- Email: [email protected]
Effect of bound-nucleotide substitution on the properties of F-actin
ARCHIVES
OF
RIOCHEMISTRY
‘Effect
of
AND
BIOPHYSICS
132,
Bound-Nucleotide
388-396
(1969)
Substitution
on the
Properties
of F-Act in JAMES Department
of Biophysics,
State
Received
E. ESTES’ University December
AND
of IVew 2, 1968;
CARL York accepted
MOOS2
at Buffalo, April
Bu$alo,
New
York
i4Si4
2, 1969
F-actins have been prepared in which the bound adenosine nucleotide has been replaced by inosine or cytidine nucleotide, and their properties have been studied. The intrinsic viscocity of IDP-containing F-actin was the same as that of ADP-containing F-actin, but the intrinsic viscosity of GDP-containing F-actin was about half this value. There were no differences in the myosin-binding affinities of the three types of I?-actin or in their ability to activate the magnesium-ATPase of myosin. On the other hand, the exchangeability of the F-actin-bound nucleotide, both in the presence and in the absence of myosin, was dependent on the type of bound nucleotide in the actin, increasing in the order IDP
While it has been well established that actin contains 1 mole of firmly bound adenosine nucleotide per mole of protein (l-4), the function of this bound nucleotide in muscle contraction is not yet understood. The bound adenosine nucleotide may be replaced by other nucleoside phosphates (5-7), and both the relative viscosities of F-a&ins containing bound inosine, cytidine, or guanosine nucleotides, and the abilities of these F-a&ins to form actomyosins which can be dissociated by ATP, are reported to be the same as those of normal F-ADPactin (8). Furthermore, an F-actin containing bound AMP, or even one lacking a bound nucleotide, retains the viscosity and myosinbinding properties of F-ADP-actin (9, 10). While none of these investigations gave any direct evidence that the actin-bound nu-
cleotide plays a role in muscle contraction, the results of mechanochemical studies of actin (11) and also the recent finding that the exchangeability of the F-actin-bound nucleotide is increased during superprecipitation of actomyosin (12, 13) have provided encouragement for continued speculation about the physiological role of actin and its bound nucleotide in contractile systems. In further pursuing this problem we have prepared F-a&ins in which the bound ADP is almost completely replaced by IDP or CDP, and have made quantitative comparisons of the properties of these actins and their interactions with myosin. The binding of the F-actin to myosin and its activation of the myosin ATPase were quite unaffected by the bound-nucleotide substitutions. On the other hand, the exchangeability of the a&in-bound nucleotide in the presence and absence of myosin, and the rate and extent of partial polymerization at low Mg++ concentration, were strongly dependent on the type of bound nucleotide in the actin, suggesting that the bound nucleotide influences
1 Present address: Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181. 2 Present address: Department of Biological Sciences, State University of New York, Stony Brook, New York 11790. 388
BOUND-NUCLEOTIDE
the interaction between F-a&in filament.
monomers
SUBSTITUTED
in the
F-ACTINS
389
measured. Blank extracts, used to correct the spectrum for contaminants in the reagents, were prepared in the same manner MATERIALS AND METHODS except that the protein sample was replaced with an equal volume of water. In mixtures Protein preparations. Myosin was prepared from rabbit skeletal muscle by a of inosine and adenosine nucleotides, the concentration of each nucleotide was calcumodification of t’he method of A. Szentlated from the absorbances at 250 260, and Gyijrgyi (14) as previously described (15). 290 rnp, while in the case of mixtures of Creatine kinase (ATP : creatine phosphoand adenosine nucleotides, t,he transferase, EC 2.7.3.2) was isolated and cytidine absorbances at 260, 280, and 300 rnp were purified by previously published procedures used. The nucleotide concentrations in t,he (16, 17). mixtures were calculated using the equations Actin was extracted from acetone-dried of Loring (20) and the published spectral muscle powder (14) at 0” to avoid contamconstants for the three nucleotides (21, 22). ination with tropomyosin (18) and purified It was assumed that the only ultraviolet according to Ulbrecht, Grubhofer, Jaisle absorbing components present in each and Walter (19). The washed F-actin extract were the t,wo nucleotides specified. pellets were stored in capped tubes in the The application of this method t’o art,ificial cold and used within six days. To replace known mixtures of ATP and ITP or of ATP the actin-bound adenosine nucleotide with and CTP showed that the concentrations in inosine or cytidine nucleotide, F-ADP-actin nucleotide mixtures can be determined pellets were suspended in 0.5 m&t ITP or CTP and 10 m&l Tris-HCI, pH 7.4, and within =t 3 %. Bound-nucleotide exchange studies. For the sonicated for two l-minute periods in an ice measurements of the exchange of the bound bath. (The ultrasonic generator used was a nucleotide with free ATP, suitable mixtures Blackstone Biosonik Model BP-l with a were prepared containing actin or actomyox-in. probe.) Free nucleotides were then removed by t’he addition of WO vol of a 50 % sin, and 3H-ATP was added. Samples were taken from these reaction mixtures at suspension of thoroughly washed Dowex lvarious times thereafter, myosin was added XS (Cl- form, 20&400 mesh) followed by to the actin samples, and all samples were filtration. The addition of nucleosidc triwashed by centrifugation and five-times-rephosphate, sonication, and treatment with peated reprecipitation (13). The bound Dowex were repeated four more times, nucleotides were then extracted as defollowed by an extra Dowex treatment to inscribed above, and the incorporation of free sure that the free nucleotides were removed. The final actin solution was then brought to adenosine nucleotide was determined in two of each ex0.1 nf in KC1 and 1 rnM in MgCl, and al- ways. First, the radioactivity tract was measured by liquid-scintillation lowed to polymerize 2-3 hr at room temperacounting and divided by the total nucleotide ture. Control F-ADP-actin was routinely prepared by the same procedure using ATP concentration to obtain the specific activity instead of ITP or CTP. All actin solutions of the extract, and the percent exchange was were used for experiments on the day they calculated as the ratio of this specific acwere prepared from the washed pellets. tivity to that of the 3H-ATP used in the Determination of bound nucleotide conapo- reaction mixture (13). Second, in the case of sition. The bound nucleotides of the actin or IDP- and CDP-actins, the extracted nuactomyosin were extracted from the prohein cleotides were analyzed spectrophotometriwith cold HC104 at a final concentration of tally as described above and the percentage 0.4-0.7 AJI, and the extract was neutralized with IZHC03 or IIOH. After freezing and of exchange was calculated as the percentage of decrease in the fraction of inosine or thawing to precipitat’e IZClO,, the extract was filtered and the ultraviolet absorption cytidine nucleotide in the total bound nuspectrum of the water-clear filtrate was cleotide as compared with the initial actin
390
ESTES
AND
or actomyosin measured before the addition of 3H-ATP. Other measurements. Protein concentrations were measured by ultraviolet absorption at 280 rnp using extinction coefficients of 0.543 ml/mg for myosin (23) and 1.15 ml/mg for actin. This extinction coefficient for actin was determined by micro-Kjeldahl nitrogen measurements, assuming 16% N. The ATPase activity of actomyosin was recorded by the pH-stat technique (15). In samples containing creatine kinase and phosphocreatine, ATP turnover was followed by calorimetric determinat’ion of the liberation of creatine (24). The viscosity of actin solutions was measured at 20 f 0.01” with Ostwald viscometers having an outflow time for water of about 70 set and a calculated velocity gradient of about 2300 se+. Reagents. ATP was purchased from Sigma Chemical Co., ITP and CTP were purchased from P-L Biochemicals, and 3H-labeled ATP was obtained from Schwarz BioResearch at a specific activity of about 1 Ci/mmole. All nucIeotides were used as purchased without further purification. RESULTS
Properties of nucleotide-substituted F-actins. As a basis for subsequent studies of F-ADP-, F-IDP-, and F-CDP-actins, it was necessary first to determine the actual amount of bound nucleotide in the various F-actin preparations used in this work and to verify the effectiveness of the procedure used to replace the original bound ADP with IDP or CDP. Table I shows that the nucleotide content of all three F-aetins varied about a mean value of about 18 pmoles/g protein, and it is evident from the last column of Table I that over 90% nucleotide replacement was achieved in our preparations of F-IDP- and F-CDP-actins. The state of polymerization of the various actins was investigated by measuring sedimentability in the preparative ultracentrifuge. As shown in Table II, both nucleotidesubstituted actins, as well as F-ADP-actin, were about 90% sedimented in 3 hr at 110,000 g, showing that the polymerizability of the actin was not lost upon substitution of the bound nucleotide.
MOOS TABLE THE
BOUND
COMPOSITION Type of actin
ADP
ADP-actin
IDP
IDP-actin
CDP
CDP-actin
Mean
I
NUCLEOTIDE
Nucleotide
content
CONTENT
AND
OF F-ACTINS’ bmoles/g
actin)
E%% CDP
ADP
mr
CDP
15.9 16.2 18.9 17.9 18.6 mean
-
-
15.9 16.2 18.9 17.9 18.6 17.5 f 1.4
-
18.3 17.3 18.0 16.6 14.5
-
19.6 17.9 18.5 17.2 16.1 17.9 f 1.3
93 96 98 96 90
-
19.0 17.7 16.1 17.1 15.9
19.6 19.3 16.9 17.7 18.1 18.3 It 1.1
97 91 95 96 88
=
17.8 f 1.2
1.3 0.6 0.5 0.6 1.6 mean
0.6 1.6 0.8 0.6 2.2 mean
value
for
=
=
=
all actins
a Values are given for five different of each type of actin.
Total
preparations
The viscosity properties of the three Factins are compared in Fig. 1. Figure la shows the specific viscosity of each actin solution plotted as a function of the concentration of F-actin, i.e., protein sedimented in the experiment of Table II. All three F-actins showed a nearly linear increase in specific viscosity with concentration, but there was clearly a pronounced difference in viscosity between F-ADPand F-IDP-actins on one hand and F-CDP actin on the other. The intrinsic viscosity of F-IDPand F-ADP-actins, determined by extrapolation of the reduced viscosity to zero concentration in Fig. lb, was about 0.93 ml/mg, in reasonable agreement with previously reported results for F-ADPactin (25, ZS), but the intrinsic viscosity of F-CDP-actin was less than half this value.
BOUND-NUCLEOTIDE TABLE SEDIMENTATION F-ACTINS
SUBSTITUTED
II
OF NWLEOTIDE-SUBSTITUTED IN THE ULTRACENTRIFUGE~
Supemate
Percent sedimented protein
ADP
1.22 1.03 0.84 0.63 0.42
0.12 0.11 0.09 0.08 0.06
90 89 89 87 86
IDP
1.27 l.OF 0.87 0.65 0.44
0.06 0.04 0.04 0.03 0.03
95 96 95 95 93
CDP
1.26 1.06 0.84 0.64 0.41
0.16 0.14 0.11 0.08 0.07
87 87 87 87 83
Type of F-actin
Protein concentration Initial
solution
&-/ml)
a Actin solutions were diluted as indicated with 0.1 M KCl, 1 mM MgCI?, 1 mM imidazole buffer, pH 7.4. (The samples were those used in the viscosity study in Fig. 1.) Protein concentrations were determined by UV spectrophotometrv before and after centrifugation ior 3 hi at llO,OO-0 9.
391
F-ACTINS
This result appears to be inconsistent with the report by Iyengar and Weber (8) that F-actin polymerized in the presence of CTP has the same reduced viscosit)y as F-ADPactin, but no bound nucleotide analysis was reported for the actin used in those measurements so the extent of replacement of its bound nucleotide with CDP is uncertain. Our finding of a difference in visco&y between I?-actin containing CDP and F-actins containing ADP and IDP indicates a difference in some hydrodynamic parameter of the polymer, such as its length or rigidity, suggesting that the type of bound nucleotide in F-actin may affect the struct)ure of the polymer. Interaction of the F-act&s with myosin. A key property of F-actin is its ability to bind to myosin. To investigate the possible effect of the bound nucleotide on the affinity of F-actin for myosin, an experiment was performed to determine whether myosin would selectively bind one of two types of F-actin in a mixture containing an excess of both types. Small amounts of myosin were added to excess amounts of actin mixtures cant sining different aroport,ions of F-ADPand FIIDP-actins or of WADPb
0 F-Aclin
CDIIE,
(mghl)
FIG. 1. Viscosity of nucleotide-substituted 1 mM imidazole buffer, pH 7.4. After completjion samples were used for the sedimentation study given here is that of sedimentable F-actin. 0, actin. (a) Dependence of specific viscosity on intrinsic viscosity by extrapolation of reduced by least-squares method.
0.25
0.50 F-octin
cont.
0.75
1.00
(mg/ml)
F-actins. Conditions: 0.1 M KCl, 1 mMMgC&, of the viscosity measurements, the same in Table II and the protein concentration F-ADP-actin; 0, F-IDP-actin; A, F-CDPF-actin concentration. (b) Determination of viscosity to zero concentration. Lines fitted
392
ESTES
AND
and F-CDP-actins, the resulting actomyosin was centrifuged down, and its bound nucleotide was analyzed as a measure of the amount of each actin bound by the myosin. Since the nucleotide replacement in the substituted actins was less than 100% (Table I), and since the actin solutions might contain some protein which was not capable of binding to myosin, the basis of comparison for each of these experimental samples was a control sample containing a small amount of the same actin mixture with an excess of myosin. In these control samples, all the actin which could be bound by myosin would be precipitated with the actomyosin. As shown in Table III, the total amount of actin-bound nucleotide in each sediment was close to the theoretically expected amount, and the bound nucleotide composition of the actin bound by a limited amount of myosin in the presence of excess actin (Column 3) was similar to that of the total actin in the mixture which could be bound by excess myosin (Column 5). In other words, when a small amount of myosin was added to a large excess of actin, the portion of the actin which was bound was essentially a random sample of the total actin mixture. Similar results were also obtained when the TABLE BINDING Experimental Actin mixture ADP
+
IDP
ADP
+ CDP
OF ACTIN
MOOS
actin and myosin were mixed in 0.5 M KCI, instead of 0.1 M KC1 as was used in Table III, and the actomyosin was precipitated by dilution. Furthermore, we have observed in other experiments that when an actomyosin containing a mixture of actins is reprecipitated as many as nine times, there is no significant change in the composition of its bound nucleotide. All of these experiments show clearly that there are no major differences in the affinity of myosin for F-actins with various bound nucleotides. Another aspect of the actin-myosin interaction is the activation of the myosin ATPase by actin in the presence of Mg++. To investigate the ATPase activation by nucleotide-substituted actins, ATPase rates were measured using a fixed amount of myosin with varying amounts of the three types of F-actin. As shown in Fig. 2, all three F-actins activated the myosin ATPase to the same extent, and maximum activation occurred at roughly a six-to-one weight ratio of myosin to actin, in reasonable agreement with published results for F-ADPactin (25, 27). This similarity of the ATPase activation by the different actins could not have resulted from replacement of the bound IDP or CDP by ADP during the III
MIXTURES
TO MYOSIN~
samplesb (Excess actin) Total nucleotide in sediment (moles X 109) Theor. value = 33.3
Control sampW
(Excess my&n)
Per cent IDP or CDP
Total nucleotide in sediment (moles X 100) Theor. value = 26.7
Per cent IDP or CDP
28.5 29.7 32.7
49 30 19
23.8 25.F 27.1
52 38 25
32.5 34.0 34.7
43 36 20
23.1 22.7 24.9
56 38 24
a Various proportions of the two actins were combined with myosin in a final volume of 10 ml containing 0.1 M KCl, 1 mM MgC12 , and 10 mM imidazole buffer, pH 7.0. The mixture was incubated 15 min diluted with 10 ml 10 mM imidazole buffer, pH 7, and centrifuged at 1700 g. The bound nucleotide in each sediment was then extracted and analyzed as described in Methods. b Erperimental da?nples contained 8.0 mg myosin and 9.0 mg total actin. The theoretical amount of bound nucleotide in these actomyosin sediments would be 33.3 X Wg moles, assuming a l-to-4 weight ratio of actin to myosin. c Control samples contained 16 mg myosin and 1.6 mg total actin. The total amount of actin-bound nucleotide, at 17.8 pmoles/g actin, was therefore 26.7 X leg moles.
BOUND-NUCLEOTIDE
SUBSTITUTED
7 .E 6 2 _f .I an $0.2 2 IA 2 8 0 0
0.1
0.2 0.3 Actin cont. (mglml)
0.4
0.5
393
F-ACTINS
measured by radioactivity determinations and also, in the case of the IDP- and CDPactins, by spectrophotometric analysis of the bound nucleot’ide. In the actomyosin experiments, a low conceruration of ATP was used, together with creatine kinase and phosphocreat,ine, so that in all cases the actomyosin superprecipitated immediately and remained superprecipitated throughout the experiment. Figure 3 shows the time course of the incorporation of free adenosine nucleotidc into t,he three different actomyosins during
Fro. 2. Activation of the myosin ATPase by nucleotide-substituted F-actins. Conditions: 2 mM ATP, 2 mrvr MgCl2, 50 mM KCI, 3 mM imidazole buffer, pH 7.0, 1.0 mg/ml myosin. Total volume, 7.5 ml. Temperature, 25”. ATPase rates measured for initial three minutes of reaction. q , F-ADPactin; 0, F-IDP-actin; A, F-CDP-actin.
experiment because only the initial ATPase rate was measured and, as will be evident from the nucleotide-exchange studies to be presented below, the amount of nucleotide exchange which would have occurred during this initial period of 3 min could not have affected the results significantly. From these results, it seems clear that the type of nucleotide bound to F-actin does not affect the ability of the actin to bind to lnyosin and to activate the myosin ATPase, in agreement with other studies which showed that substitution or removal of the a&in-bound nucleotide does not affect’ the actin-myosin interaction (S-10). Bound-nucleotide exchange studies. It was first reported by Szent-Gyorgyi (12) that the superprecipitation of actomyosin under certain conditions may lead to extensive exchange of the actin-bound nucleotide wit,h free ATP. While appreciable exchange may also occur in actin alone under similar conditions (28), the exchange in superprecipitating actomyosin is considerably greater (13). We have compared the rate of boundnucleotide exchange in F-actins containing bound ADP, IDP, and CDP, both in the presence and in the absence of myosin. Tritium-labeled ATP was added to the medium and the incorporation of free adenosine nucleotide into the actin was
r 5-
b
/
-m---g-g-
,/%
.4 4
B
/”
A/ 0
, 20
40
60 Time
80
100
120
(min)
FIG. 3. Incorporation of 3H-ATP into actomyosins containing nucleotide-substituted actins. Conditions: 0.1 mg/ml actin, 0.8 mg/ml myosin, 50 mM KCl, 4 mM MgC12, 10 mM imidazole buffer, pH 7.0, 5 mM phosphocreatine, 0.1 mg/ml creatine kinase. 50 pM 3H-ATP added at zero time. (a) Bound-nucleotide exchange: Aliquots of each suspension were removed just before and at various times after addition of 3H-ATP, each sample was centrifuged and washed by reprecipitation five times, the bound nucleotide was extracted and analyzed and the percentage of exchange calculated as described in Methods. Open symbok, exchange measured spectrophotometrically; solid symbols, exchange measured by trit)ium incorporation. n , ADPactomyosin; 0, 0, IDP-actomyosin; A, A, CDPactomyosin. (b) ATP turnover as measured by creatine liberation. 0, ADP-actomyosin; 0, IDPactomyosin; A, CDP-actomyosin.
394
ESTES
AND
superprecipitation, together with the turnover of ATP measured by the liberation of free creatine. It is quite clear that, although the rate of creatine liberation was identical in the three actomyosin suspensions, there were considerable differences in the incorporation of labeled adenosine nucleotide into the three types of actomyosin. The exchange in the ADP-actomyosin was comparable to that observed previously (12, 13), but the exchange in CDP-actomyosin was appreciably greater than this and that in the IDP-actomyosin was considerably less. When the F-actins were incubated with H3-ATP in the absence of myosin (Fig. 4) the exchange rate in every case was less than that of the corresponding actomyosin in Fig. 3, in agreement with our previous report (13), but here again the exchange rate in IDP-actin was less than that in ADPactin. Thus, in contrast with the apparent lack of any influence of the a&in-bound nucleotide on the actin-myosin interaction, the exchangeability of the F-actin-bound nucleotide is strongly dependent on the type of bound nucleotide present.
20
.-t 5 15 k P uo c 10
s 5
-A 0
20
40 Time
60
80
100
120
(mitt)
FIG. 4. Incorporation of aH-ATP into nucleotide-substituted F-actins in the absence of myosin. Conditions: 0.1 mg/ml F-actin, 50 mM KCl, 4 mM buffer, pH 7.0. 50 pM M&l 2, 10 mM imidazole 3H-ATP added at zero time. Samples removed at indicated times were immediately mixed with myosin, centrifuged, and treated as in Fig. 3. Open symbols, exchange measured spectrophotometrically; solid symbols, exchange measured by tritium incorporation. q , F-ADP-actin; 0, 0, F-IDP-actin; A, 4, F-CDP-actin.
MOOS
Since the bound nucleotide in F-actin is so much less exchangeable than that in G-actin (29), it is generally assumed that the nucleotide exchange in F-actin is restricted somehow by the polymer structure. Hence the differences in nucleotide exchange rates among the various F-actins suggest that the species of bound nucleotide may influence the interaction between monomers in the F-actin polymer. Partial polymerization studies. As another approach to the possible effect of the bound nucleotide on the formation of actin-actin bonds in F-actin, partial polymerization at low MgClz concentration was compared for actins containing inosine and adenosine nucleotides. The IDP- and ADP-actins were prepared as described in Fig. 5 and suspended in ITP or ATP solution, respectively, containing 50 PM MgCL. Each actin was then depolymerized by sonication, the solution was transferred to a viscometer, and the time course of repolymerization was followed by means of viscosity measurements. As can be seen in Fig. 5, the actin containing inosine nucleotide polymerized considerably faster and to a greater extent than that containing adenosine nucleotide. Even when no MgCL was added to the inosine nucleotide-containing actin, so that the only salt present was the small amount remaining from the Dowex treatments (see caption of Fig. 5), the partial polymerization proceeded somewhat faster and to a slightly greater extent than the polymerization of adenosine nucleotide-containing actin with 50 PM MgCL. These differences in polymerization were not due to differences in the initial sonication of the actins because t’he first viscosity values, determined three minutes after the end of sonication, were the same whether the sonication had lasted only a few seconds or more than two minutes. It is also clear that there were no differences in actin concentration because both solutions in Fig. 5 reached the same final viscosity when fully polymerized by 0.1 M KC1 and 1 InM MgClz at the end of the experiment, and according to Fig. 1, the viscosity is an equivaIent measure of concentration for both ADP- and IDP-containing F-a&ins. Hence, these partial polymerization studies
BOUND-NUCLEOTIDE
0.8
-
0.6
-
0
30
SUBSTITUTED
60 Minuler
90 dler
120 end of ronicalion
395
F-ACTINS
150
1.90
211D
FIG. 5. Partial polymerization of actins containing inosine or adenosine nucleotide. Actin solutions, prepared as usual but without added salts, were made 1 mM in ITP or ATP respectively. (The nucleotides had been treated with Chelex-100 resin to remove contaminant Ca++ or Mg”.) The actins at this point were partially polymerized as a result of the Dowex treatments (see Mefhods), and the actin was sediment,ed by 6-hr centrifugation at 105,000 g, homogenized in 10 IIIM imidazole buffer, pH 7.0, containing 1 mM ITP or ATP respectively, and clarified by filtration. Protein concentration was adjusted to 0.75 mg/ml and the pH to 7.1, and MgClz was added to a final concentration of 50 NM. The solution was sonicated 60 set in an ice bath, and the time-course of repolymerization was followed by viscosity measurements. At the end of the experiment, the solutions were made 1 mM in MgClz and 0.1 M in KCI, and the viscosity of the fully polymerized F-actin was measured. 0, actin with inosine nucleotide; l , actin with adenosine nucleotide.
reveal a clear dependence of polymerization rate on the type of nucleotide in the actin, which gives further support t’o the suggestion, made on the basis of the nucleotide exchange studies, that the bound nucleotide affects the actin-actin interaction in the F-actin polymer. DISCUSSION
In this work, F-actins have been prepared which contain bound IDP or CDP in place of the usual bound ADP, and the effects of this nucleotide substitution on a number of properties of the actin have been investigated. The replacement of the bound nucleotide did not affect the binding of the F-actin to myosin or its activation of the myosin ATPase, which is in agreement with previous studies of nucleotide-substituted or nucleotide-deficient actins (S-10). On the ot’her hand, the rate of exchange of the bound nucleotide with free ATP, in both actin and actomyosin, was strongly influ-
enced by the type of nucleotide initially bound to the actin, the exchangeability increasing in the order IDP < ADP < CDP. In view of the great increase in the rate of nucleotide exchange in F-actin brought about by ultrasonic treatment (30), it seems reasonable to suppose that the replacement of the actin-bound nucleoside diphosphate by free ATP in our experiments depends on the prior rupture of an actin-actin bond in the polymer to create an “open” site at which the bound nucleotide is temporarily exposed to the medium. These “open” sites may be either partial disruptions within the polymer, called “f-actin” by Asakura et al. (ll), or complete breaks to form new free ends or G-actin monomers. The observed nucleotide exchange rate would then depend on the steady-state fraction of such “open” sites in the F-actin. For example, the influence of myosin on the exchange rate is interpreted, on this basis, to mean that the interaction of F-actin with myosin causes
396
ESTES
AND
an increased frequency of these local disruptions in the polymer (12, 13). Applying this approach to the difference in exchange rate between IDP- and ADPactins, one interpretation would be that the IDP-actin shows a slower rate of nucleotide exchange because it has fewer “open” sites in the steady-state than ADP-actin does under the same conditions, i.e., the presence of bound IDP in the actin in place of ADP makes the polymer more stable. Similarly, the greater exchange rate in CDP-actin may indicate that breaks occur more frequently in CDP-actin than in ADP-actin. In the case of IDP-actin, this interpretation would also correlate with our observation that the presence of bound inosine nucleotide in place of adenosine nucleotide increases both the rate and extent of the partial polymerization of the actin at low magnesium concentration. Other interpretations of our observations are no doubt possible, but it is clear in any case that the species of bound nucleotide in F-actin has a definite effect on certain of its properties. ACKNOWLEDGMENTS We thank Dr. Evan Eisenberg for suggestions and discussions. This was supported by Training Grant and Research Grant GM-10249 from Institute of General Medical Sciences, lic Health Service.
many helpful investigation 5Tl-GM-718 the National U. S. Pub-
REFERENCES 1. STRAUB, phys.
F. B. .~ND FEUER,
G., Biochim.
Bio-
Acta 4, 455 (1950).
K., BOTVEN, W. J., AND CLARK, A., J. Gen. Physiol. 33, 437 (1950). 3. MOMMAERTS, W. F. H. M., J. Biol. Chem., 198,
2. LAKI,
469 (1952). 4. TSUBOI, K. K., Biochim. Biophys. Acta 180, 420 (1968). 5. GRUBHOFER, N. AND WEBER, H. H., 2. Naturforsch. 16b, 435 (1961). 6. MARTONOSI, A. AND GOUVEA, M. A., J. Biol. Chem. 236, 1345 (1961). 7. STROHMAN, R. C. AND SAMORODIN, A. J., J. Biol. Chem. 237, 363 (1962).
MOOS 8. IYENGAR,
M.
R. AND WEBER,
H. H.,
Biochim.
Biophys. Acta 86, 543 (1964). 9. BARONY,
M.
AND
FINKELMAN,
F.,
Biochim.
Biophys. Acta 78, 175 (1963). 10. BUNNY, E., J. 11. ASAKURA,
M.,
TUCCI,
A. F.,
AND CONOVER,
T.
Mol. Biol. 19, 483 (1966). S., TANIGUCHI,
M.
AND OOSAWA,
F.,
J. Mol. Biol. 7, 55 (1963). 12. SZENT-GY~RGYI, A. G. AND PRIOR, G., J. Mol. Biol. 16, 515 (1966). 13. Moos, C., EISENBERG, E., AND ESTES, J. E., Biochim. Biophys. Acta 147, 536 (1967). 14. SZENT-GY~RGYI, A., “Chemistry of Muscular Contraction,” 2nd Ed., pp. 146-150. Academic Press, New York (1951). E. AND Moos, C., J. Biol. Chem., 15. EISENBERG, 242, 2945 (1967). 16. KUBY, S. A., Nova, L., AND LARDY, H. A., J. Biol. Chem. 209, 191 (1954). 17. Moos, C., Biochim. Biophys. Acta 93,85 (1964). 18. DRABIKOWSKI, W. END GERGELY, J., J. Biol. Chem. 237, 3412 (1962). 19. ULBRECHT, M., GRUBHOFER, N., JAISLE, F., AND WALTER, S., Biochim. Biophys. Acta 45, 443 (1960). 20. LORING, H. S., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, Eds.). Vol. 1, p. 191. Academic Press, New York (1955). 21. “Circular OR-17”. Pabst Laboratories, Milwaukee, Wisconsin (1961). 22. BOCK, R. M., LING, N. S., MORELL, S. A., AND LIPTON, S. H., Arch. Biochem. Biophys. 62, 253 (1956). M. F. AND ENGLANDER, S. W., Bio23. GELLERT, chemistry 2, 39 (1963). D., ELSDEN, S. R., AND GOUGH, N., 24. EGGLETON, Biochem. J. 37,526 (1943). 25. MARUYAMA, K. AND GERGELY, J., J. Biol. Chem. 237, 1095 (1962). B. AND JENCKS, W. D., J. Am. Chem. 26. NAGY, Sot. 87, 2480 (1965). 27. BARONY, M., NAGY, B., FINICELMAN, F., AND CHRAMBACH, A., J. Biol. Chem. 236, 2917 (1961). 28. KITAGAWA, S., DRABIKOWSKI, W., AND GERGELY, J., Arch. Biochem. Biophys. 126, 706 (1968). A., GOUVEA, M. A. AND GERGELY, 29. MARTONOSI, J., J. Biol. Chem., 236, 1700 (1960). S., TANIGUCHI, M., AND OOSAWA, 30. ASAKURA, F., Biochim. Biophys. Acta 74, 140 (1963).
OF
RIOCHEMISTRY
‘Effect
of
AND
BIOPHYSICS
132,
Bound-Nucleotide
388-396
(1969)
Substitution
on the
Properties
of F-Act in JAMES Department
of Biophysics,
State
Received
E. ESTES’ University December
AND
of IVew 2, 1968;
CARL York accepted
MOOS2
at Buffalo, April
Bu$alo,
New
York
i4Si4
2, 1969
F-actins have been prepared in which the bound adenosine nucleotide has been replaced by inosine or cytidine nucleotide, and their properties have been studied. The intrinsic viscocity of IDP-containing F-actin was the same as that of ADP-containing F-actin, but the intrinsic viscosity of GDP-containing F-actin was about half this value. There were no differences in the myosin-binding affinities of the three types of I?-actin or in their ability to activate the magnesium-ATPase of myosin. On the other hand, the exchangeability of the F-actin-bound nucleotide, both in the presence and in the absence of myosin, was dependent on the type of bound nucleotide in the actin, increasing in the order IDP
While it has been well established that actin contains 1 mole of firmly bound adenosine nucleotide per mole of protein (l-4), the function of this bound nucleotide in muscle contraction is not yet understood. The bound adenosine nucleotide may be replaced by other nucleoside phosphates (5-7), and both the relative viscosities of F-a&ins containing bound inosine, cytidine, or guanosine nucleotides, and the abilities of these F-a&ins to form actomyosins which can be dissociated by ATP, are reported to be the same as those of normal F-ADPactin (8). Furthermore, an F-actin containing bound AMP, or even one lacking a bound nucleotide, retains the viscosity and myosinbinding properties of F-ADP-actin (9, 10). While none of these investigations gave any direct evidence that the actin-bound nu-
cleotide plays a role in muscle contraction, the results of mechanochemical studies of actin (11) and also the recent finding that the exchangeability of the F-actin-bound nucleotide is increased during superprecipitation of actomyosin (12, 13) have provided encouragement for continued speculation about the physiological role of actin and its bound nucleotide in contractile systems. In further pursuing this problem we have prepared F-a&ins in which the bound ADP is almost completely replaced by IDP or CDP, and have made quantitative comparisons of the properties of these actins and their interactions with myosin. The binding of the F-actin to myosin and its activation of the myosin ATPase were quite unaffected by the bound-nucleotide substitutions. On the other hand, the exchangeability of the a&in-bound nucleotide in the presence and absence of myosin, and the rate and extent of partial polymerization at low Mg++ concentration, were strongly dependent on the type of bound nucleotide in the actin, suggesting that the bound nucleotide influences
1 Present address: Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181. 2 Present address: Department of Biological Sciences, State University of New York, Stony Brook, New York 11790. 388
BOUND-NUCLEOTIDE
the interaction between F-a&in filament.
monomers
SUBSTITUTED
in the
F-ACTINS
389
measured. Blank extracts, used to correct the spectrum for contaminants in the reagents, were prepared in the same manner MATERIALS AND METHODS except that the protein sample was replaced with an equal volume of water. In mixtures Protein preparations. Myosin was prepared from rabbit skeletal muscle by a of inosine and adenosine nucleotides, the concentration of each nucleotide was calcumodification of t’he method of A. Szentlated from the absorbances at 250 260, and Gyijrgyi (14) as previously described (15). 290 rnp, while in the case of mixtures of Creatine kinase (ATP : creatine phosphoand adenosine nucleotides, t,he transferase, EC 2.7.3.2) was isolated and cytidine absorbances at 260, 280, and 300 rnp were purified by previously published procedures used. The nucleotide concentrations in t,he (16, 17). mixtures were calculated using the equations Actin was extracted from acetone-dried of Loring (20) and the published spectral muscle powder (14) at 0” to avoid contamconstants for the three nucleotides (21, 22). ination with tropomyosin (18) and purified It was assumed that the only ultraviolet according to Ulbrecht, Grubhofer, Jaisle absorbing components present in each and Walter (19). The washed F-actin extract were the t,wo nucleotides specified. pellets were stored in capped tubes in the The application of this method t’o art,ificial cold and used within six days. To replace known mixtures of ATP and ITP or of ATP the actin-bound adenosine nucleotide with and CTP showed that the concentrations in inosine or cytidine nucleotide, F-ADP-actin nucleotide mixtures can be determined pellets were suspended in 0.5 m&t ITP or CTP and 10 m&l Tris-HCI, pH 7.4, and within =t 3 %. Bound-nucleotide exchange studies. For the sonicated for two l-minute periods in an ice measurements of the exchange of the bound bath. (The ultrasonic generator used was a nucleotide with free ATP, suitable mixtures Blackstone Biosonik Model BP-l with a were prepared containing actin or actomyox-in. probe.) Free nucleotides were then removed by t’he addition of WO vol of a 50 % sin, and 3H-ATP was added. Samples were taken from these reaction mixtures at suspension of thoroughly washed Dowex lvarious times thereafter, myosin was added XS (Cl- form, 20&400 mesh) followed by to the actin samples, and all samples were filtration. The addition of nucleosidc triwashed by centrifugation and five-times-rephosphate, sonication, and treatment with peated reprecipitation (13). The bound Dowex were repeated four more times, nucleotides were then extracted as defollowed by an extra Dowex treatment to inscribed above, and the incorporation of free sure that the free nucleotides were removed. The final actin solution was then brought to adenosine nucleotide was determined in two of each ex0.1 nf in KC1 and 1 rnM in MgCl, and al- ways. First, the radioactivity tract was measured by liquid-scintillation lowed to polymerize 2-3 hr at room temperacounting and divided by the total nucleotide ture. Control F-ADP-actin was routinely prepared by the same procedure using ATP concentration to obtain the specific activity instead of ITP or CTP. All actin solutions of the extract, and the percent exchange was were used for experiments on the day they calculated as the ratio of this specific acwere prepared from the washed pellets. tivity to that of the 3H-ATP used in the Determination of bound nucleotide conapo- reaction mixture (13). Second, in the case of sition. The bound nucleotides of the actin or IDP- and CDP-actins, the extracted nuactomyosin were extracted from the prohein cleotides were analyzed spectrophotometriwith cold HC104 at a final concentration of tally as described above and the percentage 0.4-0.7 AJI, and the extract was neutralized with IZHC03 or IIOH. After freezing and of exchange was calculated as the percentage of decrease in the fraction of inosine or thawing to precipitat’e IZClO,, the extract was filtered and the ultraviolet absorption cytidine nucleotide in the total bound nuspectrum of the water-clear filtrate was cleotide as compared with the initial actin
390
ESTES
AND
or actomyosin measured before the addition of 3H-ATP. Other measurements. Protein concentrations were measured by ultraviolet absorption at 280 rnp using extinction coefficients of 0.543 ml/mg for myosin (23) and 1.15 ml/mg for actin. This extinction coefficient for actin was determined by micro-Kjeldahl nitrogen measurements, assuming 16% N. The ATPase activity of actomyosin was recorded by the pH-stat technique (15). In samples containing creatine kinase and phosphocreatine, ATP turnover was followed by calorimetric determinat’ion of the liberation of creatine (24). The viscosity of actin solutions was measured at 20 f 0.01” with Ostwald viscometers having an outflow time for water of about 70 set and a calculated velocity gradient of about 2300 se+. Reagents. ATP was purchased from Sigma Chemical Co., ITP and CTP were purchased from P-L Biochemicals, and 3H-labeled ATP was obtained from Schwarz BioResearch at a specific activity of about 1 Ci/mmole. All nucIeotides were used as purchased without further purification. RESULTS
Properties of nucleotide-substituted F-actins. As a basis for subsequent studies of F-ADP-, F-IDP-, and F-CDP-actins, it was necessary first to determine the actual amount of bound nucleotide in the various F-actin preparations used in this work and to verify the effectiveness of the procedure used to replace the original bound ADP with IDP or CDP. Table I shows that the nucleotide content of all three F-aetins varied about a mean value of about 18 pmoles/g protein, and it is evident from the last column of Table I that over 90% nucleotide replacement was achieved in our preparations of F-IDP- and F-CDP-actins. The state of polymerization of the various actins was investigated by measuring sedimentability in the preparative ultracentrifuge. As shown in Table II, both nucleotidesubstituted actins, as well as F-ADP-actin, were about 90% sedimented in 3 hr at 110,000 g, showing that the polymerizability of the actin was not lost upon substitution of the bound nucleotide.
MOOS TABLE THE
BOUND
COMPOSITION Type of actin
ADP
ADP-actin
IDP
IDP-actin
CDP
CDP-actin
Mean
I
NUCLEOTIDE
Nucleotide
content
CONTENT
AND
OF F-ACTINS’ bmoles/g
actin)
E%% CDP
ADP
mr
CDP
15.9 16.2 18.9 17.9 18.6 mean
-
-
15.9 16.2 18.9 17.9 18.6 17.5 f 1.4
-
18.3 17.3 18.0 16.6 14.5
-
19.6 17.9 18.5 17.2 16.1 17.9 f 1.3
93 96 98 96 90
-
19.0 17.7 16.1 17.1 15.9
19.6 19.3 16.9 17.7 18.1 18.3 It 1.1
97 91 95 96 88
=
17.8 f 1.2
1.3 0.6 0.5 0.6 1.6 mean
0.6 1.6 0.8 0.6 2.2 mean
value
for
=
=
=
all actins
a Values are given for five different of each type of actin.
Total
preparations
The viscosity properties of the three Factins are compared in Fig. 1. Figure la shows the specific viscosity of each actin solution plotted as a function of the concentration of F-actin, i.e., protein sedimented in the experiment of Table II. All three F-actins showed a nearly linear increase in specific viscosity with concentration, but there was clearly a pronounced difference in viscosity between F-ADPand F-IDP-actins on one hand and F-CDP actin on the other. The intrinsic viscosity of F-IDPand F-ADP-actins, determined by extrapolation of the reduced viscosity to zero concentration in Fig. lb, was about 0.93 ml/mg, in reasonable agreement with previously reported results for F-ADPactin (25, ZS), but the intrinsic viscosity of F-CDP-actin was less than half this value.
BOUND-NUCLEOTIDE TABLE SEDIMENTATION F-ACTINS
SUBSTITUTED
II
OF NWLEOTIDE-SUBSTITUTED IN THE ULTRACENTRIFUGE~
Supemate
Percent sedimented protein
ADP
1.22 1.03 0.84 0.63 0.42
0.12 0.11 0.09 0.08 0.06
90 89 89 87 86
IDP
1.27 l.OF 0.87 0.65 0.44
0.06 0.04 0.04 0.03 0.03
95 96 95 95 93
CDP
1.26 1.06 0.84 0.64 0.41
0.16 0.14 0.11 0.08 0.07
87 87 87 87 83
Type of F-actin
Protein concentration Initial
solution
&-/ml)
a Actin solutions were diluted as indicated with 0.1 M KCl, 1 mM MgCI?, 1 mM imidazole buffer, pH 7.4. (The samples were those used in the viscosity study in Fig. 1.) Protein concentrations were determined by UV spectrophotometrv before and after centrifugation ior 3 hi at llO,OO-0 9.
391
F-ACTINS
This result appears to be inconsistent with the report by Iyengar and Weber (8) that F-actin polymerized in the presence of CTP has the same reduced viscosit)y as F-ADPactin, but no bound nucleotide analysis was reported for the actin used in those measurements so the extent of replacement of its bound nucleotide with CDP is uncertain. Our finding of a difference in visco&y between I?-actin containing CDP and F-actins containing ADP and IDP indicates a difference in some hydrodynamic parameter of the polymer, such as its length or rigidity, suggesting that the type of bound nucleotide in F-actin may affect the struct)ure of the polymer. Interaction of the F-act&s with myosin. A key property of F-actin is its ability to bind to myosin. To investigate the possible effect of the bound nucleotide on the affinity of F-actin for myosin, an experiment was performed to determine whether myosin would selectively bind one of two types of F-actin in a mixture containing an excess of both types. Small amounts of myosin were added to excess amounts of actin mixtures cant sining different aroport,ions of F-ADPand FIIDP-actins or of WADPb
0 F-Aclin
CDIIE,
(mghl)
FIG. 1. Viscosity of nucleotide-substituted 1 mM imidazole buffer, pH 7.4. After completjion samples were used for the sedimentation study given here is that of sedimentable F-actin. 0, actin. (a) Dependence of specific viscosity on intrinsic viscosity by extrapolation of reduced by least-squares method.
0.25
0.50 F-octin
cont.
0.75
1.00
(mg/ml)
F-actins. Conditions: 0.1 M KCl, 1 mMMgC&, of the viscosity measurements, the same in Table II and the protein concentration F-ADP-actin; 0, F-IDP-actin; A, F-CDPF-actin concentration. (b) Determination of viscosity to zero concentration. Lines fitted
392
ESTES
AND
and F-CDP-actins, the resulting actomyosin was centrifuged down, and its bound nucleotide was analyzed as a measure of the amount of each actin bound by the myosin. Since the nucleotide replacement in the substituted actins was less than 100% (Table I), and since the actin solutions might contain some protein which was not capable of binding to myosin, the basis of comparison for each of these experimental samples was a control sample containing a small amount of the same actin mixture with an excess of myosin. In these control samples, all the actin which could be bound by myosin would be precipitated with the actomyosin. As shown in Table III, the total amount of actin-bound nucleotide in each sediment was close to the theoretically expected amount, and the bound nucleotide composition of the actin bound by a limited amount of myosin in the presence of excess actin (Column 3) was similar to that of the total actin in the mixture which could be bound by excess myosin (Column 5). In other words, when a small amount of myosin was added to a large excess of actin, the portion of the actin which was bound was essentially a random sample of the total actin mixture. Similar results were also obtained when the TABLE BINDING Experimental Actin mixture ADP
+
IDP
ADP
+ CDP
OF ACTIN
MOOS
actin and myosin were mixed in 0.5 M KCI, instead of 0.1 M KC1 as was used in Table III, and the actomyosin was precipitated by dilution. Furthermore, we have observed in other experiments that when an actomyosin containing a mixture of actins is reprecipitated as many as nine times, there is no significant change in the composition of its bound nucleotide. All of these experiments show clearly that there are no major differences in the affinity of myosin for F-actins with various bound nucleotides. Another aspect of the actin-myosin interaction is the activation of the myosin ATPase by actin in the presence of Mg++. To investigate the ATPase activation by nucleotide-substituted actins, ATPase rates were measured using a fixed amount of myosin with varying amounts of the three types of F-actin. As shown in Fig. 2, all three F-actins activated the myosin ATPase to the same extent, and maximum activation occurred at roughly a six-to-one weight ratio of myosin to actin, in reasonable agreement with published results for F-ADPactin (25, 27). This similarity of the ATPase activation by the different actins could not have resulted from replacement of the bound IDP or CDP by ADP during the III
MIXTURES
TO MYOSIN~
samplesb (Excess actin) Total nucleotide in sediment (moles X 109) Theor. value = 33.3
Control sampW
(Excess my&n)
Per cent IDP or CDP
Total nucleotide in sediment (moles X 100) Theor. value = 26.7
Per cent IDP or CDP
28.5 29.7 32.7
49 30 19
23.8 25.F 27.1
52 38 25
32.5 34.0 34.7
43 36 20
23.1 22.7 24.9
56 38 24
a Various proportions of the two actins were combined with myosin in a final volume of 10 ml containing 0.1 M KCl, 1 mM MgC12 , and 10 mM imidazole buffer, pH 7.0. The mixture was incubated 15 min diluted with 10 ml 10 mM imidazole buffer, pH 7, and centrifuged at 1700 g. The bound nucleotide in each sediment was then extracted and analyzed as described in Methods. b Erperimental da?nples contained 8.0 mg myosin and 9.0 mg total actin. The theoretical amount of bound nucleotide in these actomyosin sediments would be 33.3 X Wg moles, assuming a l-to-4 weight ratio of actin to myosin. c Control samples contained 16 mg myosin and 1.6 mg total actin. The total amount of actin-bound nucleotide, at 17.8 pmoles/g actin, was therefore 26.7 X leg moles.
BOUND-NUCLEOTIDE
SUBSTITUTED
7 .E 6 2 _f .I an $0.2 2 IA 2 8 0 0
0.1
0.2 0.3 Actin cont. (mglml)
0.4
0.5
393
F-ACTINS
measured by radioactivity determinations and also, in the case of the IDP- and CDPactins, by spectrophotometric analysis of the bound nucleot’ide. In the actomyosin experiments, a low conceruration of ATP was used, together with creatine kinase and phosphocreat,ine, so that in all cases the actomyosin superprecipitated immediately and remained superprecipitated throughout the experiment. Figure 3 shows the time course of the incorporation of free adenosine nucleotidc into t,he three different actomyosins during
Fro. 2. Activation of the myosin ATPase by nucleotide-substituted F-actins. Conditions: 2 mM ATP, 2 mrvr MgCl2, 50 mM KCI, 3 mM imidazole buffer, pH 7.0, 1.0 mg/ml myosin. Total volume, 7.5 ml. Temperature, 25”. ATPase rates measured for initial three minutes of reaction. q , F-ADPactin; 0, F-IDP-actin; A, F-CDP-actin.
experiment because only the initial ATPase rate was measured and, as will be evident from the nucleotide-exchange studies to be presented below, the amount of nucleotide exchange which would have occurred during this initial period of 3 min could not have affected the results significantly. From these results, it seems clear that the type of nucleotide bound to F-actin does not affect the ability of the actin to bind to lnyosin and to activate the myosin ATPase, in agreement with other studies which showed that substitution or removal of the a&in-bound nucleotide does not affect’ the actin-myosin interaction (S-10). Bound-nucleotide exchange studies. It was first reported by Szent-Gyorgyi (12) that the superprecipitation of actomyosin under certain conditions may lead to extensive exchange of the actin-bound nucleotide wit,h free ATP. While appreciable exchange may also occur in actin alone under similar conditions (28), the exchange in superprecipitating actomyosin is considerably greater (13). We have compared the rate of boundnucleotide exchange in F-actins containing bound ADP, IDP, and CDP, both in the presence and in the absence of myosin. Tritium-labeled ATP was added to the medium and the incorporation of free adenosine nucleotide into the actin was
r 5-
b
/
-m---g-g-
,/%
.4 4
B
/”
A/ 0
, 20
40
60 Time
80
100
120
(min)
FIG. 3. Incorporation of 3H-ATP into actomyosins containing nucleotide-substituted actins. Conditions: 0.1 mg/ml actin, 0.8 mg/ml myosin, 50 mM KCl, 4 mM MgC12, 10 mM imidazole buffer, pH 7.0, 5 mM phosphocreatine, 0.1 mg/ml creatine kinase. 50 pM 3H-ATP added at zero time. (a) Bound-nucleotide exchange: Aliquots of each suspension were removed just before and at various times after addition of 3H-ATP, each sample was centrifuged and washed by reprecipitation five times, the bound nucleotide was extracted and analyzed and the percentage of exchange calculated as described in Methods. Open symbok, exchange measured spectrophotometrically; solid symbols, exchange measured by trit)ium incorporation. n , ADPactomyosin; 0, 0, IDP-actomyosin; A, A, CDPactomyosin. (b) ATP turnover as measured by creatine liberation. 0, ADP-actomyosin; 0, IDPactomyosin; A, CDP-actomyosin.
394
ESTES
AND
superprecipitation, together with the turnover of ATP measured by the liberation of free creatine. It is quite clear that, although the rate of creatine liberation was identical in the three actomyosin suspensions, there were considerable differences in the incorporation of labeled adenosine nucleotide into the three types of actomyosin. The exchange in the ADP-actomyosin was comparable to that observed previously (12, 13), but the exchange in CDP-actomyosin was appreciably greater than this and that in the IDP-actomyosin was considerably less. When the F-actins were incubated with H3-ATP in the absence of myosin (Fig. 4) the exchange rate in every case was less than that of the corresponding actomyosin in Fig. 3, in agreement with our previous report (13), but here again the exchange rate in IDP-actin was less than that in ADPactin. Thus, in contrast with the apparent lack of any influence of the a&in-bound nucleotide on the actin-myosin interaction, the exchangeability of the F-actin-bound nucleotide is strongly dependent on the type of bound nucleotide present.
20
.-t 5 15 k P uo c 10
s 5
-A 0
20
40 Time
60
80
100
120
(mitt)
FIG. 4. Incorporation of aH-ATP into nucleotide-substituted F-actins in the absence of myosin. Conditions: 0.1 mg/ml F-actin, 50 mM KCl, 4 mM buffer, pH 7.0. 50 pM M&l 2, 10 mM imidazole 3H-ATP added at zero time. Samples removed at indicated times were immediately mixed with myosin, centrifuged, and treated as in Fig. 3. Open symbols, exchange measured spectrophotometrically; solid symbols, exchange measured by tritium incorporation. q , F-ADP-actin; 0, 0, F-IDP-actin; A, 4, F-CDP-actin.
MOOS
Since the bound nucleotide in F-actin is so much less exchangeable than that in G-actin (29), it is generally assumed that the nucleotide exchange in F-actin is restricted somehow by the polymer structure. Hence the differences in nucleotide exchange rates among the various F-actins suggest that the species of bound nucleotide may influence the interaction between monomers in the F-actin polymer. Partial polymerization studies. As another approach to the possible effect of the bound nucleotide on the formation of actin-actin bonds in F-actin, partial polymerization at low MgClz concentration was compared for actins containing inosine and adenosine nucleotides. The IDP- and ADP-actins were prepared as described in Fig. 5 and suspended in ITP or ATP solution, respectively, containing 50 PM MgCL. Each actin was then depolymerized by sonication, the solution was transferred to a viscometer, and the time course of repolymerization was followed by means of viscosity measurements. As can be seen in Fig. 5, the actin containing inosine nucleotide polymerized considerably faster and to a greater extent than that containing adenosine nucleotide. Even when no MgCL was added to the inosine nucleotide-containing actin, so that the only salt present was the small amount remaining from the Dowex treatments (see caption of Fig. 5), the partial polymerization proceeded somewhat faster and to a slightly greater extent than the polymerization of adenosine nucleotide-containing actin with 50 PM MgCL. These differences in polymerization were not due to differences in the initial sonication of the actins because t’he first viscosity values, determined three minutes after the end of sonication, were the same whether the sonication had lasted only a few seconds or more than two minutes. It is also clear that there were no differences in actin concentration because both solutions in Fig. 5 reached the same final viscosity when fully polymerized by 0.1 M KC1 and 1 InM MgClz at the end of the experiment, and according to Fig. 1, the viscosity is an equivaIent measure of concentration for both ADP- and IDP-containing F-a&ins. Hence, these partial polymerization studies
BOUND-NUCLEOTIDE
0.8
-
0.6
-
0
30
SUBSTITUTED
60 Minuler
90 dler
120 end of ronicalion
395
F-ACTINS
150
1.90
211D
FIG. 5. Partial polymerization of actins containing inosine or adenosine nucleotide. Actin solutions, prepared as usual but without added salts, were made 1 mM in ITP or ATP respectively. (The nucleotides had been treated with Chelex-100 resin to remove contaminant Ca++ or Mg”.) The actins at this point were partially polymerized as a result of the Dowex treatments (see Mefhods), and the actin was sediment,ed by 6-hr centrifugation at 105,000 g, homogenized in 10 IIIM imidazole buffer, pH 7.0, containing 1 mM ITP or ATP respectively, and clarified by filtration. Protein concentration was adjusted to 0.75 mg/ml and the pH to 7.1, and MgClz was added to a final concentration of 50 NM. The solution was sonicated 60 set in an ice bath, and the time-course of repolymerization was followed by viscosity measurements. At the end of the experiment, the solutions were made 1 mM in MgClz and 0.1 M in KCI, and the viscosity of the fully polymerized F-actin was measured. 0, actin with inosine nucleotide; l , actin with adenosine nucleotide.
reveal a clear dependence of polymerization rate on the type of nucleotide in the actin, which gives further support t’o the suggestion, made on the basis of the nucleotide exchange studies, that the bound nucleotide affects the actin-actin interaction in the F-actin polymer. DISCUSSION
In this work, F-actins have been prepared which contain bound IDP or CDP in place of the usual bound ADP, and the effects of this nucleotide substitution on a number of properties of the actin have been investigated. The replacement of the bound nucleotide did not affect the binding of the F-actin to myosin or its activation of the myosin ATPase, which is in agreement with previous studies of nucleotide-substituted or nucleotide-deficient actins (S-10). On the ot’her hand, the rate of exchange of the bound nucleotide with free ATP, in both actin and actomyosin, was strongly influ-
enced by the type of nucleotide initially bound to the actin, the exchangeability increasing in the order IDP < ADP < CDP. In view of the great increase in the rate of nucleotide exchange in F-actin brought about by ultrasonic treatment (30), it seems reasonable to suppose that the replacement of the actin-bound nucleoside diphosphate by free ATP in our experiments depends on the prior rupture of an actin-actin bond in the polymer to create an “open” site at which the bound nucleotide is temporarily exposed to the medium. These “open” sites may be either partial disruptions within the polymer, called “f-actin” by Asakura et al. (ll), or complete breaks to form new free ends or G-actin monomers. The observed nucleotide exchange rate would then depend on the steady-state fraction of such “open” sites in the F-actin. For example, the influence of myosin on the exchange rate is interpreted, on this basis, to mean that the interaction of F-actin with myosin causes
396
ESTES
AND
an increased frequency of these local disruptions in the polymer (12, 13). Applying this approach to the difference in exchange rate between IDP- and ADPactins, one interpretation would be that the IDP-actin shows a slower rate of nucleotide exchange because it has fewer “open” sites in the steady-state than ADP-actin does under the same conditions, i.e., the presence of bound IDP in the actin in place of ADP makes the polymer more stable. Similarly, the greater exchange rate in CDP-actin may indicate that breaks occur more frequently in CDP-actin than in ADP-actin. In the case of IDP-actin, this interpretation would also correlate with our observation that the presence of bound inosine nucleotide in place of adenosine nucleotide increases both the rate and extent of the partial polymerization of the actin at low magnesium concentration. Other interpretations of our observations are no doubt possible, but it is clear in any case that the species of bound nucleotide in F-actin has a definite effect on certain of its properties. ACKNOWLEDGMENTS We thank Dr. Evan Eisenberg for suggestions and discussions. This was supported by Training Grant and Research Grant GM-10249 from Institute of General Medical Sciences, lic Health Service.
many helpful investigation 5Tl-GM-718 the National U. S. Pub-
REFERENCES 1. STRAUB, phys.
F. B. .~ND FEUER,
G., Biochim.
Bio-
Acta 4, 455 (1950).
K., BOTVEN, W. J., AND CLARK, A., J. Gen. Physiol. 33, 437 (1950). 3. MOMMAERTS, W. F. H. M., J. Biol. Chem., 198,
2. LAKI,
469 (1952). 4. TSUBOI, K. K., Biochim. Biophys. Acta 180, 420 (1968). 5. GRUBHOFER, N. AND WEBER, H. H., 2. Naturforsch. 16b, 435 (1961). 6. MARTONOSI, A. AND GOUVEA, M. A., J. Biol. Chem. 236, 1345 (1961). 7. STROHMAN, R. C. AND SAMORODIN, A. J., J. Biol. Chem. 237, 363 (1962).
MOOS 8. IYENGAR,
M.
R. AND WEBER,
H. H.,
Biochim.
Biophys. Acta 86, 543 (1964). 9. BARONY,
M.
AND
FINKELMAN,
F.,
Biochim.
Biophys. Acta 78, 175 (1963). 10. BUNNY, E., J. 11. ASAKURA,
M.,
TUCCI,
A. F.,
AND CONOVER,
T.
Mol. Biol. 19, 483 (1966). S., TANIGUCHI,
M.
AND OOSAWA,
F.,
J. Mol. Biol. 7, 55 (1963). 12. SZENT-GY~RGYI, A. G. AND PRIOR, G., J. Mol. Biol. 16, 515 (1966). 13. Moos, C., EISENBERG, E., AND ESTES, J. E., Biochim. Biophys. Acta 147, 536 (1967). 14. SZENT-GY~RGYI, A., “Chemistry of Muscular Contraction,” 2nd Ed., pp. 146-150. Academic Press, New York (1951). E. AND Moos, C., J. Biol. Chem., 15. EISENBERG, 242, 2945 (1967). 16. KUBY, S. A., Nova, L., AND LARDY, H. A., J. Biol. Chem. 209, 191 (1954). 17. Moos, C., Biochim. Biophys. Acta 93,85 (1964). 18. DRABIKOWSKI, W. END GERGELY, J., J. Biol. Chem. 237, 3412 (1962). 19. ULBRECHT, M., GRUBHOFER, N., JAISLE, F., AND WALTER, S., Biochim. Biophys. Acta 45, 443 (1960). 20. LORING, H. S., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, Eds.). Vol. 1, p. 191. Academic Press, New York (1955). 21. “Circular OR-17”. Pabst Laboratories, Milwaukee, Wisconsin (1961). 22. BOCK, R. M., LING, N. S., MORELL, S. A., AND LIPTON, S. H., Arch. Biochem. Biophys. 62, 253 (1956). M. F. AND ENGLANDER, S. W., Bio23. GELLERT, chemistry 2, 39 (1963). D., ELSDEN, S. R., AND GOUGH, N., 24. EGGLETON, Biochem. J. 37,526 (1943). 25. MARUYAMA, K. AND GERGELY, J., J. Biol. Chem. 237, 1095 (1962). B. AND JENCKS, W. D., J. Am. Chem. 26. NAGY, Sot. 87, 2480 (1965). 27. BARONY, M., NAGY, B., FINICELMAN, F., AND CHRAMBACH, A., J. Biol. Chem. 236, 2917 (1961). 28. KITAGAWA, S., DRABIKOWSKI, W., AND GERGELY, J., Arch. Biochem. Biophys. 126, 706 (1968). A., GOUVEA, M. A. AND GERGELY, 29. MARTONOSI, J., J. Biol. Chem., 236, 1700 (1960). S., TANIGUCHI, M., AND OOSAWA, 30. ASAKURA, F., Biochim. Biophys. Acta 74, 140 (1963).