Functional coupling between sarcoplasmic-reticulum- bound creatine kinase and Ca2+-ATPase

Transcription

1 Eur. J. Biochem. 213, (1993) 0 FEBS 1993 Functional coupling between sarcoplasmic-reticulum- bound creatine kinase and Ca2+-ATPase Paavo KORGE, Shere K. BYRD and Kenneth B. CAMPBELL Dept VCAPP, Washington State University, Pullman WA, USA (Received November 17, 1992/January 18, 1993) - EJB /5 We investigated the role of creatine kinase bound to sarcoplasmic reticulum membranes of fast skeletal muscle in the local regeneration of ATP and the possible physiological significance of this regeneration for calcium pump function. Our results indicate that ADP produced by sarcoplasmic reticulum Ca2+-ATPase is effectively phosphorylated by creatine kinase in the presence of creatine phosphate. This phosphorylation is an important function of the membrane-bound creatine kinase because accumulation of ADP has a depressive effect on Ca2+-uptake by sarcoplasmic reticulum vesicles. The concentration-dependent depression of Ca2+-uptake by ADP was especially pronounced when there was strong back inhibition by high intravesicular [Ca ]. ATP regenerated by endogenous creatine kinase was not in free equilibrium with the ATP in the surrounding medium, but was used preferentially by Ca2+-ATPase for Ca2+-uptake. Efficient translocation of ATP from creatine kinase to Ca2+-ATPase, despite the presence of an ATP trap in the surrounding medium, can be explained by close localization of creatine kinase and Ca2+-ATPase on the sarcoplasmic reticulum membranes. These results suggest the existence of functional coupling between creatine kmase and Ca2+-ATPase on skeletal muscle sarcoplasmic reticulum membranes. Several factors (amount of membrane-bound creatine kinase, oxidation of SH groups of creatine kinase, decrease in [phosphocreatine]) can influence the ability of creatine kinase/phosphocreatine system to support a low ADP/ATP ratio and fuel the Ca2+-pump with ATP. These factors may become operative in the living cells, influencing functional coupling between creatine kinase and Ca2+-ATPase and may have an indirect effect on Ca2+-pump function before Ca2+-ATPase itself is affected. There is ample published evidence to suggest that ATP resynthesis can occur locally on myofibrills and biomembranes due to the activity of creatine kinase and glycolytic enzymes bound to myofibrills [l -41, sarcoplasmic reticulum (SR) [5-81 and plasma membranes [8-lo]. The existence of site-specific regeneration of ATP, which creates a local pool of ATP in the close vicinity of sites of ATP utilization, provides an explanation for numerous findings where changes in myofibrillar function did not correlate with the cytosolic [ATP] or ATP level in the media [l, 3, 11-13]. Reversible binding of creatine kinase to cardiac and skeletal muscle myofibrils and the involvement of this bound creatine kinase in the energy supply for contraction has been demonstrated in several laboratories [l -41. Recently, creatine kinase has been shown to be attached to highly purified SR membranes from skeletal muscle [5]. These results confirmed earlier findings [6, 71, which demonstrated that cre- Correspondence to P. Korge, Dept VCAPP, Washington State University, Pullman WA 99164, USA Fax: Abbreviation. SR, sarcoplasmic reticulum ; P-creatine, phosphocreatine ; P-pyruvate, phospho(enol)pyruvate ; Ap5A, adenosine- (5 )pentaphospho( 5 )adenosine ; Dnp-F, 1 -fluoro-2,4-dinitrobenzene; MalNEt, N-ethylmaleimide. Enzyme. CaZ+-ATPase (EC ); creatine kinase (EC ); pyruvate kinase (EC ); hexokinase (EC ); adenylate kinase (EC ); lactic dehydrogenase (EC ); glucose-6-phosphate dehydrogenase (EC ) ; apyrase (EC S); glyceraldehyde-3-phosphate dehydrogenase (EC ). atine kinase bound to SR membranes, in the presence of ADP and phosphocreatine (P-creatine), can provide a significant portion of the ATP required for maximal in vitro Ca2+-uptake rate. However, since phosphorylation of Ca2+-ATPase is saturated at [ATP] as low as 10 pm and since half-maximal stimulation of Ca2+-ATPase through the regulatory site occurs at about mm ATP [14, 151, a possible condition in vivo only within a micro-compartment, the physiological importance of a local creatine-kinase-catalyzed reaction on SR remains to be determined. One possible approach for revealing the functional coupling between Ca2+-ATPase and creatine kinase is to perform kinetic analysis of the interaction between the two enzymes under various conditions. Experiments with myofibrillar preparations from rabbit heart showed that myofibrillar ATPase has enhanced affinity for ATP rephoshorylated by creatine kinase bound to myofibrills compared with ATP synthezised by exogenous regeneration systems [4]. A plausible explanation for this kinetic enhancement of the myosin ATPase is close structural association of the ATPase and creatine kinase. This results in a reduced diffusion distance for ATP regenerated by creatine kinase compared with diffusion distances for ATP from other sources. Another important function of SR-bound creatine kinase may be connected with the maintenance of high local ATP/ADP ratios near the sites of ATP hydrolysis, as suggested by Rossi et al. [5]. In this role, creatine kinase increases the thermodynamic efficiency of ATP hydrolysis, which may be critical for Ca2+-pump function [16].

2 974 In this paper we present data which confirms earlier findings that creatine kinase participates in local phosphorylation of ADP near ATP-consuming sites of SR Ca2+-ATPase. Further, we present new data which shows that: (a) creatinekinase-produced ATP is preferentially used for Ca2+ trans- port; (b) Ca2+ -ATPase-produced ADP is preferentially available to creatine kinase for phosphorylation; (c) local creatine-kinase-catalyzed reaction is more sensitive to SH group modification than Ca2+-ATPase; and (d) local ATP regeneration is especially important for Ca2+-uptake by SR when the rate of ADP production is high and luminal free calcium starts to increase. MATERIALS AND METHODS Materials SR vesicles were isolated from horse gluteal muscle and rat fast-twitch skeletal muscle using methods described by Heilmann et al. [17]. These SR preparations are characterized by low activity of citrate synthase (<I0 nmol. mg-i. min-l) and low lactic dehydrogenasekreatine kinase ratio (0.03 in vesicles compared with 0.8 in tissue). SR vesicles were suspended in homogenization buffer (5 mm Hepes ph 7.4, 250mM sucrose, 30mM NaN,) and used immediately or quickly frozen and stored at -70 C until used. Ca2+-uptake Ca2+-uptake was measured at 37 "C by monitoring the decrease in extravesicular [Ca"] with a Ca"-sensitive minielectrode, assembled as described by Affolter and Sigel [18] using a polyvinylchloride membrane with an incorporated ion exchanger (W. Moller, Zurich). The electrode was calibrated by using Ca-EGTA solutions (1 mm EGTA) as described elsewhere [19]. The electrode response to added [Ca'+] was linear in the range of pca with a slope of mv/pca. A Scatchard plot was used to determine the apparent Ca-EGTA association constant, which in our buffer was 3.12X 106M-'. Ca2+ binding to ATP and oxalate in the uptake medium was instantaneous and the free [Ca*+] at the start of uptake was experimentally determined by subtracting the amount of Ca bound to ATP and oxalate from the total amount of Ca2+ added to the uptake buffer. The amount of contaminant Ca added with chemicals and determined with atomic absorption spectroscopy was <2 pm. Both internal and external calibrations were used to verify electrode response. The Ca2+-uptake buffer (final volume 1.25 ml) contained 1OOmM KCI, 50mM Hepes ph7.0, 1 mm MgC12, 5 mm potassium oxalate, 10 mm NaN, and 7 pm Ca2+. ATP, ADP and P-creatine were added as indicated. Enzyme activities ATPase activity was determined at 37 "C in oxalate-free uptake buffer containing 0.01 mm Ca", 0.5 mm NADH, 2 phospho(enoz)pyruvate (P-pyruvate), 1 mm ATP and 2 U/ml of pyruvate kinase and lactic dehydrogenase. Decrease in NADH level was followed spectrophotometrically (Shimadzu UV 160U). Creatine kinase activity was determined at 37 C in the same buffer containing 5 mm EGTA (to inhibit ATPase activity competing for ATP), 1OmM P-creatine, 1 mm ADP, 6.5 mm AMP, 20 mm glucose, 1 mm NAD, 2 U/ml of w 7 30pM[ADP] [P-creatine]( rnm) Fig. 1. The effect of [P-creatine] on P-creatine + ADP-stimulated ATP-dependent Caz+-uptake by SR (35 pg) isolated from horse gluteal muscle, in the presence of 5 mm oxalate and two different [ADP]. 30 pm free ADP represents a reasonable level in skeletal muscle at rest, while concentrations of 100 pm and higher are expected to occur with fatigue or during ischemia [22]. hexokinase and glucose-6-phosphate dehydrogenase. The increase in NADH level was followed with the spectrophotometer. Citrate synthase was determined by the method of Srere [20], lactic dehydrogenase activity by oxidation of NADH in the presence of 3 mm pyruvate and P, by the enzymic method described by Guynn et al. [21]. RESULTS P-creatine + ADP-stimulated Caz+-uptake by SR In accordance with earlier findings [5-91, we observed that SR vesicles isolated from horse or rat skeletal muscle accumulated Ca2+ in a buffer with the high-energy phosphate source in the form of P-creatine plus ADP instead of ATP. This P-creatine + ADP-stimulated Ca2+-uptake was not inhibited by the adenylate kinase inhibitor, adenosine(5')pentaphospho(5')adenosine (Ap,A) (10 pm), but was totally depressed by the creatine kinase inhibitor, 1 -fluoro-2,4-dinitrobenzene (Dnp-F) (20 pm). Because both inhibitors had no important effect on ATP-stimulated Ca2'-uptake, the effect of Dnp-F on P-creatine+ ADP-stimulated Ca2+-uptake must have been the result of creatine kinase inhibition and block of ADP phosphorylation by P-creatine. P-creatine + ADPstimulated Ca2+-uptake depends on the amount of creatine kinase bound to SR membranes. Creatine kinase activity determined in the direction of ATP formation was 2.05 t 0.35 pmol. mg-i. min-' in SR isolated from horse gluteal muscle (n = 12) and pmol. mg-'. min-' in SR from rat plantaris muscle (n = 10). Creatine kinase binding to SR vesicles withstood isolation procedures, confirming findings of others who demonstrated relatively strong binding of creatine kinase to isolated SR preparations using biochemical and immunohistochemical methods [5]. The effect of [P-creatine] and [ADP] on creatine-kinase-mediated Ca*+-uptake P-creatine + ADP-stimulated Caz+-uptake was found to be affected by both [P-creatine] and [ADP]. In the presence of 30 pm or 100 pm ADP, the rate of oxalate-supported P- creatine+adp-stimulated Ca2+-uptake increased in parallel with the increase in [P-creatine]. However, the concentration of P-creatine needed to obtain the half-maximal rate of Ca2+uptake was about fourfold lower in the presence of 100 FM ADP compared with 30 pm ADP (Fig. 1).

3 t 975 L Fig. 2. Time course of calcium uptake by SR isolated from horse gluteal muscle. Uptake was monitored with a Ca2+-sensitive minielectrode in the uptake buffer as described in Methods. Note that tracings were placed so that upward movement reflects decrease in free Ca". Calibration scales showing electrode output (mv) and time (s) are indicated separately in each panel. Electrode response to stepwise additions of CaCl,, shown on the left, was used as an internal standard. In A and B, uptake rate was calculated using the linear part of tracings where uptake rate reaches a constant level. Uptake was started by addition of SR (68 pg). (A) Ca2+-uptake in the presence of 5 mm oxalate: trace 1, 1 mm ATP, uptake rate 1.02 pmol. mg-'. min-'; trace 2, 5 mm P-creatine plus 0.05 mm ADP, uptake rate 0.58 pmol. mg-'. min-'; trace 3, 5 mm P-creatine plus 0.1 mm ADP, uptake rate 0.61 pmol. mg-'. min-'; trace 4, 5 mm P-creatine plus 0.4 mm ADP, uptake rate 0.54 pmol. mg-'. min-' ; trace 5, 5 mm P-creatine plus 0.6 mm ADP, uptake rate 0.47 pmol. mg-*. min-'. (B) Non-oxalate-supported Ca2+-uptake stimulated by: 1 mm ATP (trace 1); 5 mm P-creatine after ATPstimulated uptake had reached a plateau (trace 2); 1 mm ATP and 5 mm P-creatine (trace 3); 0.1 mm ADP and 5 mm P-creatine (trace 4). (C) Non-oxalate-supported Ca2+-uptake stimulated by 1 mm ATP (trace 1) or 1 mm ATP plus 50 pm (trace 2), 100 pm (trace 3) or 150 pm ADP (trace 4). (D) Mg2+, Ca2+-ATPase activity of SR preparation. ADP production under condition of Ca2+-uptake was determined following NADH oxidation. Reaction was started by addition of 1 mm ATP; ADP production was 52 nmovmin. ADP affected P-creatine+ ADP-stimulated Ca2+ -uptake in a complex way. In oxalate-containing buffer with fixed [P-creatine] (5 mm), Ca2+-uptake increased with increasing [ADP] until reaching a plateau at pm. There was a lag phase in Ca"-uptake before a steady state was reached. This lag phase was most probably due to the time required for creatine kinase to increase local [ATPI to the level necessary for steady-state Ca2+-uptake under those conditions. At 100 pm ADP, Ca2+-uptake by horse SR was 0.61 pmol. mg-'. min-' which was about 60% of the uptake rate observed when Ca2+-uptake was stimulated by 1 mm ATP. Increases in ADP above 200 pm resulted in concentration-dependent depression of Ca2+-uptake (Fig. 2A). In another study, we examined the efficacy of creatine kinase/p-creatine system in relieving ADP depression of Ca2+-uptake. In these studies, oxalate was omitted from the uptake buffer. It is well known that in the absence of Caprecipitating anions, Ca2+-uptake by SR is significantly depressed, mainly due to a back inhibition of Ca2+-ATPase by luminal Ca2+ [14, 151. This depression in ATP-stimulated Ca2+-uptake in the absence of oxalate is demonstrated in Fig. 2B. However, the presence of P-creatine in the uptake I [ADPl(mM) Fig. 3. Dependence of steady-state Caz+-uptake by SR on [ADP] and [ATP] in the presence of oxalate. (A) Dixon plots of reciprocal of steady-state Caz+-uptake rate by SR (40 pg) from horse skeletal muscle in the presence of 5mM oxalate versus [ADP] for three different [ATP]: (A) 50 pm; (0) 100 pm; (0) 400 pm. The data were fitted by least-squares linear regression analysis with no other weight of the data. (B) Time to reach steady-state rate of Caz+uptake at three different [ATP] depending on [ADPI. Data from the same experiment as in A. buffer at the start of uptake or the addition of P-creatine after ATP-stimulated uptake had ceased, resulted in significantly longer periods of Ca2'-uptake which presumably resulted in the maintenance of a higher Ca2+ gradient. The same long duration of Ca2+-uptake in these non-oxalate-supported vesicles was obtained when Ca"-uptake was stimulated by P-creatine plus ADP. The relief of suppressed Ca"-uptake when creatine kinase catalyzed the formation of ATP from ADP indicated that at high luminal free Ca2+ the inhibiting effects of ADP on Caz+-pump function are potentiated. Additional evidence to show that Ca2+-uptake is inhibited by ADP under non-oxalate-supported conditions came from measuring the changes in ATP-stimulated Ca2+-uptake with varying [ADP]: the addition of ADP (in final concentrations pm) to the uptake buffer caused a concentrationdependent decrease in Ca2+-uptake (Fig. 2C). These inhibiting [ADP] are consistent with SR preparations used in this experiment which generated about 50 nmol ADP/min under Ca2+-uptake conditions (Fig. 2D). To investigate further ADP depression of Ca2+-uptake, we conducted two other studies. In one, we examined ADP depression of oxalate-supported, ATP-stimulated Ca2+-uptake under conditions of different [ATPI. When the reciprocal of steady-state Ca2+-uptake rate (llv) was plotted against [ADP] (using concentrations from mm) at three different [ATP] (50, 100, 400 pm), three lines were obtained which intersected at a single point, showing that ADP acts as a competitive inhibitor of Ca2+-ATPase (%= 180 pm, V,, = 1.3 pmol. mg-'. min-'; Fig. 3A). Complimentary results were obtained when the time to steady-state

4 976 Ca2+-uptake rate versus [ADP] were plotted at the same three [ATP] ; with increasing [ADP], the time to steady-state uptake rate increased. However, at any [ADP], an increase in [ATP] causes a decrease in this time interval (Fig. 3B). Thus, the inhibiting action of ADP and the ADP-ATP interaction were clearly expressed at these high, unphysiological [ADP]. I f The effect of contaminant Pi in P-creatine A potential experimental problem with stimulation of Ca2+-uptake by adding P-creatine in the absence of oxalate is the possible contamination of P-creatine with P,. P, is known to act as a precipitating anion and, thus, increases Ca2+-uptake in the absence of oxalate, even when added in relatively low (1 mm) concentrations [23]. To investigate the possibility that the observed P-creatine-induced increase in Ca2+-uptake may have been due to contaminant P,, we first determined P, content in the P-creatine used in our experiments. Because P-creatine is unstable in acid media, which complicates P, determination by colorimetric methods, we used an enzymatic method for P, assay. This method is based on the reaction of glyceraldehyde-3-phosphate dehydrogenase and is recommended as most suitable for determination of P, in the presence of P-creatine [21]. By using this method we found that free P, in P-creatine was %. In other words, 5 mm P-creatine contained mm P,. The value of contaminant PI in P-creatine obtained by us is close to values reported by Sigma ( %). Sigma uses a colorimetric method, but takes readings after 30 s when contribution of P-creatine hydrolysis is not significant (information from Sigma Technical Service). In our hands, P, added as KH,PO, at a concentration that is ten times higher than is present as a contaminant in 5 mm P-creatine was unable to stimulate Ca2+-uptake when ATPstimulated uptake had ceased in oxalate-free buffer (data not shown). Clearly, the contamination of P-creatine with P, is too small to influence Ca2+-uptake by SR in the absence of oxalate. Addition of 5 mm P, into oxalate-free uptake buffer after ATP-stimulated Ca2+-uptake had ceased generated a faster Ca2+-uptake than the addition of an equimolar amount of P-creatine (Fig. 4, Ia). However, the addition of P, at a tenth the niolar concentration of P-creatine (equivalent to an enormous contamination of P-creatine with P,) was less effective in stimulating Ca2+-uptake than 5 mm P-creatine alone (compare Fig. 4, Ib and IIa). Therefore, if contaminating P, were to account for the Ca2+-uptake with P-creatine addition, contamination would have to be greater than 10%. A second experiment provided additional evidence that P-creatine-stimulated Ca2+-uptake is not due to contaminating P,. We found that P-creatine + ADP-stimulated Ca2+ transport has a much higher sensitivity to sulfhydryl modification by N-ethylmaleimide (MalNEt) than does ATP-stimulated Ca2+ transport. The effect of MalNEt on P-creatine + ADP-stimulated Ca2+-uptake is time- and concentration-dependent (Fig. 5). This particular preparation of SR from horse muscle showed half inhibition of oxalate-supported P- creatine + ADP-stimulated Ca2+-uptake at 32 pm MalNEt after a 3-min preincubation with MalNEt. After the same preincubation time, ATP-stimulated Ca2+-uptake was practically unchanged even at 2 mm MalNEt (Fig. 5B). This large difference in the sensitivity of P-creatine + ADP- and ATP- stimulated Ca2+-uptakes to MalNEt allowed us to demonstrate that P-creatine stimulates Ca2+-uptake through a mechanism other than contaminating P, present in P-creatine. Fig. 4. Caz+-uptake in oxalate-free buffer by SR isolated from rat skeletal muscle. Initial Caz+-uptake was stimulated in all cases by 1 mm ATP which was quickly inhibited as evidenced by the plateau in the recording. Second-phase Ca2+-uptake following the drop from the plateau occurred as the result of relief of inhibition by addition of either P-creatine or P,. Note, that the time scale during ATPstimulated Ca2+-uptake prior to the drop is different from the time scale of the P-creatine- or P,-induced Ca2+-uptake following the drop. (I) P, relief of inhibition of ATP-stimulated Caz+-uptake is not sensitive to SH group modification by 10 pm MalNEt. (a) Secondphase Ca -uptake was induced by the addition of 5 mm P,. (b) Second-phase Ca2+-uptake was induced by the addition of 0.5 mm P,. (c) Second-phase Ca2 -uptake stimulated by 5 mm P, was unaffected after incubation of SR for 1 min with 10 pm MalNEt. (11) P- creatine relief of inhibition of ATP-stimulated Caz+-uptake is sensitive to SH group modification by 10 pm MalNEt. (a) Second-phase Caz+-uptake was induced by the addition of P-creatine (5 mm) after ATP-stimulated uptake by SR (86 pg) had reached a plateau. (b) Second-phase Caz+-uptake did not occur in response to P-creatine addition when SR had been incubated with 10 pm MalNEt for 1 min prior to addition of P-creatine. Initial phase of ATP-stimulated Ca2 - uptake was not affected. (c) Second-phase Ca2+-uptake in response to P-creatine addition was restored when 10 mm dithiothreitol was added with the 10 pm MalNEt to the SR incubation medium. (111) MalNEt effect on P-creatine relief of inhibition of Caz+-uptake depends on SR protein content. SR protein content (172 pg) is twice that in I. (a) Second-phase Ca2+-uptake was induced by the addition of P-creatine (5 mm) after ATP-stimulated uptake had reached a plateau. (b) With this high SR protein content, second-phase Ca2+uptake induced by the addition of P-creatine varied with the concentration of MalNEt (10, 20, 40 pm) in the incubation medium. (c) Second-phase Caz+-uptake in response to P-creatine addition was restored when 10 mm dithiothreitol was added with the 40 pm Mal- NEt in the SR incubation medium. In accordance with the results presented earlier (Fig. 2B), the addition of 5 mm P-creatine to oxalate-free uptake buffer, after ATP-stimulated Ca -uptake had ceased, significantly enhanced Ca2+ accumulation by rat skeletal muscle

5 977 Evidence supporting ATP-ADP compartmentation Incubation time (min) 100 A +-,'-A lmalnetl(~m) Fig. 5. Effect of MalNEt on P-creatine + ADP-stimulated ATPdepndent Caz+-uptake by the SR. (A) The effect of 10 pm MalNEt on the rate of P-creatine+ADP-stimulated (5 mm plus 0.1 mm) Caz+-uptake by SR (35 pg) isolated from horse skeletal muscle measured after preincubation of SR with MalNEt at 37OC during the indicated time periods. (B) The effect of [MalNEt] on P-creatine+ADP-stimulated ATP-dependent CaZ' -uptake (0) and ATP-stimulated Ca"' uptake (A) by the same preparation, measured after a I-min preincubation at 37 C with 1 mm ATP and indicated indicated [MalNEt]. The rate of Caz+-uptake in the absence of Mal- NEt was taken as 100%. SR vesicles (Fig. 4, IIa). The rate of this uptake was low and depended on the amount of protein added (compare IIa and IIIa in Fig. 4). However, the main finding is that the presence of P-creatine allowed a higher Caz+ gradient to be reached and maintained in the absence of oxalate. This effect of P-creatine was totally abolished after a 1 -min preincubation with 10 pm MalNEt (Fig. 4, IIb). Preincubation with 10 pm MalNEt together with the SH group protector dithiothreitol (10 mm) had no significant effect on P-creatine-stimulated Ca2+-uptake in the presence of ATP (Fig. 4, IIc). When SR protein concentration was increased in the buffer, the rate of P-creatine-stimulated Ca2+-uptake was higher and also more MalNEit was necessary to inhibit P-creatine-stimulated uptake. Dithiothreitol again eliminated the inhibitory effect of MalNEt on P-creatine-stimulated Ca2+-uptake which had ceased due to significant back-inhibition in oxalate-free buffer (Fig. 4, IIIc). However, a 1-min preincubation with MalNEt, which totally abolished P-creatine-stimulated uptake (Fig. 4, IIb, IIIb), had no significant effect on Ca2+-uptake stimulated with 5 mm Pi (Fig. 4, Ic). Clearly, Pi and P-creatine stimulate Ca2+-uptake by different mechanisms and, thus, contaminating Pi is unlikely to be the means by which P-creatine enhances Ca2+ -uptake in oxalate-free media. Further, when the creatine kinase activity was high, as in SR from horse skeletal muscle, the effect of P-creatine was greater than when the creatine kinase activity was low as in SR from rat skeletal muscle. Therefore, P-creatine-stimulated Ca2+-uptake depends on creatine kinase activity and its highly sensitive functional SH groups, an unlikely situation if contaminating Pi were responsible for the P-creatine effect. To demonstrate functional coupling between Ca2+- ATPase and creatine kinase bound to the SR it is important to show that ADP produced in the ATPase reaction is preferentially used by bound creatine kinase and ATP synthesized in the creatine kinase reaction is preferentially used by AT- Pase. We obtained results that showed that ADP and ATP were exchanged within the interacting creatine kinase/ca2+- ATPase system and were not freely exchanged with the surrounding medium. Rather, they were locally constrained within an effective micro-environment defined by the spatial proximity of the two enzymes. In the presence of apyrase, a potato ATPase with no effect on SR Ca2+ transport [24], ATP-stimulated Ca"-uptake (0.1 mm ATP) stopped within 15 s, indicating that ATP in the medium had decreased below the level necessary to stimulate Ca2+ transport. However, when Ca2+-uptake was stimulated with P-creatine plus ADP (5 mm P-creatine, 0.1 mm ADP), Ca2+-uptake rate was maintained for 90 s (Fig. 6A). Comparison between rates of P-creatine + ADP-stimulated Ca*+-uptake in the presence and absence of apyrase indicated that about 60% of ATP generated by membrane-bound creatine kinase was not accessible to the apyrase, i.e. was not in free equilibrium with ATP in the uptake buffer, but was preferentially used by the Ca2+-pump. Similar results were obtained when hexokinase (8 U/ml) and glucose (20 mm) were used instead of apyrase. A complementary finding was obtained from experiments to examine the exchange of ADP between Ca2+-ATPase and SR-bound creatine kinase. When ADP production by ATPase was measured using the P-pyruvatelpyruvate-kinase-coupled enzyme system, it was observed that the addition of P-creatine to the reaction mixture significantly decreased ADP release into the medium during the ATPase reaction (Fig. 6B). This can only be explained by ADP rephosphorylation with P-creatine due to SR-bound creatine kinase. This creatinekinase-catalyzed phosphorylation of ADP occurred despite the fact that the activity of the added pyruvate kinase was >20 times higher than that of endogenous creatine kinase and that the K,,, of pyruvate kinase and creatine kinase for ADP are in the same range [4]. The creatine kinase dependence of this ADP phosphorylation was confirmed by adding creatine kinase inhibitor Dnp-F (20 pm), which fully reversed the effect of P-creatine (Fig. 6B). Thus, we concluded that ADP produced in the Ca2+-ATPase reaction was preferentially available to SR-bound creatine kinase. The amount of creatine kinase bound to SR has sufficient capacity to allow determination of the K,,, value for ATP at low [ATP]. In the presence of 5 mm P-creatine, oxalate-supported steady-state Ca2+-uptake by horse skeletal muscle SR was stimulated by either [ATP] or [ADP] as low as 0.2 pm. From the linear part of Lineweaver-Burk plot, both ATP and ADP gave similar concentrations for half-maximal stimulation of Ca2+-uptake (apparent K, = 1.4 pm; Fig. 7). In the absence of P-creatine, when ATP was added alone in low concentrations (0.2-5 pm) it was not possible to achieve a measurable steady-state rate of Ca2+-uptake because ATP is hydrolyzed too rapidly. When ATP was used in concentrations >10 pm, the apparent K, determined from the obtained values was at least tenfold higher than in the presence of P-creatine. The addition of exogenous P-pyruvate (5 mm) and pyruvate kinase (in an amount equal to the activity of endogenous creatine kinase), which is supposed to regenerate ATP in the uptake buffer with the efficiency of the endogenous creatine kinase/p-creatine system, was much less able

6 978 also increased (data not shown). These results additionally suggested a more efficient translocation of ATP from creatine kinase to the high-affinity binding sites of Ca +-ATPase on SR than occurs with ATP from surrounding medium or from other soluble enzymes. A Time (s) Fig. 6. Time courses of Ca++-uptake and ADP production by the SR. (A) Time course of Ca2+-uptake by SR isolated from horse skeletal muscle and measured in the presence of 3 U apyrase. The reaction was started by addition of 0.1 mm ATP (trace 1 j or 0.1 mm ADP plus 5 mm P-creatine (trace 2) after short preincubation of SR (68 pg) with apyrase. (B) Time course of ADP production determined by NADH oxidation. 72 pg SR isolated from horse skeletal muscle was used. Reaction was started by addition of ATP (1 mm). Time of additions of ApsA (lopm), P-creatine (1OmM) and Dnp-F (20 pmj are shown with arrows. \ SR / I I/ 1 I lumatp SR I z P-crealine I I Fig. 7. Double-reciprocal plot for steady-state oxalate-supported Ca*+-uptake rate by horse skeletal muscle SR (39 pg) at low concentrations of ATP (0) or ADP (A) in the presence of 5mM P-creatine. The data were fitted by least-squares linear regression analysis with no weighting of the data. Insets show Ca2+-uptake, stimulated by 1 pm ATP and either in the presence of 5 mm P-pyruvate and 0.08 U pymvate kinase (A) or 5 mh4 P-creatine and 0.08 U endogenous creatine kinase (B). Note differences in rates of Caz+-uptake depending on ATP regeneration by exogenous pyruvate kinase or endogenous creatine kinase. to increase the steady-state rate of Ca +-uptake at low [ATP] (compare insets in Fig. 7). Separate experiments demonstrated that 7 pm CaZ+ had no significant effect on pyruvate kinase activity when measured by oxidation of NADH in the presence of 5 mm P-pyruvate and 1.5 mm ADP in the CaZ+uptake buffer without oxalate. However, by increasing the amount of added pyruvate kinase, uptake rate at low [ATP] DISCUSSION This study was undertaken to investigate the functional importance of SR-bound creatine kinase in the regulation of ATP-dependent Ca transport by SR vesicles. Previously, it had been demonstrated by Rossi et al. [5] that creatine kinase binding to purified SR membranes survived isolation and was resistant to extraction by 0.6 M KC1. These authors also showed that SR vesicles, prepared from chicken pectoralis muscle, accumulated Ca + in media containing P-creatine plus ADP. This P-creatine + ADP-stimulated Ca -uptake was inhibited by the creatine kinase inhibitor DnP-F, which had no significant effect on ATP-stimulated CaZ+-uptake. However, because these previous experiments were mainly directed at characterizing the binding of the creatine kinase isoform to SR membranes, only a single high concentration of ADP (1 mm) was used in the uptake experiments. Nevertheless, an interesting hypothesis was put forward that the physiological role of SR-bound creatine kinase was in the local regeneration of ATP and, through this, the regulation of local ATP/ADP ratios in the proximity of the Ca +-pump [5]. Results obtained in our study fully support this original hypothesis. One very important result to support the hypothesis was that we found that ADP produced in the Mg, Ca2+-ATPase reaction is preferentially used by creatine kinase bound to SR. Relative to the amounts and binding affinities of the two enzymes, endogenous SR-bound creatine kinase proved to be a more effective competitor for ADP released in the ATPase reaction than exogenous pyruvate kinase in the surrounding medium. This apparent greater binding of ADP by SR-bound creatine kinase can be interpreted as a sign of close structural localization of creatine kinase and ATPase on the SR membrane. The importanance of this local phosphorylation of ADP by P-creatine is not only to supply the Caz -pump with ATP, but also to keep a low ADP level in the vicinity of ATP binding sites of the ATPase. Mechanisms for ADP inhibition of the Ca*+-pump have been described by others [25, 261. Accumulation of ADP may decrease the rate of dephosphorylation of ADP-sensitive phosphoenzyme species which are formed during ATPase turnover. As a result, the rate of reversal of the phosphorylation step may be faster than its forward rate [25]. Under these conditions, the free energy of the transmembrane Ca +-gradient may be used for resyn- thesis of ATP (ADP-ATP exchange) [25, 261. Increase in [ATP] activates the phosphoenzyme turnover [25] with concomitant increase in the rate of Ca2+-uptake. ADP+ATP exchange was found to predominate when phosphoenzyme turnover is slow [26]. These findings are consistent with our observations of the inhibitory effects of high [ADP] on both the time to achieve a steady-state rate of Ca2 -uptake and this steady-state rate itself. The inhibiting effect of ADP on Caz+-pump function is more expressed when there is back-inhibition of Ca2+ transport by high luminal Ca as in the absence of oxalate. Oxalate diffuses through the SR membrane and forms a calcium salt of low solubility. These salts precipitate when the solubility product of calcium X oxalate (about 2 X lo- MZ) is re-

7 ached inside the vesicles. Because oxalate is not actively accumulated by vesicles, formation of Ca precipitates is due to active transport of Ca2+, which in the presence of oxalate can generate a large concentration gradient of Ca2+. Low [Ca2+] in the assay media avoids precipitation of calcium oxalate outside vesicles [27]. However, our results demonstrated that in the absence of oxalate, ADP phosphorylation by P-creatine dramatically-stimulated ATP-dependent Ca2+-uptake by SR even when uptake had ceased due to back-inhibition by high luminal Ca (Figs 2 and 4). The possibility that the observed increase in Ca2+-uptake was due to contamination of P-creatine by P, was ruled out by enzymatic determination of Pi in P-creatine and also by demonstrating that P-creatineinduced stimulation of Caz+-uptake is sensitive to SH-group modifications by MalNEt, while Pi-stimulated transport was not. The high sensitivity of creatine kinase to SH-group modifications has been known for more than 30 years [28]. Creatine kinase has eight SH-groups, two of which per dimer are more reactive to thiol-blocking reagents and are therefore believed to be essential for the activity of the enzyme [29]. SR Ca2+-ATPase also has two SH-groups which are considered to be essential for Ca2+ transport: one group seems to be important for phosphoenzyme formation, another for its decomposition, when modified with MalNEt [30]. According to Kawakita et al. [30], SH-groups responsible for dephosphorylation are modified first at lower concentration of MalNEt and this early modification leads to a significant decrease in Ca2+-uptake by skeletal muscle SR. However, they found that the [MalNEt] required to produce half-maxima1 decrease in Ca2+-uptake during a 10-min incubation at 30 C is about 1 mm [30] which is close to the result we obtained. On the other hand, creatine kinase, like the P-creatine effect on Ca +-uptake, is much more sensitive to Mal- NEt. Thus, the P-creatine effect on Ca2+-uptake was mediated through creatine kinase. The synergistic inhibition of Ca2+-uptake by the accumulation of ADP along with an increase in luminal Caz+, as we observed here, is in accordance with the results by Inesi and de Meis [23]. They demonstrated that the reduction of ADP produced in the ATPase reaction by adding ATP-regenerating systems significantly improved skeletal muscle SR Ca2+-uptake in the absence of precipitating anions. According to these authors, high luminal Ca2+ and ADP accumulation during ATPase turnover may cause slippage of Ca2+ through the Ca2+-ATPase channel. Slippage of Ca within the channel is coupled to reverse events of the catalytic cycle and produces uncoupling of catalytic and transport activities. In addition, an increase in free [ADP], resulting in lower phosphorylation potential, may have a significant influence on SR Ca2+- ATPase because even the maintenance of a large Ca gradient without net transfer requires 52kJ/mol ATP [16]. This suggestion is supported by a recent study indicating that free energy of ATP hydrolysis in P-creatine-depleted isolated perfused rat hearts, calculated from 31P-NMR spectroscopy data, is probably below that required for the SR Ca2+-ATPase [31]. Whatever the mechanism, phosphorylation of ADP by the creatine kinase reaction in our preparation demonstrably and dramatically relieved ADP inhibition of Ca2+-uptake. Complementary to the preferential use of Ca -ATPaseproduced ADP by creatine kinase, we found that ATP produced in the creatine kinase reaction on SR membranes is preferentially used by the Ca2+-ATPase for Ca2+-uptake. This was demonstrated in experiments with the apyrase and hexokinase ATP traps which quickly depressed Ca2+-uptake 979 stimulated by exogenous ATP but had little effect on P-creatine + ADP-stimulated ATP-dependent Ca2+-uptake. This result, together with the fact that the apparent K, for ATP was much lower when endogenous creatine kinase was allowed to fuel the Ca2+-pump with ATP, can be explained by a local increase in [ATP] due to the close proximity of SR-bound creatine kinase and ATP binding sites of Ca2+- ATPase. A similar close association between creatine kinase bound to cardiac myofibrils and Ca2+-activated myosin AT- Pase was recently proposed to explain the higher affinity of the myofibrillar-atpase for ATP in the presence of an active endogenous creatine kinase/p-creatine system [4]. Our results are also compatible with data from others showing that glycolytic enzymes bound to smooth muscle plasma membranes [9] or red cell membranes [32] could fuel the Ca2+pump or Na+, K+-pump despite the presence of a hexokinase-based ATP trap that eliminated ion uptake stimulated by exogenously added ATP. In fact, reversible binding of glycolytic enzymes in relatively large quantities to phospholipids of purified SR membranes has been demonstrated [8] and this binding, together with binding of glycogenolytic complex to SR [33], may also have importance in local regeneration of ATP. This suggestion is supported by findings that membrane-bound glycolytic enzymes are able to generate ATP within the skeletal muscle triadic gap creating a local ATP pool separate from the cytosolic ATP pool [34]. In addition, the possibility exists that, at least in fast-twich glycolytic muscle, creatine kinase is functionally coupled to glycolysis [35]. Based on an extensive literature review, Wallimann et al. [35] have concluded that because the creatine kinase/phosphocreatine system is active not only on the sites of ATP consumption but also on the sites of ATP production, these two intracellular sites are connected by way of compartmentalized creatine kinase isoenzymes. Conceivably, the importance of local ATP regeneration near ATP-consuming sites increases when the rate of ATP hydrolysis increases. However, the functional coupling between SR-bound creatine kinase and Ca2+-ATPase may not be stable under those circumstances. A decrease of [P-creatine] is characteristic of increased consumption of ATP in muscle cells and [P-creatine] <5 mm starts to influence the local regeneration potential of the creatine kinaselp-creatine system. To a certain extent, the effect of [P-creatine] decrease on P-creatine + ADP-stimulated Ca2+-uptake is compensated for by an increase in free [ADP], which may occur during intense contractile activity, especially when oxygen supply is restricted. Besides fluctuations in substrate concentration, functional coupling between SR-bound creatine kinase and Ca2+-ATPase can be influenced by changes in creatine kinase binding to SR membranes and creatine kinase activity, which is critically dependent on the state of SHgroups on the enzyme. However, further investigations are necessary to determine the importance of disruption of thts functional coupling for Ca -pump function under conditions when cellular ATP consumption is increased together with decrease in [P-creatine], increase in [ADP], and possible production of free radicals which are able to oxidize SH-groups. The actual amount of creatine kinase bound to SR membranes in vivo is probably higher than that determined in our preparations, the difference being due to creatine kinase lost during the isolation of vesicles. The importance of isolation to obtain myofibrillar preparations with high specific activity of creatine kinase compared with that of ATPase is underlined in the recent paper by Krause and Jacobus [4]. Myofibrillar preparations isolated by these authors retained fivefold

8 980 higher specific activity of creatine kinase relative to the ATPase, which indicates that creatine kinase does not limit the rate of ADP phosphorylation and subsequent supply of ATPase with ATP. Thus, our evidence of functional coupling between these two enzymes probably underestimates the importance of this coupling in the intact cells. Taken together, the results presented in this paper indicate that regeneration of ATP by creatine kinase on SR membranes in close proximity to ATP-consuming sites of Caz+- ATPase is an important physiological mechanism which provides stable local concentrations of ATP and ADP necessary for optimal functioning of the Ca*+-pump. In accordance with this finding, factors influencing the capacity of bound creatine kinase to rephosphorylate ADP may also affect Ca -pump function. This work was supported by National Institutes of Health Grant AR REFERENCES 1. Bessman, S. P., Yang, W. C. T., Gieger, P. J. & Erikson-Viitanen, S. 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