA and ATPA showed little or no ability to displace [3H]kainate. In contrast, Scatchard analysis using [3H]glutamate showed linearity, indicating the presence of a single binding site with a KD and Bmax of 500 +/- 50 nM and 14 +/- 0.8 pmol/mg protein, respectively. These results suggest that razor clam siphon contains both a high and low affinity receptor site for kainic acid and may contain more than one subtype of glutamate receptor, thereby allowing the clam to function normally in a marine environment that often contains high concentrations of domoic acid.     

Effect of olfactory bulbectomy and chronic amitryptiline treatment in rats. 3H-imipramine binding and behavioral analysis by swimming and open field tests

An 'animal model' of depression, based on bulbectomy, followed by chronic treatment with amitryptiline was used in rats. In the synaptosomal membranes of the cerebral cortex plus hippocampus, the number of binding sites for 3H-imipramine increased significantly when bulbectomy was associated with the antidepressant. In the bulbectomized rats the tendency was toward a decrease in binding. The treatment with 0.2% Triton X-100 of the membranes revealed a large increase in postsynaptic sites in the bulbectomized treated rats. The behavioral parameters analyzed by the swimming with a water wheel and the open field test revealed a series of differences in the various groups of rats, with respect to handling, bulbectomy and antidepressant treatment. Handling resulted in an increase in swimming time in controls, while bulbectomy reduced this parameter. In both the swimming and open fields tests, chronic bulbectomy reduces the motility of the rat. In control rats chronic amitryptiline increases locomotion and exploratory activity, a behavioral effect that is even more prominent in bulbectomized treated rats.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.