- Email: [email protected]
Reaction of myosin with salicylaldehyde II. Effect of salicylalation on the ATPase activity of myosin
BIOCHIMICA ET BIOPHYSICA ACTA
35~
BBA 45932 REACTION OF MYOSIN W I T H SALICYLALDEHYDE II. E F F E C T OF SALICYLALATION ON T H E ATPase ACTIVITY OF MYOSIN A. MI~HLRAD, K. A J T A I AND F. F A B I A N
Department of Biochemistry, E6tv6s Lordnd University, Budapest (Hungary) (Received December i8th, 1969)
SUMMARY
The effect of salicylalation on the biological properties of myosin was studied. I. The ATPase activity of myosin is affected by salicylalation if the treatment is carried out at higher pH than 6.5. The MgZ+-activated ATPase shows a maximal curve with 25o-380% maximal activation when 25-70 moles of salicylaldehyde are bound per mole of myosin. The EDTA-activated ATPase decreases with increasing salicylalation. Ca~+-activated ATPase shows a small increase with increasing salicylalation. 2. Less salicylaldehyde is bound if the treatment is carried out in the presence of ATP, while that of PPi does not affect the degree of salicylalation. The enzymic properties of myosins salicylalated in the presence of ATP or PPt are not different from those of the samples treated in their absence. 3- Salicylalation decreases ATP sensitivity of ATPase and superprecipitation of actomyosins reconstituted from salicylalated myosins only if more than 50 moles of salicylaldehyde are bound per mole myosin.
INTRODUCTION
It was shown in the previous paper 1 that salicylaldehyde specifically reacts with the lysyl residues of myosin. Since it was tound in the investigations of TOSOMUgh and co-workers z-4 and of FAmIAN AND MOHLRAD~ that the trinitrophenylation of the e-amino groups of myosin b y 2,4,6-trinitrobenzenesulphonate (TBS) altered the enzymic properties of myosin and the alteration was observed to be markedly different from that caused b y the blocking of the sulphydryl groups of myosin, it seemed of interest to study the changes in enzymic activity caused by the modification of the lysyl residues with another amino group reagent. METHODS
The preparation of myosin and the treatment with s~icylaldehyde, the determination of bound salicylaldehyde and protein concentrations were performed as described in MATERIALSAND METHODSin the previous paper I. Abbreviation: TBS, 2,4,6-trinitrobenzene sulphonate.
Biochim. Biophys. Aaa, 205 (i97 o) 355-360
356
A. MUHLRAD et al.
The ATPase activity of myosin was measured in a solution containing I mg of myosin per ml, 4 mM ATP, 50 mM Tris-maleate buffer (pH 7.6) and alternatively 5 mM EDTA, 5 mM CaCI~ or 2 mM MgC1v Also present was 0.5 or o.o5 M KC1. The measurements were carried out on samples of 2 ml at 22 °. Incubation was terminated b y the addition of 2 ml of IO % trichloroacetic acid. Pi was measured b y the method of FISKE AND SUBBAROW6. The ATPase activity was evaluated as #mole Pt per mg of myosin per rain. The incubation time was chosen so as to obtain a decomposition of the terminal phosphate of ATP to less than 25 %. The ATPase activity of synthetic actomyosin was measured in a solution containing o.8 mg of myosin per ml, o.132 mg of actin per ml, 2 mM ATP, 2 mM MgClz and io mM Tris-maleate buffer (pH 7.4) at o °. The superprecipitation was measured by the method of EBASHI7 at 66o m/~ with a Spectromom 360 spectrophotometer at room temperature. The conditions of the test were 0.8 mg of myosin, 0.066 mg of actin per ml, o.I mM ATP, 5 mM Trismaleate buffer (pH 7.4) and 2 mM MgC1v Superprecipitation was evaluated from the difference between the values of the absorbance measured before, then 3 rain after, the addition of ATP. The actomyosin formation at high ionic strength was established from the measured ATP sensitivity. The measurements were carried out in an Ostwald viscometer using the method of B~.R/~.NY et al. s under the following conditions: I mg of myosin per ml, 0.33 mg of actin per ml, 0.5 M KCl-o.o2 M borate buffer (pH 7.4) at o °. RESULTS
The ATPase activities of myosins salicylalated at pH 6.5, 7.6, 8:0 and 9.0 were measured alternatively in the presence of Mg2+, Ca *+ or E D T A each time in the presence of 0.5 M KC1. At p H 8 an additional run was made in the presence of 0.05 M KCI. The three types of the ATPase activity curve measured as a function of salicylalation at various p H ' s are shown in Fig. I. The ATPase activity curves (Fig. I) measured in the presence of Mg*+ show m a x i m a as a function of salicylalation. The m a x i m a of the curves are 25-7o moles of bound salicylaldehyde per mole of myosin and appear at higher degrees of salicylalation for higher values of p H of the treatment. The maximal values of activation lie between 25 ° and 380 %. In the sample treated b y salicylaldehyde at p H 6.5 the activation was hardly appreciable and also much less, not more than 13 moles of salicylaldehyde being bound per mole of myosin (see also Fig. IO of the previous paper1). I t seems that the very poor activation cannot be related to the decrease in the complex formation at p H 6.5, since at p H 7.6 at the same degree of salicylalation (13 moles of salicylaldehyde bound per mole of myosin) the activation was measured as approx. 2oo %. I t seems more probable t h a t the lysyl residues having some role in the ATPase activity do not react with salicylaldehyde at p H 6.5. The m a x i m a of the Ca2+-activated ATPase as a function of salicylalation were relatively small as compared with those measured in the presence of Mg2+, and at higher degrees of salicylalation the activity was lower than that measured on the non-treated control. This m a y be due to unspecific conformational changes caused by the binding of numerous moles of salicylaldehyde to the myosin molecule. Biochim. Biophys. Acta, 205 (197 o) 355-36o
SALICYLALATION OF MYOSIN. I I
a
357
a
.>
2oG
a
w
.
.
.
5o
.
, ~0
mo~es of ~alicyfaMehycle bound p e r m o l e
.
.
n
. 2o0
rnyosi'n
Fig. z. The ATPase activity of myosin salicylslated at different pH's. Myosin was sslicylalated in and dialysed against a s o l u t i o n containing 0. 5 M KCI and 20 mM Tris--maleate buffer (a and b), or 20 mM borate buffer (c and d). a. pH 6.5. b. pH 7.6. c. pH 8.0. d. pH 9.0. For o t h e r c o n d i t i o n s of the salicylalation see MATERIALS AND METHODS i n t h e previous paper I. Conditions of the enzymic assay: i mg of myosin per ml; 50 mM Tris--maleate buffer (pH 7.4)i 4 mM ATP; 0. 5 (D, O, ×) or 0.05 (E, O, * ) M KCI; and alternatively 5 mM EDTA (O, O), 5 mM CaCIs ( ×, . ) or 2 mM MgCI9 (D, ss). Control (lOO%) activities (/~moles Pi per mg myosin per rain): a. Call 2, 0.29; EDTA, o.51; MgCI~, o.oo41, b. CaCI~, 0.25; EDTA, o.6o; MgCll, o.0o44, c. o.5M KCI: CaCll, 0.22; EDTA, 0.70; Mg,CIv 0.0054; 0.05 M KCI: CaCla, 0.46; EDTA, o.141MgC] v 0.0093. d. CaCIz, o.17; EDTA, 0.82; and MgC12, 0.0055. The ATPase activity measured in the presence of EDTA, i.e. K+-activated ATPase ~, m a r k e d l y decreases with increasing salicylalation. The measurements of ATPase activities at low ionic strength of the sample treated b y salicylaldehyde at p H 8 (Fig. IC) showed in each of the curves, for activity against salicylalation, lower values t h a n those measured at high ionic strength. The effect of the presence of magnesium A T P and magnesium p y r o p h o s p h a t e on the salicylalation of myosin was also studied since it was observed b y TONOMURA, et al. 1°, as well as b y F~BIAN AND MiJHLRADs, t h a t in the presence of either c o m p o u n d less lysyl residues were trinitrophenylated t h a n in their absence. A similar phenomenon was also observed b y BARANY et al. n on the reaction of myosin with i-fluoro-2,4dinitrobenzene. As a p p a r e n t from Fig. 2, the presence of A T P reduced the degree of salicylalation, but P P I did not appreciably affect the reaction between salicylaldehyde a n d myosin. I n this experiment an additional control sample was used which was treated b y salicylaldehyde in the presence of the same magnesium concentration as t h a t of the magnesium A T P or magnesium p y r o p h o s p h a t e test samples. No difference was observed between the reaction yields of the additional and the usual (see MATERIALSAND METHODS in the previous paper 1) control samples. Biochim. Biophys. Aaa, 205 (197o) 355-360
358
A. MUHLRADet al.
The K+-activated ATPase (ATPase measured in the presence of EDTA) of myosins salicylalated in the presence of magnesium ATP, magnesium pyrophosphate or Mg~+ showed the same dependence on salicylalation as the control sample treated by salicylaldehyde in the absence of these ingredients (Fig. 3). The MgS+-inhibited ATPase curves are also very similar to the control curve if magnesium pyrophosphate or Mg2+ are present during the treatment with salicylaldehyde. Higher activation at a lower degree of salicylalation was observed only in the presence of magnesium ATP during the treatment. .c ~150 E
E
~
4OC I00
3C( 2OC
~
o moles
I0-3 I0"~ of salicyloldehycle in Mcutmti~ solution
<~
0
~o
m0~-~0f sollc~ld~l'~ b0u/~ i ~ f ~
rr~,,o~in
Fig. 2. Effect of A T P a n d P P t on t h e salicytalation of myosin. Conditions of salicylalation: B o t h i n c u b a t i n g a n d dialysing solutions: o. 5 M KC1 a n d o.i M borate buffer (pH 8); o t h e r c o n s t i t u e n t s of i n c u b a t i n g solution: none ( × ] ; 6 m M MgC11 ( * ) ;'6 m M MgCI~ a n d 6 m M s o d i u m p y r o p h o s p h a t e ( 0 ) ; 6 m M MgCIj a n d 6 mM ATP (A). F o r dialysing solution: none ( × ) ; 2 m M MgC12 ( , ) ; 2 m M MgCI~ and x m M sodium p y r o p h o s p h a t e (Q) ; 2 m M MgCI~ knd i m M A T P (A). F o r other conditions see MATERIALS AND METHODS in t h e previous p a p e r 1. Fig. 3. ATPase activity of m y o s i n saticylalated in t h e presence of ATP and PPt- For conditions of saIicylalation see legend to Fig. 2. Salicylalation was carried out in t h e p r e s e n c e ' o f : A T P and Mg 2+ ( O , 0. ) ; P P I a n d Mg 2+ ( [3, I ) ; Mg t+ (iX, A ) ; ho e x t r a c o n s t i t u e n t ( × , * ). ATPase activity m e a s u r e d in t h e presence of 5 m M E D T A ( O , I , A , * ) or 2 m M MgCls (O, rT, A, X). Control ( i o o % ) activities: O , o.31; I , 0.4o; J k , o;34; * , o.64; O , o.oo48; [3, o.oo44; &, o.oo39; × , o . o o 4 3 / , m o l e Pl per m g m y o s i n per rain. T h e control E D T A - a c t i v a t e d ATPases of those samples t h a t were t r e a t e d in t h e presence of Mg I+ (O, I , A ) were low because of t h e relatively high Mg I+ c o n c e n t r a t i o n p r e s e n t during t h e m e a s u r e m e n t of ATPase.
The effect of complex formation with salicylaldehyde on the characteristic properties of actomyosin synthesized from salicylalated myosin was also studied. The experimental data on ATPase activity, actomyosin formation (evaluated from the measured ATP sensitivity) and sup~rprecipitation are listed in Table I. The actomyosin ATPase measured in the presence of Mgs+ at low ionic strength did not change on salicylalation when less than 46.5 moles of salicylaldehyde were bound per mole of myosin. At higher degrees of saliCylalation a sharp decrease in actomyosin ATPase was observed which m a y be due to the unspecific conformation change taking place on the introduction of a large number of salicylaldehyde residues into the myosin molecule. The percentage activation of ATPase b y actin decreased even on the addition of small amounts of salicylaldehyde because of the activating effect of salicylalation on myosin ATPase in the presence of Mg2+ (Table I, Column 2). (Activation b y actin was evaluated as actomyosin ATPase/myosin ATPase × IOO. Therefore, if the myosin ATPase increases without alteration of actomyosin ATPase, the activation b y actin decxeases.) A similar phenomenon was observed by F£BIAIq A~D M~dI~LRAD~ Biochim. Biophys. Acta, 205 (197 o) 355-36o
SALICYLALATION OF MYOSIN. I I
359
TABLE I T H E CHARACTERISTIC P R O P E R T I E S OF ACTOMYOSIN R E C O N S T I T U T E D FROM SALICYLALATED MYOSIN
For detailed conditions of enzymic assays, determinations of ATP sensitivity and superprecipiration see METHODS.Myosin ATPase was measured under the same conditions as actomyosin ATPase. Percentage activation by actin = actomyosin ATPase/myosin ATPase × lOO. Moles oJ salicylaldehyde bound per mole myosin
Myosin A TPase
A ctomyosin A TPase
A TP sensitivity
Superprecipitation
2
Percentage activation by actin 3
I
4
5
-3.2 5.3 13.7 46.5 82.6 122.8
0.0234 o-o314 0.0340 0.0378 o.o34o o.o192 0.0058
0.227 0.246 0.249 0.239 O.lO9 0.058 o,oo 3
97 ° 784 734 632 466 300 52
16o 154 158 156 143 I33 80
o.61 0.57 0.54 o.61 o.36 0.35 o.oo
for t r i n i t r o p h e n y l a t e d m y o s i n *. A c t o m y o s i n formation a n d superprecipitation showed a similar decrease to t h a t of a c t o m y o s i n ATPase when more t h a n 46.5 moles of salicylaldehyde were b o u n d per mole of :myosin.
DISCUSSION The effect of the blockingof amino groups with salicylaldehydeon the enzymm properties of myosin is comparable to that observed on the blocking of these groups with TBS~-~.' In both :instances th e Mg2+-inhibited ATPase of myosin showed activation, whereas the Ca~+-activated ATPase was left almost unchanged by the blocking (Figs. 2, 3 and 4, and ref. 5)- In contrast, the blocking of sulphydryl groups results in the activation of both the Ca2+-activated and Mg~+-inhibited ATPase1~-1~. The K~activated ATPase of myosin decreases on any modification of myosinS,X~,I~,5,i~,is and hence also on the blocking of sulphydryl or amino groups. The other biological properties of myosin, such as the actomyosin formation, tlle Mg~+-activated ATPase and the superprecipitation of actomyosin synthesized from the modified myosin, are also affected ]n almost the same way by the modification with salicylaldehyde as by that with TBS5. The effect ol trinitropheny!ati~n on the myosin ATPase is assumed to be due to the removal of the positive charges of one or some lysyl residues located in or near to the active site of the molecules5. The differences which can nevertheless be observed between the effects of trinitrophenylation and salicylalationon the enzymic properties suggest that trinitropheny]ation more specificallyaffects the lysyl residues having some role in the ATPase activity. This would explain the observation that the activation of the Mg~+-inhibited ATPase of myosin is 2000 %, if approx. 5 lysyl residues are trinitrophenylated per mole of myosin5, as compared with an activation of 380 % if approx. 5° lysyl residues are salicylalated per mole of myosin. The effect of the presence of the substrate is also different in the two cases. If ATP is present during trinitropheny]ation the lysyl residues having some role in the ATPase activity are not modifiedsince the change in the enzymic properties is much Biochim. Biophys. Acta, 205 (197o) 355-36o
360
A. MUHLRAD et al.
less a p p a r e n t t h a n in t h e absence of t h e s u b s t r a t e . On t h e other h a n d t h e m a x i m u m a c t i v a t i o n of t h e Mg¢+-inhibited A T P a s e is o b s e r v e d a t a lower degree of s a l i c y l a l a t i o n in t h e presence t h a n in t h e absence of ATP. The different effects of t h e two r e a g e n t s in t h e presence of t h e s u b s t r a t e can b e e x p l a i n e d if one a t t r i b u t e s to t h e l y s y l residues h a v i n g some role in t h e A T P a s e a c t i v i t y a higher affinity for T B S t h a n t h e o t h e r l y s y l residues of myosin. On t h e a d d i t i o n of A T P a n d Mg ~+, however, a c o n f o r m a t i o n a l change t a k e s place u, zg, ~o b y which t h e higher affinity of these specific l y s y l residues is levelled off. T h e l y s y l residues in question, however, h a v e no highei affinity for s a l i c y l a l d e h y d e a n d therefore t h e a d d i t i o n of t h e s u b s t r a t e , w h i c h equalizes t h e affinities of t h e residues for TBS, does n o t specifically affect t h e s a l i c y l a l d e h y d e r e a c t i o n of t h e l y s y l residues i n v o l v e d in t h e A T P a s e a c t i v i t y of m y o s i n . ACKNOWLEDGEMENTS T h e a u t h o r s are i n d e b t e d to Dr. N. A. B i t 6 a n d Mrs. J. Monori for t h e i r k i n d h e l p in t h e p r e p a r a t i o n of this p a p e r . REFERENCES I 2 3 4 5 6
7 8 9
io n I2 13 14 15 16 17 18 19 20
A. MOHLRAD, K. AJTAI AND F. FkBIJ,N, Biockim. Biophys. Acts, 205 (197o) 342. S. KUBO, S. TOKURAAND Y. TONOMURA,J. Biol. Chem., 235 (196o) 2835. S. KITAGAWA,J. YOSHIMURAAND Y. TONOMURA,J. Biol. Chem., 236 (1961) 902. H. TOKUYAMAAND Y. TONOMURA,J. Biochem. Tokyo, 62 (1967) 456. F. FXBIXN AND A. MOHLRAD,Biochim. Biophys. Aaa, 162 (i968) 596. C. H. FISKE AND Y. SUBBARow, f . Biol. Chem., 66 (I925) 375. S. EBAsm, J. Bioohem. Tokyo, 5° (I961) 236. M. BkI~NY, B. NAGY, F. FII~I~LMA~ AND A. Cm~mBACH, J. Biol. Chem., 236 (I961) 2917 A. MOHLRAD, F. F~BI~N AND N. A. BII~, Biockim. Biophys. Acts, 89 (1964) 186. Y. TONOMURA, J. YOSmMURA AND T. OmSHI, Biochim. Biophys. Aaa, 78 (I963) 698. M. BLI~NY, G. BArtoN AND K. B/~1Lt~NY,jr. Biol. Chem., 244 (z969) 648. W. W. KIELLEY AND L. B. BRADLEY, J. Biol. Chem., 218 (1956) 653. H. M. LEvY AND E. M. RYAN, Bioahim. Biopltys. Aaa, 46 (i961) 193. T. SEKINE ANn W. W. KIELLEY, Biockim. Biophys. Aaa, 81 (1964) 336. M. F. MORALESAND K. HOTTA, J. Biol. Chem., 235 (196o) 1979. S. V. PERRY AND J. COTTERILL, Biochem. J., 96 (1966) 224. N. AZUMA,J. Biochem. Tokyo, 63 (1968) z3o. G. HsGYI AND A. MOHLRAD,A S s Biochim. Biophys. Acad. Sci. Hung., 3 (1968) 425 • K. SEKIYAAND Y. TONOMURA,J. Biochem. Tokyo, 61 (1967) 787 . F. MORITA,J. Biol. Chem., 242 (I967) 45o1.
Biochim. Biophys, Acts, 205 (197o) 355-36o
35~
BBA 45932 REACTION OF MYOSIN W I T H SALICYLALDEHYDE II. E F F E C T OF SALICYLALATION ON T H E ATPase ACTIVITY OF MYOSIN A. MI~HLRAD, K. A J T A I AND F. F A B I A N
Department of Biochemistry, E6tv6s Lordnd University, Budapest (Hungary) (Received December i8th, 1969)
SUMMARY
The effect of salicylalation on the biological properties of myosin was studied. I. The ATPase activity of myosin is affected by salicylalation if the treatment is carried out at higher pH than 6.5. The MgZ+-activated ATPase shows a maximal curve with 25o-380% maximal activation when 25-70 moles of salicylaldehyde are bound per mole of myosin. The EDTA-activated ATPase decreases with increasing salicylalation. Ca~+-activated ATPase shows a small increase with increasing salicylalation. 2. Less salicylaldehyde is bound if the treatment is carried out in the presence of ATP, while that of PPi does not affect the degree of salicylalation. The enzymic properties of myosins salicylalated in the presence of ATP or PPt are not different from those of the samples treated in their absence. 3- Salicylalation decreases ATP sensitivity of ATPase and superprecipitation of actomyosins reconstituted from salicylalated myosins only if more than 50 moles of salicylaldehyde are bound per mole myosin.
INTRODUCTION
It was shown in the previous paper 1 that salicylaldehyde specifically reacts with the lysyl residues of myosin. Since it was tound in the investigations of TOSOMUgh and co-workers z-4 and of FAmIAN AND MOHLRAD~ that the trinitrophenylation of the e-amino groups of myosin b y 2,4,6-trinitrobenzenesulphonate (TBS) altered the enzymic properties of myosin and the alteration was observed to be markedly different from that caused b y the blocking of the sulphydryl groups of myosin, it seemed of interest to study the changes in enzymic activity caused by the modification of the lysyl residues with another amino group reagent. METHODS
The preparation of myosin and the treatment with s~icylaldehyde, the determination of bound salicylaldehyde and protein concentrations were performed as described in MATERIALSAND METHODSin the previous paper I. Abbreviation: TBS, 2,4,6-trinitrobenzene sulphonate.
Biochim. Biophys. Aaa, 205 (i97 o) 355-360
356
A. MUHLRAD et al.
The ATPase activity of myosin was measured in a solution containing I mg of myosin per ml, 4 mM ATP, 50 mM Tris-maleate buffer (pH 7.6) and alternatively 5 mM EDTA, 5 mM CaCI~ or 2 mM MgC1v Also present was 0.5 or o.o5 M KC1. The measurements were carried out on samples of 2 ml at 22 °. Incubation was terminated b y the addition of 2 ml of IO % trichloroacetic acid. Pi was measured b y the method of FISKE AND SUBBAROW6. The ATPase activity was evaluated as #mole Pt per mg of myosin per rain. The incubation time was chosen so as to obtain a decomposition of the terminal phosphate of ATP to less than 25 %. The ATPase activity of synthetic actomyosin was measured in a solution containing o.8 mg of myosin per ml, o.132 mg of actin per ml, 2 mM ATP, 2 mM MgClz and io mM Tris-maleate buffer (pH 7.4) at o °. The superprecipitation was measured by the method of EBASHI7 at 66o m/~ with a Spectromom 360 spectrophotometer at room temperature. The conditions of the test were 0.8 mg of myosin, 0.066 mg of actin per ml, o.I mM ATP, 5 mM Trismaleate buffer (pH 7.4) and 2 mM MgC1v Superprecipitation was evaluated from the difference between the values of the absorbance measured before, then 3 rain after, the addition of ATP. The actomyosin formation at high ionic strength was established from the measured ATP sensitivity. The measurements were carried out in an Ostwald viscometer using the method of B~.R/~.NY et al. s under the following conditions: I mg of myosin per ml, 0.33 mg of actin per ml, 0.5 M KCl-o.o2 M borate buffer (pH 7.4) at o °. RESULTS
The ATPase activities of myosins salicylalated at pH 6.5, 7.6, 8:0 and 9.0 were measured alternatively in the presence of Mg2+, Ca *+ or E D T A each time in the presence of 0.5 M KC1. At p H 8 an additional run was made in the presence of 0.05 M KCI. The three types of the ATPase activity curve measured as a function of salicylalation at various p H ' s are shown in Fig. I. The ATPase activity curves (Fig. I) measured in the presence of Mg*+ show m a x i m a as a function of salicylalation. The m a x i m a of the curves are 25-7o moles of bound salicylaldehyde per mole of myosin and appear at higher degrees of salicylalation for higher values of p H of the treatment. The maximal values of activation lie between 25 ° and 380 %. In the sample treated b y salicylaldehyde at p H 6.5 the activation was hardly appreciable and also much less, not more than 13 moles of salicylaldehyde being bound per mole of myosin (see also Fig. IO of the previous paper1). I t seems that the very poor activation cannot be related to the decrease in the complex formation at p H 6.5, since at p H 7.6 at the same degree of salicylalation (13 moles of salicylaldehyde bound per mole of myosin) the activation was measured as approx. 2oo %. I t seems more probable t h a t the lysyl residues having some role in the ATPase activity do not react with salicylaldehyde at p H 6.5. The m a x i m a of the Ca2+-activated ATPase as a function of salicylalation were relatively small as compared with those measured in the presence of Mg2+, and at higher degrees of salicylalation the activity was lower than that measured on the non-treated control. This m a y be due to unspecific conformational changes caused by the binding of numerous moles of salicylaldehyde to the myosin molecule. Biochim. Biophys. Acta, 205 (197 o) 355-36o
SALICYLALATION OF MYOSIN. I I
a
357
a
.>
2oG
a
w
.
.
.
5o
.
, ~0
mo~es of ~alicyfaMehycle bound p e r m o l e
.
.
n
. 2o0
rnyosi'n
Fig. z. The ATPase activity of myosin salicylslated at different pH's. Myosin was sslicylalated in and dialysed against a s o l u t i o n containing 0. 5 M KCI and 20 mM Tris--maleate buffer (a and b), or 20 mM borate buffer (c and d). a. pH 6.5. b. pH 7.6. c. pH 8.0. d. pH 9.0. For o t h e r c o n d i t i o n s of the salicylalation see MATERIALS AND METHODS i n t h e previous paper I. Conditions of the enzymic assay: i mg of myosin per ml; 50 mM Tris--maleate buffer (pH 7.4)i 4 mM ATP; 0. 5 (D, O, ×) or 0.05 (E, O, * ) M KCI; and alternatively 5 mM EDTA (O, O), 5 mM CaCIs ( ×, . ) or 2 mM MgCI9 (D, ss). Control (lOO%) activities (/~moles Pi per mg myosin per rain): a. Call 2, 0.29; EDTA, o.51; MgCI~, o.oo41, b. CaCI~, 0.25; EDTA, o.6o; MgCll, o.0o44, c. o.5M KCI: CaCll, 0.22; EDTA, 0.70; Mg,CIv 0.0054; 0.05 M KCI: CaCla, 0.46; EDTA, o.141MgC] v 0.0093. d. CaCIz, o.17; EDTA, 0.82; and MgC12, 0.0055. The ATPase activity measured in the presence of EDTA, i.e. K+-activated ATPase ~, m a r k e d l y decreases with increasing salicylalation. The measurements of ATPase activities at low ionic strength of the sample treated b y salicylaldehyde at p H 8 (Fig. IC) showed in each of the curves, for activity against salicylalation, lower values t h a n those measured at high ionic strength. The effect of the presence of magnesium A T P and magnesium p y r o p h o s p h a t e on the salicylalation of myosin was also studied since it was observed b y TONOMURA, et al. 1°, as well as b y F~BIAN AND MiJHLRADs, t h a t in the presence of either c o m p o u n d less lysyl residues were trinitrophenylated t h a n in their absence. A similar phenomenon was also observed b y BARANY et al. n on the reaction of myosin with i-fluoro-2,4dinitrobenzene. As a p p a r e n t from Fig. 2, the presence of A T P reduced the degree of salicylalation, but P P I did not appreciably affect the reaction between salicylaldehyde a n d myosin. I n this experiment an additional control sample was used which was treated b y salicylaldehyde in the presence of the same magnesium concentration as t h a t of the magnesium A T P or magnesium p y r o p h o s p h a t e test samples. No difference was observed between the reaction yields of the additional and the usual (see MATERIALSAND METHODS in the previous paper 1) control samples. Biochim. Biophys. Aaa, 205 (197o) 355-360
358
A. MUHLRADet al.
The K+-activated ATPase (ATPase measured in the presence of EDTA) of myosins salicylalated in the presence of magnesium ATP, magnesium pyrophosphate or Mg~+ showed the same dependence on salicylalation as the control sample treated by salicylaldehyde in the absence of these ingredients (Fig. 3). The MgS+-inhibited ATPase curves are also very similar to the control curve if magnesium pyrophosphate or Mg2+ are present during the treatment with salicylaldehyde. Higher activation at a lower degree of salicylalation was observed only in the presence of magnesium ATP during the treatment. .c ~150 E
E
~
4OC I00
3C( 2OC
~
o moles
I0-3 I0"~ of salicyloldehycle in Mcutmti~ solution
<~
0
~o
m0~-~0f sollc~ld~l'~ b0u/~ i ~ f ~
rr~,,o~in
Fig. 2. Effect of A T P a n d P P t on t h e salicytalation of myosin. Conditions of salicylalation: B o t h i n c u b a t i n g a n d dialysing solutions: o. 5 M KC1 a n d o.i M borate buffer (pH 8); o t h e r c o n s t i t u e n t s of i n c u b a t i n g solution: none ( × ] ; 6 m M MgC11 ( * ) ;'6 m M MgCI~ a n d 6 m M s o d i u m p y r o p h o s p h a t e ( 0 ) ; 6 m M MgCIj a n d 6 mM ATP (A). F o r dialysing solution: none ( × ) ; 2 m M MgC12 ( , ) ; 2 m M MgCI~ and x m M sodium p y r o p h o s p h a t e (Q) ; 2 m M MgCI~ knd i m M A T P (A). F o r other conditions see MATERIALS AND METHODS in t h e previous p a p e r 1. Fig. 3. ATPase activity of m y o s i n saticylalated in t h e presence of ATP and PPt- For conditions of saIicylalation see legend to Fig. 2. Salicylalation was carried out in t h e p r e s e n c e ' o f : A T P and Mg 2+ ( O , 0. ) ; P P I a n d Mg 2+ ( [3, I ) ; Mg t+ (iX, A ) ; ho e x t r a c o n s t i t u e n t ( × , * ). ATPase activity m e a s u r e d in t h e presence of 5 m M E D T A ( O , I , A , * ) or 2 m M MgCls (O, rT, A, X). Control ( i o o % ) activities: O , o.31; I , 0.4o; J k , o;34; * , o.64; O , o.oo48; [3, o.oo44; &, o.oo39; × , o . o o 4 3 / , m o l e Pl per m g m y o s i n per rain. T h e control E D T A - a c t i v a t e d ATPases of those samples t h a t were t r e a t e d in t h e presence of Mg I+ (O, I , A ) were low because of t h e relatively high Mg I+ c o n c e n t r a t i o n p r e s e n t during t h e m e a s u r e m e n t of ATPase.
The effect of complex formation with salicylaldehyde on the characteristic properties of actomyosin synthesized from salicylalated myosin was also studied. The experimental data on ATPase activity, actomyosin formation (evaluated from the measured ATP sensitivity) and sup~rprecipitation are listed in Table I. The actomyosin ATPase measured in the presence of Mgs+ at low ionic strength did not change on salicylalation when less than 46.5 moles of salicylaldehyde were bound per mole of myosin. At higher degrees of saliCylalation a sharp decrease in actomyosin ATPase was observed which m a y be due to the unspecific conformation change taking place on the introduction of a large number of salicylaldehyde residues into the myosin molecule. The percentage activation of ATPase b y actin decreased even on the addition of small amounts of salicylaldehyde because of the activating effect of salicylalation on myosin ATPase in the presence of Mg2+ (Table I, Column 2). (Activation b y actin was evaluated as actomyosin ATPase/myosin ATPase × IOO. Therefore, if the myosin ATPase increases without alteration of actomyosin ATPase, the activation b y actin decxeases.) A similar phenomenon was observed by F£BIAIq A~D M~dI~LRAD~ Biochim. Biophys. Acta, 205 (197 o) 355-36o
SALICYLALATION OF MYOSIN. I I
359
TABLE I T H E CHARACTERISTIC P R O P E R T I E S OF ACTOMYOSIN R E C O N S T I T U T E D FROM SALICYLALATED MYOSIN
For detailed conditions of enzymic assays, determinations of ATP sensitivity and superprecipiration see METHODS.Myosin ATPase was measured under the same conditions as actomyosin ATPase. Percentage activation by actin = actomyosin ATPase/myosin ATPase × lOO. Moles oJ salicylaldehyde bound per mole myosin
Myosin A TPase
A ctomyosin A TPase
A TP sensitivity
Superprecipitation
2
Percentage activation by actin 3
I
4
5
-3.2 5.3 13.7 46.5 82.6 122.8
0.0234 o-o314 0.0340 0.0378 o.o34o o.o192 0.0058
0.227 0.246 0.249 0.239 O.lO9 0.058 o,oo 3
97 ° 784 734 632 466 300 52
16o 154 158 156 143 I33 80
o.61 0.57 0.54 o.61 o.36 0.35 o.oo
for t r i n i t r o p h e n y l a t e d m y o s i n *. A c t o m y o s i n formation a n d superprecipitation showed a similar decrease to t h a t of a c t o m y o s i n ATPase when more t h a n 46.5 moles of salicylaldehyde were b o u n d per mole of :myosin.
DISCUSSION The effect of the blockingof amino groups with salicylaldehydeon the enzymm properties of myosin is comparable to that observed on the blocking of these groups with TBS~-~.' In both :instances th e Mg2+-inhibited ATPase of myosin showed activation, whereas the Ca~+-activated ATPase was left almost unchanged by the blocking (Figs. 2, 3 and 4, and ref. 5)- In contrast, the blocking of sulphydryl groups results in the activation of both the Ca2+-activated and Mg~+-inhibited ATPase1~-1~. The K~activated ATPase of myosin decreases on any modification of myosinS,X~,I~,5,i~,is and hence also on the blocking of sulphydryl or amino groups. The other biological properties of myosin, such as the actomyosin formation, tlle Mg~+-activated ATPase and the superprecipitation of actomyosin synthesized from the modified myosin, are also affected ]n almost the same way by the modification with salicylaldehyde as by that with TBS5. The effect ol trinitropheny!ati~n on the myosin ATPase is assumed to be due to the removal of the positive charges of one or some lysyl residues located in or near to the active site of the molecules5. The differences which can nevertheless be observed between the effects of trinitrophenylation and salicylalationon the enzymic properties suggest that trinitropheny]ation more specificallyaffects the lysyl residues having some role in the ATPase activity. This would explain the observation that the activation of the Mg~+-inhibited ATPase of myosin is 2000 %, if approx. 5 lysyl residues are trinitrophenylated per mole of myosin5, as compared with an activation of 380 % if approx. 5° lysyl residues are salicylalated per mole of myosin. The effect of the presence of the substrate is also different in the two cases. If ATP is present during trinitropheny]ation the lysyl residues having some role in the ATPase activity are not modifiedsince the change in the enzymic properties is much Biochim. Biophys. Acta, 205 (197o) 355-36o
360
A. MUHLRAD et al.
less a p p a r e n t t h a n in t h e absence of t h e s u b s t r a t e . On t h e other h a n d t h e m a x i m u m a c t i v a t i o n of t h e Mg¢+-inhibited A T P a s e is o b s e r v e d a t a lower degree of s a l i c y l a l a t i o n in t h e presence t h a n in t h e absence of ATP. The different effects of t h e two r e a g e n t s in t h e presence of t h e s u b s t r a t e can b e e x p l a i n e d if one a t t r i b u t e s to t h e l y s y l residues h a v i n g some role in t h e A T P a s e a c t i v i t y a higher affinity for T B S t h a n t h e o t h e r l y s y l residues of myosin. On t h e a d d i t i o n of A T P a n d Mg ~+, however, a c o n f o r m a t i o n a l change t a k e s place u, zg, ~o b y which t h e higher affinity of these specific l y s y l residues is levelled off. T h e l y s y l residues in question, however, h a v e no highei affinity for s a l i c y l a l d e h y d e a n d therefore t h e a d d i t i o n of t h e s u b s t r a t e , w h i c h equalizes t h e affinities of t h e residues for TBS, does n o t specifically affect t h e s a l i c y l a l d e h y d e r e a c t i o n of t h e l y s y l residues i n v o l v e d in t h e A T P a s e a c t i v i t y of m y o s i n . ACKNOWLEDGEMENTS T h e a u t h o r s are i n d e b t e d to Dr. N. A. B i t 6 a n d Mrs. J. Monori for t h e i r k i n d h e l p in t h e p r e p a r a t i o n of this p a p e r . REFERENCES I 2 3 4 5 6
7 8 9
io n I2 13 14 15 16 17 18 19 20
A. MOHLRAD, K. AJTAI AND F. FkBIJ,N, Biockim. Biophys. Acts, 205 (197o) 342. S. KUBO, S. TOKURAAND Y. TONOMURA,J. Biol. Chem., 235 (196o) 2835. S. KITAGAWA,J. YOSHIMURAAND Y. TONOMURA,J. Biol. Chem., 236 (1961) 902. H. TOKUYAMAAND Y. TONOMURA,J. Biochem. Tokyo, 62 (1967) 456. F. FXBIXN AND A. MOHLRAD,Biochim. Biophys. Aaa, 162 (i968) 596. C. H. FISKE AND Y. SUBBARow, f . Biol. Chem., 66 (I925) 375. S. EBAsm, J. Bioohem. Tokyo, 5° (I961) 236. M. BkI~NY, B. NAGY, F. FII~I~LMA~ AND A. Cm~mBACH, J. Biol. Chem., 236 (I961) 2917 A. MOHLRAD, F. F~BI~N AND N. A. BII~, Biockim. Biophys. Acts, 89 (1964) 186. Y. TONOMURA, J. YOSmMURA AND T. OmSHI, Biochim. Biophys. Aaa, 78 (I963) 698. M. BLI~NY, G. BArtoN AND K. B/~1Lt~NY,jr. Biol. Chem., 244 (z969) 648. W. W. KIELLEY AND L. B. BRADLEY, J. Biol. Chem., 218 (1956) 653. H. M. LEvY AND E. M. RYAN, Bioahim. Biopltys. Aaa, 46 (i961) 193. T. SEKINE ANn W. W. KIELLEY, Biockim. Biophys. Aaa, 81 (1964) 336. M. F. MORALESAND K. HOTTA, J. Biol. Chem., 235 (196o) 1979. S. V. PERRY AND J. COTTERILL, Biochem. J., 96 (1966) 224. N. AZUMA,J. Biochem. Tokyo, 63 (1968) z3o. G. HsGYI AND A. MOHLRAD,A S s Biochim. Biophys. Acad. Sci. Hung., 3 (1968) 425 • K. SEKIYAAND Y. TONOMURA,J. Biochem. Tokyo, 61 (1967) 787 . F. MORITA,J. Biol. Chem., 242 (I967) 45o1.
Biochim. Biophys, Acts, 205 (197o) 355-36o