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On the inhibition of myosin-ATPase by adenosine diphosphate
.~H(‘HIVES
On
Oli
the
BIWHEMISTRY
Inhibition
ANI)
BIOPHYSICS
96, 51-55
of Myosin-ATPase
(19621
by Adenosine
Diphosphate
the apparent CaATP constant in 0.1 M NaCl at pH 7.0, that pCa changes from 2.54 t,o 2.785 which still means :I 31$Z0 artivit,y drop. For ilTP = 10ea M, one calculates a pCa change from 2.67 t,o 2.88 which also means :I 31% activity drop. The ralculat,ions of free C:t are made by means of the equation : Ca = ~Clt
FIG.
centration. (C2Hs)aNBr
1. pH shift at pH 6.8-6.9 versus Ca conADP = 1.03 X lo-” M, upper curve in 0.1 M; lower curve in NaCl 0.1 M.
by approximately 2.30 [compare (S)]. Walaas’ apparent constant of 660 (pK = 2.82) was measured at pH 8.2 in 0.1 M NaCl at 23” (9) and results in a true constant of 660(1 + 5 X 0.1 + 106.90 X 10-s.“) = 1.55 X 660 = 1025, in good agreement with t,he value derived from the pH shift. One can calculate that for Ca = 3 X 1O-3 111 and ADP = 3 X 10m3 M, free Ca = 1.7 X 10m3, using the value 462 for the apparent association constant, : Ca = $$[(zCa
-
zADP
-
&) (1)
+ in which
d(zCa :
ZCa
ZADP
zADP
- K#
= total
Ca
= total
ADP
Kd = apparent
Formula
zBDP
= ADP
-
of
con-
in which Kd’ is the apparent dissociation constant of CaATP (= l/3050). The value 3050 is derived from the true association const,ant 8500 divided by 2.80 [= 1 + 10 X CNa) + 106.go X (H)], compare Ref. (8). When, however, zCa = 2 X 10m2, Ca = 1.74 X 1OV for ZADP 3 X 1O-3, so that pCa changes from I .70 to 1.76, which gives an activity increase of l-270 according to the mentioned Ca optimum curve. In t,he presence of ATP = 10-Z M, this would be about the same as CaATP < 10e3 M, or Ca + CaADP > 1.9 X IOVM. ForZCa = 1.9 X lOV, pCa changes from 1.72 to 1.78 which results, likewise, in an activit)y increase of l&2%.
IR-~1~1~10~
BT BDP
OF SI~osrx-B'l'l'.zs~
The experiments on SDP inhibition were performed in a milieu of 0.1 M NaCl, 2 X lo--* M C&l? , 0.01 !lI Tris buffer, pH 7.0 at 25”. The final mposin concent,ration was 0.1%. The ATP concentration was varied between 5 X 10 ~5 and 5 X 1O-4 JI while ADP was added to :I final concentr:+
ASSWI~TIOK
U/J@4
CaADP
CaATP
CONST.~NT SHIFT; ISITIAL
0.1 M (CaHs)&Br;
I OF CaADP pH 6.8-6.9 1 .03 X lo-”
FROM M ADP
Ca
(1) is derived Ca X ADP
CaADP
TABLE dissociation
stant, Ca = free
+ 4&ZCa]
-
by solving
Eq.
(2) for
Ca:
= Kd X CaADP
+ CaAl>P
APH
Ca
l&H
0.05 0.70 0.15
1.2 x 10-4 2.5 X IOF 4.2 X lo-’
1 .I2 1.26 1.42
K
1010 1090 1030
= CaaDP k’ = 1053 zt 23
Ca = Xh
-
CaADP
= Z.?a
-
(2)
- zADp 1+h;”
0.1 M NaCl;
1.03 X 10m4 X ADP
CLt This means that, 1)~ adding this amount of ADP the pCa (= -log Ca) changes from 2.52 to 2.77 which means an activit,y drop of 31y0 according to the Ca optimum curve, compare Fig. 8 in Ref. (1). This was calculated in the absence of ATP. If ATP = lo-* M, it can be calculat,ed, using 3050 for
0.05
0.10 0.15
1.60 3.i.5 7.00
X lo-? X lo-” x lo-~ I(
= 716 zk 40
I .12 1.26 1.42
774 718
656
pH
INHIBITIOr\’
OF
MTOSIW.4TPASE
53
SDP
7-
tion of 15.6 X 10m4 and 31.3 X 10e4 111. The results were plotted as a reciprocate activity-substrate plot according to Lineweaver and Burk (Fig. 2). K, was calculated from intercept (l/V,) and slope (K,/V,) for the experiments without ADP (J’, is maximal nct,ivit,y) .3
K,/V,
BY
= 0.65 X IOP; v, = 0.125;
Km = 0.28 X IO-& The experiments with through the same point ing competitive inhibition, the enzymic site. The
ADP on the or slope
give lines which go ordinate, indicatbinding of ADP to of these lines is:
Km/T7vn{l+ [(ADP)IK,I). 15.65 X lo-”
K,z
= 1.75 X lOV4 ;
I 0.5
I 1.0
RECIPROCATE
I& Km jyr
m(
lf
31.30
X 10-d Ki
= 9.3 x
10-d
= 2.80 x 10-4; )
Ki = 9.5 X 1O-1 In these calculations all ATP present is supposed to act as substrate and all iZDP as inhibitor; no difference is made here between the free ATP or ADP and their calcium complexes. The result is that Ki is 33.6 X K, Bs the value of Ki is independent of the ADP concentration, it follows that one molecule of ADP combines with one active site, or with one myosin molecule as one molecule contains one active sit,e, as was established by Nanninga and Mommaerts
(2). Tlnm
clourts~
OF THE
HYDROLYSIS
OF
ATP
The hydrolysis of ATP by myosin was measured as a function of time in t.he same milieu for 6.95 X lo-* .{I ATP. The myosin was precipitated four times in order to free it completely from traces of myokinase. This is done in order to come to a completion of I’ liberat,ion when all ATP is split, to ,4Dl’ [compare (IO)]. It is found (Fig. 3) that, the 9 The liberated inorganic phosphate is measured as the extinction at 610 III~ of 5 ml. TCA filtrate (one part sample plus one part 5y0 t richloroacet~ic acid) after nddit,ion of 4 ml. 2.5 b molybdenut~~ sufuric acid and 1 ml.0.2’% st)annous chloridesolution. The activity is expressed as the difference in this extinction (Eslo) for two samples taken 100 sec. apart (for the first 100 WC. this is a linear rcxlntion). The urlit of the activitg is not important as this cancels out, in the calculation of K,,, or K,
I 1.5
Xl04
ATP CONCENTRATION
FIG. 2. Reciprocat,e act.ivit,y versus reciprocate substrate concentration in absence and presence of ADP, see t,est. The activit,y is expressed as the slope of the &,I0 versus time curve (&‘slo per 100 sec.)3.
i Y 0I k a E 2
+, 1.0- ------ ----------5:X/X- +- 4= ---------------~ / . /-/’ / 0.8/ J / / / / 0.6/ x/ / /
iz 0.4 m J a w 0.2
0’ I
/
!i
/' .g'
Cr
V
7
I
5
I
I
IO
15
MIKUTES
FIG.
3. Phosphate liberated rlTP versus t,irne for 6.95 X 1OW 7.0 in 0.1 ~11 KaCl, 0.01 :I2 Tris, 0.02 Dotted curve is for Ki = K, The half of the original activity bvhen is hydrolyzed.
in moles/mole 111 ATP at pH X CaCl2 at 25”. activity is one92% of the ATP
activity decreased to one-half of the init,ial activity only after 92% of ATP is hydrolyzed. This is in good agreement with the values for K,n and Kl which were calculated above. If 2) = activity; iv,,, = maximum activity; /I,, = initi:ll activity; S,, = initial ATP ~oncrnt rat ion;
j = fraction of ATP hydrolyzed, then ,fS,, = ADP roncentrat ion and (1 - ,j)SO = .4TI’ conrent ration :tl :IIIJ’ time during hydrolqis. Therefore: 1’,n(l
(, =
- mo
(3)
Km
using the formula for competitive 2, =
inhibition.
For
0.5VO)
'1 =
0.51,,& Ic,is,
(4)
It follows from Eys. (3) and (4) that for 1’ = 0.521,,:
of the acti\+t,y. =\ reduct,ion of :2L %, in the x%ivity was cxlculated for a (In cwncentralion of 3 X 10~~ ;II and 3 X 10~~ ;1[ ADI’. Howwcr, with the optimal C’:L c*onwntraCon as used in this study, such apparent c#‘ccts are eliminated. There map hc other wsrs whcrc swmingly competitive inhibitjion may h(h dut to the binding of a hi\-nlent mrtal ion for which both the substrsk and the inhihitjor have an affinit’y, so that the kinetic cvidcncc ulorw does not, ncccssurily indicate an affinity of the inhibitor to the cnzymk sit,c. This would seem likely, (kg., for the inhihit.ion of aglyccrophosphntc: by Vcrscnc (7).
This formula gives with K,,, = 0.28 X 10-l; K, = 0.3 X 1Oe1t,he follo\ving relation Iwtwecn j and S,, is 0 ,I
I 0.804
2 0.871
5 0.0”5-.
10 20 x 1OV” O.!)-t(i 0.958
The dotted line in Fig. 3 rel)resents the :11,1)rosinutte rourse of the hydrolysis of K,,, were rqwl to Iii
In this
21 =
0.25~~
drnwrl
case f’ = 0.50 for 1’ = 0.5~~ ; 0.75 for 0.25 for I’ = 0.75~~~ The line is so thxt these corlditiorls :Iw :ipprorirll:itel~ and
met with. P' =
DISCC8810?i The reinclusion from this work is that at, pEI 7.0 ADI’ is a compc‘tit,iw inhibitor, but considerably ltw thau was npparcnt from the work of Grern and ,IIommwrts (4). Blum found that addition of an M11’ COP wntration of 10 times the initial LkTI’ couwutration gave a slight inhibitory t#kct (wlativc activity ‘71.4’ C) (3). The results in this study point to a similar low inhibition c3ffec.t at pH 7.0. &WV found that thr 11111’ inhibition at, pH 6.4 is wnsidcrably 1~:s than t.hnt, at pH 9.0 ((9.
1'
p
=
F’ =
-77700 .y
+4140
F = -8100 iref.
II)
Thcrcfore: -7700
+ .Y + 4140 = -MOO
It is utldcrstand~Lblc that i hc libtwt cd free fw’rgy of stop 2 is smnllt~r than -8400 (&ich would bc provided by the splitting) when energy is nwdcd to rwnow &cl t crm-
INHIBITIOK
OF
RITOSIK-ATPASE
nal P from t,he myosin. This supportfs therefore the theory that, in the complex the terminal P of ATP is bound to t#he myosin, probably by means of a Ca bridge since it is know that Ca binds both to myosin and to the terminal P of ATP (1, 8).
The myosin ATP-asc is inhibited by ,4DP competit~ively. The inhibition constant is 33 tilnw the hlichnelie constant. This small amount of inhibit’ion explains the product wrsus time curve of the ,4TP hydrolysis and is confirmed by this curve. The association constant of CaADP was determined as 1050. In order to establish true ADP inhibition Ca has to be in excess, so that Ca chelation by ADP does not affect Ca nvailnhlc to the myosin.
The nuthor wishes 1;. H. M. Mommnerts this work.
to :~ckr~owledgc Dr. Wilfried for all his encouragement in
BY
ADP
55
On
Oli
the
BIWHEMISTRY
Inhibition
ANI)
BIOPHYSICS
96, 51-55
of Myosin-ATPase
(19621
by Adenosine
Diphosphate
the apparent CaATP constant in 0.1 M NaCl at pH 7.0, that pCa changes from 2.54 t,o 2.785 which still means :I 31$Z0 artivit,y drop. For ilTP = 10ea M, one calculates a pCa change from 2.67 t,o 2.88 which also means :I 31% activity drop. The ralculat,ions of free C:t are made by means of the equation : Ca = ~Clt
FIG.
centration. (C2Hs)aNBr
1. pH shift at pH 6.8-6.9 versus Ca conADP = 1.03 X lo-” M, upper curve in 0.1 M; lower curve in NaCl 0.1 M.
by approximately 2.30 [compare (S)]. Walaas’ apparent constant of 660 (pK = 2.82) was measured at pH 8.2 in 0.1 M NaCl at 23” (9) and results in a true constant of 660(1 + 5 X 0.1 + 106.90 X 10-s.“) = 1.55 X 660 = 1025, in good agreement with t,he value derived from the pH shift. One can calculate that for Ca = 3 X 1O-3 111 and ADP = 3 X 10m3 M, free Ca = 1.7 X 10m3, using the value 462 for the apparent association constant, : Ca = $$[(zCa
-
zADP
-
&) (1)
+ in which
d(zCa :
ZCa
ZADP
zADP
- K#
= total
Ca
= total
ADP
Kd = apparent
Formula
zBDP
= ADP
-
of
con-
in which Kd’ is the apparent dissociation constant of CaATP (= l/3050). The value 3050 is derived from the true association const,ant 8500 divided by 2.80 [= 1 + 10 X CNa) + 106.go X (H)], compare Ref. (8). When, however, zCa = 2 X 10m2, Ca = 1.74 X 1OV for ZADP 3 X 1O-3, so that pCa changes from I .70 to 1.76, which gives an activity increase of l-270 according to the mentioned Ca optimum curve. In t,he presence of ATP = 10-Z M, this would be about the same as CaATP < 10e3 M, or Ca + CaADP > 1.9 X IOVM. ForZCa = 1.9 X lOV, pCa changes from 1.72 to 1.78 which results, likewise, in an activit)y increase of l&2%.
IR-~1~1~10~
BT BDP
OF SI~osrx-B'l'l'.zs~
The experiments on SDP inhibition were performed in a milieu of 0.1 M NaCl, 2 X lo--* M C&l? , 0.01 !lI Tris buffer, pH 7.0 at 25”. The final mposin concent,ration was 0.1%. The ATP concentration was varied between 5 X 10 ~5 and 5 X 1O-4 JI while ADP was added to :I final concentr:+
ASSWI~TIOK
U/J@4
CaADP
CaATP
CONST.~NT SHIFT; ISITIAL
0.1 M (CaHs)&Br;
I OF CaADP pH 6.8-6.9 1 .03 X lo-”
FROM M ADP
Ca
(1) is derived Ca X ADP
CaADP
TABLE dissociation
stant, Ca = free
+ 4&ZCa]
-
by solving
Eq.
(2) for
Ca:
= Kd X CaADP
+ CaAl>P
APH
Ca
l&H
0.05 0.70 0.15
1.2 x 10-4 2.5 X IOF 4.2 X lo-’
1 .I2 1.26 1.42
K
1010 1090 1030
= CaaDP k’ = 1053 zt 23
Ca = Xh
-
CaADP
= Z.?a
-
(2)
- zADp 1+h;”
0.1 M NaCl;
1.03 X 10m4 X ADP
CLt This means that, 1)~ adding this amount of ADP the pCa (= -log Ca) changes from 2.52 to 2.77 which means an activit,y drop of 31y0 according to the Ca optimum curve, compare Fig. 8 in Ref. (1). This was calculated in the absence of ATP. If ATP = lo-* M, it can be calculat,ed, using 3050 for
0.05
0.10 0.15
1.60 3.i.5 7.00
X lo-? X lo-” x lo-~ I(
= 716 zk 40
I .12 1.26 1.42
774 718
656
pH
INHIBITIOr\’
OF
MTOSIW.4TPASE
53
SDP
7-
tion of 15.6 X 10m4 and 31.3 X 10e4 111. The results were plotted as a reciprocate activity-substrate plot according to Lineweaver and Burk (Fig. 2). K, was calculated from intercept (l/V,) and slope (K,/V,) for the experiments without ADP (J’, is maximal nct,ivit,y) .3
K,/V,
BY
= 0.65 X IOP; v, = 0.125;
Km = 0.28 X IO-& The experiments with through the same point ing competitive inhibition, the enzymic site. The
ADP on the or slope
give lines which go ordinate, indicatbinding of ADP to of these lines is:
Km/T7vn{l+ [(ADP)IK,I). 15.65 X lo-”
K,z
= 1.75 X lOV4 ;
I 0.5
I 1.0
RECIPROCATE
I& Km jyr
m(
lf
31.30
X 10-d Ki
= 9.3 x
10-d
= 2.80 x 10-4; )
Ki = 9.5 X 1O-1 In these calculations all ATP present is supposed to act as substrate and all iZDP as inhibitor; no difference is made here between the free ATP or ADP and their calcium complexes. The result is that Ki is 33.6 X K, Bs the value of Ki is independent of the ADP concentration, it follows that one molecule of ADP combines with one active site, or with one myosin molecule as one molecule contains one active sit,e, as was established by Nanninga and Mommaerts
(2). Tlnm
clourts~
OF THE
HYDROLYSIS
OF
ATP
The hydrolysis of ATP by myosin was measured as a function of time in t.he same milieu for 6.95 X lo-* .{I ATP. The myosin was precipitated four times in order to free it completely from traces of myokinase. This is done in order to come to a completion of I’ liberat,ion when all ATP is split, to ,4Dl’ [compare (IO)]. It is found (Fig. 3) that, the 9 The liberated inorganic phosphate is measured as the extinction at 610 III~ of 5 ml. TCA filtrate (one part sample plus one part 5y0 t richloroacet~ic acid) after nddit,ion of 4 ml. 2.5 b molybdenut~~ sufuric acid and 1 ml.0.2’% st)annous chloridesolution. The activity is expressed as the difference in this extinction (Eslo) for two samples taken 100 sec. apart (for the first 100 WC. this is a linear rcxlntion). The urlit of the activitg is not important as this cancels out, in the calculation of K,,, or K,
I 1.5
Xl04
ATP CONCENTRATION
FIG. 2. Reciprocat,e act.ivit,y versus reciprocate substrate concentration in absence and presence of ADP, see t,est. The activit,y is expressed as the slope of the &,I0 versus time curve (&‘slo per 100 sec.)3.
i Y 0I k a E 2
+, 1.0- ------ ----------5:X/X- +- 4= ---------------~ / . /-/’ / 0.8/ J / / / / 0.6/ x/ / /
iz 0.4 m J a w 0.2
0’ I
/
!i
/' .g'
Cr
V
7
I
5
I
I
IO
15
MIKUTES
FIG.
3. Phosphate liberated rlTP versus t,irne for 6.95 X 1OW 7.0 in 0.1 ~11 KaCl, 0.01 :I2 Tris, 0.02 Dotted curve is for Ki = K, The half of the original activity bvhen is hydrolyzed.
in moles/mole 111 ATP at pH X CaCl2 at 25”. activity is one92% of the ATP
activity decreased to one-half of the init,ial activity only after 92% of ATP is hydrolyzed. This is in good agreement with the values for K,n and Kl which were calculated above. If 2) = activity; iv,,, = maximum activity; /I,, = initi:ll activity; S,, = initial ATP ~oncrnt rat ion;
j = fraction of ATP hydrolyzed, then ,fS,, = ADP roncentrat ion and (1 - ,j)SO = .4TI’ conrent ration :tl :IIIJ’ time during hydrolqis. Therefore: 1’,n(l
(, =
- mo
(3)
Km
using the formula for competitive 2, =
inhibition.
For
0.5VO)
'1 =
0.51,,& Ic,is,
(4)
It follows from Eys. (3) and (4) that for 1’ = 0.521,,:
of the acti\+t,y. =\ reduct,ion of :2L %, in the x%ivity was cxlculated for a (In cwncentralion of 3 X 10~~ ;II and 3 X 10~~ ;1[ ADI’. Howwcr, with the optimal C’:L c*onwntraCon as used in this study, such apparent c#‘ccts are eliminated. There map hc other wsrs whcrc swmingly competitive inhibitjion may h(h dut to the binding of a hi\-nlent mrtal ion for which both the substrsk and the inhihitjor have an affinit’y, so that the kinetic cvidcncc ulorw does not, ncccssurily indicate an affinity of the inhibitor to the cnzymk sit,c. This would seem likely, (kg., for the inhihit.ion of aglyccrophosphntc: by Vcrscnc (7).
This formula gives with K,,, = 0.28 X 10-l; K, = 0.3 X 1Oe1t,he follo\ving relation Iwtwecn j and S,, is 0 ,I
I 0.804
2 0.871
5 0.0”5-.
10 20 x 1OV” O.!)-t(i 0.958
The dotted line in Fig. 3 rel)resents the :11,1)rosinutte rourse of the hydrolysis of K,,, were rqwl to Iii
In this
21 =
0.25~~
drnwrl
case f’ = 0.50 for 1’ = 0.5~~ ; 0.75 for 0.25 for I’ = 0.75~~~ The line is so thxt these corlditiorls :Iw :ipprorirll:itel~ and
met with. P' =
DISCC8810?i The reinclusion from this work is that at, pEI 7.0 ADI’ is a compc‘tit,iw inhibitor, but considerably ltw thau was npparcnt from the work of Grern and ,IIommwrts (4). Blum found that addition of an M11’ COP wntration of 10 times the initial LkTI’ couwutration gave a slight inhibitory t#kct (wlativc activity ‘71.4’ C) (3). The results in this study point to a similar low inhibition c3ffec.t at pH 7.0. &WV found that thr 11111’ inhibition at, pH 6.4 is wnsidcrably 1~:s than t.hnt, at pH 9.0 ((9.
1'
p
=
F’ =
-77700 .y
+4140
F = -8100 iref.
II)
Thcrcfore: -7700
+ .Y + 4140 = -MOO
It is utldcrstand~Lblc that i hc libtwt cd free fw’rgy of stop 2 is smnllt~r than -8400 (&ich would bc provided by the splitting) when energy is nwdcd to rwnow &cl t crm-
INHIBITIOK
OF
RITOSIK-ATPASE
nal P from t,he myosin. This supportfs therefore the theory that, in the complex the terminal P of ATP is bound to t#he myosin, probably by means of a Ca bridge since it is know that Ca binds both to myosin and to the terminal P of ATP (1, 8).
The myosin ATP-asc is inhibited by ,4DP competit~ively. The inhibition constant is 33 tilnw the hlichnelie constant. This small amount of inhibit’ion explains the product wrsus time curve of the ,4TP hydrolysis and is confirmed by this curve. The association constant of CaADP was determined as 1050. In order to establish true ADP inhibition Ca has to be in excess, so that Ca chelation by ADP does not affect Ca nvailnhlc to the myosin.
The nuthor wishes 1;. H. M. Mommnerts this work.
to :~ckr~owledgc Dr. Wilfried for all his encouragement in
BY
ADP
55