Eur. J. Biochem. 6 I , 77-80 (1976)

[32P]Phosphate Incorporation into ATP during ATP Hydrolysis and Its Dependence on the Interaction of Actin and Myosin Giseia PAULSEN

Department of Cellphysiology, Ruhr-University Bochum

(Received July 26/0ctober 6, 1975)

The incorporation of 32Pi into ATP has been found to be catalyzed by myosin only when and if it interacts with actin. This exchange reaction is inhibited in natural but not in desensitized acto- myosin after removing of trace Ca2 + with ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid (EGTA). In desensitized as well as in synthetic actomyosin the exchange reaction can be fully in- hibited by the addition of troponin I (0.5 mg troponin I/mg actomyosin results in a 50% inhibition) or after replacing the Mg activator by CaCl,. The exchange rate is about 1 : 500 of the ATPase rate in presence of 2 mM phosphate. These results suggest the existence of an 'energy-rich' actin - myosin- nucleoside-diphosphate intermediate during the cross-bridge cycle.

An incorporation of inorganic ["Plphosphate into ATP catalysed by actomyosin has been demonstrated recently in a number of laboratories [l-31. This exchange reaction involves at least two steps: (a) the conversion of an enzyme-ADP intermediate and 32Pi into enzyme-["'PIATP and (b) its dissociation into actomyosin and [32P]ATP. The first step does not seem to require actin, since an incorporation of Pi into protein-bound ATP is also obtained with myosin alone [3,4]. However, the dissociation of myosin-ATP is quite improbable because a very high energy change is associated with the binding of ATP and myosin [3,5]. The dissociation of ATP from the actomyosin- ATP complex is much faster than that of the myosin- ATP complex [3]. These findings may partly account for the fact that the rate of ATP/Pi exchange is several-hundred-fold higher with actin-activated myo- sin than with myosin alone [2,3]. But preliminary results indicate that an admixture of actin to myosin is not a sufficient prequisite for this exchange reaction [1,2]; it is necessary, furthermore, that the two proteins interact in the same way as they do during contraction. In this respect it was of special interest to study the effect of specific inhibitors of actin- myosin interaction and contraction, such as EGTA, troponin I [6] and CaCl, [7].

In order to measure the free [32P]ATP formed it was useful to transfer the 32P after the desired reac- tion time into glucose 6-phosphate as described below.

MATERIALS AND METHODS

Preparation of the Proteins

Natural actomyosin, desensitized actomyosin myo- sin and troponin I were prepared from rabbit skeletal muscles according to Perry et al. [ 8 - 111. Myosin was further purified by high-speed centrifugation [ 121. Actin was extracted from an acetone dry powder [13] and purified by ultracentrifugation [14]. All proteins were stored at 2 "C in 10 mM azide.

Determination of ATPase Activity

The incubation conditions for the ATPase reaction were: 20 mM Tris-HC1, pH 7.6, 0.05 M KCl, CaZ+, MgZ+, and ATP as indicated and the temperature was 22 "C. The Ca" concentration was adjusted with EGTA [15]. The reaction was stopped with 15% trichloroacetic acid and the Pi liberated was deter- mined according to Rockstein and Herron [16].

Abbreviations. Pi, inorganic phosphate; EGTA, ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid.

Enzymes. Hexokinase, ATP : D-hexose 6-phosphotransferase (EC 2.7.1.1); myosin ATPase, ATP phosphohydrolase (EC 3.6.1.3).

Enzymic Determination of 32Pi Incorporation into A TP

The same incubation mixture as used for the ATPase activity determination was applied for the

78 "Pi Incorporation into ATP Catalyzed by Actomyosin

2.5

2.0

- 1.5 -

hl

- 1.0 d

.- I

0.5

measurement of the "Pi incorporation rate into ATP. In addition, the incubation solution usually contained 2 mM phosphate and chromatographically purified ["P]phosphate of varing specific activity. The reaction was stopped by adding 100-500 pg hexokinase (Boehringer, Mannheim) and 50 mM glu- cose in 0.05 M triethanolamine-HC1 buffer [17]; within 1 min all the y-phosphate of ATP was trans- ferred to glucose to form glucose 6-phosphate. The insoluble actomyosin gel was spun down and 40 pl of the supernatant was analysed for glucose 6-phos- phate by paper chromatography (Whatman MM3) with the solvent H20/methanol/ammonium hydroxyde (20/60/20) and 0.001 M EDTA [18]. The chromato- gram was cut into strips, the activities of which were estimated and expressed as a fraction of the total activity.

-

-

-

-

-

Direct Determination of [y3, P I A TP

The [y-32P]ATP formed was also determined with a slight modification of the method of Lindberg and Ernster [19]. After stopping the reaction with tri- chloroacetic acid the denatured protein was centri- fuged and phosphate (2 mM) and ATP (5 mM) were added as carriers to the supernatant. 0.5 ml of the supernatant was then mixed with 0.5ml lox, am- monium molybdate, 2 ml perchloric acid and 5 ml isobutanol/benzol mixture ( l / l ) for 15 s. When the phases were separated the organic phase was counted for radioactivity on a glass filter (MN 85, Macherey and Nagel) after neutralization with ammonia. Ex- periments were done in triplicate and did not usually differ by more than 5%. Samples contained more than 300 counts/min blanks less than 200 counts/min.

RESULTS

32Pi Incorporation Rate into ATP during A TP Hydrolysis

As shown previously [2], a measurable incorpora- tion of Pi into free ATP is only obtained in the presence of both actin and myosin (Table 1) and under condi- tions which favour actin-myosin interaction and contraction (low ionic strength, Mg2 + activation). If the M$+ activator is replaced by CaC1, the acto- myosin ATPase is transformed into the myosin ATPase [7] and though the specific ATPase activity is quite high under these conditions, the exchange reaction is completely inhibited (Table 1). Fig. 1 demonstrates the linear time course of ATP splitting and ''Pi incorporation into ATP during the first minutes with slopes of 580 nmol . mg-' . min-' and 3.3 nmol . mg-' + min-' respectively. Hence, the rate of the back reaction is about 500 times lower than that of the

Table 1. A TP splitting and " P i incurporaiion (Iwxokinase methud) into A TP catalyzed by uctin and nijosin uiider vurious conditions Protein concentration: 1 mgjml myosin; 0.4 mg/ml actin; 10 mM ATP; 2 m M Pi; 10 m M M g 2 + ; 0.01 mM Ca2+ or only 0.2 mM CaCI,. pH 7.6

System ATPase activity 32Pi incorporation in presence of after 5 min

in presencc of

Mg2 + CaCI, Mg2 + CaCI,

nmol. rng-'. min-' total P i

Actin 0 0 0 0 Myosin 40 480 0 0 Actin

+myosin 160 410 0.3 0

6 o o K ; ; 3 1 5 ; ; 8 0

Time (rnin)

Fig. 1. Time course of' ATP splitting and " P i incorporation into ATP cutolyred by actomyosin (direct determination). Protein concentra- tion 2.5 mg/ml; 5 mM Mgz* ; 0.01 mM Ca2+; 4 mM ATP; 2 mM Pi. (0) ATPase; (A) 32P incorporation

forward reaction under these conditions, as found by Wolcott and Boyer [3] and by Gillis and MarCchal [201.

Inhibition of the Pi-ATP Exchange Reaction by Troponin I

Instead of synthetic actomyosin prepared from purified actin and myosin it is possible to use so-called desensitized actomyosin for the study of P incorpora- tion into ATP. This protein preparation has bcen washed free from the regulatory proteins and it is, like synthetic actomyosin, insensitive to traces of Ca2+ ions. Both ATPase activity and Pi incorporation rate are similar in the presence and absence of Ca2' (Table 2). However, both reactions may be inhibited concomitantly by the addition of troponin I (Fig. 2), a component which is known to inhibit the interaction between the myosin product complex with actin

G. Paulsen 19

Table 2. The qflect of iroponin I on the M$+ and Ca2'-activated rrctom~osin A TPase and on the " P i incorporation rate into ATP 1 mgiml desensitized actomyosin; 0.3 mg/ml troponin I ; 5 mM ATP; 0.2 mM CaCI,

System ATPase activity 32Pi incorporation in presence of after 5 min

in presence of

Mg2+, CaCl,, Mg2+, CaCI,, 5 mM 0.2 mM 5 mM 0.2 mM

nmol. mg-'. min-' "/oo total pi

Desensitized

Desensitized actomyosin 130 250 1.1 0.59

actomyosin and troponin I 70 255 0.58 0.6

-no 1

-80 0 c .L .-

I 0 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Amount of troponin I (rng Img actomyosin)

Fig. 2. c f fbct oj'troponin I on the ATPase activity and 32Pi incorpora- tion rate mtalyzed hy actomyosin ( 2 mglrnl) (direct determination). 5 mM Mg2' ; 0.01 mM Ca2' ; 4 m M ATP. (0) ATPase; (A) "P incorporation

[6,21] while it does not inhibit the ATPase after replacing MgCl, by CaC1,. The ATPase of actin myosin mixtures activated with CaCl, is of the myosin type [7]. Fig.2 illustrates that 0.5 mg troponin I is required to inhibit by 50 % both the Mg-activated actomyosin ATPase and the exchange reaction cata- lysed by 1 mg actomyosin. Assuming that 1 mg actomyosin contains about 0.3 mg actin, the above results indicate that under our experimental conditions troponin I must be far in excess of actin monomers in order to induce full inhibition (cf. [22]).

Inhibition of the Exchange Reaction by EGTA

Unlike desensitized actomyosin the so-called natural actomyosin extracted from muscle at high ionic strength still contains the regulatory proteins

Table 3 . Effect of EGTA on ATP splitting and "Pi incorporation rate (hevokinase method) into A T P catalyzed by natural actomyosin or desensitized actomyosin Protein concentration 2 mg/ml; 10 mM ATP; 10 mM Mg2'; 0.01 mM Ca2+ or 5 rnM EGTA

Systcm ATPase activity 32Pi incorporation in presence of after 5 min

in presence of

Mg2+ MgZ+ M$+ Mg' Ca2' EGTA Ca2' EGTA

nmol mg-' . min-' o/oo total pi

Natural

Desensitized actomyosin 170 90 0.3 0.18

actomyosin 150 145 0.27 0.28

troponin and tropomyosin, so that its activity is sensitive to traces of Ca2+. As shown in Table 3 both the activity of the Mg-activated actomyosin ATPase and the exchange reaction rate are high in the presence of about 0.01 mM Ca2+ and drastically reduced after removal of Ca2+ ions by EGTA. The latter is known to abolish or diminish the extent of actin-myosin interaction in the presence of ATP (e.g. ~ 3 1 ) .

DISCUSSION

The reported results clearly indicate that the incorporation of Pi into ATP requires and is dependent on an interaction of actin and myosin similar to that occuring in contraction. Of particular interest is the inhibiting effect of troponin I because of its high specifity, so that it seems quite unlikely that the measured exchange reaction is due to an impurity still associated with desensitized actomyosin. It will be recalled that some previous investigators [24 - 261 failed to demonstrate an incorporation of P i into ATP and at least one of them attributed the occasional observation of an exchange to impurities [24]. It is equally obvious, however, that the small incorpora- tion detected in this and other papers [ l- 51 would not have been measurable by Inoue's method [25].

In natural Mg2 +-activated actomyosin the forma- tion of free [32P]ATP is activated by increasing the Ca2+ concentration, and in myofibrillar bundles from insect flight muscle as well as rabbit skeletal muscle it is activated by Ca2+ [2,27] and stretching [28 - 301. It is known that increasing Ca2+ concentration and stretching increase the extent of actin-myosin inter- action, or the number of cross-bridges attached to actin at anyone moment (J. Herzig, unpublished results). Since the formation of protein-bound [32P]-

G. Paulsen: 3zPi Incorporation into ATP Catalyzed by Actomyosin

ATP readily occurs with myosin alone (i.e. [3,4], it must be concluded that the interaction with actin is required to promote the dissociation of the myosin- [32P]ATP complex into myosin and free [32P]ATP. This suggests the existence of an actomyosin-ATP complex in addition to an actomyosin-ADP complex, which is similar perhaps to that postulated by Blum [32] and described by Taylor [33]. The described ex- change reaction may occur either after the combina- tion of actin with the myosin product complex or earlier in the myosin product state (cf. Lymn and Taylor’s scheme [34]). If the exchange reaction occurs in the myosin product state then high Ca” concentra- tion and troponin I also must inhibit the combination of actin with the myosin substrate complex. In this case the myosin substrate and product complex should be very similar in their conformation.

As a first approximation these results might imply that the energy transferred to the contractile system by the binding of ATP (see [3 - 51) ought to be conserved in the form of the system consisting of myosin - nucleoside-diphosphate and actin. It is tempting to speculate that this energy might be con- verted into mechanical energy during the power stroke of the cross-bridge.

The author thanks Professor J. C. Riiegg for many suggestions and helpful discussions and H. Bartel for expert technical assistance. This work was partly supported by a Grant of the Deutsche Forsrhun~~~~emeinschaff (RU 154/8-9) to Professor J. C. Riiegg.

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G . Paulsen, Institut fur Zellphysiologie der Ruhr-Universitat Bochum, D-4630 Bochum-Querenburg, Postfach 2148, Federal Republic of Germany