H2O2-induced Kinetic and Chemical Modifications of Smooth Muscle Myosin
2006; Elsevier BV; Volume: 282; Issue: 7 Linguagem: Inglês
10.1074/jbc.m609499200
ISSN1083-351X
AutoresAlan R. Penheiter, Michelle Bogoger, Patricia Ellison, Barbara Oswald, William J. Perkins, Keith A. Jones, Christine Cremo,
Tópico(s)Muscle Physiology and Disorders
ResumoThe effect of H2O2 on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of Pi release were measured by single-turnover kinetics. H2O2 treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, ∼50% of the molecules lost the ability to bind to ATP. H2O2 treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H2O2-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H2O2-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mm ATP, H2O2 caused a ∼50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H2O2 directly affects the force generating complex. Dithiothreitol treatment reversed the H2O2 inhibition of the maximal force by ∼50%. These data, when compared with the in vitro kinetic data, are consistent with a H2O2-induced loss of functional myosin heads in the muscle. The effect of H2O2 on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of Pi release were measured by single-turnover kinetics. H2O2 treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, ∼50% of the molecules lost the ability to bind to ATP. H2O2 treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H2O2-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H2O2-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mm ATP, H2O2 caused a ∼50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H2O2 directly affects the force generating complex. Dithiothreitol treatment reversed the H2O2 inhibition of the maximal force by ∼50%. These data, when compared with the in vitro kinetic data, are consistent with a H2O2-induced loss of functional myosin heads in the muscle. Contraction of smooth muscle is controlled largely by phosphorylation of the 20-kDa myosin regulatory light chain (RLC), 2The abbreviations used are: RLC, regulatory light chain; ASM, airway smooth muscle; HMM, heavy meromyosin; S1, subfragment 1; MLCK, myosin light chain kinase; SMM, smooth muscle myosin; upHMM, unphosphorylated HMM; pHMM, thiophosphorylated HMM; MantATP, 2′(3′)-methylanthranilyoyl-ATP; ELC, essential light chain; DTT, dithiothreitol; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); MOPS, 4-morpholinepropanesulfonic acid; ATPγS, adenosine 5′-O-(thiotriphosphate). resulting in the cyclic attachment and detachment of myosin to actin (cross-bridge cycling) and the hydrolysis of ATP by actin-activated myosin ATPase (1.Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar). The level of RLC phosphorylation depends on the balance between the activities of MLCK and SMM phosphatase. MLCK is primarily activated (through Ca2+-calmodulin) by an increase in [Ca2+]i from intracellular stores or influx of extra-cellular Ca2+, whereas inhibition of myosin phosphatase, which contributes to the amount of force at a given [Ca2+]i (Ca2+ sensitivity), occurs through a receptor-G protein-catalyzed signal transduction cascade (2.Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1696) Google Scholar, 3.Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (266) Google Scholar, 4.Savineau J.P. Marthan R. Fundam. Clin. Pharmacol. 1997; 11: 289-299Crossref PubMed Scopus (65) Google Scholar, 5.Chitaley K. Weber D. Webb R.C. Curr. 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Physiol. 2002; 283: L1220-L1230PubMed Google Scholar). In general, healthy smooth muscle is quite resistant to exogenous H2O2. The most extreme example is agonist activated bladder smooth muscle (bladder concentrations of H2O2 often reach levels of 0.2–0.4 mm in healthy adults (21.Hiramoto K. Kida T. Kikugawa K. Biol. Pharm. Bull. 2002; 25: 1467-1471Crossref PubMed Scopus (42) Google Scholar), which was shown to exhibit an EC50 for relaxation of 36 mm H2O2 (22.Aikawa K. Leggett R.E. Levin R.M. J. Urol. 2003; 170: 2082-2085Crossref PubMed Scopus (38) Google Scholar)). Airway smooth muscle, although considerably more sensitive than bladder, still requires nearly mm concentrations of H2O2 for relaxation of agonist induced contraction (23.Lorenz R.R. Warner D.O. Jones K.A. Am. J. Physiol. 1999; 277: L816-L822PubMed Google Scholar). Previously we showed that 0.1–3 mm H2O2-induced relaxation of ASM was caused by a reduction in the amount of force produced at given [Ca2+]i, (Ca2+ sensitivity), whereas [Ca2+]i actually increased in response to H2O2 treatment (23.Lorenz R.R. Warner D.O. Jones K.A. Am. J. Physiol. 1999; 277: L816-L822PubMed Google Scholar). Additionally, we showed that the inhibitory effect of peroxide on ASM contraction persisted after Triton X-100 permeabilization and RLC thiophosphorylation. Because the levels of RLC thiophosphorylation were unaffected by H2O2 treatment, we proposed that H2O2 directly inhibits the acto-myosin contractile apparatus (24.Perkins W.J. Lorenz R.R. Bogoger M. Warner D.O. Cremo C.R. Jones K.A. Am. J. Physiol. 2003; 284: L324-L332Google Scholar). Here we address whether or not myosin oxidation underlies the physiological effect of H2O2 treatment in ASM as mentioned above. This is an important question because in terms of contractility it may not matter what the effects of H2O2 are on signaling cascades upstream of myosin activation, because even pro-contractile-signaling would be nullified by direct inhibition of acto-myosin cycling. A better understanding of effects of peroxide on SMM will ultimately clarify the potential mechanisms at play in previous studies of H2O2-induced pathogenesis. Here we characterize the effects of H2O2 on the kinetics of purified SMM and correlate those data to changes in force generation in ASM tissue. We have examined the steady-state and single-turnover ATPase kinetics of purified SMM in both the unphosphorylated and phosphorylated states. We have used two soluble myosin subfragments that are suitable for kinetic experiments. HMM is double-headed and lacks two-thirds of the tail and therefore cannot form filaments. However, it is fully regulated by light chain phosphorylation. S1 is a single head domain that cannot form filaments and is not regulated by light chain phosphorylation. The effects of EDTA during the H2O2 treatment and the effect of DTT after the H2O2 treatment have been analyzed. In addition, we have analyzed the effect of H2O2 on the total number of reactive thiols and the formation of intersubunit disulfide bonds. Mass spectral studies revealed which residues were oxidized and to which oxidation state. The effect of H2O2 on the rate and amplitude of force development in permeabilized porcine tracheal smooth muscle strips was also examined. Remarkably, the specific effects on myosin kinetics observed for purified SMM were paralleled by the effects observed in the muscle, strongly suggesting that elevated H2O2 levels during diseased states could alter myosin kinetics and partially explain altered contractility. Protein Preparations−Protein preparations were essentially as described (25.Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). HMM and S1 were prepared by digestion of gizzard SMM (26.Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (128) Google Scholar) with Staphylococcus aureus protease V8 (Sigma). Both HMM (E (0.1%) = 0.65) and S1 (E (0.1%) = 0.75) are mixtures of full-length heavy chains and clipped heavy chains with an internal cleavage at the actin binding loop ∼26 kDa from the head-tail junction. The light chains were unaffected. F-actin was prepared from rabbit muscle according to the method of Spudich and Watt (27.Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) and was dialyzed versus 10 mm MOPS (pH 7.0), 0.1 mm EGTA, 50 mm NaCl, 0.8 mm MgCl2, 0.2 mm ATP. MLCK (E (0.1%) = 1.14) was prepared (28.Adelstein R.S. Klee C.B. Methods Enzymol. 1982; 85: 298-308Crossref PubMed Scopus (47) Google Scholar) with the modifications described (25.Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and was stored at –80 °C in single aliquots with 10 mm DTT. 10 mm DTT was added to MLCK immediately after thawing and then stored on ice. Thiophosphorylation of HMM−HMM was thiophosphorylated as described (29.Ellison P.A. DePew Z.S. Cremo C.R. J. Biol. Chem. 2003; 278: 4410-4415Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). This preparation is referred to as pHMM. The extent of thiophosphorylation was verified using 10% Tris-glycine gels (10 cm × 10 cm, 12 lanes; Invitrogen) with standard Tris-glycine running buffer. This type of gel gave superior results to urea and/or urea-glycerol gels (30.Perrie W.T. Perry S.V. Biochem. J. 1970; 119: 31-38Crossref PubMed Scopus (595) Google Scholar, 31.Facemyer K.C. Cremo C.R. Bioconj. Chem. 1992; 3: 408-413Crossref PubMed Scopus (35) Google Scholar). The samples were precipitated with 3 volumes of cold acetone prior to the addition of sample buffer (8 m urea (ultrapure, Research Organics), 33 mm Tris-glycine (pH 8.6), 0.17 mm EDTA, 10 mm DTT (added immediately before use), bromphenol blue) to 6–7 mg/ml protein. Approximately 25–30 μg of HMM was applied to the gel. The samples were not heated above room temperature. Peroxide Treatment of HMM and S1−HMM samples were thiophosphorylated where indicated and spun through a 5-ml buffer exchange column (32.Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar) prepared with Sephadex G-50–80 resin (Sigma) in nonreducing buffer (10 mm MOPS, 50 mm NaCl (pH 7.0)). The samples (typically 1–2 mg/ml) were incubated with H2O2 under specified conditions (see legends). To remove H2O2, the samples were either spun through buffer exchange columns to nonreducing buffer or treated with catalase, as indicated. Single Turnover of HMM-MantATP in the Presence of Actin−This assay measures the rate of phosphate release from the acto-HMM MantADPPi state (25.Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Because phosphate release is rate-limiting, the decrease in fluorescence as MantADP dissociates from HMM can be used to measure phosphate release rates. The single-turnover assay allows populations of molecules or heads with different turnover rates to be elucidated. The assays were performed at 25 °C in a temperature controlled stopped flow fluorimeter (Hi-Tech, SF-61DX2, Salisbury, UK) equipped with a 75-watt Xe-Hg lamp. The mixing dead time was ∼2 ms. The excitation wavelength was 365 nm, and the excitation bandwidth was 4 nm. Emission was collected through a 389 low cut-off filter (Corion). For upHMM, formycin triphosphate was used instead of MantATP. The excitation wavelength was 313 nm, and the excitation bandwidth was 4 nm. Emission was collected through a 370 low cut-off filter (Corion). The experiment was done with two syringes. The first syringe contained 0.8 μm HMM heads in 10 mm MOPS (pH 7.0), 50 mm NaCl. The second syringe contained 10 μm actin, 200 μm ATP, 10 mm MOPS (pH 7.0), 50 mm NaCl, 0.1 mm EGTA, 0.8 mm MgCl2,1mm DTT. A stopwatch was started upon adding MgMantATP (1.6 μm MgCl2, 0.8 μm MantATP) to the contents of the first syringe. The fluorophore binds maximally to pHMM in about 45 s (data not shown). After flushing the cuvette three times, 50 μl was shot from each syringe (at 45 s), and the data were collected with the anti-bleaching shutter engaged for upHMM (1000–1400 s; 1024 points) or without the shutter for shots under 60 s for pHMM and S1 (1024 points). All of the data were analyzed with Kaleidagraph software (Synergy Software, Reading, PA). The data were fit to a double exponential model (25.Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). KATPase (Km for actin) and Vmax for pHMM were determined by varying the actin concentration from 5 to 150 μm and fitting the curve to the Michaelis-Menten equation as described (29.Ellison P.A. DePew Z.S. Cremo C.R. J. Biol. Chem. 2003; 278: 4410-4415Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Steady-state ATPase Assay−Steady-state [γ-32P]ATP hydrolysis of S1 was measured in 10 mm MOPS (pH 7.0), 1 mm ATP, 2 mm MgCl2, and 50 μm F-actin at 25 °C. Released 32Pi was separated from [γ-32P]ATP with acidified, activated charcoal (33.White H.D. J. Biol. Chem. 1985; 260: 982-986Abstract Full Text PDF PubMed Google Scholar) and quantified by scintillation counting. The background [γ-32P]ATP hydrolysis rates of actin alone and S1 in the absence of actin were subtracted. Mass Spectrometry−UpS1 was treated with 1 mm H2O2 for 30 min, alkylated with 10 mm iodoacetamide for 30 min, and subjected to nonreducing SDS-PAGE. The bands were stained with Coomassie Safe Stain, excised, reduced with DTT, treated with vinyl-pyridine, and subjected to in-gel trypsinolysis with modified sequencing grade trypsin (Promega). Following digestion, the samples were subjected to nanobore LC with on-line tandem mass spectrometry performed on a ThermoFinnigan LTQ-FT. This instrument is a hybrid of a linear ion trap with a Fourier transform-infrared cyclotron resonance detector (34.Olsen J.V. Mann M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13417-13422Crossref PubMed Scopus (283) Google Scholar, 35.Syka J.E. Marto J.A. Bai D.L. Horning S. Senko M.W. Schwartz J.C. Ueberheide B. Garcia B. Busby S. Muratore T. Shabanowitz J. Hunt D.F. J. Proteome Res. 2004; 3: 621-626Crossref PubMed Scopus (333) Google Scholar). Searches for oxidative modifications of Cys and Met, alkylation of Cys, and N-acetyl Cys (ELC) were conducted with Mascot (Matrix Science). Tracheal Smooth Muscle Experiments−See the supplemental materials and the legend for Fig. 9 for information regarding the tracheal smooth muscle experiments. To evaluate the effects of H2O2 on the ATPase kinetics of pHMM (Fig. 1) and upHMM (Fig. 2), samples were heated for 30 min at 37 °C in nonreducing buffer with H2O2 or H2O2 plus 5mm EDTA. After rapidly removing H2O2, the transient kinetics of phosphate release was measured in the presence of 5 μm actin. The data were fit to a double exponential model. This assay has two advantages over the more typical steady-state assay. First, it can monitor populations of molecules with different turnover rates, and second, it can monitor the total number of ATP-binding sites by analyzing the extent of fluorescence increase upon binding of a fluorescent ATP analog. The rate of the fast phase of turnover (as a percentage of an identically treated control without H2O2) for pHMM is plotted versus H2O2 concentration in Fig. 1A. Unheated controls (4 °C) behaved essentially identically to heated controls (37 °C exposure without H2O2, data not shown). The fast rate decreased to ∼50%, and the slow rate increased to about 120% of control at 2.5 mm H2O2. An identical H2O2 treatment in the presence of 1 mm MgADP (data not shown) gave a similar result, suggesting that nucleotide binding at the active site did not protect against the inactivation. Fig. 1B shows that the fast rate contributes ∼80% and the slow rate contributes ∼ 20% of the total turnover amplitude prior to H2O2 treatment. H2O2 treatment caused the fractional amplitude of the fast and slow rate to decrease and increase, respectively. These data might suggest that untreated HMM contains ∼20% oxidized heads or heads that behave like H2O2-oxidized heads. However, we have found that the highly similar nonmuscle IIB HMM (expressed in insect cells) (36.Cremo C.R. Wang F. Facemyer K. Sellers J.R. J. Biol. Chem. 2001; 276: 41465-41472Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and the single head fragment S1 (see later) also show the 80% fast to 20% slow turnover behavior. This suggests that the biphasic turnover is an intrinsic property of smooth and nonmuscle myosin heads. The molecular basis of this phenomenon is under further investigation. The weighted rate plotted in Fig. 1C takes into account both rates and amplitudes and is indicative of the steady-state ATPase rate of the population of molecules that bind to nucleotide. In the absence of EDTA, the weighted rate decreases to ∼40% of control (squares). Similarly, the total amplitude (Fig. 1D), which reflects the total number of nucleotide-binding sites, decreases to ∼50% of control (squares). This was confirmed in a separate experiment monitoring the rate and amplitude of MantATP binding under pseu-do-first order conditions (data not shown). Only the amplitude and not the rate of binding was altered by H2O2. Therefore treatment with 2.5 mm peroxide results in two populations of heads, 50% with 40% activity and 50% with ∼0 activity. If this sample had been measured by a steady-state assay, the activity would be 20% of control. In summary, H2O2 affects the kinetics of pHMM in two ways. First, for those molecules that still bind to nucleotide, it decreases the steady-state ATPase by decreasing the rate and the fractional amplitude of a fast component and increasing the rate and fractional amplitude of a slow component. This is an effect upon Vmax, not Km. Actin titrations showed that the KATPase (of Km for actin) of control pHMM was 83 ± 18 μm, and that of peroxide-treated pHMM was 80 ± 21 μm (data not shown). Second, H2O2 causes ∼50% of the molecules to either lose the ability to bind to nucleotide or drastically weaken their affinity for nucleotide.FIGURE 2Formycin triphosphate single-turnover kinetics of H2O2-treated upHMM. A, rates of fast (squares) and slow (circles) phases versus H2O2 concentration. B, corresponding amplitudes of the fast (squares) and slow (circles) phases. C, weighted rate as described in Fig. 1. D, total amplitude (percentage of control) versus H2O2 concentration. The error bars and replicates are similar to those in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The presence of EDTA during the H2O2 treatment (5.0 mm) generally diminished the H2O2-induced inactivation of pHMM with respect to the fast rates and diminished the H2O2-induced activation with respect to the slow rates (Fig. 1A), as reflected in the weighted rates (Fig. 1C). EDTA also protected against the loss of ATP-binding sites (Fig. 1D), although the effect was not large. The effect of H2O2 on the kinetics of upHMM is shown in Fig. 2. The assay was the same as in Fig. 1, except that formycin triphosphate instead of MantATP was used as the fluorescent nucleotide. Fig. 2 (A and B) shows that the effect of H2O2 is 2-fold. Without H2O2, the turnover data can be fit to a single exponential with a rate constant of ∼0.0015 s–1. H2O2 causes this rate to increase slightly, and a new phase appears with a rate at about 10 times the control rate. The relative contribution of this faster rate increases with increasing H2O2 concentration (Fig. 2B). Fig. 2C shows that H2O2 increases the weighted rate by ∼15-fold at 2 mm peroxide. Fig. 2D shows that treatment with 2 mm peroxide caused a loss of ∼50% of the total ATP-binding sites as was found for pHMM (Fig. 1D). Peroxide-treated upHMM was capable of being phosphorylated (data not shown). As in pHMM, the effect of peroxide on upHMM was to alter the kinetics of a population of molecules that can still bind to nucleotide and to render about 50% of the molecules incapable of binding to nucleotide. The effect of H2O2 on the kinetics of upS1 is shown in Fig. 3. This subfragment has no tail and only a single head domain. It is unregulated in that both the unphosphorylated and phosphorylated forms have a high activity. The pattern of H2O2-induced kinetic effects is very similar to those observed for pHMM (Fig. 1). This is expected, as pHMM and upS1 have similar high catalytic activity and other similarities with respect to RLC structure (37.Mazhari S.M. Selser C.T. Cremo C.R. J. Biol. Chem. 2004; 279: 39905-39914Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The observed changes in the two rate constants are similar, although the changes in amplitudes (Fig. 3B) are not as great. Again, ∼50% of the total nucleotide-binding sites have been destroyed (Fig. 3D). Fig. 4 shows the protective effect of EDTA on the peroxide-mediated inactivation of upS1 as measured with a steady-state ATPase assay. At 3 mm H2O2 without EDTA, about 20% of the activity remains. This is consistent with the single-turnover data in Fig. 3, which predicts that at 2.5 mm H2O2 the steady-state activity should be ∼30% of control. EDTA (5 mm) protected against the inactivation, with about 70% of the activity remaining after a 3.0 mm H2O2 treatment. These data are similar to those observed for pHMM (Fig. 1). Fig. 5 shows a time course of the reaction of upS1 with 3 mm H2O2. The percentage of inhibition of the steady-state ATPase activity correlated with the total number of thiols as measured by Ellman's reagent, DTNB (38.Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar). There is an initial rapid inactivation phase in which about two thiols are modified, followed by a slower phase in which approximately six or seven thiols (about one-half of the total) are modified after 30 min. At the same time the ATPase was inhibited to ∼70% of control. The tight correlation between the inactivation and the loss of reactive thiols suggests that oxidized thiols cause the ATPase inactivation. The final thiol oxidation states could be disulfides or higher oxidation states such as sulfenic or sulfonic acids. Fig. 6 shows the effect of treatment with the reducing agent, DTT, on H2O2-inhibited upS1. Treatment with DTT (50 mm) for 1 or 2 h caused reversal of the inhibition from 40% of control to ∼66 and 77%, respectively, and suggests that the mechanism for at least some of the loss of ATPase activity is by thiol oxidation. Because it is known that DTT readily reduces disulfides and sulfenic acid(s) (R-SOH) (39.Denu J.M. Tanner K.G. Biochemistry. 1998; 37: 5633-5642Crossref PubMed Scopus (828) Google Scholar) to thiols, it is likely that these modifications are responsible for some of the inhibited ATPase activity. Under these robust DTT treatment conditions the ATPase activity was not completely reactivated. This suggests that H2O2 treatment causes some of the thiols to become oxidized further to DTT-unreactive states such as sulfinic (R-SO2H) or sulfonic acids (R-SO3H) or that methionine(s) is also oxidized. Fig. 7 shows the effects of DTT on the single-turnover kinetics of H2O2-treated pHMM. In this example the H2O2 treatment inhibited the ATPase activity (of those heads that can still bind nucleotide) to ∼30% of control (Fig. 7C). At the same time, ∼54% of the heads lost the ability to bind to nucleotide and therefore have 0 activity (Fig. 7D). Treatment with up to 40 mm DTT did not increase the total ATP-binding sites (Fig. 7D) but did increase the weighted rate (Fig. 7C) to ∼55% of control and partially reversed the pattern of kinetic rates (Fig. 7A) and amplitudes (Fig. 7B) characteristic of the H2O2-treated S1. These data suggest that DTT can partially restore the native kinetics of molecules still capable of binding nucleotide but has no effect upon the molecules that lost the ability to bind nucleotide. This suggests that the loss of ATP binding ability is not correlated to disulfide bond formation but perhaps to further thiol oxidation or to methionine oxidation (40.Levine R.L. Mosoni L. Berlett B.S. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15036-15040Crossref PubMed Scopus (893) Google Scholar). To test for H2O2-induced intersubunit disulfide bond formation, we analyzed a nonreducing SDS gel of H2O2-treated upS1 (Fig. 8A). S1 prepared in this manner has most of the heavy chain internally cleaved into a ∼65-kDa N-terminal fragment and a C-terminal 26-kDa fragment. A small amount of uncleaved heavy chain still remains at ∼90 kDa. H2O2 internally cross-linked the ELC, causing it to smear to higher Rf. In addition, the 26-kDa heavy chain fragment was cross-linked to the 65-kDa heavy chain fragment (C-terminal heavy chain to N-terminal heavy chain). This latter cross-link was identified by excising, reducing, and subsequently electrophoresing the band onto a reducing gel (Fig. 8A, right lane). Mass spectral analysis of a tryptic digest of the ∼90-kDa band identified heavy chain peptides from both the 65- and 26-kDa fragments (data not shown). The nonreducing gel in Fig. 8B shows that the presence of EDTA during H2O2 treatment largely inhibits disulfide cross-linking of the 65/26-kDa peptide pair. Under these conditions EDTA does not fully protect against the loss of ATPase activity. Therefore peroxide-treated S1 can be partially inactivated without the above disulfide bonding, suggesting that the disulfide bond is not the only modification to the protein. Fig. 8C shows that the reductant in the sample buffer was sufficient to largely reverse the cross-link, as expected for a disulfide bond. Therefore, gel analysis revealed that there are at least two SH groups, one on the 65-kDa fragment and the other on the 26-kDa fragment that become disulfide-bonded (reversible by DTT) upon H2O2 treatment and that EDTA c
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