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Adenosine triphosphatase activity and superprecipitation of actomyosin from the frog, Rana temporaria
ht. J. Biochem. [Scientechnica
324
ADENOSINE
TRIPHOSPHATASE
SUPERPRECIPITATION FROG, J. J.
ACTIVITY
OF ACTOMYOSIN
RANA
A. HEFFRON*
(Publishers)
Ltd.]
AND
FROM
THE
TEMPORARIA and P. F. DUGGAN
Department of Biochemistry, University College, Dublin, Ireland (Received26 May, 1970) ABSTRACT Magnesium, as well as calcium, was found (contrary to some published reports) to activate natural actomyosin adenosine triphosphatase (ATPase) from Rana temwaria. The optimal level of Mg*+ was I mM at p=o.o5, pH 7-2. With increasing ATP concentration substrate inhibition occurred with Mg*+ but not with Ca*+. 2. Contamination with mitochondrial or sarcoplasmic reticulum fragments was not sufficient to account for the Mg*+-activation of actomyosin ATPase. 3. Mga+, but not Car+, was essential for super-precipitation. 4. Mgl+ -activated ATPase activity and superprecipitation showed a similar but slight dependence upon the free Ca-+ concentration. 5. A comparison was made with rabbit natural actomysin. I.
MUCH of the present knowledge of the physiology and energetics of muscular contraction and relaxation has been obtained on frog skeletal muscle (see Wilkie, 1966, for review). The enzymatic events which form the basis of the contraction-relaxation cycle have been characterized in considerable detail by biochemical studies on mammalian muscle, primarily rabbit muscle. The chemical properties of frog actomyosin are similar in many respects to the comparable system in other animals (de Villafranca and Hochgraf, 1962 ; de Villafranca, 1964; Heffron, 1969). Nass ( 1g62), in a developmental study of actomyosin ATPase in Rana tipiens skeletal muscle, remarked that the effects of magnesium ions on the ATPase activity of adult actomyosin were similar to the results reported for other species, i.e., Mg2+ and Ca2+ ions activated actomyosin ATPase at low ionic strength, whereas Ca2+ alone activated the enzyme at high ionic strength. de Villafranca and Hochgraf (1962) reported that
* Present address: Animal Sciences Division, The Agricultural Institute, Castleknock, Dublin, Irrland.
they were unable to find Mg2+ activation of frog (R. pipiens) actomyosin ATPase even at low ionic strength. Similar results were found with actomyosin from the horseshoe crab Limulus polyphemus (de Villafranca and Naumann, 1964). Bailey (1942)) in a study of myosin from various species (rabbit, chicken, pig, frog), observed that Mg2 +-activation of myosin ATPase was significant only in the case of frog myosin. In view of the difficulty in preparing myosin free from actin from frog skeletal muscle (Hamoir and Reuter, 1956 ; de Villafranca and Hochgraf, 1962)) and since actin was discovered in 1942 it seems probable that the Mg2 +-activation observed by Bailey was due to the presence of actin in the myosin preparations. In the course of a search for the Na +-K +ATPase in a sarcolemmal preparation from frog (R. temporaria) muscle (Heffron and Duggan, 1967) it became necessary to establish the effects of Mg2+ and Ca2+ on the frog actomyosin system, in particular on the ATPase activity. Furthermore, the effect of Mg2 + on the enzyme activity is of interest in view of the high levels of Mg2- in frog muscle (Conway, 1957) and the postulate of Schaub,
‘971, 29324-336
ACTOMYOSINATPaSe AND
Hartshorne, and Perry (1967) that the role of Mg2 + in contraction is as a complex with ATP, and that Ca2+ acts indirectly through the troponin-tropomyosin complex. The results presented here on R. temporaria actomyosin show, contrary to some published reports, that frog actomyosin is activated by Mg* + as well as Ca2 + and that ethylene glycol bis-( P-aminoethyl)&, N-tetra-acetic acid (EGTA), the Ca2 + chelating agent, has a weak inhibitory effect -on frog actomyosin ATPase and superprecipitation compared with rabbit actomyosin. MATERIALS
AND METHODS
FROGPROTEINS Adult Rana ten&raria were stored in plastic water tanks at x2:16’ C. and occasionally-forcefed with liver. The chilled. minced unner lea muscles were homogenized in’4 volumes $WeberI Edsall solution (0.6 M KCl, 0.04 M NaHCOs, o-01 M Na, CO,) containing I mM dithiothreitol and extracted for 24 hours at 2’ C. The extract was diluted with an equal volume of Weber-Edsall solution, filtered through terylene net, and centrifuged at 20~~x1g for 20 minutes at o0 C. The actomyosin was precipitated by diluting the supernatant with IO volumes of water (adjusted to pH 7.4 with Tris), and collected by centrifugation at 2ooog for ro minutes. The actomyosin was redissolved in an equal volume of t-2 M KCl, adjusted to pH 7.4 with Tris, and clarified at 20,ooog. It was precipitated and collected as before, washed once with jo mM KCI-IO mM Tris-HCl pH 7.4, and suspended in the same medium with a protein concentration of 2-5 mg. per ml. The actomyosin was stored in polythene tubes at 2’ C. Fresh suspensions of natural actomyosin (NAM) were prepared each week. In some experiments, indicated in the text, the actomyosin was purified by 3 cycles of clarification and precipitation, and centrifugation at 10,ooog for 20 minutes. Incorporation of dithiothreitol into the Weber-Edsall solution increased the specific activity of the Mg-activated ATPase of frog NAM by 20 per cent. Frog myofibrils were prepared from freshly excised leg muscle accordin to the method of Perry and Grev ( 1g56a) with minor modification, and stored in 25 mM KCl. 20 mMsodium borate bH 7. I at 2°C. “Myosin w&” prepared from n&CCd fm? k&l muscle by extraction for Io minutes with 4 volumes of Guba-Straub solution (0.4 M KCl, 0.05 M KH, PO,, o-10 M Ks HPO,) or Hanson-Huxley solution (0.6 M KCl, 0.01 M Na,PsO,, 0.10 M phosphate buffer, oao~ M MgCl,, pH 6.4) at 2’ C. The mvosin was nurified accordinn to the method described by Mommaerts (‘958) fey rabbit myosin.
SUPERPRECIPITATION
325
The final preparation was kept in 50 mM KC1 at 2’ C: These preparations-had significant Mes+-activated ATPase activitv and ATP-sensitiv%y and were therefore more like actomyosin than myosin. A protein resembling mammalian myosin more closely in enzymic and physical properties was prepared from frog actomyosin by the dissociation procedure of Weber (1956). Frog actomyosin, dissolved in 0.6 M KCl, IO mM hiitidine-HCl, IO mM ATP, IO mM MgSO,, pH 7.0 with a protein concentration of 3 mg. per ml., was centrifuged at ~~oo,ooo g for 2 hours at o” C. The upper half of the supernatant, which was clear, w& removed with a plastic syringe, dialvsed a2ainst a lame volume of IO mM KCl. and’the m&n collectid by centrifugation. After washing 3 times with 50 mM KCl, the myosin was dissolved in 0.6 M KCl/lo mM histidineHCl PH 7.0 (2 mg. per ml.) and stored at 2’ C. RABBITA~TOMYOSIN Rabbit actomyosin was prepared according to Mommaerts (1958) by extraction with WeberEdsall solution. It was clarified by centrifugation at 20,ooo g for 30 minutes, and precipitated by dilution 3 times. ASSAYS
ATPase activities were measured in a final volume of I ml. at 30” C. in the following media: (I) 1.5 mM ATP-Tris, 1.5 mM MgCIs, (2) 5 mM ATP-Tris, 5 mM CaCl,, buffered with 25 mM Tris-HCl pH 7’2-7’3. Enzyme activities were also measured in the presence of varying concentrations of ATP, MgCI,, CaCl,, and are indicated. The liberated inorganic phosphate was determined by the method of Taussky and Shorr (1853) using FeSO, as reducing agent. Protein was measured by the biuret method using crystalline bovine serum albumin as standard (Lavne, 1957). The biuret reaction was standard&d by the Kieldahl method described bv Conwav f I 06~). Superprecipitation of frog NAM was ‘c&t& mined by following the ahsorbancy increase at 410 mn in a medium containing 2.5 mM M&l,, 25 mM Tris-HCI PH 7.3, and 0.3-0’5 mg. NAM per ml. at about 25” C. with a r-cm. light path. The extent of superprecipitation is expressed as absorbancy units per mg. NAM (‘A’ per mg. protein). ATP-sensitivity (Weber and Portzehl, 1952) was measured wnh an Ostwald viscometer at 16” C. using 0.5 mM ATP, final concentration. The actomyosin was dissolved in 0.6 mM KCl, 25 mM Tris-HCl PH 7.2 with a concentration of 2-3 mg. NAM per ml. When present, MgCl, and C&&l, were 3 mM. ATP-sensitivity was also determined with I mM Na,P,O, and 3 mM MgCl, as dissociating agent. All solutions were ‘ AnalaR ’ grade (British Drug Houses Ltd.) in glass-double-distilled water. Disodium ATP, from Sigma, London, was
Znr. J, Bidan.
HFFFRON AND DUGGAN
326
converted to the Tris salt using the cation exchange Dithiothreitol was resin Amberlite IR-120.
obtained from Calbiochem, London, oligomycin from Mann, New York, pyridoxal phosphatefrom Sigma, and EGTA from K. & K. Labs., New York, RESULTS Attempts were made to prepare myosin as well as natural actomyosin from fr;g leg
It is shown, in Table I, that the standard myosin extraction media, the Guba-Straub and Hanson-Huxley solutions, yield a protein with significant ATP-sensitivity and a Mg2 factivated ATPase activity at low ionic strength indicating the presence of some actin with the myosin. NAM, when dissociated by the procedure of Weber ( 1956))
Table 1.-T= REI.ATIONSH~P BETWEEN THE COMPOSITION OF THE EXTRACTION MII~XIIN ANDTHE ATP-s~~snnrr~ ANDATP~E Acrrvrrv OFTHEPROTEJN EXTRA-D FROM FROG Muscz.a AT IONICSTRENOTH o-6
EXTRACTION MEDIUM
Guba-Straub (IO minutes)
Hanson-Huxley (IO minutes) Weber (1956) dissociation method for myosin Weber-Edsali (I) 10 minutes (2) 24 hours
ATPASE Acr~vrrv
ATP(pmoles Pi per mg. protein per minute) SENSITNITY Ca Mg 24 35
o.050 0’047
0’433 0’325
19
o.058
0’345
o=142 ~467
0.483 0.550
50 108
The extraction time is shown in parentheses. The ATPase activity in the absence of divalent cations was subtracted from that in their presence in order to show the effect of the added Mg or Ca. ATP-sensitivity per cent = (log II,=,--log nAn)jlog aAlpx roe. Tab& ZZ.-THE Mg-ACTNATEDATPA~E Acrmn
ACTOMYOSIN AT -RENT
A. B. C. D.
OF NATURAL STAGESOF PURIFICATION
PURIFICATION PROCEDURE
ATPA~E ACTMTY (nmoles Pi per mg protein per minute
I x 20,ooo g per 30 minutes
o-467
32
o-408
27’5
u-375
28
o-350
24
I 2 2 3 3 I I 2
precipitation x 20,000 g per 30 minutes, precipitations x 20,ooo g per 30 minutes, precipitations x 20,000 g per 30 minutes x 100,000 g per 20 minutes, precipitations
EGTASENsITNITY (per cent)
ATPase activity was measured in 2 mM ATP-Tris, 2 mM MgCl,, 25 mM Tris-HCI pH 7’2, in the presence and absence of I mM EGTA. muscle, since a comparison of the ATPsensitivity and effect of Mgs+ ions on the ATPase activity of the different preparations would allow a decision to be made on the nature of the extracted protein (SzentGyiirgyi, rggx ; Weber and Portzehl, 1952).
gives a protein with the lowest ATP-sensitivity whilst a low degree of Mg8+-activated ATPase persists. A lo-minute extraction with Weber-Edsall solution yields a protein which has a lower ATP-sensitivity and a lower Mg*+-activated ATPase activity than
1971, 2
ACTOMYOSIN
ATPaW
AND
the protein obtained by a 2q-hour extraction with the same medium. The results strongly indicate that the system extracted was actomyosin, some preparations (myosin extraction procedures) being actin-poor and others being actin-rich and generally resembling mammalian actomyosins. It is of interest that the ATP-sensitivity of the 2q-hour extract with Weber-Edsall solution is similar to the value reported by de Villafranca and Wochgraf (1962) for R. @@ens. The ATPsensitivity of the IO-minute extracts reported here is, however, considerably less than that obtained by the above workers. Contamination of the NAM preparations with particulate material such as mitochondrial or sarcoplasmic reticulum fragments was investigated by centrifuging the
327
SUPERPRECIPITATION
activity activated equally by Mg* + or Ca* + at fiH 7.3 and u=o*og, and unaffected by EGTA; the specific activity was of the same order as that of the EGTA-insensitive ATPase of frog sarcoplasmic reticulum (Duggan, I 968). The succinic dehydrogenase activity of the sediment, measured calorimetrically with tetrazolium at 4go rnl.t and 25’ C., was 0.175 A per mg. protein per hour or 16 per
00
IO-'
IO-'
[MgChlor PChl 0”)
Fro I.-The activation of the ATPase activity of NAM by Mg*+ (0) and Ca*+ (A) in the presence of equimolar ATP, at ionic strength 0.05 and PH 7.5. NAM dissolved in 0.6 M KC1 at ~oo,ooo g. The sediment was discarded and the protein Table II shows that precipitated by dilution. a 25 per cent decrease in the Mg*+-activated ATPase activity occurs during ultra-centrifugation. The inhibition of this enzyme activity by EGTA is low compared with that of sarcoplasmic reticulum Mg* +-dependent ATPase (Duggan, x968) whilst it remains essentially constant throughout the different Two cycles of precipicentrifugal procedures. tation and dilution, and centrifugation at 20,000 g were as effective in clarifying the extracted actomyosin as 3 cycles. It was found also that the sediment obtained by centrifugation at 20,000 g of once-centrifuged and once-precipitated NAM had an ATPase
._
It-l-l
FIG. I.-Effect of varying the MgCl, and CaCl, concentrations on the ATPase activity of NAM at ionic strengths ~05 and o-6. Assays were conducted in 25 mM Tris-HCl pH 7-2. 0, Mg*+ (~=0*05); 0, Mg*+ (p=o-6); ATP concentration was 1-5 mM. A, Ca*+ (p=o-05); 0, Ca*+, ~=0*6; ATP concentration was 5 mM.
cent of that of a pure mitochondrial fraction. Results obtained by Duggan (x964) with frog mitochondrial ATPase showed that Mg* + was at least 3 times more effective than Ca2+ as an activator, indicating that the NAM obtained here was more significantly contaminated by sarcoplasmic reticulum than mitochondrial fragments. It therefore seems reasonable to conclude that frog NAM ATPase is activated by Mg*+ at low ionic strength like actomyosins from other species. COMPARISON
OF THE
ACTIVATING
EFFECTS
OF
M~*+ANDC~*+ONNAMATPASE
At low ionic strength (approximately 0.05) both Mg* + and Ca*+ are potent activators (Fig. I) : when Mg*+ or Ca*+ and
HEFFRON
328
AND DUCCAN
ATP are added in equimolar amounts, optima1 activity with the former cation occurs at 1-2 mM, and 4-8 mM with the latter. Considerable inhibition occurs with Mg2 + and ATP concentrations greater than 3 mM, activity being reduced by 2g per cent at 3 mM and 7g per cent at 15 mM. When Mg2 + is varied from I-I o mM, in the presence of a constant concentration of ATP (1.5 mM) optimal activity occurs at 1-2.5 mM (Fig. 2) ; when Ca2 + is varied with the 0.5 -
Y s
Int. J. Biochem.
ratio was found to be in the range I : 0.4-2, no substrate inhibition being observed. Gas f, when added to the assay medium in the concentration range 0.25-2.5 mM, has no significant effect on Mgs+-activated ATPase activity; however, a slight activation occurs when the ATP level is increased to 4.5 mM with Mgz+ constant at 1.5 mM (Fig. 3B). It is clear that Ca2+ does not abolish the substrate inhibition caused by an excess of ATP over Mgs+. The fiH optimum with
A
0.4 -
,i k C ?: 0.3 e, ri ; d 0.2 3 4 I.1
1
o0
1
4
6
8
IO
FIG. 3.-A, Effect of increasing ATP concentrations on the ATPase activity of NAM in the presence A, 1.5 mM MgCl,; 0, 6 mM MgCI,; 0, 5 mM CaCl,. of constant levels of MgCl, and CaCl,. 6, Effect of added CaCl, on the Mg-activated ATPase activity of NAM. 0, 4’5 mM ATP only; a, 4’5 mM ATPf1.5 mM MgCI,; A, t-5 mM ATPfr.5 mM MgCl,. All assays were performed in 25 mM Tris-HCI pH 7.3, ATP level constant at 5 mM, optimal activity was found at 5-10 mM (Fig. I). Furthermore, the optimal level of Ca2+ is the same at high and low ionic strengths whereas at high increasing the Mg2+ concentration ionic strength causes an inhibition of the basal ATPase activity. Varying the ATP concentration from I ‘5 to g-o mM with constant Mg2+ levels reveals that the optimal ATP: Mg ratio is I : I (Fig. 3A); inhibition of enzyme activity is very pronounced when this ratio is exceeded. The optimal ATP : Ca
Mg2+ as activator at low ionic strength is in the range 6.5-7.6 with a peak at 6.8. At high ionic strength Mg2+ is inhibitory at all pH values examined (Fig. 4). Optimal activity with Ca2 + is at about pH g* I at both high and low ionic strengths; a smaller activity peak is seen also at pH 6.5 (Fig. 4) at low ionic strength. The enzyme activity in the absence of added divalent cations at u=o*o5 is also maximal at pH 9.1, indicating that endogenous Ca* + is responsible for the basal level of activity.
‘971,fa
ACTOMYOSIN
AND SUFERPRECIP~ATION
ATPaX!
329
Fig. 5 demonstrates that Mge+activation of the frog enzyme has been reduced to zero at 200 mM KC1 whilst the Ca2 +-activated activity has been only slightly
system.
EFFECT OF KC1 ON ATPASE ACTIVITY
The inhibition of the Mgs+-activated ATPase activity of NAM by monovalent cations is a characteristic of the actomyosin
0.1 -
6.0
74
9.0
8.0
10.0
Fxo. 4,--The@activity curves of the (A) MgP+-activated and (B) Caf+-activated ATPase activities of NAM at ionic strengths ~05 and 0.6. Assays were carried out with I.5 mM AT&T% + I ‘5 mM MgCI, in A and 5 mM ATP-Tris+5 mM CaCl, in 6. Buffers were imidaaoie-HCI Below pH ;*I, Tris-HCI between 7.2 and g-r, and Tris-acetate at pH 9’1 and 10.2, at a concentration of 50 mh4. 0, n=o*o5; A, n~~o.6; 0, l,t=o*o5; A, u=o*6; 0, l1=o+o5 in the absence of added divaltnt cations.
0
: 0
I
200
I
4oil
1
600
WI WI) FIO. 5.-Effect of increasing KC1 concentration on the. Mg*+ and Ca’+- activated ATPase activities of NAM. Assays were carried out in 25 mM Tris-HCI pH 7.3 with x-5 mM ATP-Tris + 1.5 mM MgCl, (a).; 5mM ATF-Tris+5 mM C&Cl, (0) ; and with 5 mM AT%‘-Tris only ( A).
depressed. Fifty mkf KC1 inhibited the Mgs+-activated enzyme by 16 per cent but had no effect on the Cast-activated enzyme. The latter retained 30 per cent of its activity at l.t=o.o5 in the presence of 600 mM KCl, whilst Mg2+ was inhibitory above 200 mM (Fig. 5). Na+ was found to have a similar but less ~nhjbito~ effect than K+ on the Mgs+and Car+-activated ATPase activities. The complete inhibition of the Mgs+-activated ATPase activity at 9=0*6 is consistent with the explanation offered for this effect in the NAM of other species, namely, that at high ionic strength ATP causes dissociation of the actomyosin complex into its constituent proteins and the enzymic characteristics change to those of myosin. ATP-SENSITIVITY AND SUPERPRECIPITATIONOF
Ac~~osx~ Frog actomyosin, dissolved in ~6 A4 KCl, had a high viscosity as indicated by the range
HEFFRONANDDUGGAN
330
of the viscosity number from 0.27 to o-4’. ATP-sensitivity was variable and lower than that of rabbit NAM (Table. ZZZ). ATP on its own was as effective in causing the viscosity drop as in the presence of Mg*+ or Gas+. The viscosity returned almost to the initial value 2 hours after ATP addition. MgCl, and inorganic pyrophosphate, but not pyrophosphate on its own, also elicited the viscosity drop but in an irreversible manner. The reduction in viscosity caused by ATP at u=o+6 (Weber and Portzehl, 1952) and the phenomenon of superprecipitation (SzentGyorgyi, 1951) are characteristics of the Table
III-THE
Znt. J. Biochem.
However, there is a marked fall-off in the turbidity response produced by a third aliquot ofo-25 mM ATP and with a fourth aliquot no further super-precipitation occurs (Fig. 6B). Fig. 6B also shows that 4 aliquots of ATP added to give concentrations in the medium of 0.25 mM at ~-minute intervals produce a greater turbidity change than I aliquot of I mM ATP. Frog actomyosin, therefore, is much less sensitive to ATP than rabbit actomyosin which has been found to superprecipitate at u=o~o5-o~15 w.ith ATP levels as low as 5 x 10-~.44 (Levy and Fleisher, 1965). One mM EGTA was found to inhibit
ATP-~ENSITX~~Y OF FROGI-+TURALAcro~uosm
ADDXWON
0.5 mM ATP 0.5 mM ATP, and 3 mA4 MgCl, or CaCl I mM Na,P,O, I mM Na,P,O, and 3 mM MgCI, Control z-value
a* ‘45 0.150 w27.5 o-150 o.275
After 2 Hours
Initial
After 2 Hours
0’255
90
8
0’240
83
I.5
o-270 0’145 0.275
0
83
0
90
is defined as log (n,,,)/C; C=mg. NAM per ml. (Weber and Portzehl, 1952). The concentration of NAM was 2.4 mg. per ml.
actomyosin system and are used here as criteria for the presence of actomyosin in high ionic strength extracts of frog. Frog actomyosin was found to superprecipitate at p=o*o8 if relatively high concentrations of ATP were used. ATP, in in the range 5 x IO-~--I x IO-=M in the presence of Mg*+, elicited optimal superprecipitation at a concentration of 5 x 1om4M as measured by the increase in optical density at 41 o mn (Fig. 6). ATP alone and in the presence of Ca*+ caused a much smaller degree of synaeresis, this being about 25 per cent of the response to ATP and Mg*+. The dependence of the extent of superprecipitation of actomyosin on the initial ATP concentration is shown in Fig. 6A. Superprecipitation is complete in 60-90 seconds after ATP addition in each case. A second aliquot of ATP at this stage elicits a further response.
the Mg* +-ATP-induced superprecipitation by 37 per cent with an average of 31 per cent for 4 different preparations. EFFECTS OF EGTA, PYRIDOXAL PHOSPHATE, ANDOLIGOMYCINONACTOMYOSIN ATPASE The profound inhibitory effects of EDTA and EGTA on both myofibrillar and NAM Mg* +-activated ATPase activities of rabbit skeletal muscle reported by Perry and Grey ( 1g56a, b) is due to the removal of small amounts of Ca*+ present in the actomyosin preparations and necessary for expression of maximal ATPase activity. Fig. 7 demonstrates the effect of EGTA on frog myofibrillar and actomyosin Mg* +-activated ATPases. Actomyosin is inhibited to a lesser degree than myofibrillar ATPase by EGTA concentrations in the range 0.1-10 mM. The initial sharp decrease in activity by O-I mM EGTA
‘971,
2
ACTOMYOSIN
ATPa%
is due to the removal of trace amounts of con-
taminating Ca2 +, but even with IO m&f EGTA added the inhibition does not exceed 45 per cent. The effect of EGTA on the Ca2 +activated ATPase of myofibriis and actomyosin is also shown in Fig. 7. The pattern of inhibition indicates that it is due to the simple 0.5
33’
AND SL~PERPRECIPITATION
phosphate was a potent inhibitor of both rabbit NAM and myofibriilar Mg2+-activated ATPases at concentrations of 1-2 mM. The Mgs+-activated ATPase activities of frog NAM and myofibri~s are equally sensitive, being inhibited by 49 per cent and 6n per cent respectively by 2 mM pyridoxal phosphate I *o
4
0.4
0‘8
2 0.3
04
“E3
0.4
0.2
t 0 0
Time
(wxonds)
1
2
3
fimt
(minutes)
4
5
The dependence of the extent of supe~recipita~on of NAM on ATP concentration. I, 0.50 mM ATP; A, 0.25 M ATP; 0, o-10 mM ATP. 6, Comparison of the effects of adding 4 aliquots of ATP (each) (0.25 mM final concentration) at r-minute intervals, and I aliquot (I mM final concentration) at zero time, on the extent of superprecipitation. 0, o-25 mM ATP; A, I mM ATP. ATP was added at times indicated by arrows. FIG.
6.-A,
chelation of the activator, Since EGTA has a great affinity for Ca*+ ($JK~ I I *o) and a low affinity for Mgz+ (p&, 5.2) it is reasonable to conclude that the partial inhibition of the Mg2+-activated ATPase of frog actomyosin (and myofibrils) is due to removal of small amounts of Ca2+ rather than Mge+. EGTA appears to be equally effective in inhibiting su~rprecipjtation and Mgz+-activated ATPKaldor ase activity of frog actomyosin. and Gergely (1959) found that pyridoxal
(Fig* 8). At this concentration the Mgg+activated ATPase of the microsomal fraction from frog muscle is inhibited by only 19 per cent-it is less sensitive to pyridoxal phosphate than the contractile proteins. Oligomycin, an inhibitor of transport ATPase (Jobsis and Vreman, 1963) and mitochondrial ATPase (Schatz and Saltzgraber, rg6g), had negligible inhibitory effect on the Mge*-activated ATPase of NAM {5 per cent inhibition), whilst the myofibrillar enzyme
HEFFRONAND DUGGAN
332
int. J. Bio&?n.
(Fig. 8) at 125 pg. oligomycin per mg. protein. In this respect frog actomyosin ATPase resembles the microsomal Mg2 +-activated ATPase described by Duggan ( 1965).
#I’sof
COMPARWN OF IMPORTANTCHARACTERISTICS OF FROG AND RABBIT ACTOMYOSIN
goes twice the degree of superprecipitation
was inhibited by 22 per cent
frog and rabbit NAM are compared in Table IV. The ATP-sensitivity of frog NAM
is considerably less than that of rabbit under the same conditions. Superprecipitation of NAM from both species takes place under similar conditions but rabbit NAM underof
ATP-sensitivity, superprecipitation, ATPase activity, EGTA-sensitivity, and optimal
I
z
3
4
5
f
I
A t
0
”
0
IO
I
0
[EGTA] (mM)
Fro. 7.-Effect of EGTA on the Mg*+- and Ca’+-activated ATPase activities of NAM and myofibrils at ionic strength 0.05 and pH 7.3. Substrate concentrations were 2 mM ATP-Tris+ and 5 mM ATP-Trisf-5 mM 2 mM MgCl, CaC1,. Mg”+-ATPase: 0, NAM; a, myofibrils. CaP+-ATPase: 0, NAM; A, myofibrils.
3
2 mM Pyridoxrl 62.5
ATP-sensitivity (per cent) Superprecipitation (PH 7.3,25” C., u=ovo8) ATPase activity (#H 7.2,30” C., n=o+o5) 2 mM ATP, MgCls 2 mM ATP, CaCl,
~75
Ce%TPase Effect of EGTA (I mM) on Mg-ATPase activity
FROG
RABBIT
84 (6)
x72 (2) 1.8 A Per mg.
A per mg.
o-471 (12) 0.385 (3)
CPtMm;;{Ee(p=o.05)
6.8-7.4 9o-9’5 Inhibition Per cent (I I)
22
125
r&s. Oligompin Fro 8.-Effects of pyridoxal phosphate and oligomycin on the Mg “+-activated ATPase activity of NAM and myofibrils. Assay conditions as in Fig., 5. Pyridoxal phosphate: 0, NAM; A, myofibrils. Oligomycin: a, NAM; A., myofibrils.
Table IV.-COMP~RLWN OF FROGANDRABBIT NATURAL ACTOMYOSIN PROPERTY
4
phosphate
Inhibition 78 Per cent (3)
Figures in parentheses denote number of preparations tested. * Szent-Gyiirgyi (x960),
‘971, 2
ACTOMYOSIN
ATPaSe
AND SUPERPRECIPITATION
frog NAM as indicated by an absorbancy increase on ATP addition of I *8 A per mg. protein compared with 0.75 A per mg. protein. Levy and Fleisher (1965) have obtained absorbancy changes as great as qA per mg. protein for rabbit NAM with an operating concentration of ATP as low as 6.6 x IO-~ M at ~=o*I. Initial ATP concentrations as low as this did not induce superprecipitation in suspensions of frog NAM. The Mgs+- and Ca2+-activated ATPase activities of frog NAM are about one-half and two-thirds respectively of the corresponding rabbit actomyosin ATPase activities. On the other hand, optimal PH values with both activators are similar. Optimal Mg2 +-activated ATPase activity of frog NAM occurs near neutral PH similar to the value given by Szent-Gyorgyi (1960) for rabbit NAM. The low EGTAsensitivity of frog Mg+2-activated ATPase compared with that of rabbit is surprising as both actomyosins were prepared by the same method. EGTA-sensitivity values of 60-85 per cent with 0.1 mM EGTA for rabbit NAM have been reported by several workers while the average EGTA-sensitivity of frog NAM is 22 per cent with I mM EGTA. This low EGTA-sensitivity of frog NAM may be explained by (I) insufficient Ca2+ present in the assay medium for maximal Mg2+activation of the ATPase or (2) possible loss of the EGTA-sensitizing factor (ESF) [described by Ebashi (1g63)] during the course of the preparative procedure resulting in a form of partially desensitized actomyosin (DAM) (see Schaub and others, 1967). Added Ca2+, in the range o*o~-o* I mM, increased the EGTA-sensitivity from 25 per cent to 32 per cent with a corresponding increase in the specific activity of the ATPase of frog actomyosin. Higher levels of Ca2+, in the presence of Mg2+, did not enhance the ATPase activity further (Fig. 38). Lack of Ca2+ is therefore not responsible for the low EGTAsensitivity of frog actomyosin. The second suggestion will be reported in detail elsewhere (Heffron and Duggan, 1971). Briefly, rabbit EGTA-sensitizing factor, prepared according to Hartshorne and Mueller (rg67), did not increase the EGTA-sensitivity of frog NAM or frog NAM which had been extracted with
333
the low ionic strength solution (n=o*oo2) used by Schaub and others (1967). DISCUSSION Maruyama (1966) and de Villafranca and Campbell (1969) have found, upon reexamination of insect actomyosin ATPase and Limulus actomyosin ATPase, that Mg2+ behaves as an activator at KC1 concentrations below 30 mM and 60 mM respectively. In their previous investigations Mg2+-activation was occluded by the high levels of KC1 added to the assay medium with the enzyme solution. Frog actomyosin, as prepared here from R. temporaria leg muscle, was not quite as sensitive as insect actomyosin to KCl, but was more sensitive than rabbit actomyosin. Mgz+activation of rabbit actomyosin takes place in up to 200 mM KC1 (Banga and SzentGyorgyi, I 943 ; Mommaerts and Seraydarian, I 947)) whilst frog actomyosin, like insect and horseshoe crab actomyosins, is completely inhibited at this level of KCl. Contamination of the insect actomyosin by granular Mg2+activated ATPase was ruled out by Maruyama (1g66), who found that sodium azide, even at I o mM, had no effect on the Mg2 +-activation. de Villafranca and Campbell ( 1g6g), detected paramyosin but no submicroscopic particles upon analytical ultracentrifugation of Limulus actomyosin, also indicating that Mg2+-activation was an intrinsic property of the actomyosin. In this study reliance was placed on ultracentrifugation at IOO,OOO g at u=o.6 to remove mitochondrial and sarcoplasmic reticulum (SR) fragments. This procedure did not remove the Mg2 +activated ATPase activity, demonstrating that frog actomyosin ATPase, although contaminated to a small degree with other Mg*+enhanced ATPase(s), was activated by Mgs+ at low p. Nass (1962) used 2 cycles of clarification at 20,000 g and precipitation by dilution to obtain actomyosin from R. pipiens, and found that the preparation was Mg2+activated at low n only. Here it was found that this procedure removed most of the contaminating particulate material; 3 cycles of purification resulted in a slight decrease in the specific activity of the Mg2+-activated ATPase but the EGTA-semi tivity remained
334
HEFFRON
AND DUGCAN
constant at 28 per cent. The PH activity curves further indicate that one enzyme is responsible for the Mg2+-activated ATPase activity (cf. Duggan, 1965). The lack of effect of oligomycin levels up to 125 ylg. per mg. protein demonstrates that mitochondrial fragments are not important contaminants (Duggan, 1965). The Mg*+-activated ATPase of frog sartorius sarcoplasmic reticulum is strongly activated by K+ and inhibited by low levels of EGTA (Duggan, rg67a, b). It is therefore reasonable to conclude that this enzyme system is not responsible for the overall Mg*+activation of frog actomyosin. In view of the inhibitory effect of KC on frog actomyosin Mgs +-ATPase, contamination of the actomyosin with SR fragments would tend to reduce this inhibitionowing to the activation of the SR Mgs +-activated ATPase by K + which is optimal at about IOO m&f. The low EGTAsensitivity of the ATPase suggests that SR is a minor contaminant, if present at all. The basal M$ f- or Cast-enhanced ATPase also present in frog SR (Duggan, 1968) is not a contaminant of actomyosin, as shown by the inhibition of this ATPase above 2 mM Gas+ and the lack of inhibition by Mga+ up to 12 m&L The basal ATPase is also rather insensitive to KC1 being 40 per cent inhibited by 800 rnfi1 KCl, whilst actomyosin Mg2+ATPase does not express itself above 200 mill KCl. Although de Villafranca and Hochgraf (1962) used the ATP-sensitivity test as the criterion for the presence of the actomyosin complex in their extracts, they point out that myosin in various st*ates of polymerization may have been present in some of their preparations (cf. Noda and Ebashi, 1960). Interaction of myosin with itself may take place reversibly at ionic strengths less than 0.35 and irreversibly at high ionic strength in the physiological PH range (see Perry, 1967). The latter is probably accompanied by changes in the tertiary structure of the myosin and loss of biological activity. The former type of aggregation is more likely to have occurred here where the actomyosin was stored in 50 rnitl KC1 at PH 7-4, whilst the ’ myosin ’ prepared by the Weber dissociation
ht. J. ~~~C~f~*
method and stored at p=o*6, pH 7.0, should exhibit irreversible aggregation. However, the latter still showed some Mg2+- activated ATPase activity, indicating that high ionic strength storage is not detrimental to this enzymic function. Such forms of aggregation of myosin are probably common to all proteins and result from non-specific molecular properties of the myosin. However, complex formation between actin and myosin and its modification by ATP is due to a highly specific protein-protein interaction involving centres specialized for this function on both molecules. In view of the rather specific conditions under which Noda and Ebashi ( I 960) obtained dissociation of myosin aggregates by ATP it is reasonable to conclude that the viscosity drop upon ATP addition, taken together with a positive superprecipitation response, is a sufficient criterion for the presence of actomyosin. It now appears certain that synaeresis of actomyosin suspensions requires ATP hydrolysis and the presence of Mg2+ and Gas+ ions in the medium (see Perry, 1967). Frog actomyosin exhibits a relatively strong superprecipitation with almost complete dependence on Mg*+ ions; as with the Mg*+-activated ATPase activity, trace amounts of Ca2+ do not control the degree of superprecipitation to the same extent that Gas + controls superprecipitation of rabbit actomyosin. Limulus actomyosin superprecipitates upon ATP addition in the presence of Mgs+-, whereas Ca*+ exerts no control over Limuhs actomyosin synaeresis (de Villafranca and Campbell, 1969). These workers have therefore suggested that the lack of sensitivity to Gas+ is most likely due to the absence of the modifying proteins (EGTAsensitizing factor) from their preparations. Maruyama and Allen (1967) demonstrated & requirement for minor amounts of Mgz f, but not of Ca2+, for the onset of superprecipitation of insect actomyosin even though the Mgs+-activated ATPase activity showed a similar dependence on trace amounts of CaZ + to rabbit actomyosin. The EGTA-sensitivity of the MgZfactivated ATPase and superprecipitation of frog actomyosin therefore occupies an intermediate position between the high sensitivity
197’9 2
ACTOMYOSINATPaSe AND SUPERPRECIPITATION
of rabbit actomyosin and the insensitivity of insect actomyosin. This agrees with the evolutionary position of the frog relative to rabbit and insect, in that the calcium binding of EGTA mimics the same property of the troponin-tropomyosin system, which complex has so far not been shown to occur in lower organisms. ACKNOWLEDGEMENTS This work was supported in part by grants from the Muscular Dystrophy Associations of America and the Irish Cancer Society.
REFERENCES ‘ Myosin and adenosine RAIL?=, K. (rgq), triphosphatase ‘, Biochem.J., 36, 12 1-139. BANGA,I., and SZENT-GYGRGYI, A. (1843)~ ‘ The influence of salts on the phosphatase action of myosin ‘, Stud. Inst. med. Gem. Univ. Szeged., 3, 72-75.
CONWAY,E. J. (rg57),, ‘ Nature and significance of concentration relattons of potassium and sodium ions in skeletal muscle ‘, Physiol. Rev., 37, 84I 32. CONWAY, E. J. (x963), Micr~~~s~on Ano&& and Volumetric Error, 3rd ed., p. 137. New York: Chemical Publishers. DIJGGAN,P. F. (19641, ‘ Adenosine triphosphatase activity in fractions separated from frog sartorius homogenates ‘, Ir. J. med. Sci., ser. 3, September, 393-402. DUGGAN,P. F. (rg65), ‘ Some properties of the monovalent-cation-stimulated adenosinetriphosphatase of fro8 sartorius microsomes ‘, Biochim. biophys. Acta, 9g, 14._4-155.
DUGGAN, P. F. (tg67a), ‘ Potassium-activated adenosinetriphosphatase and calcium uptake by sarcoplasmic reticulum ‘, L@ Sci., 6, 561567.
DUCGAN, P. F. (rg67b), ‘ The monovalentcation-stimulated calcium numb in fro2 skeletal muscle ‘, Life Sci., 7,913-&g. 1 ” DUGGAN, P. F. ( rg68), ‘ ~lcium-inde~ndent adenosine~ph~pha~e activity in fro2 muscle microsomes r, L$? Sci., 7, tz65~1271. ” EBASHI, S. (rg63), ‘ Third component participating in superprecipitation of “natural actomyosin” ‘, Jvatut-e, Lond., zoo, 1010. HAMOIR, G., and REVTER, A. ( rg56), ‘ Isolement et proprittts de la myosine de grenouille ‘, Biochim. biophys. Acta, PI, 24-34.
HARTSHORNE,D. J., and MUELLER, H. (1967)~ ‘ Separation and recombination of the ethvlene glycol bis ( 8-aminoethyl ether)-& N’-&t&l acetic acid-sensitizing factor obtained from a low ionic strength extract of natural actomyosin ‘, 3. biol. C&em., 241, 308g-3092.
335
HEFFRON,J. J. A. (1g6g), ’ The AT&se activity of the particulate fractions of fro2 skeletal muscle ‘. Ph.D. Thesis, National University of Ireland; Dublin. HEFFRON, J. J. A., and DUGGAN,P. F. (x967), ‘ The adenosine triphosphatase activity of skeletal muscle cell membranes ‘, Biochem. J,, 103,46P.
HEFFRON,J. J. A., and DUGGAN,P. F. ( 197 I), in preparation. JOBSIS, F. F., and VREMAN,H. J. (Ig63), ‘ Inhibition of a Na+- and K+-stimulated adenosine triphosphathe by oligomycin ‘, Biochim. biophys. Act&, 73* 346-348.
KALDOR, G., and GERGELY, J. (rg5g), ‘ Effect of pyridoxal phosphate and its inhibition by camosine on the ATPase activitv and svneresis of myofibrils ‘, A&s Biochem. ‘Biuphys.: 80, 3g3399. LAYi’%
‘ Speetrophotometric and E. (19571, turbidimetric methods for measuring proteins’, in Methods in Enzymology (ed. COLOWICK, S. P., and KAPLAN, N. O.), vol. 3, pp. 447-454. New York: Academic Press. LEVY, H. M., and FLEISHER, M. (tg65), ‘ Studies on the superprecipitation of actomyosin suspensions by the change in turbidity. I. Effects of adenosine triphosphate concentration and temperature ‘, Biochim. biophy5. Acta, xoo, 479-490. MARUYAMA,K. (rg66), ‘ Effect of magnesium on the aden~inet~phosphat~e activity of insect actomyosin at low ionic strength ‘, Co@. Biothem. Physiol., x8, 48x-487.
MARVYAMA,K., and ALLEN, S. R. (1~67)~ ‘ Effects of magnesium and calcium ions on the adenosinetriphosphatase activity of insect actomyosin at low ionic strength ‘, Camp. Biochem. Physiol., OX, 713-718. MOMMAERTS, W. F. H. M. (x958), Methods in Medical Research (ed. WARREN,J. V.), vol. 7, pp. I68. Chicano: Yearbook Publishers.
MOMMAERTS,~W.F. H. M., and SERAYDARIAN,K.
(1g47), ‘ A study of the adenosine triphosphatase activity of myosin and actomvosin ‘,,” 7. -pen.
Phpsiol. ~~401-422.
NASS,M. M. K. (rg6z), ‘ Developmental changes in fro8 actomyosin characteristics ‘, .Devl Biol., 412~320. NOVA, I-I.: and EBASHI, S. ( ES@), ‘ Aggregation of myosin A’, B&him. biophys. Acta, 4x,386-392. PERRY, S. V. (1967), ‘ The structure and interactions of myosin ‘, Prog. Biopl~ys. mol. Biol., x7, 327-381.
PERRY, S. V., and GREY, T. C. (rg56a), ‘ A study of the effects of substrate concentration and certain relaxing factors on the magnesiumactivated myofibrillar adenosine triphosphatase ‘, Biochem.
J., &
184-I gz.
PERRY, S. V., and GREY, T. C. (tg56b), ‘ Ethylenediaminetetraacetate and the adenosinctriphosphatase . _- activity - of actomyosin systems ‘, Bio&Em. .j., %, 5Y.
336
HEFFRON
SCHATZ, G., and SALTZGRABER, J. (rg6g),
AND DUGGAN
‘ Identification of denatured mitochondrial ATPase in “structural protein” from beef heart mitochondria ‘, B&him. biophys. Acta, 180, 186189. SCHAUB, M. C., HARTSHORNE, D. J., and PERRY, S. V. (x967), ‘ The adenosine-triphosphatase activity of desensitized actomyosin ‘, Biochem. j., xoe,263-269. SZENT-GYGRGYI,A. ( 1951), The Chemishy of Muscular Contraction, 2nd ed. New York: Academic Press. SZENT-GY~RGYI, A. G. ( I 960)) Muscle (ed. BOURNE, G. H.), vol. 2, pp. I s-16. New York: Academic Press, Inc. TAU~~KY,H. H., and SHORR,E. (x953), ‘ A microcalorimetric method for the determination of inorganic phosphorus ‘, 3. biol. Gem., rrop, 675-685. DE V~LLAF~ANCA, G. W. (x964), ‘ Frog myofibrillar adenosinetriphosphatase activity ‘, Comp. B&hem. Physiol., q, 87-95. DE VILLAFRANCA, G. W., and CAMPBELL,L. K. (1969)> ‘ Magnesium activation of natural
actomvosin ATPase from horseshoe crab ‘, Comp. biochem. Physiol., zg, 775-783. DE VILLAFRANCA.G. W.. and HOCHGRAF.H. L. (M2L ‘ Adenosinetriphosphatase activity of frog myosin B ‘, Camp. Biochem. Physiol., 6, 147-163. DE VILLAPRANCA,G. W., and NAUMANN,D. C. (I 964)) ‘ Some properties of the myosin B from ATPase from Limuhs ‘, Comp. Biochem. Physiol., 12, 143-156. WEBER, A. (x956), ‘ The ultracentrifugal separation of L-myosin and actin in an actomyosin sol under the influence of ATP ‘, Biochim. biophys. Acta, 1% 345-351. WEBER, H. H., and PORTZEHL, H. (tg52), ’ Muscle contraction and fibrous muscle proteins ‘, Ado. Prot. Gem., 7, 161-252. WILKIE,D. R. ( rg66), ‘ Muscle ‘, A. Rev. Physiol., 28, 17-38. Kg Word In&x: Frog, Rana temporaria, natural actomyosin, magnesium and calcium activation, rabbit, ATPand EGTA-sensitivity, superprecipitation, Oryctolagus cunicdus domesticus.
324
ADENOSINE
TRIPHOSPHATASE
SUPERPRECIPITATION FROG, J. J.
ACTIVITY
OF ACTOMYOSIN
RANA
A. HEFFRON*
(Publishers)
Ltd.]
AND
FROM
THE
TEMPORARIA and P. F. DUGGAN
Department of Biochemistry, University College, Dublin, Ireland (Received26 May, 1970) ABSTRACT Magnesium, as well as calcium, was found (contrary to some published reports) to activate natural actomyosin adenosine triphosphatase (ATPase) from Rana temwaria. The optimal level of Mg*+ was I mM at p=o.o5, pH 7-2. With increasing ATP concentration substrate inhibition occurred with Mg*+ but not with Ca*+. 2. Contamination with mitochondrial or sarcoplasmic reticulum fragments was not sufficient to account for the Mg*+-activation of actomyosin ATPase. 3. Mga+, but not Car+, was essential for super-precipitation. 4. Mgl+ -activated ATPase activity and superprecipitation showed a similar but slight dependence upon the free Ca-+ concentration. 5. A comparison was made with rabbit natural actomysin. I.
MUCH of the present knowledge of the physiology and energetics of muscular contraction and relaxation has been obtained on frog skeletal muscle (see Wilkie, 1966, for review). The enzymatic events which form the basis of the contraction-relaxation cycle have been characterized in considerable detail by biochemical studies on mammalian muscle, primarily rabbit muscle. The chemical properties of frog actomyosin are similar in many respects to the comparable system in other animals (de Villafranca and Hochgraf, 1962 ; de Villafranca, 1964; Heffron, 1969). Nass ( 1g62), in a developmental study of actomyosin ATPase in Rana tipiens skeletal muscle, remarked that the effects of magnesium ions on the ATPase activity of adult actomyosin were similar to the results reported for other species, i.e., Mg2+ and Ca2+ ions activated actomyosin ATPase at low ionic strength, whereas Ca2+ alone activated the enzyme at high ionic strength. de Villafranca and Hochgraf (1962) reported that
* Present address: Animal Sciences Division, The Agricultural Institute, Castleknock, Dublin, Irrland.
they were unable to find Mg2+ activation of frog (R. pipiens) actomyosin ATPase even at low ionic strength. Similar results were found with actomyosin from the horseshoe crab Limulus polyphemus (de Villafranca and Naumann, 1964). Bailey (1942)) in a study of myosin from various species (rabbit, chicken, pig, frog), observed that Mg2 +-activation of myosin ATPase was significant only in the case of frog myosin. In view of the difficulty in preparing myosin free from actin from frog skeletal muscle (Hamoir and Reuter, 1956 ; de Villafranca and Hochgraf, 1962)) and since actin was discovered in 1942 it seems probable that the Mg2 +-activation observed by Bailey was due to the presence of actin in the myosin preparations. In the course of a search for the Na +-K +ATPase in a sarcolemmal preparation from frog (R. temporaria) muscle (Heffron and Duggan, 1967) it became necessary to establish the effects of Mg2+ and Ca2+ on the frog actomyosin system, in particular on the ATPase activity. Furthermore, the effect of Mg2 + on the enzyme activity is of interest in view of the high levels of Mg2- in frog muscle (Conway, 1957) and the postulate of Schaub,
‘971, 29324-336
ACTOMYOSINATPaSe AND
Hartshorne, and Perry (1967) that the role of Mg2 + in contraction is as a complex with ATP, and that Ca2+ acts indirectly through the troponin-tropomyosin complex. The results presented here on R. temporaria actomyosin show, contrary to some published reports, that frog actomyosin is activated by Mg* + as well as Ca2 + and that ethylene glycol bis-( P-aminoethyl)&, N-tetra-acetic acid (EGTA), the Ca2 + chelating agent, has a weak inhibitory effect -on frog actomyosin ATPase and superprecipitation compared with rabbit actomyosin. MATERIALS
AND METHODS
FROGPROTEINS Adult Rana ten&raria were stored in plastic water tanks at x2:16’ C. and occasionally-forcefed with liver. The chilled. minced unner lea muscles were homogenized in’4 volumes $WeberI Edsall solution (0.6 M KCl, 0.04 M NaHCOs, o-01 M Na, CO,) containing I mM dithiothreitol and extracted for 24 hours at 2’ C. The extract was diluted with an equal volume of Weber-Edsall solution, filtered through terylene net, and centrifuged at 20~~x1g for 20 minutes at o0 C. The actomyosin was precipitated by diluting the supernatant with IO volumes of water (adjusted to pH 7.4 with Tris), and collected by centrifugation at 2ooog for ro minutes. The actomyosin was redissolved in an equal volume of t-2 M KCl, adjusted to pH 7.4 with Tris, and clarified at 20,ooog. It was precipitated and collected as before, washed once with jo mM KCI-IO mM Tris-HCl pH 7.4, and suspended in the same medium with a protein concentration of 2-5 mg. per ml. The actomyosin was stored in polythene tubes at 2’ C. Fresh suspensions of natural actomyosin (NAM) were prepared each week. In some experiments, indicated in the text, the actomyosin was purified by 3 cycles of clarification and precipitation, and centrifugation at 10,ooog for 20 minutes. Incorporation of dithiothreitol into the Weber-Edsall solution increased the specific activity of the Mg-activated ATPase of frog NAM by 20 per cent. Frog myofibrils were prepared from freshly excised leg muscle accordin to the method of Perry and Grev ( 1g56a) with minor modification, and stored in 25 mM KCl. 20 mMsodium borate bH 7. I at 2°C. “Myosin w&” prepared from n&CCd fm? k&l muscle by extraction for Io minutes with 4 volumes of Guba-Straub solution (0.4 M KCl, 0.05 M KH, PO,, o-10 M Ks HPO,) or Hanson-Huxley solution (0.6 M KCl, 0.01 M Na,PsO,, 0.10 M phosphate buffer, oao~ M MgCl,, pH 6.4) at 2’ C. The mvosin was nurified accordinn to the method described by Mommaerts (‘958) fey rabbit myosin.
SUPERPRECIPITATION
325
The final preparation was kept in 50 mM KC1 at 2’ C: These preparations-had significant Mes+-activated ATPase activitv and ATP-sensitiv%y and were therefore more like actomyosin than myosin. A protein resembling mammalian myosin more closely in enzymic and physical properties was prepared from frog actomyosin by the dissociation procedure of Weber (1956). Frog actomyosin, dissolved in 0.6 M KCl, IO mM hiitidine-HCl, IO mM ATP, IO mM MgSO,, pH 7.0 with a protein concentration of 3 mg. per ml., was centrifuged at ~~oo,ooo g for 2 hours at o” C. The upper half of the supernatant, which was clear, w& removed with a plastic syringe, dialvsed a2ainst a lame volume of IO mM KCl. and’the m&n collectid by centrifugation. After washing 3 times with 50 mM KCl, the myosin was dissolved in 0.6 M KCl/lo mM histidineHCl PH 7.0 (2 mg. per ml.) and stored at 2’ C. RABBITA~TOMYOSIN Rabbit actomyosin was prepared according to Mommaerts (1958) by extraction with WeberEdsall solution. It was clarified by centrifugation at 20,ooo g for 30 minutes, and precipitated by dilution 3 times. ASSAYS
ATPase activities were measured in a final volume of I ml. at 30” C. in the following media: (I) 1.5 mM ATP-Tris, 1.5 mM MgCIs, (2) 5 mM ATP-Tris, 5 mM CaCl,, buffered with 25 mM Tris-HCl pH 7’2-7’3. Enzyme activities were also measured in the presence of varying concentrations of ATP, MgCI,, CaCl,, and are indicated. The liberated inorganic phosphate was determined by the method of Taussky and Shorr (1853) using FeSO, as reducing agent. Protein was measured by the biuret method using crystalline bovine serum albumin as standard (Lavne, 1957). The biuret reaction was standard&d by the Kieldahl method described bv Conwav f I 06~). Superprecipitation of frog NAM was ‘c&t& mined by following the ahsorbancy increase at 410 mn in a medium containing 2.5 mM M&l,, 25 mM Tris-HCI PH 7.3, and 0.3-0’5 mg. NAM per ml. at about 25” C. with a r-cm. light path. The extent of superprecipitation is expressed as absorbancy units per mg. NAM (‘A’ per mg. protein). ATP-sensitivity (Weber and Portzehl, 1952) was measured wnh an Ostwald viscometer at 16” C. using 0.5 mM ATP, final concentration. The actomyosin was dissolved in 0.6 mM KCl, 25 mM Tris-HCl PH 7.2 with a concentration of 2-3 mg. NAM per ml. When present, MgCl, and C&&l, were 3 mM. ATP-sensitivity was also determined with I mM Na,P,O, and 3 mM MgCl, as dissociating agent. All solutions were ‘ AnalaR ’ grade (British Drug Houses Ltd.) in glass-double-distilled water. Disodium ATP, from Sigma, London, was
Znr. J, Bidan.
HFFFRON AND DUGGAN
326
converted to the Tris salt using the cation exchange Dithiothreitol was resin Amberlite IR-120.
obtained from Calbiochem, London, oligomycin from Mann, New York, pyridoxal phosphatefrom Sigma, and EGTA from K. & K. Labs., New York, RESULTS Attempts were made to prepare myosin as well as natural actomyosin from fr;g leg
It is shown, in Table I, that the standard myosin extraction media, the Guba-Straub and Hanson-Huxley solutions, yield a protein with significant ATP-sensitivity and a Mg2 factivated ATPase activity at low ionic strength indicating the presence of some actin with the myosin. NAM, when dissociated by the procedure of Weber ( 1956))
Table 1.-T= REI.ATIONSH~P BETWEEN THE COMPOSITION OF THE EXTRACTION MII~XIIN ANDTHE ATP-s~~snnrr~ ANDATP~E Acrrvrrv OFTHEPROTEJN EXTRA-D FROM FROG Muscz.a AT IONICSTRENOTH o-6
EXTRACTION MEDIUM
Guba-Straub (IO minutes)
Hanson-Huxley (IO minutes) Weber (1956) dissociation method for myosin Weber-Edsali (I) 10 minutes (2) 24 hours
ATPASE Acr~vrrv
ATP(pmoles Pi per mg. protein per minute) SENSITNITY Ca Mg 24 35
o.050 0’047
0’433 0’325
19
o.058
0’345
o=142 ~467
0.483 0.550
50 108
The extraction time is shown in parentheses. The ATPase activity in the absence of divalent cations was subtracted from that in their presence in order to show the effect of the added Mg or Ca. ATP-sensitivity per cent = (log II,=,--log nAn)jlog aAlpx roe. Tab& ZZ.-THE Mg-ACTNATEDATPA~E Acrmn
ACTOMYOSIN AT -RENT
A. B. C. D.
OF NATURAL STAGESOF PURIFICATION
PURIFICATION PROCEDURE
ATPA~E ACTMTY (nmoles Pi per mg protein per minute
I x 20,ooo g per 30 minutes
o-467
32
o-408
27’5
u-375
28
o-350
24
I 2 2 3 3 I I 2
precipitation x 20,000 g per 30 minutes, precipitations x 20,ooo g per 30 minutes, precipitations x 20,000 g per 30 minutes x 100,000 g per 20 minutes, precipitations
EGTASENsITNITY (per cent)
ATPase activity was measured in 2 mM ATP-Tris, 2 mM MgCl,, 25 mM Tris-HCI pH 7’2, in the presence and absence of I mM EGTA. muscle, since a comparison of the ATPsensitivity and effect of Mgs+ ions on the ATPase activity of the different preparations would allow a decision to be made on the nature of the extracted protein (SzentGyiirgyi, rggx ; Weber and Portzehl, 1952).
gives a protein with the lowest ATP-sensitivity whilst a low degree of Mg8+-activated ATPase persists. A lo-minute extraction with Weber-Edsall solution yields a protein which has a lower ATP-sensitivity and a lower Mg*+-activated ATPase activity than
1971, 2
ACTOMYOSIN
ATPaW
AND
the protein obtained by a 2q-hour extraction with the same medium. The results strongly indicate that the system extracted was actomyosin, some preparations (myosin extraction procedures) being actin-poor and others being actin-rich and generally resembling mammalian actomyosins. It is of interest that the ATP-sensitivity of the 2q-hour extract with Weber-Edsall solution is similar to the value reported by de Villafranca and Wochgraf (1962) for R. @@ens. The ATPsensitivity of the IO-minute extracts reported here is, however, considerably less than that obtained by the above workers. Contamination of the NAM preparations with particulate material such as mitochondrial or sarcoplasmic reticulum fragments was investigated by centrifuging the
327
SUPERPRECIPITATION
activity activated equally by Mg* + or Ca* + at fiH 7.3 and u=o*og, and unaffected by EGTA; the specific activity was of the same order as that of the EGTA-insensitive ATPase of frog sarcoplasmic reticulum (Duggan, I 968). The succinic dehydrogenase activity of the sediment, measured calorimetrically with tetrazolium at 4go rnl.t and 25’ C., was 0.175 A per mg. protein per hour or 16 per
00
IO-'
IO-'
[MgChlor PChl 0”)
Fro I.-The activation of the ATPase activity of NAM by Mg*+ (0) and Ca*+ (A) in the presence of equimolar ATP, at ionic strength 0.05 and PH 7.5. NAM dissolved in 0.6 M KC1 at ~oo,ooo g. The sediment was discarded and the protein Table II shows that precipitated by dilution. a 25 per cent decrease in the Mg*+-activated ATPase activity occurs during ultra-centrifugation. The inhibition of this enzyme activity by EGTA is low compared with that of sarcoplasmic reticulum Mg* +-dependent ATPase (Duggan, x968) whilst it remains essentially constant throughout the different Two cycles of precipicentrifugal procedures. tation and dilution, and centrifugation at 20,000 g were as effective in clarifying the extracted actomyosin as 3 cycles. It was found also that the sediment obtained by centrifugation at 20,000 g of once-centrifuged and once-precipitated NAM had an ATPase
._
It-l-l
FIG. I.-Effect of varying the MgCl, and CaCl, concentrations on the ATPase activity of NAM at ionic strengths ~05 and o-6. Assays were conducted in 25 mM Tris-HCl pH 7-2. 0, Mg*+ (~=0*05); 0, Mg*+ (p=o-6); ATP concentration was 1-5 mM. A, Ca*+ (p=o-05); 0, Ca*+, ~=0*6; ATP concentration was 5 mM.
cent of that of a pure mitochondrial fraction. Results obtained by Duggan (x964) with frog mitochondrial ATPase showed that Mg* + was at least 3 times more effective than Ca2+ as an activator, indicating that the NAM obtained here was more significantly contaminated by sarcoplasmic reticulum than mitochondrial fragments. It therefore seems reasonable to conclude that frog NAM ATPase is activated by Mg*+ at low ionic strength like actomyosins from other species. COMPARISON
OF THE
ACTIVATING
EFFECTS
OF
M~*+ANDC~*+ONNAMATPASE
At low ionic strength (approximately 0.05) both Mg* + and Ca*+ are potent activators (Fig. I) : when Mg*+ or Ca*+ and
HEFFRON
328
AND DUCCAN
ATP are added in equimolar amounts, optima1 activity with the former cation occurs at 1-2 mM, and 4-8 mM with the latter. Considerable inhibition occurs with Mg2 + and ATP concentrations greater than 3 mM, activity being reduced by 2g per cent at 3 mM and 7g per cent at 15 mM. When Mg2 + is varied from I-I o mM, in the presence of a constant concentration of ATP (1.5 mM) optimal activity occurs at 1-2.5 mM (Fig. 2) ; when Ca2 + is varied with the 0.5 -
Y s
Int. J. Biochem.
ratio was found to be in the range I : 0.4-2, no substrate inhibition being observed. Gas f, when added to the assay medium in the concentration range 0.25-2.5 mM, has no significant effect on Mgs+-activated ATPase activity; however, a slight activation occurs when the ATP level is increased to 4.5 mM with Mgz+ constant at 1.5 mM (Fig. 3B). It is clear that Ca2+ does not abolish the substrate inhibition caused by an excess of ATP over Mgs+. The fiH optimum with
A
0.4 -
,i k C ?: 0.3 e, ri ; d 0.2 3 4 I.1
1
o0
1
4
6
8
IO
FIG. 3.-A, Effect of increasing ATP concentrations on the ATPase activity of NAM in the presence A, 1.5 mM MgCl,; 0, 6 mM MgCI,; 0, 5 mM CaCl,. of constant levels of MgCl, and CaCl,. 6, Effect of added CaCl, on the Mg-activated ATPase activity of NAM. 0, 4’5 mM ATP only; a, 4’5 mM ATPf1.5 mM MgCI,; A, t-5 mM ATPfr.5 mM MgCl,. All assays were performed in 25 mM Tris-HCI pH 7.3, ATP level constant at 5 mM, optimal activity was found at 5-10 mM (Fig. I). Furthermore, the optimal level of Ca2+ is the same at high and low ionic strengths whereas at high increasing the Mg2+ concentration ionic strength causes an inhibition of the basal ATPase activity. Varying the ATP concentration from I ‘5 to g-o mM with constant Mg2+ levels reveals that the optimal ATP: Mg ratio is I : I (Fig. 3A); inhibition of enzyme activity is very pronounced when this ratio is exceeded. The optimal ATP : Ca
Mg2+ as activator at low ionic strength is in the range 6.5-7.6 with a peak at 6.8. At high ionic strength Mg2+ is inhibitory at all pH values examined (Fig. 4). Optimal activity with Ca2 + is at about pH g* I at both high and low ionic strengths; a smaller activity peak is seen also at pH 6.5 (Fig. 4) at low ionic strength. The enzyme activity in the absence of added divalent cations at u=o*o5 is also maximal at pH 9.1, indicating that endogenous Ca* + is responsible for the basal level of activity.
‘971,fa
ACTOMYOSIN
AND SUFERPRECIP~ATION
ATPaX!
329
Fig. 5 demonstrates that Mge+activation of the frog enzyme has been reduced to zero at 200 mM KC1 whilst the Ca2 +-activated activity has been only slightly
system.
EFFECT OF KC1 ON ATPASE ACTIVITY
The inhibition of the Mgs+-activated ATPase activity of NAM by monovalent cations is a characteristic of the actomyosin
0.1 -
6.0
74
9.0
8.0
10.0
Fxo. 4,--The@activity curves of the (A) MgP+-activated and (B) Caf+-activated ATPase activities of NAM at ionic strengths ~05 and 0.6. Assays were carried out with I.5 mM AT&T% + I ‘5 mM MgCI, in A and 5 mM ATP-Tris+5 mM CaCl, in 6. Buffers were imidaaoie-HCI Below pH ;*I, Tris-HCI between 7.2 and g-r, and Tris-acetate at pH 9’1 and 10.2, at a concentration of 50 mh4. 0, n=o*o5; A, n~~o.6; 0, l,t=o*o5; A, u=o*6; 0, l1=o+o5 in the absence of added divaltnt cations.
0
: 0
I
200
I
4oil
1
600
WI WI) FIO. 5.-Effect of increasing KC1 concentration on the. Mg*+ and Ca’+- activated ATPase activities of NAM. Assays were carried out in 25 mM Tris-HCI pH 7.3 with x-5 mM ATP-Tris + 1.5 mM MgCl, (a).; 5mM ATF-Tris+5 mM C&Cl, (0) ; and with 5 mM AT%‘-Tris only ( A).
depressed. Fifty mkf KC1 inhibited the Mgs+-activated enzyme by 16 per cent but had no effect on the Cast-activated enzyme. The latter retained 30 per cent of its activity at l.t=o.o5 in the presence of 600 mM KCl, whilst Mg2+ was inhibitory above 200 mM (Fig. 5). Na+ was found to have a similar but less ~nhjbito~ effect than K+ on the Mgs+and Car+-activated ATPase activities. The complete inhibition of the Mgs+-activated ATPase activity at 9=0*6 is consistent with the explanation offered for this effect in the NAM of other species, namely, that at high ionic strength ATP causes dissociation of the actomyosin complex into its constituent proteins and the enzymic characteristics change to those of myosin. ATP-SENSITIVITY AND SUPERPRECIPITATIONOF
Ac~~osx~ Frog actomyosin, dissolved in ~6 A4 KCl, had a high viscosity as indicated by the range
HEFFRONANDDUGGAN
330
of the viscosity number from 0.27 to o-4’. ATP-sensitivity was variable and lower than that of rabbit NAM (Table. ZZZ). ATP on its own was as effective in causing the viscosity drop as in the presence of Mg*+ or Gas+. The viscosity returned almost to the initial value 2 hours after ATP addition. MgCl, and inorganic pyrophosphate, but not pyrophosphate on its own, also elicited the viscosity drop but in an irreversible manner. The reduction in viscosity caused by ATP at u=o+6 (Weber and Portzehl, 1952) and the phenomenon of superprecipitation (SzentGyorgyi, 1951) are characteristics of the Table
III-THE
Znt. J. Biochem.
However, there is a marked fall-off in the turbidity response produced by a third aliquot ofo-25 mM ATP and with a fourth aliquot no further super-precipitation occurs (Fig. 6B). Fig. 6B also shows that 4 aliquots of ATP added to give concentrations in the medium of 0.25 mM at ~-minute intervals produce a greater turbidity change than I aliquot of I mM ATP. Frog actomyosin, therefore, is much less sensitive to ATP than rabbit actomyosin which has been found to superprecipitate at u=o~o5-o~15 w.ith ATP levels as low as 5 x 10-~.44 (Levy and Fleisher, 1965). One mM EGTA was found to inhibit
ATP-~ENSITX~~Y OF FROGI-+TURALAcro~uosm
ADDXWON
0.5 mM ATP 0.5 mM ATP, and 3 mA4 MgCl, or CaCl I mM Na,P,O, I mM Na,P,O, and 3 mM MgCI, Control z-value
a* ‘45 0.150 w27.5 o-150 o.275
After 2 Hours
Initial
After 2 Hours
0’255
90
8
0’240
83
I.5
o-270 0’145 0.275
0
83
0
90
is defined as log (n,,,)/C; C=mg. NAM per ml. (Weber and Portzehl, 1952). The concentration of NAM was 2.4 mg. per ml.
actomyosin system and are used here as criteria for the presence of actomyosin in high ionic strength extracts of frog. Frog actomyosin was found to superprecipitate at p=o*o8 if relatively high concentrations of ATP were used. ATP, in in the range 5 x IO-~--I x IO-=M in the presence of Mg*+, elicited optimal superprecipitation at a concentration of 5 x 1om4M as measured by the increase in optical density at 41 o mn (Fig. 6). ATP alone and in the presence of Ca*+ caused a much smaller degree of synaeresis, this being about 25 per cent of the response to ATP and Mg*+. The dependence of the extent of superprecipitation of actomyosin on the initial ATP concentration is shown in Fig. 6A. Superprecipitation is complete in 60-90 seconds after ATP addition in each case. A second aliquot of ATP at this stage elicits a further response.
the Mg* +-ATP-induced superprecipitation by 37 per cent with an average of 31 per cent for 4 different preparations. EFFECTS OF EGTA, PYRIDOXAL PHOSPHATE, ANDOLIGOMYCINONACTOMYOSIN ATPASE The profound inhibitory effects of EDTA and EGTA on both myofibrillar and NAM Mg* +-activated ATPase activities of rabbit skeletal muscle reported by Perry and Grey ( 1g56a, b) is due to the removal of small amounts of Ca*+ present in the actomyosin preparations and necessary for expression of maximal ATPase activity. Fig. 7 demonstrates the effect of EGTA on frog myofibrillar and actomyosin Mg* +-activated ATPases. Actomyosin is inhibited to a lesser degree than myofibrillar ATPase by EGTA concentrations in the range 0.1-10 mM. The initial sharp decrease in activity by O-I mM EGTA
‘971,
2
ACTOMYOSIN
ATPa%
is due to the removal of trace amounts of con-
taminating Ca2 +, but even with IO m&f EGTA added the inhibition does not exceed 45 per cent. The effect of EGTA on the Ca2 +activated ATPase of myofibriis and actomyosin is also shown in Fig. 7. The pattern of inhibition indicates that it is due to the simple 0.5
33’
AND SL~PERPRECIPITATION
phosphate was a potent inhibitor of both rabbit NAM and myofibriilar Mg2+-activated ATPases at concentrations of 1-2 mM. The Mgs+-activated ATPase activities of frog NAM and myofibri~s are equally sensitive, being inhibited by 49 per cent and 6n per cent respectively by 2 mM pyridoxal phosphate I *o
4
0.4
0‘8
2 0.3
04
“E3
0.4
0.2
t 0 0
Time
(wxonds)
1
2
3
fimt
(minutes)
4
5
The dependence of the extent of supe~recipita~on of NAM on ATP concentration. I, 0.50 mM ATP; A, 0.25 M ATP; 0, o-10 mM ATP. 6, Comparison of the effects of adding 4 aliquots of ATP (each) (0.25 mM final concentration) at r-minute intervals, and I aliquot (I mM final concentration) at zero time, on the extent of superprecipitation. 0, o-25 mM ATP; A, I mM ATP. ATP was added at times indicated by arrows. FIG.
6.-A,
chelation of the activator, Since EGTA has a great affinity for Ca*+ ($JK~ I I *o) and a low affinity for Mgz+ (p&, 5.2) it is reasonable to conclude that the partial inhibition of the Mg2+-activated ATPase of frog actomyosin (and myofibrils) is due to removal of small amounts of Ca2+ rather than Mge+. EGTA appears to be equally effective in inhibiting su~rprecipjtation and Mgz+-activated ATPKaldor ase activity of frog actomyosin. and Gergely (1959) found that pyridoxal
(Fig* 8). At this concentration the Mgg+activated ATPase of the microsomal fraction from frog muscle is inhibited by only 19 per cent-it is less sensitive to pyridoxal phosphate than the contractile proteins. Oligomycin, an inhibitor of transport ATPase (Jobsis and Vreman, 1963) and mitochondrial ATPase (Schatz and Saltzgraber, rg6g), had negligible inhibitory effect on the Mge*-activated ATPase of NAM {5 per cent inhibition), whilst the myofibrillar enzyme
HEFFRONAND DUGGAN
332
int. J. Bio&?n.
(Fig. 8) at 125 pg. oligomycin per mg. protein. In this respect frog actomyosin ATPase resembles the microsomal Mg2 +-activated ATPase described by Duggan ( 1965).
#I’sof
COMPARWN OF IMPORTANTCHARACTERISTICS OF FROG AND RABBIT ACTOMYOSIN
goes twice the degree of superprecipitation
was inhibited by 22 per cent
frog and rabbit NAM are compared in Table IV. The ATP-sensitivity of frog NAM
is considerably less than that of rabbit under the same conditions. Superprecipitation of NAM from both species takes place under similar conditions but rabbit NAM underof
ATP-sensitivity, superprecipitation, ATPase activity, EGTA-sensitivity, and optimal
I
z
3
4
5
f
I
A t
0
”
0
IO
I
0
[EGTA] (mM)
Fro. 7.-Effect of EGTA on the Mg*+- and Ca’+-activated ATPase activities of NAM and myofibrils at ionic strength 0.05 and pH 7.3. Substrate concentrations were 2 mM ATP-Tris+ and 5 mM ATP-Trisf-5 mM 2 mM MgCl, CaC1,. Mg”+-ATPase: 0, NAM; a, myofibrils. CaP+-ATPase: 0, NAM; A, myofibrils.
3
2 mM Pyridoxrl 62.5
ATP-sensitivity (per cent) Superprecipitation (PH 7.3,25” C., u=ovo8) ATPase activity (#H 7.2,30” C., n=o+o5) 2 mM ATP, MgCls 2 mM ATP, CaCl,
~75
Ce%TPase Effect of EGTA (I mM) on Mg-ATPase activity
FROG
RABBIT
84 (6)
x72 (2) 1.8 A Per mg.
A per mg.
o-471 (12) 0.385 (3)
CPtMm;;{Ee(p=o.05)
6.8-7.4 9o-9’5 Inhibition Per cent (I I)
22
125
r&s. Oligompin Fro 8.-Effects of pyridoxal phosphate and oligomycin on the Mg “+-activated ATPase activity of NAM and myofibrils. Assay conditions as in Fig., 5. Pyridoxal phosphate: 0, NAM; A, myofibrils. Oligomycin: a, NAM; A., myofibrils.
Table IV.-COMP~RLWN OF FROGANDRABBIT NATURAL ACTOMYOSIN PROPERTY
4
phosphate
Inhibition 78 Per cent (3)
Figures in parentheses denote number of preparations tested. * Szent-Gyiirgyi (x960),
‘971, 2
ACTOMYOSIN
ATPaSe
AND SUPERPRECIPITATION
frog NAM as indicated by an absorbancy increase on ATP addition of I *8 A per mg. protein compared with 0.75 A per mg. protein. Levy and Fleisher (1965) have obtained absorbancy changes as great as qA per mg. protein for rabbit NAM with an operating concentration of ATP as low as 6.6 x IO-~ M at ~=o*I. Initial ATP concentrations as low as this did not induce superprecipitation in suspensions of frog NAM. The Mgs+- and Ca2+-activated ATPase activities of frog NAM are about one-half and two-thirds respectively of the corresponding rabbit actomyosin ATPase activities. On the other hand, optimal PH values with both activators are similar. Optimal Mg2 +-activated ATPase activity of frog NAM occurs near neutral PH similar to the value given by Szent-Gyorgyi (1960) for rabbit NAM. The low EGTAsensitivity of frog Mg+2-activated ATPase compared with that of rabbit is surprising as both actomyosins were prepared by the same method. EGTA-sensitivity values of 60-85 per cent with 0.1 mM EGTA for rabbit NAM have been reported by several workers while the average EGTA-sensitivity of frog NAM is 22 per cent with I mM EGTA. This low EGTA-sensitivity of frog NAM may be explained by (I) insufficient Ca2+ present in the assay medium for maximal Mg2+activation of the ATPase or (2) possible loss of the EGTA-sensitizing factor (ESF) [described by Ebashi (1g63)] during the course of the preparative procedure resulting in a form of partially desensitized actomyosin (DAM) (see Schaub and others, 1967). Added Ca2+, in the range o*o~-o* I mM, increased the EGTA-sensitivity from 25 per cent to 32 per cent with a corresponding increase in the specific activity of the ATPase of frog actomyosin. Higher levels of Ca2+, in the presence of Mg2+, did not enhance the ATPase activity further (Fig. 38). Lack of Ca2+ is therefore not responsible for the low EGTAsensitivity of frog actomyosin. The second suggestion will be reported in detail elsewhere (Heffron and Duggan, 1971). Briefly, rabbit EGTA-sensitizing factor, prepared according to Hartshorne and Mueller (rg67), did not increase the EGTA-sensitivity of frog NAM or frog NAM which had been extracted with
333
the low ionic strength solution (n=o*oo2) used by Schaub and others (1967). DISCUSSION Maruyama (1966) and de Villafranca and Campbell (1969) have found, upon reexamination of insect actomyosin ATPase and Limulus actomyosin ATPase, that Mg2+ behaves as an activator at KC1 concentrations below 30 mM and 60 mM respectively. In their previous investigations Mg2+-activation was occluded by the high levels of KC1 added to the assay medium with the enzyme solution. Frog actomyosin, as prepared here from R. temporaria leg muscle, was not quite as sensitive as insect actomyosin to KCl, but was more sensitive than rabbit actomyosin. Mgz+activation of rabbit actomyosin takes place in up to 200 mM KC1 (Banga and SzentGyorgyi, I 943 ; Mommaerts and Seraydarian, I 947)) whilst frog actomyosin, like insect and horseshoe crab actomyosins, is completely inhibited at this level of KCl. Contamination of the insect actomyosin by granular Mg2+activated ATPase was ruled out by Maruyama (1g66), who found that sodium azide, even at I o mM, had no effect on the Mg2 +-activation. de Villafranca and Campbell ( 1g6g), detected paramyosin but no submicroscopic particles upon analytical ultracentrifugation of Limulus actomyosin, also indicating that Mg2+-activation was an intrinsic property of the actomyosin. In this study reliance was placed on ultracentrifugation at IOO,OOO g at u=o.6 to remove mitochondrial and sarcoplasmic reticulum (SR) fragments. This procedure did not remove the Mg2 +activated ATPase activity, demonstrating that frog actomyosin ATPase, although contaminated to a small degree with other Mg*+enhanced ATPase(s), was activated by Mgs+ at low p. Nass (1962) used 2 cycles of clarification at 20,000 g and precipitation by dilution to obtain actomyosin from R. pipiens, and found that the preparation was Mg2+activated at low n only. Here it was found that this procedure removed most of the contaminating particulate material; 3 cycles of purification resulted in a slight decrease in the specific activity of the Mg2+-activated ATPase but the EGTA-semi tivity remained
334
HEFFRON
AND DUGCAN
constant at 28 per cent. The PH activity curves further indicate that one enzyme is responsible for the Mg2+-activated ATPase activity (cf. Duggan, 1965). The lack of effect of oligomycin levels up to 125 ylg. per mg. protein demonstrates that mitochondrial fragments are not important contaminants (Duggan, 1965). The Mg*+-activated ATPase of frog sartorius sarcoplasmic reticulum is strongly activated by K+ and inhibited by low levels of EGTA (Duggan, rg67a, b). It is therefore reasonable to conclude that this enzyme system is not responsible for the overall Mg*+activation of frog actomyosin. In view of the inhibitory effect of KC on frog actomyosin Mgs +-ATPase, contamination of the actomyosin with SR fragments would tend to reduce this inhibitionowing to the activation of the SR Mgs +-activated ATPase by K + which is optimal at about IOO m&f. The low EGTAsensitivity of the ATPase suggests that SR is a minor contaminant, if present at all. The basal M$ f- or Cast-enhanced ATPase also present in frog SR (Duggan, 1968) is not a contaminant of actomyosin, as shown by the inhibition of this ATPase above 2 mM Gas+ and the lack of inhibition by Mga+ up to 12 m&L The basal ATPase is also rather insensitive to KC1 being 40 per cent inhibited by 800 rnfi1 KCl, whilst actomyosin Mg2+ATPase does not express itself above 200 mill KCl. Although de Villafranca and Hochgraf (1962) used the ATP-sensitivity test as the criterion for the presence of the actomyosin complex in their extracts, they point out that myosin in various st*ates of polymerization may have been present in some of their preparations (cf. Noda and Ebashi, 1960). Interaction of myosin with itself may take place reversibly at ionic strengths less than 0.35 and irreversibly at high ionic strength in the physiological PH range (see Perry, 1967). The latter is probably accompanied by changes in the tertiary structure of the myosin and loss of biological activity. The former type of aggregation is more likely to have occurred here where the actomyosin was stored in 50 rnitl KC1 at PH 7-4, whilst the ’ myosin ’ prepared by the Weber dissociation
ht. J. ~~~C~f~*
method and stored at p=o*6, pH 7.0, should exhibit irreversible aggregation. However, the latter still showed some Mg2+- activated ATPase activity, indicating that high ionic strength storage is not detrimental to this enzymic function. Such forms of aggregation of myosin are probably common to all proteins and result from non-specific molecular properties of the myosin. However, complex formation between actin and myosin and its modification by ATP is due to a highly specific protein-protein interaction involving centres specialized for this function on both molecules. In view of the rather specific conditions under which Noda and Ebashi ( I 960) obtained dissociation of myosin aggregates by ATP it is reasonable to conclude that the viscosity drop upon ATP addition, taken together with a positive superprecipitation response, is a sufficient criterion for the presence of actomyosin. It now appears certain that synaeresis of actomyosin suspensions requires ATP hydrolysis and the presence of Mg2+ and Gas+ ions in the medium (see Perry, 1967). Frog actomyosin exhibits a relatively strong superprecipitation with almost complete dependence on Mg*+ ions; as with the Mg*+-activated ATPase activity, trace amounts of Ca2+ do not control the degree of superprecipitation to the same extent that Gas + controls superprecipitation of rabbit actomyosin. Limulus actomyosin superprecipitates upon ATP addition in the presence of Mgs+-, whereas Ca*+ exerts no control over Limuhs actomyosin synaeresis (de Villafranca and Campbell, 1969). These workers have therefore suggested that the lack of sensitivity to Gas+ is most likely due to the absence of the modifying proteins (EGTAsensitizing factor) from their preparations. Maruyama and Allen (1967) demonstrated & requirement for minor amounts of Mgz f, but not of Ca2+, for the onset of superprecipitation of insect actomyosin even though the Mgs+-activated ATPase activity showed a similar dependence on trace amounts of CaZ + to rabbit actomyosin. The EGTA-sensitivity of the MgZfactivated ATPase and superprecipitation of frog actomyosin therefore occupies an intermediate position between the high sensitivity
197’9 2
ACTOMYOSINATPaSe AND SUPERPRECIPITATION
of rabbit actomyosin and the insensitivity of insect actomyosin. This agrees with the evolutionary position of the frog relative to rabbit and insect, in that the calcium binding of EGTA mimics the same property of the troponin-tropomyosin system, which complex has so far not been shown to occur in lower organisms. ACKNOWLEDGEMENTS This work was supported in part by grants from the Muscular Dystrophy Associations of America and the Irish Cancer Society.
REFERENCES ‘ Myosin and adenosine RAIL?=, K. (rgq), triphosphatase ‘, Biochem.J., 36, 12 1-139. BANGA,I., and SZENT-GYGRGYI, A. (1843)~ ‘ The influence of salts on the phosphatase action of myosin ‘, Stud. Inst. med. Gem. Univ. Szeged., 3, 72-75.
CONWAY,E. J. (rg57),, ‘ Nature and significance of concentration relattons of potassium and sodium ions in skeletal muscle ‘, Physiol. Rev., 37, 84I 32. CONWAY, E. J. (x963), Micr~~~s~on Ano&& and Volumetric Error, 3rd ed., p. 137. New York: Chemical Publishers. DIJGGAN,P. F. (19641, ‘ Adenosine triphosphatase activity in fractions separated from frog sartorius homogenates ‘, Ir. J. med. Sci., ser. 3, September, 393-402. DUGGAN,P. F. (rg65), ‘ Some properties of the monovalent-cation-stimulated adenosinetriphosphatase of fro8 sartorius microsomes ‘, Biochim. biophys. Acta, 9g, 14._4-155.
DUGGAN, P. F. (tg67a), ‘ Potassium-activated adenosinetriphosphatase and calcium uptake by sarcoplasmic reticulum ‘, L@ Sci., 6, 561567.
DUCGAN, P. F. (rg67b), ‘ The monovalentcation-stimulated calcium numb in fro2 skeletal muscle ‘, Life Sci., 7,913-&g. 1 ” DUGGAN, P. F. ( rg68), ‘ ~lcium-inde~ndent adenosine~ph~pha~e activity in fro2 muscle microsomes r, L$? Sci., 7, tz65~1271. ” EBASHI, S. (rg63), ‘ Third component participating in superprecipitation of “natural actomyosin” ‘, Jvatut-e, Lond., zoo, 1010. HAMOIR, G., and REVTER, A. ( rg56), ‘ Isolement et proprittts de la myosine de grenouille ‘, Biochim. biophys. Acta, PI, 24-34.
HARTSHORNE,D. J., and MUELLER, H. (1967)~ ‘ Separation and recombination of the ethvlene glycol bis ( 8-aminoethyl ether)-& N’-&t&l acetic acid-sensitizing factor obtained from a low ionic strength extract of natural actomyosin ‘, 3. biol. C&em., 241, 308g-3092.
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HEFFRON,J. J. A. (1g6g), ’ The AT&se activity of the particulate fractions of fro2 skeletal muscle ‘. Ph.D. Thesis, National University of Ireland; Dublin. HEFFRON, J. J. A., and DUGGAN,P. F. (x967), ‘ The adenosine triphosphatase activity of skeletal muscle cell membranes ‘, Biochem. J,, 103,46P.
HEFFRON,J. J. A., and DUGGAN,P. F. ( 197 I), in preparation. JOBSIS, F. F., and VREMAN,H. J. (Ig63), ‘ Inhibition of a Na+- and K+-stimulated adenosine triphosphathe by oligomycin ‘, Biochim. biophys. Act&, 73* 346-348.
KALDOR, G., and GERGELY, J. (rg5g), ‘ Effect of pyridoxal phosphate and its inhibition by camosine on the ATPase activitv and svneresis of myofibrils ‘, A&s Biochem. ‘Biuphys.: 80, 3g3399. LAYi’%
‘ Speetrophotometric and E. (19571, turbidimetric methods for measuring proteins’, in Methods in Enzymology (ed. COLOWICK, S. P., and KAPLAN, N. O.), vol. 3, pp. 447-454. New York: Academic Press. LEVY, H. M., and FLEISHER, M. (tg65), ‘ Studies on the superprecipitation of actomyosin suspensions by the change in turbidity. I. Effects of adenosine triphosphate concentration and temperature ‘, Biochim. biophy5. Acta, xoo, 479-490. MARUYAMA,K. (rg66), ‘ Effect of magnesium on the aden~inet~phosphat~e activity of insect actomyosin at low ionic strength ‘, Co@. Biothem. Physiol., x8, 48x-487.
MARVYAMA,K., and ALLEN, S. R. (1~67)~ ‘ Effects of magnesium and calcium ions on the adenosinetriphosphatase activity of insect actomyosin at low ionic strength ‘, Camp. Biochem. Physiol., OX, 713-718. MOMMAERTS, W. F. H. M. (x958), Methods in Medical Research (ed. WARREN,J. V.), vol. 7, pp. I68. Chicano: Yearbook Publishers.
MOMMAERTS,~W.F. H. M., and SERAYDARIAN,K.
(1g47), ‘ A study of the adenosine triphosphatase activity of myosin and actomvosin ‘,,” 7. -pen.
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NASS,M. M. K. (rg6z), ‘ Developmental changes in fro8 actomyosin characteristics ‘, .Devl Biol., 412~320. NOVA, I-I.: and EBASHI, S. ( ES@), ‘ Aggregation of myosin A’, B&him. biophys. Acta, 4x,386-392. PERRY, S. V. (1967), ‘ The structure and interactions of myosin ‘, Prog. Biopl~ys. mol. Biol., x7, 327-381.
PERRY, S. V., and GREY, T. C. (rg56a), ‘ A study of the effects of substrate concentration and certain relaxing factors on the magnesiumactivated myofibrillar adenosine triphosphatase ‘, Biochem.
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PERRY, S. V., and GREY, T. C. (tg56b), ‘ Ethylenediaminetetraacetate and the adenosinctriphosphatase . _- activity - of actomyosin systems ‘, Bio&Em. .j., %, 5Y.
336
HEFFRON
SCHATZ, G., and SALTZGRABER, J. (rg6g),
AND DUGGAN
‘ Identification of denatured mitochondrial ATPase in “structural protein” from beef heart mitochondria ‘, B&him. biophys. Acta, 180, 186189. SCHAUB, M. C., HARTSHORNE, D. J., and PERRY, S. V. (x967), ‘ The adenosine-triphosphatase activity of desensitized actomyosin ‘, Biochem. j., xoe,263-269. SZENT-GYGRGYI,A. ( 1951), The Chemishy of Muscular Contraction, 2nd ed. New York: Academic Press. SZENT-GY~RGYI, A. G. ( I 960)) Muscle (ed. BOURNE, G. H.), vol. 2, pp. I s-16. New York: Academic Press, Inc. TAU~~KY,H. H., and SHORR,E. (x953), ‘ A microcalorimetric method for the determination of inorganic phosphorus ‘, 3. biol. Gem., rrop, 675-685. DE V~LLAF~ANCA, G. W. (x964), ‘ Frog myofibrillar adenosinetriphosphatase activity ‘, Comp. B&hem. Physiol., q, 87-95. DE VILLAFRANCA, G. W., and CAMPBELL,L. K. (1969)> ‘ Magnesium activation of natural
actomvosin ATPase from horseshoe crab ‘, Comp. biochem. Physiol., zg, 775-783. DE VILLAFRANCA.G. W.. and HOCHGRAF.H. L. (M2L ‘ Adenosinetriphosphatase activity of frog myosin B ‘, Camp. Biochem. Physiol., 6, 147-163. DE VILLAPRANCA,G. W., and NAUMANN,D. C. (I 964)) ‘ Some properties of the myosin B from ATPase from Limuhs ‘, Comp. Biochem. Physiol., 12, 143-156. WEBER, A. (x956), ‘ The ultracentrifugal separation of L-myosin and actin in an actomyosin sol under the influence of ATP ‘, Biochim. biophys. Acta, 1% 345-351. WEBER, H. H., and PORTZEHL, H. (tg52), ’ Muscle contraction and fibrous muscle proteins ‘, Ado. Prot. Gem., 7, 161-252. WILKIE,D. R. ( rg66), ‘ Muscle ‘, A. Rev. Physiol., 28, 17-38. Kg Word In&x: Frog, Rana temporaria, natural actomyosin, magnesium and calcium activation, rabbit, ATPand EGTA-sensitivity, superprecipitation, Oryctolagus cunicdus domesticus.