Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents.

The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine through an exchange reaction. This unique property distinguishes this cysteine residue from the remaining cysteine residues of the (H+)-ATPase. Using this reaction, we selectively labeled the cystine-reactive cysteine residue of subunit A with fluorescein-maleimide. After complete digestion of the labeled subunit A by V8 protease, a single labeled fragment of molecular mass 3.9 kDa was isolated and the amino-terminal sequence was determined. This fragment contains 2 cysteine residues, Cys240 and Cys254. Since Cys254 is conserved among all vacuolar (H+)-ATPases whereas Cys240 is not, it is likely that Cys254 is the residue which is responsible for the sensitivity of the vacuolar (H+)-ATPase to sulfhydryl reagents.     

Vicinal dithiol-disulfide distribution in the Escherichia coli mannitol specific carrier enzyme IImtl

Escherichia coli mannitol specific EII in membrane vesicles can be inhibited by the action of the oxidizable substrate-reduced phenazine methosulfate (PMS) in a manner similar to E. coli enzyme IIGlc [Robillard, G. T., & Konings, W. (1981) Biochemistry 20, 5025-5032]. The fact that reduced PMS and various oxidizing agents protect the enzyme from inactivation by the sulfhydryl reagents N-ethylmaleimide and bromopyruvate suggests that the active form possesses a dithiol which can be protected by conversion to a disulfide. The sulfhydryl-disulfide distribution has been examined in purified EIImtl by labeling studies with N-[1-14C]ethylmaleimide ( [14C]NEM). EIImtl can be alkylated at three positions per peptide chain. When alkylation takes place in 8 M urea, only two positions are labeled. The third position becomes labeled in urea only after treatment with DTT, suggesting that the native enzyme is composed of two subunits linked by a disulfide bridge. The remaining two sulfhydryl groups per peptide chain appear to undergo changes in oxidation state as indicated by the following results. (1) Treatment of the active enzyme with NEM leads to complete inactivation and incorporation of 1 mol of [14C]NEM per peptide chain. Oxidizing agents protect the activity and prevent labeling presumably by forming a disulfide. (2) Phosphorylating the enzyme (one phosphoryl group per peptide chain) fully protects the activity, but 1 mol of NEM per peptide chain is still incorporated. Subsequent dephosphorylation by adding mannitol causes a second mole of [14C]NEM to be incorporated and results in complete inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.     

Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase

The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.     

Soluble human core 2 beta6-N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity

Human UDP-GlcNAc: Galbeta1-3GalNAc- (GlcNAc to GalNAc) beta1,6-GlcNAc-transferase (C2GnT1) is a member of a group of beta6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these beta6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. beta-Mercaptoethanol (beta-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K(M) and V(max) values for acceptor substrate Galbeta1-3GalNAcalpha-p-nitrophenyl (GGApnp), but the K(M) value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.     

Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with [14C]DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump.     

Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.     

Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.     

Effect of N-ethylmaleimide on beef and rat liver vitamin K1 epoxide reductase

There is little difference in the extent of inactivation of beef liver microsomal vitamin K1 epoxide reductase by N-ethylmaleimide (NEM) whether or not the microsomes are pre-treated with dithiothreitol (DTT). The rat liver microsomal enzyme, however, is inactivated by NEM to a much greater extent if the microsomes are pre-treated with DTT. The beef liver enzyme activity is protected from NEM inactivation by the substrate, vitamin K1 epoxide. Ping-pong kinetics are exhibited by the beef liver enzyme. These results support a mechanism for vitamin K1 epoxide reductase in which the function of the required dithiol is to reduce an active site disulfide bond; however, the geometry of the active sites of the enzyme from rat and beef may be different.     

The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action

Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.     

Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

The cysteines of the membrane-bound mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity [Roossien, F. F., & Robillard, G. T. (1984) Biochemistry 23, 211-215]. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [14C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing.     

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.     

Ouabain-binding site of (Na+ + K+)-ATPase in right-side-out vesicles has not an externally accessible SH group

The fluorescing sulfhydryl reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) inactivates purified (Na+ + K+)-ATPase at 20 microM. This inactivation results in a decrease of the ouabain-binding capacity of the enzyme. Treatment of (Na+ + K+)-ATPase, embedded in right-side-out-oriented vesicles, by DACM does not affect ouabain binding to the enzyme. Incorporation of DACM into the alpha subunit of (Na+ + K+)-ATPase embedded in right-side-out vesicles is also not affected by the presence or absence of 100 microM ouabain. It is therefore concluded that a sulfhydryl group does not reside within the ouabain-binding site of (Na+ + K+)-ATPase.     

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H(2)O(2) or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H(2)O(2), evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H(2)O(2) production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.     

Oxidative inactivation of purified plasma membrane Ca2+-ATPase by hydrogen peroxide and protection by calmodulin

Calmodulin (CaM)-regulated plasma membrane Ca(2+)-ATPase (PMCA) is critical for the regulation of free intracellular Ca(2+) levels. PMCA activity and levels in neuronal membranes are decreased with aging, possibly due to oxidation-induced inactivation. In the present studies, inhibition of PMCA by H(2)O(2) was characterized in enzyme purified from human erythrocyte membranes. Basal and CaM-stimulated PMCA activities were inhibited by exposure to H(2)O(2) (25-100 microM). However, neither the concentration-dependent enhancement of PMCA activity by CaM nor the binding of CaM to H(2)O(2)-exposed PMCA was disrupted by treatment with H(2)O(2). Rates of inactivation by H(2)O(2) of basal and CaM-stimulated PMCA were nearly identical. The addition of CaM after exposure to H(2)O(2) did not protect enzyme activity, although the binding of CaM to PMCA before exposure to H(2)O(2) protected the enzyme completely, indicating a CaM-induced conformational state resistant to oxidation. H(2)O(2) quenched Trp fluorescence in PMCA, an index of conformational changes, with a rate similar to that observed for enzyme inactivation. H(2)O(2) enhanced the solvent accessibility of Trp residues in PMCA, whereas accessibility of the only Trp residue in the CaM-binding domain peptide was unaltered. Exposure of PMCA to H(2)O(2) led to aggregate formation partially reversible by dithiothreitol (DTT) but not to recovery of activity. Amino acid analysis indicated Cys modification following H(2)O(2) exposure but no Cys oxyacids. Because DTT did not reverse inactivation by H(2)O(2), it appears that the disulfide bond formation led to conformational changes that were not fully reversed when the bonds were reduced. Preincubation of PMCA with CaM protected the enzyme from undergoing this conformational change.     

N-Ethylmaleimide Modifies the Conformation of the Plasma Membrane H+-ATPase,Strengthening the Inhibitory Action of the C-terminal Domain1

Abstract: The effect of cysteine modification with N-ethylmalei-mide (NEM) on the activity of the plasma membrane (PM) H+-ATPase and on its activation state was investigated in PM isolated from aged red beet parenchyma slices. Treatment of PM with increasing concentrations of NEM (0.1–1mM) drastically reduced H+-ATPase activity. The inhibiting effect of PM treatment with NEM was stronger when the H+-ATPase activity was assayed at pH values (7.1–7.2) higher than that optimal for enzyme activity (6.3). If the PM H+-ATPase was activated by proteolytic cleavage of the C-terminal domain or by its displacement by fusicoccin prior to NEM treatment, the inhibitory effect of NEM on the W-ATPase activity became independent of the pH of the assay medium. Moreover, inhibition by NEM of H+-ATPase activity also became independent of the pH of the assay medium if the C-terminal was proteolytically cleaved or displaced by lysophosphatidylcholine after NEM treatment of the PM. Controlled trypsin treatment of NEM-treated PM produced, beside the 90 kDa truncated PM H+-ATPase, fragments of 60 to 30 kDa of the enzyme that were undetectable after trypsin treatment of control PM. These results indicate that PM treatment with NEM modifies the H+-ATPase conformation, exposing trypsin cleavage sites scarcely accessible in control PM and strengthening the autoinhibitory action of the C-terminal domain.     

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.