Distinction of thiols involved in the specific reaction steps of the Ca2+-ATPase of the sarcoplasmic reticulum.

The reaction of N-ekhylmaleimide (MaWI%) with the -SH groups of purified Caa+-ATPase has been investigated as a function of calcium concentration. Several classes of thiols can be distinguished on the basis of the Ca2+ dependence of the reaction and the effects of thiol blocking on various steps of the ATPase reaction. Initially, MalNEt is rapidly incorporated with no effect on enzyme activity into the first thiol (-SH1, -1 mol/lO” g) at a rate which is independent of [Ca”]. The rate of further MalNEt incorporation which leads to inactivation of enzyme activity varies with [Ca2+]. At Ca2+ 5 lo-” M during MalNEt incorporation, the rate of ATP hydrolysis (u), the rate of phosphoenzyme decomposition (&.), and the phosphoenzyme level when the phosphoenzyme decomposition is prevented @PC*) are all inhibited at about the same rate. Upon increasing [Ca2+], a second thiol (-SHa, -1 mol/lO’ g) increases its reactivity with MalNEt. This leads to the rapid decrease of v and km but the rate of decrease of EPc. is about the same as that at low [Ca”‘]. These results indicate that blocking of the -SHe results in the preferential inhibition of reaction steps in which phosphoenzyme decomposition occurs, and that besides -SHz, there is the other type of thiol or thiols (SF) whose reactivity with MalNEt is independent of [Ca2+] and whose blocking inhibits the phosphoenzyme formation. The [Ca”] at a half-maximum change in the reactivity of -SHz as determined from the [Ca”+] dependence of the inhibition of u is 1.6 X lo-’ M. This is basically the same as that of Ca2+ binding to the high affinity a sites. It is suggested that the change of the MalNEt reactivity with -SHz reflects the change in enzyme conformation produced by the Ca” binding to the high affinity sites.

The reaction of N-ekhylmaleimide (MaWI%) with the -SH groups of purified Caa+-ATPase has been investigated as a function of calcium concentration. Several classes of thiols can be distinguished on the basis of the Ca2+ dependence of the reaction and the effects of thiol blocking on various steps of the ATPase reaction.
Initially, MalNEt is rapidly incorporated with no effect on enzyme activity into the first thiol (-SH1, -1 mol/lO" g) at a rate which is independent of [Ca"]. The rate of further MalNEt incorporation which leads to inactivation of enzyme activity varies with [Ca2+]. At Ca2+ 5 lo-" M during MalNEt incorporation, the rate of ATP hydrolysis (u), the rate of phosphoenzyme decomposition (&.), and the phosphoenzyme level when the phosphoenzyme decomposition is prevented @PC*) are all inhibited at about the same rate. Upon increasing [Ca2+], a second thiol (-SHa, -1 mol/lO' g) increases its reactivity with MalNEt. This leads to the rapid decrease of v and km but the rate of decrease of EPc. is about the same as that at low [Ca"'].
These results indicate that blocking of the -SHe results in the preferential inhibition of reaction steps in which phosphoenzyme decomposition occurs, and that besides -SHz, there is the other type of thiol or thiols (SF) whose reactivity with MalNEt is independent of [Ca2+] and whose blocking inhibits the phosphoenzyme formation.
The [Ca"] at a half-maximum change in the reactivity of -SHz as determined from the [Ca"+] dependence of the inhibition of u is 1.6 X lo-' M. This is basically the same as that of Ca2+ binding to the high affinity a sites. It is suggested that the change of the MalNEt reactivity with -SHz reflects the change in enzyme conformation produced by the Ca" binding to the high affinity sites.
The blocking of thiols of the sarcoplasmic reticulum (SR)' with various covalently or noncovalently reacting reagents results in inhibition of ATPase activity and Ca'+ uptake (l-lo). Analysis of the pseudo-first order kinetics of the thiol reaction with MalNEt or Nb% has shown that there are * This work was supported by Grant AM-16922 from the National Institutes of Health and grants from the National Science Foundation, the American Heart Association and the Muscular Dystrophy Association of America, Inc. The costs of nubbcation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "'advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ several distinguishable classes of thiols (3,6,8). Earlier work by Hasselbach and Seraydarian (3) has shown that blocking of four thiols/105 g of SR protein with MalNEt results in complete inhibition of ATPase and Ca2+ uptake and that ATP specifically prevents the reaction of one of these thiols with concomitant protection of ATPase activity and Ca2+ uptake. According to Yoshida and Tonomura (ll), blocking of at most two thiols with MalNEt results in complete inhibition of ATPase. It was suggested that only one thiol might be essential to the enzyme activity (6) since other nonfunctional thiols having the same reactivity cannot be distinguished.
A number of studies have been carried out on the ATPase of SR with a variety of types of thiol-directed reagents in attempts to monitor the conformation of various intermediate enzyme complexes (8,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Although nucleotides and metals cause some changes in the ESR and fluorescence spectra of the thiol-attached reagents, a definitive interpretation of these observations has not yet been possible. The crucial requirement for the interpretation of these results is the information concerning the number and the type of thiols to which these reagents are attached. Several attempts have been made to specifically label a single functionally important thiol after blocking the other tbiols in the presence of ATP (3,4). However, according to recent reports, the reaction with Nbg of a large number of thiols is reduced by ATP (6)(7)(8). The thiol reactivity appears to be considerably influenced by the Ca" concentration (6,10,21). Our recent stopped flow studies (22) of the reaction of the thiol-directed fluorescent reagent, S-mercuric N-dansyl cysteine (23), with the purified Ca'+-ATPase of SR suggest that Ca2+ may exert a more selective effect on thiol reactivity than does ATP. Therefore, it appeared worthwhile to investigate whether selective blocking of certain types of thiols might be achieved by controlling [Ca"] during the reaction with MalNEt.
As shown here, upon increasing the [Ca"'] from lo-@ to 10m5 M, the reactivity of one of the thiols of purified ATPase with MalNEt increases in parallel with the binding of Ca2+ to the high affinity (Y sites (24-26). Blocking of this thiol inhibits the decomposition of phosphoenzyme with little or no inhibition of its formation.
Of the other thiols, there appears to be at least one that reacts with MalNEt at the same rate regardless of [Ca2'] and whose blocking inhibits EP formation.
These results suggest that there are at least two functionally important thiols, the blocking of which results in inhibition of different steps of the ATPase reaction. The results also suggest that the change of thiol reactivity with [Ca2+] reflects a change in enzyme conformation controlled by Ca2+ binding.

Preparations
Fragments of the sarcoplasmic reticulum (SR) were prepared from skeletal white (fast) muscles of rabbit as described previously (27).

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Thiols Involved in Enzyme Reaction of SR-ATPase Ca'+-ATPase was purified from fragmented SR solubilized with Triton X-100 (26). of ATPase activity depend on the concentration of Ca" during the incorporation. Fig. 1 depicts the results of an experiment in which purified ATPase was allowed to react with an excess of ['?]MalNEt (50 mol of MalNEt/l mol of enzyme protein) at the two different concentrations of Ca'+, viz. pCa 8.3 and 5.0. In the initial phase of incorporation (5 min after starting the reaction), the rapid MalNEt incorporation takes place at about the same rate at both pCa values ( Fig. la). In the subsequent phase, the rate of incorporation is somewhat slower at pCa 5.0 than at pCa 8.3 (Fig. la). The extent of inhibition of ATPase activity induced by blocking the thiol (Fig. lc) shows the opposite [Ca"'] dependence, viz. at pCa 5.0, the ATPase activity is inhibited to a considerably larger extent than at pCa 8.3. These results indicate that at pCa 5.0, certain thiols whose blocking reduces the enzyme activity react more selectively.

Determination of Thiol Blocked with [%JMalNEt
The semilogarithmic plots shown in Fig. 1, b and d permit further analysis of the results described above. Approximately one thiol (0.5 to 0.8, depending on the preparation) per lo" daltons of ATPase peptide reacts during the initial rapid phase (-5 min) as estimated from the extrapolation of the plots shown in Fig. lb. From a comparison of Fig. 1 (Fig. 2). The fast phase is completed in 20 to 30 min regardless of [Ca2+]. However, the extent of the resultant inhibition of ATPase activity increases with [Ca"'], and the maximal extent of inhibition occurs at pCa 5 6. Thus, after the reaction with MalNEt at pCa 5.0 for 30 min, approximately 80% of the original ATPase activity is inhibited (Fig. Id), by which time 2.0 thiols are blocked (Fig. lb). Since -SHr accounts for -1 as described above, the actual number of the thiol which is responsible for the fast phase of Fig. Id seems to be -1. These data suggest that Ca2+ increases the reactivity of a thiol whose blocking reduces the ATPase activity by 60%, as determined from the extrapolation of the final slope to t = 0. In order to facilitate further discussion, this thiol is designated as -SH,.
At low [Ca'+], e.g. at pCa 8.3, the plot can be fitted by a single line; this indicates that -SH2 reacts with MalNEt at about the same rate as do the other thiol or thiols, whose blocking inhibits ATPase activity.
The effect of the reaction with MalNEt on the phosphorylation of the enzyme was also studied under the two different conditions: 1) the same conditions as the experiment of Fig. 1 (EPM~) and 2) the conditions under which the decomposition of phosphoenzyme is prevented in the presence of 5 mM CaClz and 0.2 mM MgClz @PC,). If the reaction with MalNEt is carried out at pCa 8.3 (Fig. 3a), the inhibition of the ATP hydrolysis rate (v) roughly parallels that of EPM,. At pCa 5.0 (Fig. 3b), however, EPM~ actually increases in the earlier phase of the reaction concomitant with the reduction of v. This suggests that the blocking of -SH2 reduces in fact the rate of those steps of the ATPase reaction in which the phosphoenzyme is decomposed with no inhibitory effect on the phosphoenzyme formation step. The rate of phosphoenzyme decomposition can be estimated from the ratio v/[EP&J, assum-  (Table I), indicating that the fast phase of reduction of v at pCa 5.0 is primarily due to the inhibition of phosphoenzyme decomposition which is accounted for by the blocking of -SH2. In contrast to EPM~, the EPca which represents the phosphoenzyme level with the prevented phosphoenzyme decomposition step is reduced at about the same rate regardless of [Ca"] during the reaction with MalNEt (Fig. 4b). This indicates that the reduction of EPc, is due to the blocking of one or more kinetically indistinguishable thiols other than -SH2. This thiol class is designated as SF; F implies that blocking of this class results in the inhibition of phosphoenzyme formation.
Previous studies on the number of thiols involved in the ATPase activity of SR have led to contradictory results (3,11 1 (bound MalNEt per lo5 g of ATPase protein) are plotted in the way used in the previous reports (Fig. 5). At pCa 5.0, blocking of approximately 1.7 thiols produces 80% inhibition of ATPase. This is consistent with the report that there are one or two "essential" thiols (11). It appears that to inhibit the remaining 20% activity, further blocking of 3.5 thiols is required. At pCa 8.3, blocking of the fast 0.5 thiol does not inhibit ATPase activity, but it is inhibited in proportion to the blocking of additional 4.5 thiols; this is consistent with the reported 4 essential thiols (3) t is the time of reaction, and Al and AZ are the constants defined below. If kr > kz, plots are fitted by two sets of lines: one expressed by In A = In A0 -((A&, + A2k2)/(A1 + AZ))& and the other, In A = In AZ -kzt, Ao = A1 + AZ. If k, = kp = h, data plots are on the single line expressed by In A = In AO -ht. In b, A1 and Al are the numbers of thiol having the rates of MalNEt incorporation, k, and h2, respectively.
Since 12 thiols were titrated with Nbsn and Smercuric N-dansyl cysteine in the native conformation of the enzyme (22) 6 shows the experiment in which the ATPase activity was determined after the enzyme was allowed to react with MalNEt for 20 min at various [Ca"']. The major change in the reactivity of -SHz with MalNEt takes place in the range of 8 > pCa > 6, and there is little or no change in the range of pCa d 6. The & (association constant) value estimated from the [Ca"'] at the half-maximum change is 6.33 x 10" Mm', which is basically identical with the KA value of the high affinity (Y sites (3 X 10" M-l, Refs. 24 to 26). This suggests that the increase in the reactivity of -SHz reflects the change of enzyme conformation induced by Cazf binding to the a sites. Contrary to the increased reactivity of -SH2 deduced from the inactivation of ATPase, the number of blocked thiols at 20 min decreases as [Ca"'] increases (Fig. 6a, also see Fig. la). This would indicate that upon increasing [Ca"], the reactivity with MalNEt of some thiols other than -SHI, -SHz, and SF is reduced.
As a consequence of the fact that blocking of -SHz inhibits the phosphoenzyme decomposition step, but not the phos- I  I  I  I  I  I   0  IO  20  30  40  TIME  phoenzyme formation step, EPhlg is rather increased during the reduction of u as described above. Therefore, the [Ca"'] dependence of EPhlg on blocking of -SH2 is exactly the opposite of that of u (Fig. 6b). On the other hand, reduction of EPc. which reflects the reactivity of class SF shows no Ca2' dependence in the range of 8 > pCa > 2 (Fig. 6b).
Factors Affecting [Ca2+]-Dependent Thiol Reactiuity-We have studied the effects of various concentrations of MgClz and KC1 on the extent of thiol blockage and the inhibition of ATPase activity (Figs. 7 and 8). Although upon increasing the concentration of MgClc the ATPase inhibition is somewhat increased at both pCa 5.0 and 8.3 (Fig. 7a), the ratio of the extent of ATPase inhibition to the amount of bound MalNEt, or the specific reactivity of -SH, is virtually independent of MgClz concentrations. Fig. 8, a and b shows the results of similar experiments with KCl. Again, the specific reactivity of -SHz is basically independent of the KC1 concentrations (Fig. 7b). This indicates that [Ca'+]-dependent reactivity of -SHz is not influenced by the other ions.
In many of the previous studies on thiol reaction with MalNEt, blocking of the thiol has been carried out at higher pH (e.g. pH 8.5,Refs. 3 and 4;pH 7.5,Ref. 11). The same type of experiment as shown in Fig. 1 was carried out at pH 8.  Fig. 3a) and pCa 5.0 (Fig. 3b). assay @PM,); 0, n , rates of Pi liberation determined in the same The reaction with MalNEt was stopped by adding a 30-fold excess of assay reaction as A Fig. 3  thiols with a nonradioactive reagent in the presence of ATP with a view to selectively protecting the essential thiol. However, as described in the introduction, recent reports (6-8, 10, 11) suggest that the protective effect of nucleotide would be produced by a conformational change in the enzyme molecule induced by the nucleotide binding, The analysis of the process of thiol blocking with MalNEt at various [Ca2+] has allowed us in this study to distinguish several types of thiols (uiz. --SH1, --SH2, and class Sr) even under conditions when MalNEt blocks less than three thiols per lo5 daltons of purified ATPase protein. Blocking of -SH1 (-l/lo" daltons) has no effect on ATPase. Blocking of -SH, and class SF produces inhibition of different steps of the ATPase reaction, uiz. phosphoenzyme decomposition and phosphoenzyme formation, respectively. Upon increasing [Ca"'] from lo-@ M to lo-" M, the extent of rapid inhibition of ATPase activity assessed from the extrapolation of the logarithmic plot of u (A,/Ao, cf legend to Fig. 1) increases from 0 to the maximum value of 0.6 to 0.7. This can be explained by the assumption that as [Ca'+] increases a larger fraction of -SH2 becomes highly reactive. The reactivity of class SF with MalNEt is independent of [Ca"'] and is approximately the same as that of -SH2 in a less reactive form. Thus, if analysis is made on the plot of In($) versus t at pCa z 8, -SH2 and class SF are kinetically indistinguishable and the plot can be fitted by a single line. At pCa 5 6, --SHz appears as a fast kinetic phase. Since class Sr is crucial for phosphoenzyme formation but --SHz is not, the slope of the plot of ln(EPcJ versus t, is independent of [Ca"'] and is roughly identical with that of ln(k,,) uersus t plots at low [Ca*'].
Although it seems clear that --SHl and --SHz are approximately 1 per lo5 daltons of ATPase peptide, respectively, the present study does not permit us to decide the number of thiols in class SF. It appears that the thiol reactivity is considerably different depending upon the type of reagent. In order to favor the selective blockage of class SF, therefore, it is probably necessary to use different types of thiol reagent.
As seen in the experiments shown in Figs