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The effect of adenosine triphosphate, inorganic pyrophosphate and inorganic tripolyphosphate on the stability of cod myosin
160
~BA
BIOCHIMICA ET BIOPHYSICA ACTA
25451
T H E E F F E C T OF A D E N O S I N E T R I P H O S P H A T E , I N O R G A N I C P Y R O P H O S P H A T E AND INORGANIC T R I P O L Y P H O S P H A T E ON T H E S T A B I L I T Y OF COD MYOSIN 1. M. M A C K I E
Torry Research Station, l]linistry of Technology, Aberdeen (Great Britain) (Received J u n e 28th, 1965)
SUMMARY
A very unstable form of cod myosin, free of actomyosin, has been isolated from an extract of pre-rigor muscle. This preparation aggregates approximately four times as fast as cod myosins previously studied. ATP, PPI and inorganic tripolyphosphate but not I T P inhibit the aggregation reaction. The second-order rate constants for these reactions have been determined from analyses of sedimentation velocity diagrams in the ultracentrifuge. The relationship of this myosin to the myosin extracted from post-rigor tissue is discussed.
INTRODUCTION
Previous papers on cod myosin 1-3 have emphasized the very unstable nature of this myosin as compared with myosin from mammalian sources. Indeed, this very instability was the reason for abandoning attempts to prepare myosin in the monomeric form by the classical method developed by SZENT-GY6RG¥I 4 because the protein appeared to aggregate spontaneously and irreversibly on precipitation at low ionic strength. HaMoII¢5,6 found that carp myosin also aggregated rapidly on precipitation. To isolate myosin in the monomeric form it was necessary to devise methods which did not involve a precipitation step. The most successful one involved the dissociation of actomyosin by ATP followed by the separation of the actin and myosin components by preparative ultracentrifugation, While this method gives myosin in a pure form (the actomyosin is stable and can be purified by repeated precipitation) the low solubility of actomyosin itself a n d the incomplete separation of undissociated actomyosin from myosin in the preparative ultracentrifuge limits the final concentration of myosin obtained to approx, o.4 %. An alternative method, which gives myosin in greater yield, although less pure, was introduced by CONNELL1; instead of separating the sarcoplasmic proteins from the myosin extract in the usual precipitation manner, he washed the muscle tissue with buffer solution (I, o.o5, pH 7.5) before extracting the myosin. In this method PPI and Mg 2+ are added to the extracting solution in order to break the actin-myosin links and so solubilize the myosin. This procedure has recently been refined 7 and now A b b r e v i a t i o n : P P P j , inorganic t r i p o l y p h o s p h a t e .
BiocLim. Biophys. Acta, I I 5 (1966) 16o-172
THE STABILITY OF COD MYOSIN
161
includes one precipitation step as a matter of routine. While aggregation undoubtedly does take place on repeated precipitation it is now clear that detectable amounts of aggregate do not form during a single precipitation step when the myosin is isolated b y this PPl-extraction procedure. In view of this discrepancy between the aggregation behaviour of the PPIextracted myosin and myosin extracted by the SZENT-GY{SRGYImethod, a more extensive investigation of the latter has been carried out. For the sake of simplicity the myosin prepared b y the SZENT-GY6RGYI method will be referred to as "pre-rigor" myosin to distinguish it from myosin prepared from washed muscle by extraction with PPi (ref. x). MATERIALS AND METHODS
ATP, ADP and I T P were obtained from the Sigma Chemical Company. All other compounds were reagent grade. Phosphate was determined by a modification of the method of ROCKSTEIN AND HERRONs. Tris [tris(hydroxymethyl)aminomethane] (0.05 M, p H 7.5) was used as buffer throughout.
Preparation of "pre-rigor" cod myosin The myosin was prepared b y the SZENT-GY6RGYI method 4 with minor modifications as follows : Immediately after killing and skinning the cod, fillets were cut out and chilled in ice before mincing. All subsequent operations were carried out as near o ° as possible. The muscle mince (IOO g) was extracted with 15o ml of 0.7 ° M KC10.05 M Tris (pH 7.5) for 15 min at o ° with occasional stirring. The residual mince was removed b y centrifuging at 4000 × g and the supernatant solution was diluted with 5 volumes of water. After standing for 15-3o min the precipitated myosin was collected by centrifugation, redissolved in 0.95 M KCl-o.o5 M Tris and the actomyosin impurity was then removed by reducing the ionic strength to 0.23 as previously described 7. The myosin was finally obtained as a water clear solution of zero ATPsensitivity in a concentration varying from 0.3-0.8 % protein. Because of the very unstable nature of this myosin the concentration had to be estimated rapidly before further dilutions were made, and for this, the approximate value obtained from the ~% absorbance at 276 m/, (assuming an E ..... of 8.0) was found to be satisfactory. Otherwise the protein concentrations were determined by the Folin-Ciocalteu method 9.
Preparation of PPl-extracted myosin The method previously described 7 was used.
Sedimentation methods Sedimentation velocity runs were carried out on a Spinco Model E ultracentrifuge as previously described1, ~°. All sedimentation coefficients and area measurements were obtained, after projecting the photographs of the schlieren diagrams onto the screen (magnification × IO) of a Shadowmaster Projector, Model CRP Mk 1I (supplied b y Buck and Hickman Ltd.) which was fitted with a two-way micrometeroperated stage. The areas under the peaks were determined by tracing the proiected schlieren diagrams onto paper, cutting out and weighing relative to a standard area. Biochim. Biophys. dcta, I15 (I966) 16o-172
162
I. M. MACKIE
The concentration of protein corresponding to this value was obtained by dividing by an area/concentration factor of o.76 (ref. I~). In order to obtain the true concentrations of the components of the mixtures in the uncentrifuged solutions these concentrations were corrected for two effects. (a) Radial dilution. (b) The "pile up" effect of the slower component at the boundary between the slow and the fast component '~. Correction of this effect by the method of JOHNSTON AND OGSTON12 has been applied previously by CONNELLn to aggregation studies on PPt-extraeted cod myosin, but in those studies the correction factor was never more than 50 %. In the present experiments on "pre-rigor" myosin the area of the dimer peak relative to the monomer is usually greater than in the experiments described by CONNELL and consequently a greater correction is required. In this situation also the sedimenting boundaries move very close together and any small error in the determination of the sedimentation coefficients in any one run has the effect of greatly magnifying errors in the correction factor. Occasional corrections which were obviously erroneous were in fact obtained using the method of JOHNSTON AND OGSTON. More consistent corrections were, however, obtained using the method of TRAUTMAN et al. ~ which has been applied by L o w E r AND HOLTZER14 to mixtures of the H and L meromyosins in digests of rabbit myosin using the following expressions: o
Cs - -
obs
obs,
Xf
r =
2
obs
(Xs /Xo) Cs
(1)
x2(l-d)
/Xo]
-
-
I
x~bS/xObS
(2)
where c°bS is the uncorrected concentration of the slower component obtained directly from the area measurement, Co ~ is the actual concentration of the slower component, x °bs and x~bs are the maximum ordinate positions is of the slow and fast components, respectively and xo is the meniscus distance. 8 is given to a "close approximation ''1* by soS~So t where Sos and Sot represent the infinite dilution sedimentation coefficients of the slow and fast components, respectively as obtained from a plot of s v e r s u s the concentration of the given component. As there is some uncertainty about the value of s o for the dimer n a value of 8.57 was selected as this gave the closest agreement with the corrections calculated by the JOttNSTON-OGSTON method for systems with only a small amount of visible aggregate. This value for So dimer in actual fact is not very different from the extrapolated value of 8.28 previously given by CONNELLn for cod myosin. Since s o for the monomer = 6.331°, 8 = 6.33/8.57 = 0.74. For the purposes of the "pile-up" correction, the approximation was made that the system was a two-component one, the faster moving boundary being assumed to be entirely dimer. Measurements were not normally made beyond the point where a discrete third component could be discerned. Using the method of TRAUTMANet al. 13 values of c~bS/c cal~" often as high as 3.0 were obtained but these are not unexpected for mixtures containing a relatively large amount of faster moving component as demonstrated by GCHACI-IMANAND HARRINGTON16 for artificial mixtures of two components whose sedimentation coefficients were Biochim. Biophys. dcta, 115 (1966) 26o-172
THE STABILITY OF COD MYOSIN
163
not widely different. While in the latter case the ratio of sol/So s was as low as 1.o9 the difference between the rates of sedimentation of the two components was sufficient to allow complete separation of the sedimenting boundaries over the concentrations studied. In the case of cod myosin, however, even although the Sol~Sos is greater, clearcut separation of the sedimenting boundaries was not obtained at much lower protein concentrations suggesting that the sedimentation coefficients of the two components do not have the same concentration dependence. In the myosin system then, the "pile-up" effect may be modified and, until it is proved empirically, that the correction of JOHNSTON AND OGSTON or of TRAUTMAN et al., applies to this situation also, the values for the "true" monomer concentration must be viewed with caution. This uncertainty over the true concentration of the components of the mixtures does not detract from the validity of the conclusions drawn about the relative effectiveness of the polyphosphates in stabilizing cod myosin.
RESULTS
Sedimentation coe~cient The sedimentation coefficient of the "pre-rigor" myosin was determined over a wide range of concentrations and the following equation was calculated from a plot of s versus concentration by the method of least squares, (s = s o - - k c ; s = 6.4o ( ± o.Io)* - - 4 . 7 5 ( i o.55)* c. The values of s o and k are not significantly different from previously determined values. It should be noted that these results were obtained on preparations containing aggregates and that the concentration used was that of the slow component. Stability of pre-rigor myosin When the "pre-rigor" myosin was examined immediately after preparation it invariably showed a distinct faster-moving peak, presumably a dimer (Fig. Ia), and after standing overnight at o ° the area of the second peak was almost equal in area to that of the monomer (Fig. ib and Table II). For comparison the pattern obtained
Fig. I. S e d i m e n t a t i o n d i a g r a m s of cod m y o s i n in 0. 5 !Vi KC1, Tris (pH 7-5) at 59980 r e v . / m i n a n d p h a s e - p l a t e a n g l e 5 o°. a a n d b, u n s t a b i l i z e d " p r e - r i g o r " m y o s i n : c, " p r e - r i g o r " m y o s i n stabilized b y A T P - M g 2+ (I,2 mlV[); d, P P t - e x t r a c t e d m y o s i n ; c o n c e n t r a t i o n of p r o t e i n : a, b a n d c, o . 3 2 % ; d, o . 2 3 % . D u r a t i o n of s t o r a g e a t o°; a, 5 m i n ; b, 18 h; c, 2o h; d, 23 h. T i m e in m i n a f t e r r e a c h i n g full speed: a, b a n d c, 48; d, 64. T e m p e r a t u r e : a, 6.7°; b, 4.7°; c, 5.7°; d, 2.2 °. Sedim e n t a t i o n fronl R to L. * 95 % confidence limits.
Biochim. Biophys. Acta, 115 (1966) I6O-I72
164
I.M. MACKIE
for the PPt-extracted myosin is shown after storage overnight (Fig. Id) in which case the dimer peak can just be detected.
Modification of the aggregation reaction A measure of the effectiveness of ATP, PPI, PPPI, ADP and I T P as modifiers of myosin stability was obtained by comparing the rates of transformation of myosin into higher aggregates in the presence and absence of these compounds. The reaction was followed by determining the concentration of myosin corresponding to the area of the slow component at intervals of time usually over 2-3 days. In order to make a direct comparison, between the modified and unmodified myosin it was necessary to run a control for each preparation as myosins prepared by the same method do vary somewhat in stability 11.
Effect of A TP The "pre-rigor" myosin could be stabilized considerably if ATP was added to the system. Fig. IC shows the effect of adding equimolar amounts of ATP and Mg 2+ (1.2 raM) to a 0.32 % solution of myosin; after storage overnight the peak corresponding to the faster moving dimer component has barely increased in area as compared with the control. As shown in Table I stabilization was observed after 24 h when equimolar amounts of Mg ~+, as opposed to Ca 2+ were present. In a separate experiment it was found that m a x i m u m stabilization was obtained when at least equimolar amounts of Mg 2+ were added to the solution (Fig. 2). Even under the conditions described in Fig. i where there is sufficient Mg2+ to reduce ATPase activity to a very low level under the usual conditions of estimation, significant hydrolysis of ATP, as measured by the formation of inorganic phosphate, did take place over the considerable period of storage studied, that is, several days at o °. It was therefore of interest to follow the aggregation reaction beyond the point where ATP was completely hydrolysed. A 0.42 % solution of myosin was stored at o ° in the presence of I, 3 and 5 mM ATP respectively, each solution containing an amount of Mg 2+ equimolar with the ATP concentration. Fig. 3 shows that ATP at all the concentrations added initially, reduced the rate of aggregation to the same TABLE
I
T H E E F F E C T OF T H E P R E S E N C E OF C& 2+ A N D M g 2+ ON T H E S T A B I L I Z I N G ACTION OF A T P SAME P R E P A R A T I O N OF " P R E - R I G O R " MYOSIN
Solvent
ON THE
Duration of storage (h)
% of aggregates (Trautman correction)
°/o of aggregates correctedfor radial dilutions only
o. 5 M K C 1 - T r i s o. 5 M K C 1 - T r i s
o 18
63 76
31 5°
0. M K C 1 - T r i s 1.2 i n M A T P 1.2 m M M g 2+
20
64
33
22
77
51
o. M K C 1 - T r i s 1.2 m M A T P 1.2 m M C a 2+
Biochim. Biophys. Acta,
1I 5 (1966) I6O-I72
165
THE STABILITY OF COD MYOSIN
level but after a time which was dependent on the initial ATP concentration there was a return to the "rapid rate" of aggregation. The effect of increasing the initial concentration of ATP above I mM was merely to extend the period of time over which the protein remained in the "stabilized state". As shown in Fig. 4, the rates Q150
0125
o
E 0.10C ,g
8 >, ~6
0075~
g ~i~" i ~ ! i l,~:i i ~i~ii~/!iiii
8 0050
~
b
iiiiii!i:iil
0.025
0
I
I
i
40
80
120 C°(h)
Storage
time
at
i
160
Fig. 2. Sedimentation diagrams of "pre-rigor" cod m y o s i n in 0. 5 IV[ KC1, Tris (pH 7.5) at 59780 rev./min and phase-plate angle 6o °. Protein concentration in all cases, o . 2 4 % . Additions: a (upper), ATP (I.o raM) only; a (lower L A D P (i.o mM) only; b (upper), ATP (i.o mM), MgCI 2 (4.0 raM); b (lower/, ATP (I.O raM), MgCI2 (i.o mM). Duration of storage at o°: a, 42 h; b, 45 h. Time after reaching full speed: 80 rain in both cases. Temperature: a, 5.0°; b, 4.3 °. Sedimentation from R to L. Fig. 3. Concentration of "pre-rigor" m y o s i n m o n o m e r measured after various times of storage at o ° and with different initial concentrations of A T P - M g 2+. V - - V , no A T P - M g 2+ added; [ ] - - [ ] , I.o mM ATP-Mg~+; © - - O , 3.0 mM ATP-Mg~+; & - - & , 5.0 m M A T P - M g z+.
of aggregation appear to conform to second-order kinetics regardless of the stage of the reaction, myosin in the "stabilized state" having a rate constant of 3.2"1o -2 (ml/g per h) and in the "unstabilized state", 25.4"1o -2 (ml/g per h). It should be noted that these second-order plots of the transformation of cod myosin monomer do not go through the origin as the initial material already contains aggregates.
Effect of PPi PP, alone. Fig. 5a shows that in the absence of added Ca2+ or Mg2+, I mM PP1 exerted a stabilizing effect. This effect was not increased by increasing the PPt concentrations above I mM (Table II). For a o.51% solution of myosin, the stabilizing effect Biochim, Biophys. Acta, 1I 5 (1966) 16o--172
166
I. M. MACKIE
4QOI"
10o
0L 0
V
~
I I I 40 80 120 Storeg time ot 0° (h)
I
160
Fig. 4. Second-order plot of t h e a g g r e g a t i o n of " p r e - r i g o r " m y o s i n m o n o m e r at o ° in t h e presence of different initial c o n c e n t r a t i o n s of ATP-Mg2+. N o t a t i o n as for Fig. 3Fig. 5. S e d i m e n t a t i o n d i a g r a m s of " p r e - r i g o r " cod m y o s i n in 0. 5 iV[ KC1, Tris (pH 7.5) a t 59 780 r e v . / m i n a n d p h a s e - p l a t e angle 5 o°. P r o t e i n c o n c e n t r a t i o n in all cases o. 19 %. A d d i t i o n s : a (upper), nil; a (lower), P P t (I.O mM) o n l y ; b (upper), PPI (i o raM) MgC12 (0. 5 mM); b (lower), PPI (i.o mM), CaC12 (0. 5 raM). D u r a t i o n of s t o r a g e a t o ° ; a, 24 h; b, 19 h. T i m e a f t e r r e a c h i n g full speed ; 64 rain in b o t h eases. T e m p e r a t u r e : a, 4.8; b, 5.o °. S e d i m e n t a t i o n from R to L.
250
2QO
2QO
150
150
0 ~5
iO0
100
50
50
O0
10
810 I 120 Storage time at O°(h)
• 160
0
I 40
I I 80 120 Storage time at Q°(h)
I 160
Fig. 6. Second-order plot of t h e a g g r e g a t i o n of "pre-rigor'" m y o s i n in t h e presence of different c o n c e n t r a t i o n s of PPt. / ~ - - D , no PPI a d d e d ; V - - V , o.i m M PPI; O - - O 0. 5 m M P P t ; 5 - - 5 , I.o m M PP,. Fig. 7. Second-order plot of t h e a g g r e g a t i o n of "pre-rigor" m y o s i n in t h e presence of different modifiers. O - - O , no a d d i t i o n ; U - - V , I.o m M I T P - M g ~ + ; A - - A , I.o m M P P P I ; [ ] - - D , I.o m M ATP-Mg2+; i - - I l l , i.o m M ADP-MgZ+.
Biochim. Biophys. Acta, 115 (1966) 16o--172
167
T H E STABILITY OF COD MYOSIN
of PPI increased from nil at o.I mM to a maximum at i.o mM PPt (Table II). The aggregation reaction appears to conform to second-order kinetics which give a rate constant of 7.0" lO-2 ml/g per h for the fully stabilized myosin at I.O mM PPI as compared with 31.o-lO -3 ml/g per h for the control without PP1 (Fig. 6). P P plus M g ~+ or Ca 2+. In contrast to the effect produced with ATP, Mg~+ inhibited the protective action of PPI and as the concentration of this cation was increased from o.I to i.o mM at a constant PPI concentration of I.O mM the stabilizing effect of PPI was correspondingly reduced (Fig. 5b and Table II). TABLE
II
SECOND-ORDER AGGREGATION RATE CONSTANTS OF COD MYOSIN IN 0. 5 M K C 1 - T r i s ( p H 7.5)
Preparation No.
Concentration of protein (g/xoo ml)
Addilion (m21l)
Rate constant h × lO 3 (ml/g per h)
27
0.32
nil 1.2 A T P , 1.2 M g 2+ 1.2 A T P , 1.2 C a 2+
23.2 4.1 25.6
28
0.42
nil I.O A T P , I.O M g 2+ 3.0 A T P , 3.0 M g 2+ 5.0 A T P , 5.o M g 2+
19.1 2. 4 2.4 2.4
3°
0.35
nil I.O I.o I.O 5.o
25.6 25.6 5-7 25.6 5.7
"Pre-rigor" myosins
M g 2+ PPt P P t , I.O M g 2+ PPt
35
°-51
nil o.I PPI o.5 P P I I.O P P t
31.4 31.4 17.1 7 .0
39
o.38
nil I.O I.O I.O i.o
I T P , I.O M g 2+ PPPt A T P , i . o M g '~+ A D P , I.O M g 2+
29.8 29.8 lO.8 3.8 3.8
PPt PPt, PPt, PPt, PPI,
28.5 7.3 7-3 15.6 16-7 28.5
4°
o.19
nil I.O
I.O I.O i.o I.O
0-5 o.I o.3 0.5
C a 2+ M g 2+ M g ~+ Mg ~+
Post-rigor P P t extracted myosins 24
0.25
nil i . o M g 2+
6.9 6.9
32
o.17
nil i.o PPt 2.0 A T P , 2.0 M g 2+
7.0 7 .0 3 .1
Biochim. Biophys. Acta, 115 (1966) 1 6 o - 1 7 2
168
I.M. MACKIE
When Mg2+ was replaced by Ca ~+, no such inhibition was observed (Fig. 5b and Table II).
Effect of PPPi, I T P and ADP The investigations were extended to include PPPi, ITP and ADP to determine if the stabilizing action of polyphosphates was in any way specific (Fig. 7 and Table II). PPPI (I.O raM) in the absence of Mg=+ produced maximum stabilization in a 0.38 % myosin solution and behaved in an analogous manner to PPi by stabilizing myosin to a similar extent and by maintaining it in the "stabilized" form throughout the observations. Under those conditions significant hydrolysis of PPPi by the cod myosin took place as shown by the gradual increase in free phosphate in the solution. Thus of the i.o mM PPPi initially present, 0.63 mM was hydrolysed after 48 h at o °. ITP (I.O raM) in the presence of Mg2+ (I.O mM), produced insignificant stabilization of the myosin at o ° as compared with the marked increase in stability produced by the same concentration of ATP (I.o raM) under the same conditions. Nevertheless the increase of free phosphate in this experiment (o.I6 mM after 4 h) was comparable with that in the ATP solution (o.II raM), indicating that the protein was as effective an ITPase as it was an ATPase under those conditions. In a separate experiment it was found that the presence or absence of M g 2+ made little, if any difference to the aggregation behaviour of the protein in the presence of ITP (Fig. 8a). For comparison the patterns are shown for the control and the ATP-stabilized myosin after the same time of storage (Fig. 8b). ADP on the other hand appeared to be an effective stabilizer and as shown in Table II, the rate of the aggregation reaction of the "stabilized myosin" was the same as that obtained with ATP as stabilizer under the same conditions (k -- 3.8" IO-'° (ml/g per h)). In addition the free phosphate content of the solution increased to 1.6 mM above the control after 48 h (cf. 1.8 mM for the ATP containing solution after the same time) and like ATP stabilization there was a return to conditions of rapid aggregation after a time which depended on the initial nucleotide concentration.
Fig. 8. S e d i m e n t a t i o n d i a g r a m s of " p r e rigor" m y o s i n in o. 5 M KC1, Tris (pH 7-5) a t 59 78o r e v . / m i n a n d p h a s e - p l a t e a n g l e 60 °. P r o t e i n c o n c e n t r a t i o n in all cases, 0.25 %. A d d i t i o n s : a (upper), I T P (I.O raM) only; a (lower), I T P (~.o raM), MgC12 (I.O raM); b (upper), A T P (I.o raM), MgCI 2, (i.o raM); b (lower), nil. D u r a t i o n of s t o r a g e at o°; a, 21 h; b, 19 h. T i m e a f t e r r e a c h i n g full speed; 64 m i n in b o t h cases. T e m p e r a t u r e : a, 3.3°; b, 3.7 °. S e d i m e n t a t i o n from R to L. Fig. 9. S e d i m e n t a t i o n d i a g r a m s of P P t - c x t r a c t e d m y o s i n in 0. 5 M KC1, Tris (pH 7.5) at 5978o r e v . / m i n a n d p h a s e - p l a t e angle 50 °. P r o t e i n c o n c e n t r a t i o n in all cases o. I 7 % . U p p e r p a t t e r n s , m y o s i n w i t h o u t ATP-Mg2+; lower p a t t e r n s m y o s i n w i t h A T P (2.0 raM), MgC12 (2.0 mM) a d d e d initially. D u r a t i o n of s t o r a g e a t o°: a, 48 h; b, 69 h. T i m e after r e a c h i n g full speed; 64 rain in b o t h cases. T e m p e r a t u r e : a, 4.3°; b, 4.1 °. S e d i m e n t a t i o n from R to L.
Biochim. Biophys. dcta, 115 (1966) 16o-172
THE STABILITY OF COD MYOSIN
16 9
Comparison of the stabilizing substances In Table I I are collected several determinations of the second-order rate constants for the aggregation reaction of the new type of myosin under different conditions of stabilization. Recently determined values for the rate constants of this reaction for untreated PPl-extracted myosin are included. It is evident that the PPIextracted myosin has a stability comparable with that of "pre-rigor" myosin to which PPI is added at a concentration of I raM. ATP has the effect of increasing the stability of myosin beyond this level, giving the most stable cod myosin so far prepared. It was of interest to see if ATP would increase the stability of the PPl-extracted myosin to a similar level. As shown in Fig. 9 when a o.1 7 % solution of PPi-extracted myosin was stored in the presence of 2.0 mM A T P - M g 2+ visible aggregates did not appear until the third day while the control solution without ATP had aggregated to approximately the same extent after only 2 days. PPt on the other hand at I.O mM concentration did not produce any increase in the stability of this myosin (Table II). DISCUSSION
It is now clear that there is a basic difference between cod myosin extracted from pre-rigor muscle by a method analogous to the conventional SZENT-GY6RGYI method 4 ("pre-rigor myosin") and cod myosin isolated b y PPt-extraction of muscle previously washed free of sarcoplasmic proteins 7. As reported by CONNELL11, the latter protein can be precipitated at low ionic strength without the formation of detectable amounts of aggregates. In the case of this "pre-rigor" myosin, however, the damaging effect of precipitation invariably produces aggregates visible in the ultracentrifuge because this myosin is inherently more unstable. In fact, when the crude myosin extract is examined even before precipitation, the schlieren diagram often shows the presence of a small amount of aggregate (presumably dimer). It has also been noted that in aggregated "pre-rigor" myosins the dimer peak is sharper than that observed by CONNELL11 for the PPl-extracted myosin and is more like the dimer peak of rabbit myosin as observed by JOHNSON AND ROWE15 during heat aggregation studies at room temperature. ATP and PPt have often been regarded as analogous compounds as far as their effects on actomyosin systems are concerned. It has been established that both polyphosphates are strongly bound to rabbit myosin and that for both there is one "binding site" per protein moleculelT, ls,2~. In some situations ATP and PPI protect the ATPase activity of myosin against inactivation by denaturants such as 2,4-dinitrophenol 2~ and in other situations where the ATPase activity has been destroyed (by N-ethylmaleimide, for example), these compounds still protect the ATP and PPI binding site against inactivation by high concentrations of this reagent 19. In addition, optical-rotatory dispersion studies by TONOMURA et al. 21, indicate that the secondary and tertiary structures of the molecule change slightly on binding ATP or PPI. While the mechanism of binding has not been elucidated, it appears that these polyphosphates modify the configuration of the molecule in such a way that it is protected against inactivation of one kind and another1% 2~, and it seems likely that in the present studies similar configurational changes serve to protect the myosin against heat aggregation. The results obtained with ATP as modifier suggest that stabilization is only Biochim. Biophys. Acta, 115 (1966) 16o-172
170
I. M. MACKIE
effected as long as ATP as distinct from a hydrolysis product of this nucleotide is present. Only two rates of aggregation are observed; an initial slow one with a rate constant of 2.4' lO-2-4.1 • lO-2 (ml/g per h) which eventually changes over to a much faster one with a rate constant of 19.1" lO-2-31.o • IO-e (ml/g per h). It is likely that the slow rate corresponds to the aggregation of myosin stabilized by ATP and that the fast one corresponds to tile aggregation of myosin in the unstabilized form. It is significant that increasing amounts of ATP do not produce myosins with progressively smaller aggregation rates suggesting that only a small critical concentration of ATP is required to effect maximum stabilization and that once the concentration falls below this critical level the rate of aggregation returns to the value observed without ATP. By increasing the amount of ATP-Mg ~+ added, the myosin can be kept in this stabilized state for a longer period of time, because more time is required for enzymic hydrolysis to proceed to the point where the ATP concentration falls below the critical level. It is tempting to equate this "critical level" with that occurring when I mole of ATP binds to I mole of myosin; initially in these experiments about 200 moles of ATP are typically present per myosin molecule. However, it was not possible to observe analytically whether, in fact, the rate of aggregation increased at the point where a mole to mole ratio had been reached because ultimately more free phosphate was produced than was equivalent to the hydrolysis of only the terminal phosphate group of ATP. This indicates that the myosin is contaminated with myokinase, so that the concentration of ATP left in the solution cannot be calculated merely from an analysis of free phosphate. It is necessary to add at least an equimolar concentration of Mg2+ together with the ATP before maximum stabilization is obtained, and when Mg2÷ is replaced by Ca 2+ no stabilization can be observed after 24 h at the concentrations of protein and nucleotide studied. Both of these effects are in line with the known behaviour of myosin ATPase under the influence of these cations. No attempt was made to determine the minimum concentration of ATP which had to be added to effect stabilization of the myosin. This was because the slow rate of aggregation of myosin in the "stabilized state" meant that measurable changes in area of the monomer peak would only be detected over at least 24 h and to maintain myosin in the stabilized state for this length of time it was necessary to add I mM ATP-Mg e+. When ADP is added instead of ATP, maximum stabilization is effected when Mg~+ and not Ca 2+ is present in at least equimolar concentrations. This observation can also be explained on the basis that the myosin is contaminated by myokinase which converts the ADP into ATP which then functions as a stabilizer. It appears that this stabilizing effect on myosin is not a general property of polyphosphates. Further support for this idea is obtained from the studies with ITP which is relatively ineffective under the conditions used for ATP stabilization. Strength of binding may be important here as ATP is very strongly bound to myosin as compared with ITP 22. In contrast to previous studies by MARTONOSI AND MEYER19 on the protective action of PPI on the "binding site" of myosin, the present studies show that, as far protection against aggregation is concerned, Mg~+ is not required. In fact, increasing concentrations of Mg2+ apparently inhibit the protective action of PPI, yet on replacing Mge+ by Ca 2+, no such inhibition is observed. Whatever the mechanism of this inhibition it is unlikely to be simply a removal of PPt by binding with the heavy metal as the association constants with Ca 2+ and Mg~+ are similar 23. To be certain on this Biochim. Biophys. At/a, 115 (1966) 16o-172
THE
S T A B I L I T Y O F COD M Y O S I N
171
point, however, it would be necessary to know the association constants of the other complexes present under those conditions, namely the metal-protein and PPl-protein complexes. As there is no increase in the rate of aggregation of either "pre-rigor" myosin or PPl-extracted myosin in the presence of Mg 2+ alone (Table II), it is assumed that this ion itself does not act as a denaturant. The minimum amount of PPl required to effect a measurable change in the stability of myosin is high (at least 20 moles PPI per mole of myosin, increasing to about 200 moles PPI at m a x i m u m stabilization). It is not obvious why such relatively large amounts of PPI are required to effect stabilization. This m a y be due in part at least, to the inhibiting effect of contaminating Mg2+ in the myosin preparation. When the "pre-rigor" myosin is maximally stabilized by PPt its rate of aggregation is closely similar to that of the PPl-extracted myosin (approx. 7.0" I0 ~ ml/g perh) and it is suggested that the latter myosin is already in the stabilized form as a consequence of the extraction procedure; as shown by the work of C O R S I et al. 24 the stro,lgly bound PPI is unlikely to be removed during the purifying process and must therefore stabilize the myosin. When the ATP is used as stabilizer the rate constants of both myosins are similar (Table II); in the case of the PPl-extracted myosin, the PPI ion is presumably replaced by the more strongly bound ATP ion 2° which acts as a more effective stabilizer. PPPt which has a similar binding constant to myosin as PPI (ref. 2o), stabilizes myosin to a similar extent but in contrast to the behaviour observed with ATP, there is no return to the condition of rapid aggregation once the polyphosphate has been hydrolysed, presumably because the PPt produced is equally effective as a stabilizer as PPPi. When the kinetics of aggregation of this myosin are considered, all indications are that second-order kinetics are observed throughout so that if one assumes the scheme postulated b y JO~INSON A~D ROWE15 Fast 2M
Slow ~ 2M*
(~)
> M2
(2)
where M is monomer, M* is modified intermediate and M 2 is dimer, then the rate of dimerisation (Reaction 2) must at first sight be that affected when ATP, PPI or PPPI are present. However, reactions (I) could also be modified without affecting the overall rate in so far as the presence of the polyphosphates could change the configuration of the species M* in such a way that the rate of Reaction 2 is reduced. As the ATPstabilized myosin has a rate constant of approximately half that of the PPl-stabilized myosin, M* for the former would then be in a more stable configuration than M* of the latter. Equimolar amounts of ATP and Mg 2+ have been added previously to the more stable rabbit myosin by JO~INSON AND ROWE~5 who reported a negative effect on its aggregation behaviour at room temperature. With the same protein HOLTZER AND L O W E Y 2~ reported a similar finding although they observed a slight stabilizing effect if Mg2+ was omitted from the system, This apparent discrepancy m a y simply be another point of difference between cod and rabbit myosins, the more stable configuration of rabbit myosin being such that shape changes produced by ATP do not have such marked effects on its aggregation behaviour.
Biochim. Biophys. dcta, 115 (1966) 1 6 o - 1 7 2
I72
i. M. M A C K I E
ACKNOWLEDGEMENTS
The author wishes to thank Dr. J. J. CONNELL and Mr. P. F. HOWGATE for valuable discussions and Mr. B. THOMSON for technical assistance. The statistical analysis carried out by Miss J. E. DUNCAN is gratefully acknowledged. The work described in this paper was carried out as part of the programme of the Ministry of Technology. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
J. J. CONNELL, Bioehem. J., 75 (196o) 530. J. J. CONNELL, Biochem. J., 69 (1958) 5" J. J. CONNELL, Biochem. J., 80 (1961) 503 . A. SZENT-GYCRGYI, Inst. Med. Chem. Univ. Szeged, 3 (1943) 544. G. HAMO1R, Arch. Intern. Physiol. Biochirn. Suppl., 63 (1955). G. HAMO1R, Advan. Protein Chem., IO (1955) 227. I. M. MACKIE AND J. J. CONNELL, Biochim. Biophys. Acta, 93 (1964) 544M. I{OCKSTEIN AND P. W. HERRON, Anal. Chem., 23 (1951) I5OO. V. 1. OYAMA AND H. EAGLE, Proc. Soc. Exptl. Biol. Med., 91 (1956) 305 . J. J. CONNELL, Biochem. ]., 7 ° (1958) 8I. J. J. CONNELL, Biochim. Biophys. Aeta, 74 (1963) 374. J. P. JOHNSTON AND A. G. OGSTON, Trans. Faraday Soc., 42 (1946) 789 . R. TRAUTMAN, V. •. SCHUMAKER, ~V. F. HARRINGTON AND ]7I. K. SCHACHMAN, J. Chem. Phys., 22 (1954) 555. S. LOwEY AND A. HOLTZER, Biochim. Biophys. Acta, 34 (1959) 47 TM P. JOHNSON AND A. J. t{o~,vE, Biochim. Biophys. Acta, 53 (1961) 343, "W. F. HARRINGTON AND H. K. SCHACHMAN,J. Am. Chem. Soc., 75 (1953) 3533. J. GERGELY, A. MARTONOSI AND M. A. GOUVEA, in R. BENESCH, Symposium on Sulphur in Proteins, Academic Press, New York, 1959, p. 297. L. B. NANNINGA AND \V. F. 1-I. M. MOMMAERTS, Proc. Natl. Aead. Sci. U.S., 46 (196o) 1155. A. MARTONOSI AND H. MEYER, J. Biol. Chem., 239 (1964) 640. H. M. LEVY, P. D. LEBER AND E. M. RYAN, J. Biol. Chem., 238 (1963) 3654 . Y. TONOMURA, i(. SEKIYA, K. IMAMURA AND T. TOKIWA, Biochim. Biophys. Acta, 69 (1963) 305 • H. M. LEVY, M. SHARON, E. M. P~YAN .aND D. E. KOSHLAND JR., Biochim. Biophys. Acta, 56 (1962) 118. E. E. DANIEL AND J. IRWIN, Can. J. Physiol. Pharmacol., 43 (1965) 89. A. CORSI, P. :BARGELLINI AND V. GALLUCCI, Experientia, 15 (1962) 12o. A. HOLTZER AND S. LOXVEY,J. Am. Chem. Soc., 81 (1959) 137o. L. B. NANNINGA AND ~V. F. H. M. MOMMAERTS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 11o6.
Biochim. Biophys. Acta, 115 (1966) 16o-172
~BA
BIOCHIMICA ET BIOPHYSICA ACTA
25451
T H E E F F E C T OF A D E N O S I N E T R I P H O S P H A T E , I N O R G A N I C P Y R O P H O S P H A T E AND INORGANIC T R I P O L Y P H O S P H A T E ON T H E S T A B I L I T Y OF COD MYOSIN 1. M. M A C K I E
Torry Research Station, l]linistry of Technology, Aberdeen (Great Britain) (Received J u n e 28th, 1965)
SUMMARY
A very unstable form of cod myosin, free of actomyosin, has been isolated from an extract of pre-rigor muscle. This preparation aggregates approximately four times as fast as cod myosins previously studied. ATP, PPI and inorganic tripolyphosphate but not I T P inhibit the aggregation reaction. The second-order rate constants for these reactions have been determined from analyses of sedimentation velocity diagrams in the ultracentrifuge. The relationship of this myosin to the myosin extracted from post-rigor tissue is discussed.
INTRODUCTION
Previous papers on cod myosin 1-3 have emphasized the very unstable nature of this myosin as compared with myosin from mammalian sources. Indeed, this very instability was the reason for abandoning attempts to prepare myosin in the monomeric form by the classical method developed by SZENT-GY6RG¥I 4 because the protein appeared to aggregate spontaneously and irreversibly on precipitation at low ionic strength. HaMoII¢5,6 found that carp myosin also aggregated rapidly on precipitation. To isolate myosin in the monomeric form it was necessary to devise methods which did not involve a precipitation step. The most successful one involved the dissociation of actomyosin by ATP followed by the separation of the actin and myosin components by preparative ultracentrifugation, While this method gives myosin in a pure form (the actomyosin is stable and can be purified by repeated precipitation) the low solubility of actomyosin itself a n d the incomplete separation of undissociated actomyosin from myosin in the preparative ultracentrifuge limits the final concentration of myosin obtained to approx, o.4 %. An alternative method, which gives myosin in greater yield, although less pure, was introduced by CONNELL1; instead of separating the sarcoplasmic proteins from the myosin extract in the usual precipitation manner, he washed the muscle tissue with buffer solution (I, o.o5, pH 7.5) before extracting the myosin. In this method PPI and Mg 2+ are added to the extracting solution in order to break the actin-myosin links and so solubilize the myosin. This procedure has recently been refined 7 and now A b b r e v i a t i o n : P P P j , inorganic t r i p o l y p h o s p h a t e .
BiocLim. Biophys. Acta, I I 5 (1966) 16o-172
THE STABILITY OF COD MYOSIN
161
includes one precipitation step as a matter of routine. While aggregation undoubtedly does take place on repeated precipitation it is now clear that detectable amounts of aggregate do not form during a single precipitation step when the myosin is isolated b y this PPl-extraction procedure. In view of this discrepancy between the aggregation behaviour of the PPIextracted myosin and myosin extracted by the SZENT-GY{SRGYImethod, a more extensive investigation of the latter has been carried out. For the sake of simplicity the myosin prepared b y the SZENT-GY6RGYI method will be referred to as "pre-rigor" myosin to distinguish it from myosin prepared from washed muscle by extraction with PPi (ref. x). MATERIALS AND METHODS
ATP, ADP and I T P were obtained from the Sigma Chemical Company. All other compounds were reagent grade. Phosphate was determined by a modification of the method of ROCKSTEIN AND HERRONs. Tris [tris(hydroxymethyl)aminomethane] (0.05 M, p H 7.5) was used as buffer throughout.
Preparation of "pre-rigor" cod myosin The myosin was prepared b y the SZENT-GY6RGYI method 4 with minor modifications as follows : Immediately after killing and skinning the cod, fillets were cut out and chilled in ice before mincing. All subsequent operations were carried out as near o ° as possible. The muscle mince (IOO g) was extracted with 15o ml of 0.7 ° M KC10.05 M Tris (pH 7.5) for 15 min at o ° with occasional stirring. The residual mince was removed b y centrifuging at 4000 × g and the supernatant solution was diluted with 5 volumes of water. After standing for 15-3o min the precipitated myosin was collected by centrifugation, redissolved in 0.95 M KCl-o.o5 M Tris and the actomyosin impurity was then removed by reducing the ionic strength to 0.23 as previously described 7. The myosin was finally obtained as a water clear solution of zero ATPsensitivity in a concentration varying from 0.3-0.8 % protein. Because of the very unstable nature of this myosin the concentration had to be estimated rapidly before further dilutions were made, and for this, the approximate value obtained from the ~% absorbance at 276 m/, (assuming an E ..... of 8.0) was found to be satisfactory. Otherwise the protein concentrations were determined by the Folin-Ciocalteu method 9.
Preparation of PPl-extracted myosin The method previously described 7 was used.
Sedimentation methods Sedimentation velocity runs were carried out on a Spinco Model E ultracentrifuge as previously described1, ~°. All sedimentation coefficients and area measurements were obtained, after projecting the photographs of the schlieren diagrams onto the screen (magnification × IO) of a Shadowmaster Projector, Model CRP Mk 1I (supplied b y Buck and Hickman Ltd.) which was fitted with a two-way micrometeroperated stage. The areas under the peaks were determined by tracing the proiected schlieren diagrams onto paper, cutting out and weighing relative to a standard area. Biochim. Biophys. dcta, I15 (I966) 16o-172
162
I. M. MACKIE
The concentration of protein corresponding to this value was obtained by dividing by an area/concentration factor of o.76 (ref. I~). In order to obtain the true concentrations of the components of the mixtures in the uncentrifuged solutions these concentrations were corrected for two effects. (a) Radial dilution. (b) The "pile up" effect of the slower component at the boundary between the slow and the fast component '~. Correction of this effect by the method of JOHNSTON AND OGSTON12 has been applied previously by CONNELLn to aggregation studies on PPt-extraeted cod myosin, but in those studies the correction factor was never more than 50 %. In the present experiments on "pre-rigor" myosin the area of the dimer peak relative to the monomer is usually greater than in the experiments described by CONNELL and consequently a greater correction is required. In this situation also the sedimenting boundaries move very close together and any small error in the determination of the sedimentation coefficients in any one run has the effect of greatly magnifying errors in the correction factor. Occasional corrections which were obviously erroneous were in fact obtained using the method of JOHNSTON AND OGSTON. More consistent corrections were, however, obtained using the method of TRAUTMAN et al. ~ which has been applied by L o w E r AND HOLTZER14 to mixtures of the H and L meromyosins in digests of rabbit myosin using the following expressions: o
Cs - -
obs
obs,
Xf
r =
2
obs
(Xs /Xo) Cs
(1)
x2(l-d)
/Xo]
-
-
I
x~bS/xObS
(2)
where c°bS is the uncorrected concentration of the slower component obtained directly from the area measurement, Co ~ is the actual concentration of the slower component, x °bs and x~bs are the maximum ordinate positions is of the slow and fast components, respectively and xo is the meniscus distance. 8 is given to a "close approximation ''1* by soS~So t where Sos and Sot represent the infinite dilution sedimentation coefficients of the slow and fast components, respectively as obtained from a plot of s v e r s u s the concentration of the given component. As there is some uncertainty about the value of s o for the dimer n a value of 8.57 was selected as this gave the closest agreement with the corrections calculated by the JOttNSTON-OGSTON method for systems with only a small amount of visible aggregate. This value for So dimer in actual fact is not very different from the extrapolated value of 8.28 previously given by CONNELLn for cod myosin. Since s o for the monomer = 6.331°, 8 = 6.33/8.57 = 0.74. For the purposes of the "pile-up" correction, the approximation was made that the system was a two-component one, the faster moving boundary being assumed to be entirely dimer. Measurements were not normally made beyond the point where a discrete third component could be discerned. Using the method of TRAUTMANet al. 13 values of c~bS/c cal~" often as high as 3.0 were obtained but these are not unexpected for mixtures containing a relatively large amount of faster moving component as demonstrated by GCHACI-IMANAND HARRINGTON16 for artificial mixtures of two components whose sedimentation coefficients were Biochim. Biophys. dcta, 115 (1966) 26o-172
THE STABILITY OF COD MYOSIN
163
not widely different. While in the latter case the ratio of sol/So s was as low as 1.o9 the difference between the rates of sedimentation of the two components was sufficient to allow complete separation of the sedimenting boundaries over the concentrations studied. In the case of cod myosin, however, even although the Sol~Sos is greater, clearcut separation of the sedimenting boundaries was not obtained at much lower protein concentrations suggesting that the sedimentation coefficients of the two components do not have the same concentration dependence. In the myosin system then, the "pile-up" effect may be modified and, until it is proved empirically, that the correction of JOHNSTON AND OGSTON or of TRAUTMAN et al., applies to this situation also, the values for the "true" monomer concentration must be viewed with caution. This uncertainty over the true concentration of the components of the mixtures does not detract from the validity of the conclusions drawn about the relative effectiveness of the polyphosphates in stabilizing cod myosin.
RESULTS
Sedimentation coe~cient The sedimentation coefficient of the "pre-rigor" myosin was determined over a wide range of concentrations and the following equation was calculated from a plot of s versus concentration by the method of least squares, (s = s o - - k c ; s = 6.4o ( ± o.Io)* - - 4 . 7 5 ( i o.55)* c. The values of s o and k are not significantly different from previously determined values. It should be noted that these results were obtained on preparations containing aggregates and that the concentration used was that of the slow component. Stability of pre-rigor myosin When the "pre-rigor" myosin was examined immediately after preparation it invariably showed a distinct faster-moving peak, presumably a dimer (Fig. Ia), and after standing overnight at o ° the area of the second peak was almost equal in area to that of the monomer (Fig. ib and Table II). For comparison the pattern obtained
Fig. I. S e d i m e n t a t i o n d i a g r a m s of cod m y o s i n in 0. 5 !Vi KC1, Tris (pH 7-5) at 59980 r e v . / m i n a n d p h a s e - p l a t e a n g l e 5 o°. a a n d b, u n s t a b i l i z e d " p r e - r i g o r " m y o s i n : c, " p r e - r i g o r " m y o s i n stabilized b y A T P - M g 2+ (I,2 mlV[); d, P P t - e x t r a c t e d m y o s i n ; c o n c e n t r a t i o n of p r o t e i n : a, b a n d c, o . 3 2 % ; d, o . 2 3 % . D u r a t i o n of s t o r a g e a t o°; a, 5 m i n ; b, 18 h; c, 2o h; d, 23 h. T i m e in m i n a f t e r r e a c h i n g full speed: a, b a n d c, 48; d, 64. T e m p e r a t u r e : a, 6.7°; b, 4.7°; c, 5.7°; d, 2.2 °. Sedim e n t a t i o n fronl R to L. * 95 % confidence limits.
Biochim. Biophys. Acta, 115 (1966) I6O-I72
164
I.M. MACKIE
for the PPt-extracted myosin is shown after storage overnight (Fig. Id) in which case the dimer peak can just be detected.
Modification of the aggregation reaction A measure of the effectiveness of ATP, PPI, PPPI, ADP and I T P as modifiers of myosin stability was obtained by comparing the rates of transformation of myosin into higher aggregates in the presence and absence of these compounds. The reaction was followed by determining the concentration of myosin corresponding to the area of the slow component at intervals of time usually over 2-3 days. In order to make a direct comparison, between the modified and unmodified myosin it was necessary to run a control for each preparation as myosins prepared by the same method do vary somewhat in stability 11.
Effect of A TP The "pre-rigor" myosin could be stabilized considerably if ATP was added to the system. Fig. IC shows the effect of adding equimolar amounts of ATP and Mg 2+ (1.2 raM) to a 0.32 % solution of myosin; after storage overnight the peak corresponding to the faster moving dimer component has barely increased in area as compared with the control. As shown in Table I stabilization was observed after 24 h when equimolar amounts of Mg ~+, as opposed to Ca 2+ were present. In a separate experiment it was found that m a x i m u m stabilization was obtained when at least equimolar amounts of Mg 2+ were added to the solution (Fig. 2). Even under the conditions described in Fig. i where there is sufficient Mg2+ to reduce ATPase activity to a very low level under the usual conditions of estimation, significant hydrolysis of ATP, as measured by the formation of inorganic phosphate, did take place over the considerable period of storage studied, that is, several days at o °. It was therefore of interest to follow the aggregation reaction beyond the point where ATP was completely hydrolysed. A 0.42 % solution of myosin was stored at o ° in the presence of I, 3 and 5 mM ATP respectively, each solution containing an amount of Mg 2+ equimolar with the ATP concentration. Fig. 3 shows that ATP at all the concentrations added initially, reduced the rate of aggregation to the same TABLE
I
T H E E F F E C T OF T H E P R E S E N C E OF C& 2+ A N D M g 2+ ON T H E S T A B I L I Z I N G ACTION OF A T P SAME P R E P A R A T I O N OF " P R E - R I G O R " MYOSIN
Solvent
ON THE
Duration of storage (h)
% of aggregates (Trautman correction)
°/o of aggregates correctedfor radial dilutions only
o. 5 M K C 1 - T r i s o. 5 M K C 1 - T r i s
o 18
63 76
31 5°
0. M K C 1 - T r i s 1.2 i n M A T P 1.2 m M M g 2+
20
64
33
22
77
51
o. M K C 1 - T r i s 1.2 m M A T P 1.2 m M C a 2+
Biochim. Biophys. Acta,
1I 5 (1966) I6O-I72
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THE STABILITY OF COD MYOSIN
level but after a time which was dependent on the initial ATP concentration there was a return to the "rapid rate" of aggregation. The effect of increasing the initial concentration of ATP above I mM was merely to extend the period of time over which the protein remained in the "stabilized state". As shown in Fig. 4, the rates Q150
0125
o
E 0.10C ,g
8 >, ~6
0075~
g ~i~" i ~ ! i l,~:i i ~i~ii~/!iiii
8 0050
~
b
iiiiii!i:iil
0.025
0
I
I
i
40
80
120 C°(h)
Storage
time
at
i
160
Fig. 2. Sedimentation diagrams of "pre-rigor" cod m y o s i n in 0. 5 IV[ KC1, Tris (pH 7.5) at 59780 rev./min and phase-plate angle 6o °. Protein concentration in all cases, o . 2 4 % . Additions: a (upper), ATP (I.o raM) only; a (lower L A D P (i.o mM) only; b (upper), ATP (i.o mM), MgCI 2 (4.0 raM); b (lower/, ATP (I.O raM), MgCI2 (i.o mM). Duration of storage at o°: a, 42 h; b, 45 h. Time after reaching full speed: 80 rain in both cases. Temperature: a, 5.0°; b, 4.3 °. Sedimentation from R to L. Fig. 3. Concentration of "pre-rigor" m y o s i n m o n o m e r measured after various times of storage at o ° and with different initial concentrations of A T P - M g 2+. V - - V , no A T P - M g 2+ added; [ ] - - [ ] , I.o mM ATP-Mg~+; © - - O , 3.0 mM ATP-Mg~+; & - - & , 5.0 m M A T P - M g z+.
of aggregation appear to conform to second-order kinetics regardless of the stage of the reaction, myosin in the "stabilized state" having a rate constant of 3.2"1o -2 (ml/g per h) and in the "unstabilized state", 25.4"1o -2 (ml/g per h). It should be noted that these second-order plots of the transformation of cod myosin monomer do not go through the origin as the initial material already contains aggregates.
Effect of PPi PP, alone. Fig. 5a shows that in the absence of added Ca2+ or Mg2+, I mM PP1 exerted a stabilizing effect. This effect was not increased by increasing the PPt concentrations above I mM (Table II). For a o.51% solution of myosin, the stabilizing effect Biochim, Biophys. Acta, 1I 5 (1966) 16o--172
166
I. M. MACKIE
4QOI"
10o
0L 0
V
~
I I I 40 80 120 Storeg time ot 0° (h)
I
160
Fig. 4. Second-order plot of t h e a g g r e g a t i o n of " p r e - r i g o r " m y o s i n m o n o m e r at o ° in t h e presence of different initial c o n c e n t r a t i o n s of ATP-Mg2+. N o t a t i o n as for Fig. 3Fig. 5. S e d i m e n t a t i o n d i a g r a m s of " p r e - r i g o r " cod m y o s i n in 0. 5 iV[ KC1, Tris (pH 7.5) a t 59 780 r e v . / m i n a n d p h a s e - p l a t e angle 5 o°. P r o t e i n c o n c e n t r a t i o n in all cases o. 19 %. A d d i t i o n s : a (upper), nil; a (lower), P P t (I.O mM) o n l y ; b (upper), PPI (i o raM) MgC12 (0. 5 mM); b (lower), PPI (i.o mM), CaC12 (0. 5 raM). D u r a t i o n of s t o r a g e a t o ° ; a, 24 h; b, 19 h. T i m e a f t e r r e a c h i n g full speed ; 64 rain in b o t h eases. T e m p e r a t u r e : a, 4.8; b, 5.o °. S e d i m e n t a t i o n from R to L.
250
2QO
2QO
150
150
0 ~5
iO0
100
50
50
O0
10
810 I 120 Storage time at O°(h)
• 160
0
I 40
I I 80 120 Storage time at Q°(h)
I 160
Fig. 6. Second-order plot of t h e a g g r e g a t i o n of "pre-rigor'" m y o s i n in t h e presence of different c o n c e n t r a t i o n s of PPt. / ~ - - D , no PPI a d d e d ; V - - V , o.i m M PPI; O - - O 0. 5 m M P P t ; 5 - - 5 , I.o m M PP,. Fig. 7. Second-order plot of t h e a g g r e g a t i o n of "pre-rigor" m y o s i n in t h e presence of different modifiers. O - - O , no a d d i t i o n ; U - - V , I.o m M I T P - M g ~ + ; A - - A , I.o m M P P P I ; [ ] - - D , I.o m M ATP-Mg2+; i - - I l l , i.o m M ADP-MgZ+.
Biochim. Biophys. Acta, 115 (1966) 16o--172
167
T H E STABILITY OF COD MYOSIN
of PPI increased from nil at o.I mM to a maximum at i.o mM PPt (Table II). The aggregation reaction appears to conform to second-order kinetics which give a rate constant of 7.0" lO-2 ml/g per h for the fully stabilized myosin at I.O mM PPI as compared with 31.o-lO -3 ml/g per h for the control without PP1 (Fig. 6). P P plus M g ~+ or Ca 2+. In contrast to the effect produced with ATP, Mg~+ inhibited the protective action of PPI and as the concentration of this cation was increased from o.I to i.o mM at a constant PPI concentration of I.O mM the stabilizing effect of PPI was correspondingly reduced (Fig. 5b and Table II). TABLE
II
SECOND-ORDER AGGREGATION RATE CONSTANTS OF COD MYOSIN IN 0. 5 M K C 1 - T r i s ( p H 7.5)
Preparation No.
Concentration of protein (g/xoo ml)
Addilion (m21l)
Rate constant h × lO 3 (ml/g per h)
27
0.32
nil 1.2 A T P , 1.2 M g 2+ 1.2 A T P , 1.2 C a 2+
23.2 4.1 25.6
28
0.42
nil I.O A T P , I.O M g 2+ 3.0 A T P , 3.0 M g 2+ 5.0 A T P , 5.o M g 2+
19.1 2. 4 2.4 2.4
3°
0.35
nil I.O I.o I.O 5.o
25.6 25.6 5-7 25.6 5.7
"Pre-rigor" myosins
M g 2+ PPt P P t , I.O M g 2+ PPt
35
°-51
nil o.I PPI o.5 P P I I.O P P t
31.4 31.4 17.1 7 .0
39
o.38
nil I.O I.O I.O i.o
I T P , I.O M g 2+ PPPt A T P , i . o M g '~+ A D P , I.O M g 2+
29.8 29.8 lO.8 3.8 3.8
PPt PPt, PPt, PPt, PPI,
28.5 7.3 7-3 15.6 16-7 28.5
4°
o.19
nil I.O
I.O I.O i.o I.O
0-5 o.I o.3 0.5
C a 2+ M g 2+ M g ~+ Mg ~+
Post-rigor P P t extracted myosins 24
0.25
nil i . o M g 2+
6.9 6.9
32
o.17
nil i.o PPt 2.0 A T P , 2.0 M g 2+
7.0 7 .0 3 .1
Biochim. Biophys. Acta, 115 (1966) 1 6 o - 1 7 2
168
I.M. MACKIE
When Mg2+ was replaced by Ca ~+, no such inhibition was observed (Fig. 5b and Table II).
Effect of PPPi, I T P and ADP The investigations were extended to include PPPi, ITP and ADP to determine if the stabilizing action of polyphosphates was in any way specific (Fig. 7 and Table II). PPPI (I.O raM) in the absence of Mg=+ produced maximum stabilization in a 0.38 % myosin solution and behaved in an analogous manner to PPi by stabilizing myosin to a similar extent and by maintaining it in the "stabilized" form throughout the observations. Under those conditions significant hydrolysis of PPPi by the cod myosin took place as shown by the gradual increase in free phosphate in the solution. Thus of the i.o mM PPPi initially present, 0.63 mM was hydrolysed after 48 h at o °. ITP (I.O raM) in the presence of Mg2+ (I.O mM), produced insignificant stabilization of the myosin at o ° as compared with the marked increase in stability produced by the same concentration of ATP (I.o raM) under the same conditions. Nevertheless the increase of free phosphate in this experiment (o.I6 mM after 4 h) was comparable with that in the ATP solution (o.II raM), indicating that the protein was as effective an ITPase as it was an ATPase under those conditions. In a separate experiment it was found that the presence or absence of M g 2+ made little, if any difference to the aggregation behaviour of the protein in the presence of ITP (Fig. 8a). For comparison the patterns are shown for the control and the ATP-stabilized myosin after the same time of storage (Fig. 8b). ADP on the other hand appeared to be an effective stabilizer and as shown in Table II, the rate of the aggregation reaction of the "stabilized myosin" was the same as that obtained with ATP as stabilizer under the same conditions (k -- 3.8" IO-'° (ml/g per h)). In addition the free phosphate content of the solution increased to 1.6 mM above the control after 48 h (cf. 1.8 mM for the ATP containing solution after the same time) and like ATP stabilization there was a return to conditions of rapid aggregation after a time which depended on the initial nucleotide concentration.
Fig. 8. S e d i m e n t a t i o n d i a g r a m s of " p r e rigor" m y o s i n in o. 5 M KC1, Tris (pH 7-5) a t 59 78o r e v . / m i n a n d p h a s e - p l a t e a n g l e 60 °. P r o t e i n c o n c e n t r a t i o n in all cases, 0.25 %. A d d i t i o n s : a (upper), I T P (I.O raM) only; a (lower), I T P (~.o raM), MgC12 (I.O raM); b (upper), A T P (I.o raM), MgCI 2, (i.o raM); b (lower), nil. D u r a t i o n of s t o r a g e at o°; a, 21 h; b, 19 h. T i m e a f t e r r e a c h i n g full speed; 64 m i n in b o t h cases. T e m p e r a t u r e : a, 3.3°; b, 3.7 °. S e d i m e n t a t i o n from R to L. Fig. 9. S e d i m e n t a t i o n d i a g r a m s of P P t - c x t r a c t e d m y o s i n in 0. 5 M KC1, Tris (pH 7.5) at 5978o r e v . / m i n a n d p h a s e - p l a t e angle 50 °. P r o t e i n c o n c e n t r a t i o n in all cases o. I 7 % . U p p e r p a t t e r n s , m y o s i n w i t h o u t ATP-Mg2+; lower p a t t e r n s m y o s i n w i t h A T P (2.0 raM), MgC12 (2.0 mM) a d d e d initially. D u r a t i o n of s t o r a g e a t o°: a, 48 h; b, 69 h. T i m e after r e a c h i n g full speed; 64 rain in b o t h cases. T e m p e r a t u r e : a, 4.3°; b, 4.1 °. S e d i m e n t a t i o n from R to L.
Biochim. Biophys. dcta, 115 (1966) 16o-172
THE STABILITY OF COD MYOSIN
16 9
Comparison of the stabilizing substances In Table I I are collected several determinations of the second-order rate constants for the aggregation reaction of the new type of myosin under different conditions of stabilization. Recently determined values for the rate constants of this reaction for untreated PPl-extracted myosin are included. It is evident that the PPIextracted myosin has a stability comparable with that of "pre-rigor" myosin to which PPI is added at a concentration of I raM. ATP has the effect of increasing the stability of myosin beyond this level, giving the most stable cod myosin so far prepared. It was of interest to see if ATP would increase the stability of the PPl-extracted myosin to a similar level. As shown in Fig. 9 when a o.1 7 % solution of PPi-extracted myosin was stored in the presence of 2.0 mM A T P - M g 2+ visible aggregates did not appear until the third day while the control solution without ATP had aggregated to approximately the same extent after only 2 days. PPt on the other hand at I.O mM concentration did not produce any increase in the stability of this myosin (Table II). DISCUSSION
It is now clear that there is a basic difference between cod myosin extracted from pre-rigor muscle by a method analogous to the conventional SZENT-GY6RGYI method 4 ("pre-rigor myosin") and cod myosin isolated b y PPt-extraction of muscle previously washed free of sarcoplasmic proteins 7. As reported by CONNELL11, the latter protein can be precipitated at low ionic strength without the formation of detectable amounts of aggregates. In the case of this "pre-rigor" myosin, however, the damaging effect of precipitation invariably produces aggregates visible in the ultracentrifuge because this myosin is inherently more unstable. In fact, when the crude myosin extract is examined even before precipitation, the schlieren diagram often shows the presence of a small amount of aggregate (presumably dimer). It has also been noted that in aggregated "pre-rigor" myosins the dimer peak is sharper than that observed by CONNELL11 for the PPl-extracted myosin and is more like the dimer peak of rabbit myosin as observed by JOHNSON AND ROWE15 during heat aggregation studies at room temperature. ATP and PPt have often been regarded as analogous compounds as far as their effects on actomyosin systems are concerned. It has been established that both polyphosphates are strongly bound to rabbit myosin and that for both there is one "binding site" per protein moleculelT, ls,2~. In some situations ATP and PPI protect the ATPase activity of myosin against inactivation by denaturants such as 2,4-dinitrophenol 2~ and in other situations where the ATPase activity has been destroyed (by N-ethylmaleimide, for example), these compounds still protect the ATP and PPI binding site against inactivation by high concentrations of this reagent 19. In addition, optical-rotatory dispersion studies by TONOMURA et al. 21, indicate that the secondary and tertiary structures of the molecule change slightly on binding ATP or PPI. While the mechanism of binding has not been elucidated, it appears that these polyphosphates modify the configuration of the molecule in such a way that it is protected against inactivation of one kind and another1% 2~, and it seems likely that in the present studies similar configurational changes serve to protect the myosin against heat aggregation. The results obtained with ATP as modifier suggest that stabilization is only Biochim. Biophys. Acta, 115 (1966) 16o-172
170
I. M. MACKIE
effected as long as ATP as distinct from a hydrolysis product of this nucleotide is present. Only two rates of aggregation are observed; an initial slow one with a rate constant of 2.4' lO-2-4.1 • lO-2 (ml/g per h) which eventually changes over to a much faster one with a rate constant of 19.1" lO-2-31.o • IO-e (ml/g per h). It is likely that the slow rate corresponds to the aggregation of myosin stabilized by ATP and that the fast one corresponds to tile aggregation of myosin in the unstabilized form. It is significant that increasing amounts of ATP do not produce myosins with progressively smaller aggregation rates suggesting that only a small critical concentration of ATP is required to effect maximum stabilization and that once the concentration falls below this critical level the rate of aggregation returns to the value observed without ATP. By increasing the amount of ATP-Mg ~+ added, the myosin can be kept in this stabilized state for a longer period of time, because more time is required for enzymic hydrolysis to proceed to the point where the ATP concentration falls below the critical level. It is tempting to equate this "critical level" with that occurring when I mole of ATP binds to I mole of myosin; initially in these experiments about 200 moles of ATP are typically present per myosin molecule. However, it was not possible to observe analytically whether, in fact, the rate of aggregation increased at the point where a mole to mole ratio had been reached because ultimately more free phosphate was produced than was equivalent to the hydrolysis of only the terminal phosphate group of ATP. This indicates that the myosin is contaminated with myokinase, so that the concentration of ATP left in the solution cannot be calculated merely from an analysis of free phosphate. It is necessary to add at least an equimolar concentration of Mg2+ together with the ATP before maximum stabilization is obtained, and when Mg2÷ is replaced by Ca 2+ no stabilization can be observed after 24 h at the concentrations of protein and nucleotide studied. Both of these effects are in line with the known behaviour of myosin ATPase under the influence of these cations. No attempt was made to determine the minimum concentration of ATP which had to be added to effect stabilization of the myosin. This was because the slow rate of aggregation of myosin in the "stabilized state" meant that measurable changes in area of the monomer peak would only be detected over at least 24 h and to maintain myosin in the stabilized state for this length of time it was necessary to add I mM ATP-Mg e+. When ADP is added instead of ATP, maximum stabilization is effected when Mg~+ and not Ca 2+ is present in at least equimolar concentrations. This observation can also be explained on the basis that the myosin is contaminated by myokinase which converts the ADP into ATP which then functions as a stabilizer. It appears that this stabilizing effect on myosin is not a general property of polyphosphates. Further support for this idea is obtained from the studies with ITP which is relatively ineffective under the conditions used for ATP stabilization. Strength of binding may be important here as ATP is very strongly bound to myosin as compared with ITP 22. In contrast to previous studies by MARTONOSI AND MEYER19 on the protective action of PPI on the "binding site" of myosin, the present studies show that, as far protection against aggregation is concerned, Mg~+ is not required. In fact, increasing concentrations of Mg2+ apparently inhibit the protective action of PPI, yet on replacing Mge+ by Ca 2+, no such inhibition is observed. Whatever the mechanism of this inhibition it is unlikely to be simply a removal of PPt by binding with the heavy metal as the association constants with Ca 2+ and Mg~+ are similar 23. To be certain on this Biochim. Biophys. At/a, 115 (1966) 16o-172
THE
S T A B I L I T Y O F COD M Y O S I N
171
point, however, it would be necessary to know the association constants of the other complexes present under those conditions, namely the metal-protein and PPl-protein complexes. As there is no increase in the rate of aggregation of either "pre-rigor" myosin or PPl-extracted myosin in the presence of Mg 2+ alone (Table II), it is assumed that this ion itself does not act as a denaturant. The minimum amount of PPl required to effect a measurable change in the stability of myosin is high (at least 20 moles PPI per mole of myosin, increasing to about 200 moles PPI at m a x i m u m stabilization). It is not obvious why such relatively large amounts of PPI are required to effect stabilization. This m a y be due in part at least, to the inhibiting effect of contaminating Mg2+ in the myosin preparation. When the "pre-rigor" myosin is maximally stabilized by PPt its rate of aggregation is closely similar to that of the PPl-extracted myosin (approx. 7.0" I0 ~ ml/g perh) and it is suggested that the latter myosin is already in the stabilized form as a consequence of the extraction procedure; as shown by the work of C O R S I et al. 24 the stro,lgly bound PPI is unlikely to be removed during the purifying process and must therefore stabilize the myosin. When the ATP is used as stabilizer the rate constants of both myosins are similar (Table II); in the case of the PPl-extracted myosin, the PPI ion is presumably replaced by the more strongly bound ATP ion 2° which acts as a more effective stabilizer. PPPt which has a similar binding constant to myosin as PPI (ref. 2o), stabilizes myosin to a similar extent but in contrast to the behaviour observed with ATP, there is no return to the condition of rapid aggregation once the polyphosphate has been hydrolysed, presumably because the PPt produced is equally effective as a stabilizer as PPPi. When the kinetics of aggregation of this myosin are considered, all indications are that second-order kinetics are observed throughout so that if one assumes the scheme postulated b y JO~INSON A~D ROWE15 Fast 2M
Slow ~ 2M*
(~)
> M2
(2)
where M is monomer, M* is modified intermediate and M 2 is dimer, then the rate of dimerisation (Reaction 2) must at first sight be that affected when ATP, PPI or PPPI are present. However, reactions (I) could also be modified without affecting the overall rate in so far as the presence of the polyphosphates could change the configuration of the species M* in such a way that the rate of Reaction 2 is reduced. As the ATPstabilized myosin has a rate constant of approximately half that of the PPl-stabilized myosin, M* for the former would then be in a more stable configuration than M* of the latter. Equimolar amounts of ATP and Mg 2+ have been added previously to the more stable rabbit myosin by JO~INSON AND ROWE~5 who reported a negative effect on its aggregation behaviour at room temperature. With the same protein HOLTZER AND L O W E Y 2~ reported a similar finding although they observed a slight stabilizing effect if Mg2+ was omitted from the system, This apparent discrepancy m a y simply be another point of difference between cod and rabbit myosins, the more stable configuration of rabbit myosin being such that shape changes produced by ATP do not have such marked effects on its aggregation behaviour.
Biochim. Biophys. dcta, 115 (1966) 1 6 o - 1 7 2
I72
i. M. M A C K I E
ACKNOWLEDGEMENTS
The author wishes to thank Dr. J. J. CONNELL and Mr. P. F. HOWGATE for valuable discussions and Mr. B. THOMSON for technical assistance. The statistical analysis carried out by Miss J. E. DUNCAN is gratefully acknowledged. The work described in this paper was carried out as part of the programme of the Ministry of Technology. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
J. J. CONNELL, Bioehem. J., 75 (196o) 530. J. J. CONNELL, Biochem. J., 69 (1958) 5" J. J. CONNELL, Biochem. J., 80 (1961) 503 . A. SZENT-GYCRGYI, Inst. Med. Chem. Univ. Szeged, 3 (1943) 544. G. HAMO1R, Arch. Intern. Physiol. Biochirn. Suppl., 63 (1955). G. HAMO1R, Advan. Protein Chem., IO (1955) 227. I. M. MACKIE AND J. J. CONNELL, Biochim. Biophys. Acta, 93 (1964) 544M. I{OCKSTEIN AND P. W. HERRON, Anal. Chem., 23 (1951) I5OO. V. 1. OYAMA AND H. EAGLE, Proc. Soc. Exptl. Biol. Med., 91 (1956) 305 . J. J. CONNELL, Biochem. ]., 7 ° (1958) 8I. J. J. CONNELL, Biochim. Biophys. Aeta, 74 (1963) 374. J. P. JOHNSTON AND A. G. OGSTON, Trans. Faraday Soc., 42 (1946) 789 . R. TRAUTMAN, V. •. SCHUMAKER, ~V. F. HARRINGTON AND ]7I. K. SCHACHMAN, J. Chem. Phys., 22 (1954) 555. S. LOwEY AND A. HOLTZER, Biochim. Biophys. Acta, 34 (1959) 47 TM P. JOHNSON AND A. J. t{o~,vE, Biochim. Biophys. Acta, 53 (1961) 343, "W. F. HARRINGTON AND H. K. SCHACHMAN,J. Am. Chem. Soc., 75 (1953) 3533. J. GERGELY, A. MARTONOSI AND M. A. GOUVEA, in R. BENESCH, Symposium on Sulphur in Proteins, Academic Press, New York, 1959, p. 297. L. B. NANNINGA AND \V. F. 1-I. M. MOMMAERTS, Proc. Natl. Aead. Sci. U.S., 46 (196o) 1155. A. MARTONOSI AND H. MEYER, J. Biol. Chem., 239 (1964) 640. H. M. LEVY, P. D. LEBER AND E. M. RYAN, J. Biol. Chem., 238 (1963) 3654 . Y. TONOMURA, i(. SEKIYA, K. IMAMURA AND T. TOKIWA, Biochim. Biophys. Acta, 69 (1963) 305 • H. M. LEVY, M. SHARON, E. M. P~YAN .aND D. E. KOSHLAND JR., Biochim. Biophys. Acta, 56 (1962) 118. E. E. DANIEL AND J. IRWIN, Can. J. Physiol. Pharmacol., 43 (1965) 89. A. CORSI, P. :BARGELLINI AND V. GALLUCCI, Experientia, 15 (1962) 12o. A. HOLTZER AND S. LOXVEY,J. Am. Chem. Soc., 81 (1959) 137o. L. B. NANNINGA AND ~V. F. H. M. MOMMAERTS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 11o6.
Biochim. Biophys. Acta, 115 (1966) 16o-172