Effects Of Sulfhydryl Reagents Research Articles

Effects of Sulfhydryl Reagents on Nitric Oxide Release from a Nitric Oxide Donor NOR 3 in the Presence of Rat Hepatocytes

Effects of Sulfhydryl Reagents on Nitric Oxide Release from a Nitric Oxide Donor NOR 3 in the Presence of Rat Hepatocytes

A possible effect of sulfhydryl reagents on the contractile activity of the rat detrusor muscle

We aimed to investigate the effect of sulfhydryl (SH) inactivating agents, ethacrynic acid and N-ethylmaleimide, on the contractile activity of rat detrusor muscle. Wistar Kyoto rats weighing 150–250 g were anaesthetized with ketamine and bled to death. The urinary bladders were surgically removed and detrusor strips were mounted under 0.5 g tension in organ baths. The responses were recorded with isotonic transducers on polygraph paper. After an equilibrium period, the tissues were contracted by electrical field stimulation, acetylcholine, ethacrynic acid or N-ethylmaleimide and the effects of l-cysteine, glutathione, verapamil, Ca 2+-free solution, sodium nitroprusside or atropine were then examined on these contractions. Verapamil, Ca 2+-free solution or atropine significantly reduced the contractions elicited by electrical field stimulation and acetylcholine whereas l-cysteine, glutathione or sodium nitroprusside had no effect on the contractions in response to these stimuli. l-Cysteine, glutathione, verapamil or Ca 2+-free solution significantly inhibited the contractions induced by ethacrynic acid or N-ethylmaleimide. Sodium nitroprusside slightly inhibited only the contraction induced by ethacrynic acid but not that with N-ethylmaleimide. Atropine has no action on the contractions in response to these SH reagents. These findings suggest that SH reagents may play a role in the contractile activity of rat detrusor muscle and this action seems to be related to the gating of Ca 2+ channels. Further experiments are needed to determine the cellular mechanism(s) of action by which these SH reagents act on the detrusor smooth muscle.

Effects of sulfhydryl reagents on nitric oxide release from a nitric oxide donor NOR 3 in the presence of rat hepatocytes.

Effects of sulfhydryl reagents on nitric oxide release from a nitric oxide donor NOR 3 in the presence of rat hepatocytes.

Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes.

The effects of sulfhydryl (SH) reagents on the L-type Ca current (ICa) were studied in frog ventricular myocytes using the whole-cell patch-clamp method. Methanethiosulfonate ethylammonium (MTSEA+) was found to enter the cell through the membrane and cause a remarkable increase in Ica from the intracellular side. Methanethiosulfonate ethyltrimethylammonium (MTSET+) and methanethiosulfonate ethylsulfonate (MTSES-) could not penetrate the membrane and were effective only when directly applied to the intracellular side. In addition, suppressive effects on ICa of these MTS reagents were indicated by the following observation. A progressive decay in the peak amplitude of ICa after establishing maximal ICa, stimulated by intracellular MTSET+, was prevented by adding extracellular dithiothreitol (DTT). The SH-oxidizing agents N-ethylmaleimide (NEM), chloramine-T (CL-T), 2,2'-dithiodipyridine (DTDP) and 2,2'-dithio-bis-5-nitropyridine (DTBNP) also exerted a stimulatory effect on Ica. The effect of SH reagents persisted even when cAMP production was inhibited with Rp-cAMP-S, or when G-protein was inhibited with 1 mM GDPbetaS, indicating that the effect is not due to cAMP production or G-protein stimulation. It is concluded that there are sites on the Ca channels that are subject to direct modification by SH reagents.

Involvement of Hg 2+-sensitive sulfhydryl groups in regulating noradrenaline release induced by S-nitrosocysteine in rat brain slices

Nitric oxide has been shown to regulate neurotransmitter release. Previously, we reported that S-nitrosothiols such as S-nitrosocysteine (SNC) stimulate noradrenaline (NA) release in rat hippocampus in vivo and in vitro. To examine the role of sulfhydryl groups in SNC-induced NA release, the effects of metal ions such as Hg 2+ and N-ethylmaleimide (NEM, a sulfhydryl alkylating agent) on [ 3H]NA release from labeled rat brain slices (hippocampus and cerebral cortex) were studied and compared with the effects of SNC. The addition of 200 μM HgCl 2, but not Pb 2+, Zn 2+, or Cd 2+, stimulated [ 3H]NA release from both types of slices in the presence of extracellular CaCl 2. p-Chloromercuribenzoic acid ( p-CMBA) also stimulated [ 3H]NA release. NEM stimulated [ 3H]NA release from both types of slices in the presence and absence of extracellular CaCl 2. The effect of 200 μM NEM was enhanced, but the effect of 200 μM SNC was inhibited by co-addition of 200 μM p-CMBA in the absence of extracellular CaCl 2. The concentration–response curve of SNC shifted to the right after co-addition of 200 μM p-CMBA or 100 μM HgCl 2, although the effect of 200 μM NEM was additive to the effect of SNC. These findings demonstrate that SNC acts as a sulfhydryl agent on proteins that regulate NA release, and that SNC may share the same sulfhydryl groups with Hg compounds. The effect of T-588 {( R)-(-)-(benzo[ b]thiophen-5-yl)-2-[2-( N, N-diethylamino)ethoxy]ethanol hydrochloride}, a novel cognitive enhancer and a stimulator of NA release, was compared with the effects of sulfhydryl reagents.

Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes

Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface.

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.

Functional significance of cysteine residues in the δ opioid receptor studied by site-directed mutagenesis

Previous work suggested that sulfhydryl groups and disulfide bridges have important functions in opioid binding to the delta opioid receptor. The question regarding which cysteines are essential for ligand binding was approached by replacement of cysteine residues in the cloned delta opioid receptor using site-directed mutagenesis. The wild-type and mutant receptors were expressed stably in Chinese hamster ovary cells. The two extracellular cysteine residues and the six located in transmembrane domains were mutated to serine or alanine, one at a time. Replacement of either of the extracellular cysteines produced a receptor devoid of delta agonist and antagonist binding activity. Immunofluorescence cytochemistry, performed with anti delta opioid receptor antibodies in washed cell monolayers in one of these mutants (Cys-Ser121), and immunoblots, performed on cell extracts, indicate that the receptor was expressed and seems to be associated with the cell membrane. The existence of an essential extracellular disulfide bridge, previously postulated by analogy to other G protein coupled receptors, is strongly supported by our results. Replacement of any one of the six transmembrane cysteines was virtually without effect on the ability of the receptor to bind delta agonists and antagonists. Since there is strong evidence that the transmembrane domains are involved in ligand binding, these results suggest that the cysteine residues, even those near or at the binding site, are not essential for receptor binding. Furthermore, these results support the idea that the striking effects of sulfhydryl reagents on ligand binding of opioid receptors are likely to be due to steric hindrance by the large moieties transferred to the sulfhydryl groups of cysteine residues by these reagents.

Role in fast inactivation of the IV/S4-S5 loop of the human muscle Na+ channel probed by cysteine mutagenesis.

1. In order to investigate the role in fast inactivation of the cytoplasmic S4-S5 loop of the fourth domain (IV/S4-S5) within the alpha-subunit of the adult human muscle Na+ channel, every single amino acid from R1469 to G1486 was substituted by a cysteine and the mutants were studied by functional expression in human embryonic kidney cells (tsA201) using whole-cell patch clamping. Effects following intracellular application of the sulfhydryl reagents MTSET and MTSES on the mutants were investigated. 2. Sixteen of eighteen mutants resulted in the formation of functional channels. For P1480C and N1484C, no Na+ currents could be detected in transfected cells. In the absence of sulfhydryl reagents, F1473C and A1481C slowed fast Na+ channel inactivation by 2- and 1.5-fold, respectively, and L1482C induced a steady-state Na+ current (Iss) of 3% of peak current (Ipeak) (1% for wild-type). 3. Upon application of MTSET and MTSES, changes in fast inactivation gating occurred for most of the mutants. The most dramatic destabilizing effects on fast inactivation were observed for M1476C (9-fold slowing of inactivation; Iss/Ipeak, 3.6%; +15 mV shift in steady-state inactivation; 2- to 3-fold acceleration of recovery from inactivation), A1481C (3-fold; 14%; +20 mV; no change) and F1473C (2.5-fold; 2.4%; +8 mV; 1.5-fold). Less pronounced destabilizing effects were observed for M1477C and L1479C. Strongly stabilizing effects on the inactivated state, that is a 20-30 mV hyperpolarizing shift of the inactivation curve associated with a 3- to 4-fold decrease in the rate of recovery from inactivation, occurred for T1470C, L1471C and A1474C. Almost all effects were independent of the membrane potential; however, A1474C only reacted when cells were depolarized. Significant effects on activation were not observed. 4. We conclude that the IV/S4-S5 loop plays an important role in fast inactivation of the muscle Na+ channel and may contribute to the formation of a receptor for the putative inactivation particle. The effects of sulfhydryl reagents on the various mutations suggest an alpha-helical structure of IV/S4-S5 (up to P1480) with destabilizing effects on inactivation for one cluster of amino acids (1473/76/77/79) and a stabilized inactivation at the opposite side of the helix (1470/71/74).

Sulfhydryl modification inhibits K + (M) current with kinetics close to acetylcholine in rodent NG108-15 cells

The effects of sulfhydryl reagents on M-type voltage-dependent potassium currents ( I K(M)) were examined in NG108-15 cells transformed to express m1 muscarinic acetylcholine receptors (mAChRs), a NGPM1-27 clone. Focal application of glutathione at millimolar concentrations dissolved in acidic solutions caused a transient inward current in NGPM1-27 cells at holding potentials of −30 mV, associated with an inhibition of I K(M). The glutathione-induced response was mimicked by cysteine. These effects were also reproduced by superfusion with micromolar concentrations of HgCl 2, AgNO 3, N-methylmaleimide and p-chloromercuribenzoic acid ( pCMB), agents which target protein thiols. Glutathione, HgCl 2, AgNO 3 and pCMB inhibited the peak conductance of I K(M) without shifting the half activating voltage ( V 1/2), which was comparable to the acetylcholine (ACh)-induced response. The voltage dependence of time constants for I K(M) deactivation in sulfhydryl reagent-, ACh- and non-treated cells resembled, but differed from that in Ba 2+-treated cells. These results reveal that there is an accessible cysteine moiety, but not a disulfide bond, either on the M channel protein itself or on a protein directly involved in agonist-M channel coupling.

Effects of sulfhydryl reagents on the regulation of cytosolic pH in rat sublingual acini.

The effects of sulfhydryl (SH) reagents on the regulation of cytosolic pH (pHi) in rat sublingual mucous acini were examined using pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The membrane permeable SH reagent N-ethylmaleimide (NEM) resulted in a cytosolic acidification (0.19 +/- 0.02 pH unit after 5-min treatment). In contrast, the impermeable SH reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (PCMB) and Cu2+ did not affect the resting pHi. Muscarinic stimulation with carbachol (CCh) induced a cytosolic acidification (0.15 +/- 0.01 pH unit after 5 min). Cu2+ enhanced the CCh-stimulated acidification 2-fold (0.30 +/- 0.02 pH unit after 5 min). Preincubation with SH reducing agent dithiothreitol (DTT) prevented the NEM-induced and Cu2+ -enhanced pHi decreases. The Cu2+ -enhanced but not the NEM-induced acidifications were reversed by addition of DTT after stimulation. Na+/H+ exchange was not influenced by Cu2+ or NEM, suggesting that the SH reagent-induced acidification is not caused by inhibition of exchanger activity. Anion channel inhibitor diphenylamine-2-carboxylate (DPC) abolished the NEM-induced and the Cu2+ -enhanced acidifications, suggesting that the acidification was mediated by HCO3- efflux via anion channels. Cu2+ did not affect the CCh-evoked increase in cytosolic free Ca2+ concentration ([Ca2+]i). In contrast, NEM triggered an elevation in [Ca2+]i, and DTT completely prevented this increase. The CCH-stimulated [Ca2+]i increases were prevented by blocking Ca2+ release from the intracellular pool with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), and the NEM-stimulated [Ca2+]i increases were abolished by chelating cytosolic Ca2+ with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). TMB-8 prevented the CCh-stimulated, but not the Cu2+ -enhanced pHi decrease, and the NEM-induced pHi decrease was not blocked by BAPTA, suggesting that the effects of Cu2+ and NEM on anion channels are independent of Ca2+. Taken together, these results indicate that SH groups play a critical role in pHi regulation and the reactive SH groups are probably located on cytoplasmic site(s) of anion channels or an associated regulatory protein.

Effects of Sulfhydryl Reagents, Retinoids, and Solubilization on the Activity of Microsomal Retinol Dehydrogenase

A microsomal retinol dehydrogenase (RoDH) that recognizes hole-cellular retinol binding protein (CRBP) as substrate is inhibited by phenylarsine oxide (IC 50 = 3 μM) in the presence of 10 mM cysteine. Inhibition was reversible with dithiothreitol, indicating that two cysteine residues in close proximity are essential for RoDH activity. Bromophenylarsine oxide was an irreversible inhibitor (IC 50 = 0.2 μM)) suggesting that a nucleophile lies close to the two cysteine residues. N-Ethylmaleimide inhibited reactions supported by holo-CRBP, but not from free retinol, suggesting that it obstructed holo-CRBP access to RoDH without affecting the catalytic site. RoDH activity was similar in microsomes from vitamin A-sufficient or vitamin A-deficient rats and was not inhibited by relatively high concentrations (5 μM) of all- trans-retinoic acid, holo-cellular retinoic acid binding protein, ape-cellular retinoic acid binding protein, or 9- cis-retinoic acid. Triton X-100 stimulated RoDH activity eightfold at a detergent to protein ratio of 0.25 to 1 (w/w). A combination of Tween 80, Brij 92, and Triton X-100 (2:1:2) stimulated RoDH activity eightfold at a detergent to protein ratio of 2.5 to 1 (w/w). Detergent-solubilized RoDH, partially purified through a PAO-Sepharose resin, preferred NADP(H) as cofactor, had a K m for retinal synthesis from holo-CRBP of 0.6 μM ( V max = 115 pmol/min/mg protein) and a K m for reduction of retinal bound to CRBP of 0.6 μM ( V max = 613 pmol/min/mg protein). This work provides further insight into microsomal RoDH and strengthens the evidence of an interaction between RoDH and holo-CRBP.

Effects of cysteine modification on the activity of the cGMP-gated channel from retinal rods.

The effect of sulfhydryl reagents on the activity of the cGMP-gated channel from bovine retinal rods was studied by measurements of 8-Br-cGMP-(cGMP)-induced calcium efflux from rod membrane vesicles and records of 8-Br-cGMP-dependent sodium currents through channels incorporated into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol blockers) as well as diamide (dithiol-disulfide conversion agent) have a dual effect on the channels activity: at low concentration, they increase the apparent affinity for cyclic nucleotide ("activation") at the same time inducing a loss of cooperativity for nucleotide binding; at higher concentration, N-ethylmaleimide and diamide produce a reduction of the amplitude and initial rate of the calcium release at saturating nucleotide concentration, while mersalyl is shown to reduce the activity of the channels in bilayer experiments ("inhibition"). Nitric oxide precursors have no effect. The results suggest that blocking at least 1 of the 3 cytoplasmic cysteine residues situated close to the cGMP-binding site in each channel subunit by N-ethylmaleimide, mersalyl, or diamide (forming a dimer between 2 subunits) increases the affinity for the nucleotide. Inhibition is produced by blocking at least one of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersalyl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridge), and may concern a process of channel inactivation. The 3 cytoplasmic sulfhydryl groups are accessible when the channels are in the open state, but not (or much less) accessible when the channels are in the closed state.

Fusicoccin antagonizes the effect of sulfhydryl reagents in inducing an oxidative burst inElodea densaleaves

Fusicoccin antagonizes the effect of sulfhydryl reagents in inducing an oxidative burst in<i>Elodea densa</i>leaves

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes.

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.

Effect of Sulfhydryl Reagents on Spectrin States on the Erythrocyte Membrane

The unique properties of erythrocytes are largely determined by its fibrillar network under the plasma membrane. Spectrin, one major component of the membrane skeleton, has been suggested to play a central role in this process. To understand the mechanism underlying this process, the effect of the sulfhydryl groups of erythrocyte membrane on spectrin structure and function was studied. By using non-denaturing gel analysis, dithiothreitol was found to protect spectrin in its tetramer state. In contrast, iodoacetamide and N-ethylmaleimide enhanced conversion of the spectrin tetramer to dimer and decreased its binding to the membrane. Moreover, when the membrane was treated with cadmium, the tetramer was converted to the dimer on the membrane, while zinc had no effect. Hemolysis experiments showed that cadmium could lyse erythrocytes in vitro. These results indicated that preservation of the spectrin tetramer or even higher oligomer states, by the sulfhvdryl groups may be important to the membrane integrity and the intact cell functions.

Interaction of sulfhydryl reagents with A-type channels of Lymnaea neurons

The effect of sulfhydryl reagents on macroscopic inactivation of A-current in internally perfused Lymnaea neurons under voltage-clamp conditions was investigated. It was found that the binding of Hg 2+ rather than PHMB with channel proteins resulted in a strong decrease of the peak current and the inactivation rate. Hg 2+ markedly influenced the steady-state inactivation but did not change the rate of recovery from inactivation. It was found that both reagents reacted with the same groups of the channel protein and that those are most likely sulfhydryl groups. These groups seemed not to be involved in the gating charge movement. Hg 2+ ions can immobilize some part of the gating charge thereby resulting in strong changes of the steady-state inactivation.

Studies on Gastro Intestinal Absorption of Mercury Compounds. I. Effect of Sulfhydryl Reagents on Rat Small Intestinal Absorption of Mercuric Chloride.

The effect of SH reagents on the absorption of mercuric chloride through the rat small intestine was investigated by the in situ recirculation method. The concentrations of mercury in the perfusion solutions, intestine, blood, kidney and liver were determined at each time intervals. Without SH reagents, the mercury transfer into the blood increased slightly with the time proceeds and mercury was absorbed through the intestine. The addition of L-cysteine, glutathione, 3-mercapto-1, 2-propanediol, dimercaptosuccinic acid, D (-)-penicillamine, diethyldithiocarbamate (DDTC), pyridoxine-5-thiol and thiola did not promote the mercury absorption, nor decreased the mercury content in the perfusion solutions. On the other hand, 2, 3-dimercapto-1-propanol (BAL) and 2-mercaptoethanol (MET) distinctly increased the mercury concentration in the blood, and then promoted the absorption of mercury through the intestine. To investigate the mechanism of the mercury absorption, the effect of SH compounds on the partition coefficients of mercuric chloride between octanol and water was determined. BAL and MET greatly increased the partition coefficient, but most of the other SH reagents except DDTC rather decreased it. Therefore we conclude that the lipophilic chelate formation with SH reagents is the main mechanism for the promotion of mercury absorption through the intestine.

Sulfhydryl Reagents Alter Epidermal Growth Factor Receptor Affinity and Association with the Cytoskeleton

Sulfhydryl (SH) reagents are known to influence the characteristics of many ligand-receptor systems. The SH reagent N-ethylmaleimide has been demonstrated to interact with EGF receptors, and to inhibit EGF receptor kinase activity. The data presented in this paper concern the effect of SH reagents on two intriguing features of the EGF receptor system, namely the presence of low and high affinity EGF binding sites, and the interaction of EGF receptors with the cytoskeleton. SH reagents were observed to induce a disappearance of high, but not low, affinity EGF receptors from the cell surface, and an increase in receptor-cytoskeleton interaction. Comparison of the effects of membrane-permeant and membrane-impermeant SH reagents on wild type and structurally modified EGF receptors suggested that sulfhydryl groups on the cytoplasmic, rather than the extracellular, receptor domain are involved. This indicates that the cytoplasmic domain of the EGF receptor plays a role in the high affinity binding of EGF, and in the interaction of EGF receptors with the cytoskeleton. Experiments with an anti-EGF receptor antibody that specifically blocks the binding of EGF to low affinity receptors indicated that EGF induces a shift in the EGF receptor from low to high affinity. SH reagents probably affect EGF binding by inhibiting this EGF-induced receptor conversion.

Effect of thiol reagents on Ca-ATPase in rabbit lens epithelium

The inhibitory effects of sulfhydryl reagents on Ca-ATPase activity in the rabbit lens epithelium were assessed. Test compounds used in this study were selected on their ability to cause a calcium increase in cultured lenses. Under conditions in which lenses were cultured in the presence of the test compound, epithelial Ca-ATPase was inhibited markedly by diamide, t-BHP, IAA and slightly by selenite. The findings demonstrated that hydrogen peroxide caused little inhibition of Ca-ATPase in the lens epithelium, both when the intact lens was cultured in the presence of the oxidant or when the epithelial homogenate contained peroxide during the assay of enzyme activity. The study suggests that if a thiol-modifying compound can reach Ca-ATPase or its critical SH groups, inhibition is likely.