Volume 18, number 1

MOLECULAR t~ CELLULAR BIOCHEMISTRY

November 25, 1977

THE POLYMERIZATION REACTION OF MUSCLE ACTIN* J. ENGEL, H. FASOLD, F. W. HULLA, F. W A E C H T E R & A. W E G N E R

Abteilung fiJr Biophysikalische Chemie Biozentrum, Basel, Switzerland and lnstitut fiJr Biochemie, Frankfurt a Main, GFR. (Received June 8, 1977)

Summary Recent advances in the studies of the aggregation of G-actin monomers, containing one molecule of ATP, to long filaments of F-actin, with a concomitant hydrolysis of the nucleotide to ADP, are reviewed. With the aid of e-ATP, the association and dissociation rate constant of the nucleotide could be determined. The binding of the nucleotide is enhanced by the binding of one Ca ++ ion, probably at a different site. The AG value of the Mg ++ or Ca ++ induced polymerization has been determined to - 3 9 to - 5 9 kJ/mole, the critical protein concentration for the ATP-G-actin to ADP-F-actin conversion is very strongly influenced by the concentration of bivalent cations. The rate constants of the protein monomers, and the rate and equilibrium constants for the propagation step show the process to be extremely cooperative. Actin shows the interesting phenomenon of translocational head-to-tail polymerization, which may be regulated by ATP. The contact sites between the monomers in F-actin have been labeled by chemical modification. Two tryosine residues, 53 and 69, are probably close to one of the two sites. The ATP binding site has been labeled by an ATP analog, and there is evidence that it is close to the contact site.

Introduction Actin is the main protein of the thin filament of muscle. During the last few years it has attracted additional interest because of its wide * A n invited article

distribution in all eucaryotic cells in the form of microfilaments which are involved in many locomotional processes. The protein exists in a monomeric and in a polymeric state. Monomeric G-actin (G stands for globular) is usually prepared by depolymerization of F-actin (F stands for filament). G-actin contains one molecule of ATP and may be repolymerized to F-actin bearing ADP. A detailed review on actin was presented by OOSAWA & K~SA~1. Recent research on actin to be reviewed in the present paper concentrated on the mechanism of polymerization, on the nucleotide binding and its hydrolysis and on the mapping of the important binding sites of actin.

Binding of Nucleotides to G-actin and Their Stabilizing Effect on Actin Contormation Monomeric actin (G-actin) has a molecular weight of 418002 . It forms tight 1:1 complexes with many nucleotides, under physiological conditions with ATP 3. The native conformation of actin is only stable when nucleotide is bound and nucleotide-free G-actin denatures irreversibly TM. It was found recently that the denaturation step is faster than preceding dissociation of the nucleotide under physiological conditions 5. This instability of nucleotide-free actin prevents a determination of the nucleotide binding constants by equilibrium methods. Only relative binding constants in which one nucleotide is replaced by another may be determined easily. For example, it was found by fluorimetric titration that the fluroescent ATP

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands

3

analog 1 :N6-ethenoadenosine 5'-triphosphate (eATP) binds only 2 to 5 times weaker than A T P 6"7. A fluorescence change connected with the dissociation of eATP from the actin moiety provided a convenient signal for a detailed kinetic study of nucleotide dissociation and association 5. Dissociation was induced by dilution to very low concentrations (5 nM to 1 /ZM). U n d e r normal conditions the rate limiting step was the dissociation of the complex and the concentration of free actin was always near to zero during the course of the kinetics. A n important finding was that addition of 1 mM E D T A highly accelerated the dissociation step. U n d e r these conditions nucleotide-free actin accumulates transiently during the course of the kinetics. Addition of Ca ++ within 20 sec after E D T A addition induced a re-association of the nucleotide-free but still native actin with eATP. The association was kinetically resolved by a multimixing stopped-flow apparatus and the association rate constant was determined to be 6 x 10 6 M-a sec -~. A combination of this value with the data derived from the dissociation kinetics yield the following scheme:

e A T P - G-actin

2.4×10-~sec '

~×IOaM -~ sec -~

5_5×,~ .... ,

e A T P + G-actin

~ denaturation

(1)

The values are for Tris buffer, p H 8.2 containing 0.8 mM Ca ++ at 21 °C. It was now possible to calculate the equilibrium constant of binding for e - A T P to be 2.5 x 10 9 M 1. With the relative binding constant of e - A T P and A T P the binding constant of A T P to G-actin was calculated to be K = 1.4 x 10 l° M-1. Earlier values which were derived from m e a s u r e m e n t s of the dissociation arid denaturation kinetics alone 46 are three to four orders of magnitude lower, probably because of the invalid assumption that a b o u t equal concentrations of free actin and nucleotide are present during the first phase of the dissociation kinetics. It m a y be noted that the binding constant of A T P to the subfragments of myosin K = 10 ~1 M-I ~ is even higher than that to actin. The very high binding constant of A T P to G-actin

explains that, despite the rapid denaturation rate, m o n o m e r i c actin is relatively stable in solutions containing nucleotides. For the biochemist working with actin it is of interest how fast the native actin-ATP complex is destroyed by denaturation. In the presence of an excess of A T P ([ATP]o >>[actin]o) the rate of formation of denatured actin D is d[D]/dt = k o [ A T P ] o - l K - l [ A T P - G - a c t i n ] . For [ATP]o--.1 mM only a few percent of the actin will denature at r o o m t e m p e r a t u r e during one day. The rate constant of dissociation of A T P from actin is strongly influenced by A T P itself, p r o b a b l y via an effector-like mechanism 7. The rate constant increases by a factor of about 30 in the range of [ATP] = 0.1 to 10 mM. It has been concluded f r o m equilibrium dialysis experiments that actin contains two A T P binding sites per subunit, with distinctly different binding constants 9. T h e r e exists also an interaction between the binding of Ca ++ or Mg ++ to G-actin and the binding of the nucleotide. The dissociation rate constant increases by a factor of about 2 when Ca ++ is r e m o v e d from its binding site on G-actin with which it forms a 1:1 complex with a binding constant of 105 M-1. In fact the binding of Ca ++ to e - A T P - G - a c t i n can be conveniently monitored by its effect on the dissociation at low concentrations of actin and e - A T P 1°. A t high e - A T P or A T P concentrations the effects of the nucleotide and of Ca ++ cannot be separated, since nucleoside triphosphates themselves bind Ca ++. Although direct m e a s u r e m e n t s are missing it is likely that the equilibrium constants of A T P binding are influenced by Ca ++ and A T P by about the same factors as the dissociation rate constants. There is evidence that Ca ++ and the nucleotide have separate binding sites. By careful proteolitic cleavage it was possible to isolate a large fragment which was still able to bind A T P but could no longer bind Ca ++ t L Binding constants of other nucleotides than A T P were usually determined by ASAKURA'S method 4 from the denaturation kinetics at various nucleotide concentrations. Although the published values for A D P 1"12, A T P (/3, 7 - N H ) 13 and others ~ scatter and m a y be affected by the limitations of the method, it seems to be clear that their binding is much weaker than that of ATP.

Association ot G-actin to F-actin and the role of ATP hydrolysis G-actin with nucleotide bound forms long filaments (F-actin) upon addition of bivalent cations such as Ca ++ or Mg ÷÷. In electronmicrographs the aggregates are seen as doublestranded helical polymers x4. The subunits appear as globular particles with a diameter of about 55 •. The half pitch of the helix is about 355 ~ . Heavy meromyosin molecules attached to actin filaments can be observed in electronmicrographs 15. They appear as prolate molecules each binding at the same angle with respect to the helix axis ("arrow heads"). The arrowhead structure underscores the polar character of actin filaments. The thin filaments of muscle were to be made up of a strongly distracted double helix of roughly uniform actin subunits, with the slender two tropomyosin strands attached in oppositional contacts on the outside of the fiber, as in a skewed rope ladder 16. Tropomyosin is not a necessary constituent of actin filaments which apparently have the same geometry without it. All kinetic studies reviewed below were performed without tropomyosin. Schematically the structure of F-actin is drawn in Figure 1. The asymmetric structure of the protomers which is required for the formation of polar filaments is indicated by the chevron symbol. There are at least four contact sites A to D with neighbouring protomers. In addition each actin protomer must contain at least one binding site for the nucleotide, one for tropomyosin and one for myosin. As for any linear association the product Kcto t of the propagation binding constant K and the total protomer concentration ctot must exceed a critical value K c t o t ~ 1 before association takes place. Since K depends on the Mg +÷ or Ca ÷÷ concentration 17 this leads to the phenomenon of

Fig. 1. Schematic drawing of an actin filament with two pairs of c o m p l e m e n t a r y contact sites A - - • B and C -. - D. The polar character of the filament is indicated by using chevron symbols for the protomers.

a critical necessary salt concentration at a given protein concentration and to a critical actin concentration under otherwise constant conditions 1s'19. A magnesium concentration of 1 mM is sufficient to decrease the concentration of ATP-G-actin coexisting with polymeric actin below 1 x 10 -6 M. Monovalent cations such as potassium are less effective. The ATP bound to monomeric actin takes part in the polymerization of actin3. One ATP molecule is hydrolyzed during the association of one molecule monomeric actin and is incorporated into the filament as ADP, whereas the inorganic phosphate is released into solution. The hydrolysis of ATP appears to be irreversible. A resynthesis of ATP on dissociation of a subunit from the polymer was never observed. Most studies of actin polymerization were performed with an almost constant "buffered" ATP concentration in high excess over actin. The excess is necessary in order to prevent denaturation of G-actin and it also reflects the physiological situation. Under this condition the association and dissociation reactions form a cycle- driven by ATP hydrolysis- with all partners in steady state (scheme (2)). This scheme

I ATP-G-actin

Pi Jr ,

ADP-F-actin

ADP / III ~x--~ ATP ADP J \ \ ATP ADP-G-actin

II

(2)

refers to the propagation steps by which most of the actin monomers are transformed to F-actin. Therefore reaction I stands for the addition of a monomer to an existing filament and reaction II for the dissociation of a protomer from the filament. It is not known whether ATP splitting takes also place during nucleation. This process - although of great importance for the kinetics and for other properties- involves only an extremely small portion of the total actin. Its possible contribution to the hydrolysis is therefore unmeasurably small. For the steady state equilibrium in scheme (2) the critical concentration is equal to the reciprocal of a propagation steady state constant whereas in the case of a true equilibrium (e.g. for the polymerization of ADP-G-actin to ADP-F-actin) it corresponds to

the reciprocal of the equilibrium binding constant of propagation. Recently the critical concentrations of the polymerization reaction I and II in scheme 2 were compared under identical conditions: in Tris buffer pH 8.0, containing 0.1 mM Mg C12 and 0.4 na~ ATP or ADP respectively13. A value of 1.4 × 1 0 6 M-1 was obtained for the steady state propagation constant of the ATP-G-actin to ADP-F-actin conversion as compared to an equilibrium binding constant of propagation K = 3 x 105 M-~ for reaction II. Since these values cannot be compared it is of interest to calculate the standard energy for reaction I. This is possible with the following thermodynamic cycle which is not related to the reaction mechanism but for which the standard free energies AG° are known at pH 8 and 25 °C. a) A T P - G - a c t i n ~ A T P + G-actin, 58 kJ/mol 5, b) A T P ~ A D P + P , - 3 5 kJlmol, c) G - a c t i n + A D P ~ A D P - G - a c t i n , - 5 0 to - 3 0 kJ/mol 1'8'12 d) ADP-G-actin,-~-ADP-F-actin, - 3 2 kJ/moP 3. The sum of reaction (a) to (d) yields reaction I of scheme (2) and a AG° value of - 3 9 to - 5 9 kJ/mol. The numerical values may be rather inaccurate due to the fact that the values of the individual steps were not determined under exactly identical conditions and because of the uncertainties involved in the determination of the binding of ADP to G-actin (see preceding section). The lower limit for reaction (c) of - 5 0 kJ/mol was estimated on the basis of the absolute binding constant for ATP 5 and published values for the relative binding constants 1. From the AG°-values the equilibrium constant K= [(ADP-F-actin-~÷ 1][P,] [ADP-F-actin)~][ATP-G-actin] = 9x106 to 3 x 1 0 ~° is calculated. K is approximately equal to the ratio of the concentrations of phosphate to monomeric ATP-G-actin in equilibrium with the filaments because the concentrations of filaments whose length differs by one protomer only are almost equal. Under experimental conditions, however, the ratio of phosphate concentrations to the critical monomer concentration is normally much lower than K. Also the critical monomer concentration is apparently not influenced by addition of phosphate 13. This 6

again demonstrates that the concentrations of the reaction partners are far from the equilibrium concentrations in reaction2. The observation2° that addition of ATP helps to depolymerize ADP-F-actin under so-called "depolymerizing conditions" e.g. at low Ca ++ or Mg +÷ concentrations may be rationalized on the basis of schemes by the higher affinity of ATP to G-actin as compared to ADP. When reaction I is less effective or shut off, reaction III will shift the equilibrium towards ATP-G-actin in the presence of an excess of ATP. With a relative binding constant of ATP to ADP of 100 (which corresponds to AG°= 47 kJ/mol for reaction (c)) a 10000 fold excess of ATP over ADP would be required for almost complete depolymerization. Another possible depolymerization effect of high concentrations of ATP may be the lowering of the concentration of free Ca ÷+ and Mg ÷+ via complex formation with these ions. The possibility to form F-actin with an accompanying hydrolysis of ATP and the opposing effect of ATP reaction III may be of critical importance for the regulation of actin association especially in the case of actin of nonmuscle cells in which a high amount of monomers coexisting with filaments was observed 21. Recently it was found that polymerization of actin is inhibited by a small protein named profiline and it was suggested that the profiline-G-actin complex38 is a storage form of monomeric actin in nonmuscle cells (L. CARLSON, presented at the 2 na Alpach Meeting on Muscle, March 1977). The effect of Ca ++ and Mg ÷+ on the critical concentration of the ATP-G-actin to ADP-Factin conversion is extremely large. A threefold increase of the Mg ++ concentration from 0.4 to 1.2 mM decreases the critical concentration by a factor of 10 ~7. It is unlikely that the high affinity Ca ÷÷ binding site at G-actin is involved. This site is expected to be fully saturated at salt concentrations much higher than its dissociation constant of 10 -5 M (see preceding paragraph). MARTONOSI et al. ~Ta found more than seven other low affinity binding sites for Mg +÷ and Ca +* (with binding constants of about 103 M-~). It was concluded that about half of these must be saturated in order to prevent electrostatic repulsion and in order to induce polymerization. Following the lines of thoughts on the regulatory action of ATP it may be speculated that

Ca ++ and Mg +÷ primarily influence reaction I in scheme (2). For the above considerations it would be important to know the thermodynamic parameters of the polymerization of ATP-Gactin to ATP-F-actin without hydrolysis. An attempt was made to mimic this reaction by the use of the nonhydrolyzable ATP analog ATP (~, T - N H ) 13'22. Surprisingly it was found that the propagation binding constant for G-actin saturated with this derivative was larger than for ADP-G-actin 13. Conflicting data were presented by other authors 22.

Kinetics of Actin Association The aggregation of actin was investigated by light scattering and electronmicroscopy23. The polymerization was started by a sudden increase of the calcium chloride concentration from 0.2 to I mM in a monomeric actin solution (in 5 mM Tris buffer, pH 7.5 containing 0.5 mM ATP at 20 °C). The angular dependence of the light scattering intensity indicated that a short period after the beginning of the polymerization long rods were formed which contained several hundred subunits. The time dependence of this light scattering was sigmoidal. A quantitative analysis of the experimental data was possible on the basis of a model of cooperative polymerization. The filaments were assumed to grow and shorten by sequential binding and release of monomers. This appears to be a reasonable first approximation because the dissociation into filament fragments involves the splitting of at least three contacts of complementary binding sites as compared to only two for the dissociation of a protomer (see Fig. 1). Similarly the association of fragments is expected to be a rare event because of the steric difficulties and because of the low filament concentration as compared to the monomer concentration. On account of the uniform structure of actin filaments, the rate of association and dissociation were thought to be independent of the length with the exception of dimerization. Dimerization differs from propagation because two monomers form merely a single contact, whereas a monomer forms contact with two polymer subunits on propagation. Consequently four kinetic parameters and two equilibrium parameters were introduced. Association

and dissociation rate constants (k, k') and an equilibrium constant (K = k/k') for propagation and corresponding constants for dimerization (kN, k~, KN = kN/k~). The reaction scheme is given by 2A ~

A2 ~

A3""An

"-U- A.+I,

(3) where A represents an actin subunit. As pointed out above due to the coupling of the ATP to ADP conversion KN and K are not true equilibrium constants. They are defined only for a given buffered concentration of ATP. Also it has to be recalled that k' does not simply correspond to the reverse reaction of ATP-G-actin association but to the dissociation of an ADP-actin protomer (see scheme (2)). The equilibrium concentration of monomers (el, critical monomer concentration) was easily calculated from the condition that the rate of association k. cl was equal to the rate of dissociation k' :Cl = k'/k = K -~. The critical monomer concentration was obtained from a plot of total actin concentration versus light scattering intensity. An extrapolation of the scattering intensity to zero concentration yielded a critical monomer concentration of 6 x 10 .6 M. The observed formation of long filaments was explained by slow nucleation and fast propagation. A few nuclei were formed and subsequently elongated by rapid growth to long filaments. Slow nucleation could have two reasons: The rate of dimerization was slow compared to the rate of propagation (kNk . cl, where cl is the monomer concentration). The cases of slow dimerization and fast dimer dissociation could be distinguished by measuring the kinetics of polymerization at different total actin concentrations. Slow formation of dimers was excluded, and the best fit of the experimental data was obtained for fast dimer dissociation. As a consequence of the fast dimer dissociation monomers and dimers were in equilibrium (fast pre-equilibrium). For an evaluation of the kinetic parameters an additional knowledge of the concentration of filaments was necessary which was achieved by electronmicroscopy. The following rate and equilibrium constants were 7

obtained for propagation: k = 5 × 103 M-1 see-1, k' = 3 × 10 -2 sec-1, K = ~-x = 1.7x 105 M-~. On account of the fast monomer-dimer pre, equilibrium only KN and no explicit values for kN and k~ could be extracted from the experimental data. KN was 5 × 10 6 times smaller than K. Actin association was extremely cooperative.

Kinetically Controlled Length Distributions The equilibrium length distributions predicted for a linear association with propagation binding constants independent of length are exponential. The average length naverag e is the higher the more cooperative the system is ~s'19. By electronmicroscopy almost exponential size distributions were observed already at unexpectedly short times of polymerization in vitro 24. The exponential distributions may however also be due to random breakages of filaments during specimen preparation. According to mechanism(3) with association dissociation steps occurring at the ends of filaments only and with the experimental

kinetic constants it follows that extremely long times are needed to establish equilibrium distributions. Intermediate length distributions of actin filaments were calculated with the experimentally determined parameters ~-5. The distributions showed a maximum for long polymers with a sharp decrease on the side of longer polymers and a moderate decrease for shorter polymers (Fig. 2). The concentration of that species of filaments with the highest concentration corresponded to the concentration of dimers at the start of polymerization. The degree of polymerization of these filaments corresponded to the average number of monomers bound to these initially existent dimers. Also the concentration of the other species of polymers were approximated well by assuming that the concentration of nuclei at time tl was equal to the concentration of polymers at time t2 with the average number of subunits incorporated within the interval of time t 2 - h . The moderate decrease of the concentration of shorter polymers reflected the continuous decrease in the number of nuclei.

10 -s. t=104 sec

t=2.104sec t= 3.104sec

Ci

Ctot

t-,co |

'

II

0

i

400

II

all

800 i

il

|

1200

|

~

|

1600

Fig. 2. Size distributions after various times t and at equilibrium (t--> ~). The fraction of filaments with degree of association i is p l o t t e d v e r s u s i. T h e d i s t r i b u t i o n s w e r e c a l c u l a t e d b y n u m e r i c a l i n t e g r a t i o n of t h e r a t e e q u a t i o n s c o r r e s p o n d i n g to s c h e m e 1 w i t h t h e e x p e r i m e n t a l p a r a m e t e r s Ctota 1= 2 . 2 x 10 -5, k = 5 × 10 3 M -x s e c - 1 , k ' = 3 × 1 0 - 2 s e c - 1 a n d K N = 4 × 1 0 - 2 M -1 a n d f o r f a s t monomer ~ dimer equilibrium.

8

The intermediate distributions were kinetically rather stable. The time which was needed to relax the intermediately formed length distribution into the exponential equilibrium distribution was about n ..... g~ times longer than the time necessary to reach the critical m o n o m e r concentration. This appearance of two kinetic phases was derived earlier in a general way 25"26,27. Under the conditions used for actin association the time needed to establish the critical m o n o m e r concentration was about 10 hours, which means that establishment of the equilibrium distribution is expected only after several years. These considerations demonstrate that any length distribution, regardless of the way in which it was formed, will persist for extremely long periods of time. The defined length of the thin filament in muscle is determined by a still unknown mechanism. Their length may be maintained although it may not accord to the equilibrium size distribution.

Role of the ATP to A D P Conversion in Polymerization Kinetics Since the polymerization is accompanied by the irreversible hydrolysis of A T P some restrictions

for the growth direction are removed which would be valid for systems in which the dissociation is the reverse reaction of association. In the case or reversibility the growth direction of a filament is either outwards from or inwards to the center at both ends of an aggregate. If the m o n o m e r concentration is at equilibrium (critical m o n o m e r concentration established), the distance between the ends and a particular subunit remains constant on the average. The growth rate may be different at both ends on account of the polarity of filaments and in a limiting case it may be unidirectional. In contrast, the irreversible ATP-driven associationdissociation cycle (scheme 2) enable actin polymers to lengthen at one end and to shorten simultaneously at the other 28. The distance between one end and any particular subunit becomes smaller while the distance between the other end and that subunit increases. This leads to a translocation of actin filaments (translocational head-to-tail polymerization) (see Fig. 3). Experimentally, the translocational head-to-tail polymerization mechansim was confirmed by measurements of the exchange rate of monomers and polymer subunits. Actin was polymerized starting from monomers alone. At the final state when the m o n o m e r concentration had reached the critical concentration a trace of o

P

'

ATP

P

P

~o

'

ATP

ATP

ADP

ADP

AOP 7

Fig. 3. Scheme of the process of translocational head-to-tail polymerization. Newly incorporated protomers are drawn in black. Some individual protomers at the shortening end are marked with numbers.

monomeric actin radioactively labeled with [14C]-N-ethylmaleimide was added to the solution. The incorporation of labeled actin monomers into filaments and the release of unlabeled subunits from filaments was followed by taking samples, separating monomers and filaments by centrifugation, and determining the distribution of the labeled material between monomers and filaments. The translocational head-to-tail polymerization has a strongly accelerating effect on the rate of exchange. In a limiting case of translocational head-to-tail polymerization association occurs only at one end of the filament and dissociation only at the other. In this case each association step leads to an incorporation of a labeled monomer at one end and each dissociation step to a release of an unlabeled subunit from the other end of the filament. However, if dissociation is the reverse reaction of association, association steps and dissociation steps occur at each end with the same probability. Subunits are incorporated and released in a diffusion-like process by fluctuation of filament length. The time which is needed to exchange the n-th subunit from the end of a filament in a diffusion-like process is about n times longer than in the case of translocational polymerization. As actin filaments contain several hundred subunits, measurements of subunit exchange were a useful experiment to distinguish between the diffusional and directed translocational type of subunit exchange. On the basis of the kinetic parameters of actin polymerization the rate of subunit exchange was calculated for the translocational case with the following assumptions: Association and dissociation steps may occur at either end and the simultaneous lengthening of one end and shortening at the other is the net result of different frequencies of association and dissociation steps at each end. The best fit was achieved for the case in which four association and four dissociation steps lead to a lengthening of the filament by one subunit at one end and a shortening by one subunit at the other. The irreversible hydrolysis of ATP connected with the polymerization gives the actin filaments more possibilities for the regulation of the direction of growth. It may be significant in the assembly and length determination of thin muscle filaments, in the translocation of actin filaments and in cell shape determination. 10

Mapping of the Contact Sites and of the ATP Binding Site The sequence of rabbit striated muscle actin has been determined by the group of Elzinga2. It contains 374 residues; the molecular weight based on the amino acid composition is 41825. G-actin contains 5 cysteins in positions 10, 217, 256, 284 and in the penultimate position 373. Only Cys 373 seems to be at the surface of the protein because it is the only one which is easily accessible even to bulky sulfhydryl reagents. Two others, in positions 10 and 284 may be modified by some reagents TM,with only partial inhibition of polymerization and of myosin binding. The protein contains one unusual amino acid, 3-methyl-histidine in position 73.

Labeling Experiments Both affinity labeling and unspecific side chain modification have been performed on the protein with success. Some loci in the known sequence that are involved in the contact formation between subunits have been identified, also the ATP binding site seems to evolve. On the other hand, the primary structure regions pertaining to troposin, tropomyosin, or myosin binding are as yet not clear. Perhaps the observation that Cys 373 is not accessible in the actomyosin complex is pertinent to the myosin interaction site zSa. However the modification of this sulfhydryl with quite bulky substituents does not change any of the physiological functions of polymerization, myosin binding, or tropomyosin bindingTM. A relevant observation concerning one of the contact sites was the finding of HEG¥I e t al. 29 that out of several histidines His 40 was inaccessible to diethylpyrocarbonate in F-actin but not in G-actin. A study of the nitration of G-actin with tetranitromethane3° showed a preferential modification of Tyr 69. This residue was identified by isolation of the nitrotyrosine containing peptides. Polymerization, but apparently not ATP binding, was inhibited by these modifications. A third side chain in this portion of the sequence, tyrosine 53, is the only one which reacts with diazonium-lH-tetrazole under appropriate conditions31. The surface SH group

of Cys 373 was protected by N-ethylmaleimide before addition of the tetrazole. The reagent was synthesized in the 14C-labeled form from sodium azide and laC-cyanamide. This permitted the evaluation of a linear relationship of polymerization inhibition and the degree of labelling of a single labeled tryptic peptide in fingerprints and autoradiographs• In this investigation, special care was taken to establish the intactness of other functions of native actin. An ATP content of 0.95 moles of the nucleotide per mole of modified actin was found• The modified protein still bound myosin subfragment 1, as was shown by ultracentrifugation. The modified protein may even be crystallized• Very thin needles of up to 2 mm length were formed. From the sum of these polymerization inhibiting modifications, one may conclude that an area of the actin molecule, comprising parts of the sequence between positions 40 and 69 builds up one of the contact sites of actin. The unusal amino acid, 3-methylhistidine 73, is close to this site, although a possible involvement, or at least an adjacent position in the tertiary structure is, of course, speculative. In this context it is noteworthy that the tryptic or chyrnotryptic fragmentI° starting from position 69 or 68 does not polymerize but still binds ATP. The non-polymerizing G-actin derivative modified with diazonium-lH-tetrazole in position Tyr 53 is precipitated at rather low salt concentrations when dissolved in 2 mM TrisHCl-buffer at pH 7.0, containing the usual 0.1 mM Mg++-ATP and 0.1 mM CaC12. The protein starts to aggregate at 0.3 M potassium phosphate concentration, and already at 0.06 M ammonium sulfate. This effect is distinctly counteracted by magnesium ions; in 25 mM m~tgnesium acetate solution the concentration of ammonum sulfate may be raised to 0.12 M. On the other hand, relatively large amounts of organic solvent may be added to its solution without precipitation or denaturation, as 10% of dimethylformamide, or 5-10% of dioxane. This derivative, like unmodified G-actin, will not bind phalloidin at all, which is avidly taken up by F-actin32. The derivative will however bind to and inhibit DNase, as F-actin does ~. Although it will bind to myosin, as indicated above, it is not able to activate the myosin ATPase. Since an artificial dimer of diazoniumtetrazol modified actin cross-linked by tartryl•

I

bisglycine azide, do activate the myosin ATPase to some extent 33, either a changed conformation of the protein in the dimer or a binding site for the myosin head extended over the boundaries of one actin subunit must be postulated• Some information on the architecture of the nucleotide binding site may be obtained from studies of the binding of ATP analoga. As already described, the binding constant of eATP is only 2 to 5 times smaller than that of ATP. This shows that a rather large modification of the adenine moiety is tolerated by its binding site. Also nucleotides with other bases like GTP, CTP and ITP were found to bind to actin ~. On the other hand modifications in the phosphate region appear to be more critical• ADP binds more weakly than ATP. The nonhydrolyzable analoges ATP (/3, 3,-NH) and ATP (/3, 3'-CH2) may take the place of ATP or ADP, although with much smaller binding constants. A covalent label for the ATP binding site was discovered in (S-dinitrophenyl)-6mercaptopurin riboside triphosphate ~9. After protection of Cys 373 by N-ethylmaleimide, incubation of the reagent with F-actin leads to rapid depolymerization, and during several hours' subsequent reaction, to a covalent attachment of 0.8 moles of the nucleotide moiety per mole of actin (determined after a /3,,y32p_ labeled synthesis of the analogue), while dinitrophenole is split off. Addition of i mM Mg ÷+ and 0.1 MKC1 induces the normal polymerization reaction, half of the radioactive phosphate being split off. When the newly formed F-actin is washed twice by centrifugation 0.95 moles of the labeled diphosphate per mole of actin remained covalently bound• These findings indicate that the ATP binding site is indeed occupied by the analogue. This is in agreement with our previous explanation of the depolymerizing action of ATP. Since the ADP analog is covalently attached to the actin protomers the exchange reaction III in scheme (2) cannot take place. The modified F-actin could not be depolymerized again under the usual conditions, using up to 50 mM ATP concentrations in the depolymerization buffer which contained less than 10 -s M Ca ++ and no Mg +÷ 34. Moreover, only a single tryptic peptide was found labeled in autoradiographs of fingerprints of the 11

modified protein. Preliminary analyses of the purified peptide seem to indicate that the nucleotide is bound to the same tyrosine 53 that was labeled by diazonium-lH-tetrazole, which would put the ATP binding site in close vicinity to the probable location of one intersubunit contact. These analyses have been difficult to carry out, as model ractions between the analogue and tyrosine derivatives gave no well defined product, and the label on the peptide apparently was not very stable. On the other hand, previous labelling of actin with the ATP analogue prevents subsequent attachment of diazonium-lH-tetrazole on tyrosine 53. From the experiments summarized above, one might envisage a role of the nucleotide in the construction of the contact site. The hypothesis would also explain the very slow exchange rates of ADP and ATP in the F-actin nucleotide binding site. Among others, this has been measured by GERGELYet al. 35, who incubated F-actin with radioactively labeled ADP. Under these conditions, a half life time of several hours was found for ADP-exchange. Recently it was demonstrated by fluorescence polarization measurements of e-ADP bound to F-actin which was oriented by flow that the adenine plane of the bound nucleotide is almost perpendicular to the long axis of the filaments36. The contact sites as well as the binding sites for the nucleotide, metal ions, and the proteins interacting with actin can be located with much higher precision than by the methods described above when the threedimensional structure of actin becomes available. As it was the case for many other systems an elucidation of the structure by x-ray crystallographic methods will open new dimensions in the understanding of the function of actin. Since G-actin is designed to form actin filaments whenever it associates, it is very difficult to find conditions under which it forms crystals. A French g r o u p 37 w a s able to crystallize G-actin with polyethylene glycol by which polymerization is suppressed. Another approach is the inhibition of polymerization by specific modifications of the protein such as the blocking of an SH-group by diazonium-1 Htetrazole described above. Also the proteolytic fragment which lost the polymerizing activity11 appears to be a good candidate for crystallization. Also complexes of G-actin with profiline38 and DNAse 139 have been crystallized. Promis12

ing crystallographic work with such derivatives proceeds in various laboratories and it may be hoped that the structure of G-actin will be known within a few years from now. Since it is relatively easy to prepare oriented gels from F-actin, an application of x-ray crystallographic methods to this structure is also feasible. In this case the problem of phasing is a much more difficult one than in the case of oriented gels of TMV virus because of the very small diameter of the filaments. This problem could however be solved when the structure of G-actin is known. Re|erences 1. Oosawa, F., and Kasai, M. 1971. Biological Macromolecules, Vol. 5, Subunits in Biological Systems, Marcel Dekker, New York, pp. 261-322. 2. Elzinga, M., Collins, J. H., Kuehl, W. M., and Adelstein, R. S. 1973. Proc. Nat. Acad. Sci., USA, 70, 26872691. 3. Straub, F. B., and Feuer, G. 1950. Biochim. Biophys. Acta 4, 455-470. 4. Asakura, S. 1961. Arch. Biochem. Biophys. 92, 140149. 5. Waechter, F., and Engel, J. 1977. Eur. J. Biochem., 74, 227-232. 6. Thames, K. E., Chenug, H. C., and Harvey, S. C. (1974), Biochem. Biophys. Res. Commun. 60, 12521261. 7. Waechter, F., and Engel, J. 1975. Eur. J. Biochem. 57, 453-459. 8. Mannherz, H. G., and Goody, R. S. 1976. Ann. Rev. Biochem. 45, 427. 9. Mannherz, H. G., Barrington Leigh, J., Lebermann, R., Pfrang, H. 1975. FEBS Letters 60, 34. 10. Waechter, F. 1975. Hoppe-Seyler's Z. Physiol. Chem. 356, 1821-1822. 11. Jaeobson, G., and Rosenbusch, J. 1976. PNAS 73, 2742-2746. 12. West, J. J. 1970. Biochemistry 9, 1239. 13. Cooke, R. 1975. Biochemistry 14, 3250. 14. Hanson, J., and Lowy, J. 1963, J. Mol. Biol. 6, 46. 15. Huxley, H. E. 1963. J. Mol. Biol. 7, 281-308. 16. Wakabayashi, T., Huxley, H. E., Amos, L. A., and King, A. 1975. J. Mol. Biol. 93,477. 17. Oosawa, F., Asakura, S., Hotta, K., Imai, N., and Ooi, T. 1959. J. Polym. Sci. 37, 323. 17a. Martonosi, A., Molino, C. M., and Gergely, J. 1964. J. Biol. Chem. 239, 1057. 18. Winklmair, D. 1971. Arch. Biochem. Biophys. 147, 509. 19. Engel, J., and Winklmair, D. 1972. In Enzymes: Structure and Function (ed. Drenth, J., Oosterbaan, R. A. and Veeger, C., North Holland, p. 29. 20. Bender, N., Fasold, H., and Rack, M. 1974. FEBS Letters, 44, 209. 21. Bray, D., and Thomas, C. 1976. J. Mol, Biol. 105, 527-544.

22. Schenk, H., Mannherz, H. G., and Goody, R. S. 1973. Hoppe-Seyler's Z. Physiol. Chem. 354, 234. 22a Mannherz, H. G., Brehme, H., and Lamp U. 1975. Eur. J. Biochem. 60, 109-116. 23. Wegner, A., and Engel, J. 1975. Biophysical Chemistry 3, 215-225. 24. Kawamura, M., and Maruyama, K. 1970. J. Biochem. 67, 437-457. 25. Engel, J., and Wegner, A. 1976. Studia biophysica 57, 179-184. 26. Oosawa, F. 1970. J. theor. Biol. 27, 69. 27. Oosawa, F., and Asakura, S. 1975. Thermodynamics of the Polymerization of Protein, Academic Press, London, p. 51. 28. Wegner, A. 1976. J. Mol. Biol. 108, 139-150. 28a Lusty, C. U., and Fasold, H. 1969. Biochemistry 8, 2933. 29. Hegyi, G., Premecz, G., Sain, B., and Miihlrad, A. 1974. Eur. J. of Biochemistry 44, 7. 30. Elzinga, M. 1971. Abstr. Full, Meet. Am. Chem. Soc., Washington D. C., p. 61. 31. Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G., and Volk, K. E. 1976. Eur. J. of Biochemistry 64, 215. 32. Lengsfeld, A. M., L6w, I., Wieland, T., Dancker, P., and Hasselbach, W. 1974. PNAS 71, 2803. 33. Fasold, H., B~iumert, H., and Bender, N. 1975. Biochem. Soc. Transactions 3, 935. 34. Faust, U., Fasold, H., and Ortanderl. F. 1974. J. of Europ. Biochemistry 43, 273. 35. Kuehl, W. M., and Gergely, J. 1969. YBCZ 44, 4720. 36. Mild, M., and Mihashi, K. 1977. Biophysical Chemistry 6, 101-106. 37. Oriol, Ch., Dubord, Ch., and Landon, F. 1977, FEBS Letters 73, 89. 38. Carlsson, L., Nystr6m, L.-E., Lindberg, U., Kannan, K. K., Cid-Dresdner, L6vgren, S., and J6rnvall, H. 1976. J. Mol. Biol. 105, 353. 39. Mannherz, H. G., Kabsch, W., and Leberman, R. 1977. FEBS. Letters 73, 141.

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