Detection of conformational changes in actin by proteolytic digestion: Evidence for a new monomeric species

J. Mol. Biol. (1976) 104, 777-792

Detection of Conformational Changes in Actin by Proteolytic Digestion : Evidence for a New Monomeric Species STEVEX

A.

RICH? AND JAMES

E. ESTES$

ljepartment of Biology Renssekzer Polytechnic Institute Troy, N. Y. 12181, I ‘.&‘.A, (Received 24 Januwy

1.975, and in revised form, II February

1.976)

When KC1 is added to a solution of G-a&in to induce full polymerization, a decrease in the rate at which actin undergoes enzymatic proteolysis occurs. This decrease cannot, he accounted for by factors affecting the enzymes employed, but rather appears to be due to a change in the conformation of G-actin. Partially polymerized actin solutions also show a reduction in digest,ibility which is dependent on the F-actin content, suggesting that F-actin is essentially indigestible. Moreover, low rates of digestion were also observed at sub-critical actin concentrations, where act,in in the presence of 0.1 M-KC] does not polymerize. This indicates that’ a conformational change occurs in G-a&in before the polymerization strep. At sub-critical concentrations in 0.1 M-KC& actin is in a truly monomeric state it’s inability to enhance the rate as judged by its viscosity characteristics, of polymerization of G-a&in and it,s possession of ATP as the actin-bound nucleotide. These data support t,he existence of a new species of actin, called F-ATP-actin monomer, which has the same physical properties and the same hound nucleotide as G-a&in, but digestion charact,eristics like F-actin. Since F-ATP-actin monomers have the same low susceptibility to proteolysis as F-ADP-actin polymers, and because both G-ATP-actin and G-ADP-actin have similar high rates of digestion, the observed change in the conformation of actin cannot be due to the phosphorylat#ed state of the actin-bound nucleotide. Instead, the conformational change appears to he caused by the addition of KC1 to G-actin. The newly-detected monomeric species is considered to be an intermediate in the polymerization process where F-ATP-actfin monomers form a population of polymerizable molecules which must reach a critical concentration before llucleat,ion and F-actin polymer formation begin.

1. Introduction In X-ray diffraction studies on whole muscle at rest, in rigor and during contraction. the reflections from F-actin appear to remain constant, suggesting that,, in vivo, F-actin does not undergo any large or detectable structural changes (Huxley & Brown, 1967; Elliott et al., 1967). There is in vitro evidence, however, that conformational changes can occur in F-actin during actin-myosin interaction. The t Present

address:

Division

of Lahoretories

and Rewxitrch, Nrw

Health, Albany, N.Y. 12206, U.S.A. $ To whom reprint requests should be addressed. 777

York

State Department

of

778

s . A.

IilC’H

.\SI)

J.

15. ICS’I’ES

exchangeability of t#he XDP bound t#o t hr> monomer subunit’s in t II(~ ac:t’irr polymctr is increased significantly during act’omyosin suI)erprecipita,t,ioll (SzrtntGyorgyi & Prior, 1966; Moos et al., 1967; Estes & Moos. 1969). Furthermore. conformat’ional changes in F-actin have been proposed to account’ for the ability of F-actin to behave as an enzymatic ATPase during ultrasonic vibration (Asakura, 1961; Asakura et al., 1963) and during treatment at elevated temperatures (Asai & Tawada, 1966). These conformational changes in F-act’in may be due t’o a loosening or rupturing of portions of the double-stranded polymer in some manner so that the monomer units within the polymer behave more like G-actin molecules. Such localized changes might., therefore, resemble the conformational changes that reportedly accompany hhe transformation of G-a&in into F-actin. The existence of changes in the terbiary structure of G-actin during polymerization is supported by the occurrence of ultraviolet difference spectra, which apparently originate because t,he environment of the tyrosine and bryptophan residues in actin is different in the monomer and polymer states (Higashi & Oosawa, 1965; West, 1970). Polymerization is reportedly also accompanied by a decrease in the magnitude of the electron paramagnetic resonance spectra of a maleimide derivative covalently bound to G-actin (Stone et al., 1970). Further evidence for a different conformation in the two states has come from comparing the circular dichroic spectra of G- and F-actin (Murphy, 1971) and from the difference in the intrinsic fluorescence of Gand F-actin reported by Lehrer & Kerwar (1972). These spectroscopic investigations are supported by the enzymatic study of Offer et (11. (1972), which showed that the ability of G-actin to activate the ATPase of subfragment 1 of myosin was considerably less than the activation obtained with F-actin under identical conditions. This result implies that the conformation of G-actin is different from the conformation of the subunit in F-actin. At least some of the difference in the structure of actin in the two states has been considered to be associated with t)he dephosphorylation of the G-actin-bound ATP that accompanies the G t’o F transformation. Recently, however, Cooke & Murdoch (1973) f ound t’hat replacement’ of the bound ATP with AMP-PNP (adenylylimidodiphosphate), an analog of ATP which is not hydrolyzed by actin during polymerization, does not effect either t’he rat)e or extent of F-actin formation. Thus, the energy released by bound nucleotide hydrolysis does not appear to facilitate polymerization or induce conformational changes that could enhance the polymerization process. Therefore, questions remain not only as to the necessity of conformational changes during polymerization, but also as to t)he nature and cause of such changes. To understand better t’he changes that occur in actin upon polymerization, the susceptibilities of the monomer and polymer states of actin to proteolytic digestion were examined. Because the different conformational states would be expect,ed to have different susceptibilities to proteolytic digestion, a change in the rate of proteolysis of the protein would be a sensitive, direct indication of a conformation change (Markus, 1965; Mihalyi, 1972). Previous attempt,s to subject actin to proteolysis have shown that F-actin requires treat’ment with very high trypsin concentrations before a measurable decrease in its viscosity occurs (Mihalyi, 1953; Laki, 1964). Nagy & Jencks (1962) correlated optical rotatory dispersion measurements with the rate of digestion of G-ATP-actin and of denatured, nucleotide-free G-a&in, and concluded that the loss of the actin-bound nucleotide caused a change in protein conformation.

EVIDENCE

FOR

AN

F-ATP-ACTIN

MONOMER

779

The results of this current study show that monomeric G-ATP-actin undergoes a change in digestibility upon addition of KC1 prior to formation of nuclei or polymers. The stable F-ATP-actin monomer thus formed is an intermediate in the polymerization process and will readily form F-ADP-actin polymers. Because of the similarity between the digestion properties of G-ATP- and G-ADP-actin and between F-ATP-actin monomers and F-ADP-actin polymers, it, appears that only the addition of salt,, and not the phosphorylated state of the actin-bound adenosine nucleotide, causes t’he conformational change in G-actin to form t’he F-a&n monomer species.

2. Materials and Methods (a)

Preparation

of activ

Actin was extracted from an acetone powder (Szent-Gyorgyi, 1951) at 0°C for 20 min with 25 vol. of 0.2 mm-ATP and 2 mM-Tris buffer (pH 7.8), filtered through a fritted glass funnel, and purified using the procedure of Mommaerts (1952) modified as follows. After centrifugation of the filtrate at 64,000 g for 1 h at 4”C, the supernatant containing G-actin was made 0.1 M in KC1 and 0.1 mM in CaCl, and allowed to polymerize before being centrifuged at 170,000 g for 2 h at 4°C. The resulting F-actin pellets were homogenized in 0.2 mM-ATP, 0.1 mM-CaCl, and 2 mM-Tris buffer (pH 7.8), dialyzed at 0°C against this same solution for three 12-h intervals, and then dialyzed for 2 additional intervals against 0.1 mM-CaCl, and 0.2 mM-ATP (pH 7.8). Buffer was omitted from the latter dialysis changes to allow more sensitive measurement of the digestion rates. Following three 10-s periods of ultrasonic vibration at O”C, thtx G-act,in was again centrifuged at 64,000 g for 1 h at 4°C. The resulting G-ATP-actin solution routinely gave only a single band when analyzed on 5 and 10% polyacrylamide gels containing O.l”;, sodium dodecyl sulfate according t,o t,he procedure of Weber & Osborn (1969). For comparison, actin extracted at room temperature was analyzed by this same procedure and showed 3 bands. Furthel characterization of the purified actin revealed that 984; (i 29’,) of the G-actin-bound nucleotide was ATP and that slightly more than a stoichiometric amount of ATP was assuming a molecular weight of hydrolyzed to ADP upon polymerization to F-actin, 43,000 for the actin monomer (Straub & Feuer, 1950; Tsuboi & Hayashi, 1963; Tsuboi. 1968). At 25°C a 1 mg/ml solution of G-ATP-a&n had a reduced viscosity of 6.0 -:I 3.0 ml/g, typical of its globular, monomeric state (Drabikowski & Gergely, 1962; of F-ADP-actin in Lewis et al., 1963; Grant et aE., 1964), while a 1 mg/ml solution 0.1 ~-Kc1 formed from G-ATP-actin had a redurpd viscosity of 1050 $ 200 ml/g. similar to other reported determinations (Maruyarna & Gergcly, 1962; Nagy & Jencks, 1965; Estes & Moos, 1969). G-ADP-actin was prepared by first, removing free nucleotide from purified G-ATPactin by treatment with Dowex resin (AC; 1 x 8, 200 to 400 mesh, Cl- form) followed by filtration. After polymerization in 3.5 mM-MgCl, (final conm) the F-act,in was treated again with Dowex, filtered, and centrifuged at 177,000 g for 2 h at 4°C. The actin pellets and cent,rifuged at 177,000 g for were t,hen softened in 0.04 m&I-ADP, homogenized, 2 h at 4°C before being diluted to nearly 1 mg/ml with 0.04 mM-ADP and stored on ice. Nuclcotide analysis of G-ADP-actin thus prepared showed its bound nucleotide cornposition t.o he 82:& (h 40,:) ADP with t’he remainder btling mostly ATP and trace amount,?: of AMP. The reduced viscosity of a 1 mg G-ADP-actin/ml solution at 25°C was usually around 10 f 1 ml/g immediately following the final centrifugat’ion, and the digestion st,udies were performed on preparations having this low viscosity. However, it should he noted that the viscosity of the preparat’ion gradually increased upon standing at. 4°C for 24 to 48 h indicating that nucleation and polymer formation were occurring, possibly of fully due to the presence of small amounts of residual MgCl,. The reduced viscosity polymerized 1 mg F-ADP-actin/ml in 0.1 I\I-KC1 formed from G-ADP-actin had a valuta of 1020 + 94 ml/g.

l&ch proteolytic enzyme employed was dissolved in 0.1 Infir-CaC’l, and 0.2 mrvr-ATI’ (pH 7.8), on its day of use, and the specific act’ivity of the cnzymc preparation demrminetl from its rate of digestion of synthrt,ic subst)rate before being st,orcd on ice. All rates of digestion were measured by pH-st’at, tit,rat#ion at, 25°C wit,h an automat,ic Hadiometcr titrator. An auxiliary recolder was used t*o monitor t,hr act,& pH of t,hr sample during a t,itration and t.o aid in determining t’hc region of smady-state kinetics. The digestion reaction was initiamd by the addition of a small volume of t’hr proteolytic enzyme solution to a samplr which had been adjust,ed t,o pH 7.8 at 25°C. Steady-stat,e conditions were determined from t,hc region of linear t’itrnnt addition at constant. pH and normally allowed to cont,inur for about 5 min in order t)o accurately determine the digcst.ion rat,c. The strength of t#hc titmnt, (N&H) used was mea~surrtl at, t,hc beginning of each day of 11s~.

(c) Protein

concentration

determination

The concentration of G- or F-actin was determined by u.v. absorption at 290 nm where any interfering absorption by free adenine nucleotides is essentially absent (Lehrer & Kerwar, 1972). An extinction coefficient of 6.37 at 290 nm for 1% solution was used which was based on micro-Kjeldahl determinations. The concentrations of the proteolytic enzymes used were also determined by U.V. a.bsorption using the 15.0 (Pechere 8: Neurath, 1957); following extinction coofficient~s at, 280 nm: t,rypsin, subtilisin, 11.7 (Matsubara et al., 1965); Pronasr, 11.1 and chymotrypnin, 20.0 (Smillie et al., 1966).

(d)

i’iscosity

w~ea.wrementa

All viscosity determinations were done at 25°C. Most of the measurements employed Ostwald viscometers having outflow times of about 60 s for water. Where greater sensit’ivity was desired, an Ubbelohde shea,r viscomet,er was used which permitted risrositydoterminations at 4 different shear rates.

(c) Bowtttl

n ldeotide

arhalyaia

Perchloric acid extracts (5% final concn) of actin solutions were first filtered t,o remove precipitated material, and the free nucleotides in the extract absorbed onto acid-washed charcoal. After collecting the charcoal by filtration, it was washed with water and the nucleotides elutod with a solution of 0.75 N-NH,OH in 257; ethanol (Tsuboi & Price, 1959; Martonosi et al., 1960). The separation and quantitative identification of the nucleotides in the extract were accomplished by applying the ethanol/ammonia eluate onto a 1 cm3 Dowex resin column and eluting adenosine, adenine, AMP, ADP, and ATP according to the method of Cohn & Carter (1950). Nucleotide identification was also confirmed by paper chromatography (P-L Biochemicals, Circular OR-17, 1961).

All chemicals were of reagent grade. ATP and AMP were purchased from P-L Biochemicals and ADP from Sigma Chemical Co. The 4 enzymes used and their suppliers were: trypsin (Worthington, twice crystallized, salt’-free); a-chymotrypsin (Schwarz/Mann, bovine pancreas, 3 times crystallized, salt-frer); subtilisin (Schwarz/Mann, Bacillus subtilis, crystalline); and Pronase (Cnlbiochem, sohlblr, B grade). The synthetic substrates employed were N-benzoyl-r,-arginine ethylenter.HCI, N-acetyl-L-tyrosine ethylester and p-tosyl-n-argininc metZhylester, and all were product)s of Schwarz/Mann. Glass distilled water was used in making all solutions.

EVIDENCE

FOR

AN

F-ATP-A(‘TIX

MONOMER

7x1

3. Results (a) Proteolytic digestion

of

acth

Typical initial rates of digestion of G-actin and of F-actin by trypsin, chymotrypsin, Pronase and subtilisin are presented in Figure 1. Regardless of the enzyme employed, the digestibility of G-actin was always found to be four to six times greater than that of F-act,in. The low digestion rates of both G- and F-act,in with t,rypsin are in agreement with earlier attempbs to digest actin with the enzynrc’ (Mihalyi, 1953; Laki, 1964) and also consistent wit,h the high degree of specificit), exhibited by t’rypsin. Since the enzymes used var\’ widely in their specificities. this

Time (min)

FIR. I. Enzymatic digestion of G-actin and of F-ac%in. Typical rate data from pH-stat titration of the proteolytic digestion of I mg G-actin/ml solutions with no KC1 added (---) and 1 mg F-a&in/ml solutions in 0.1 M-KC1 (). in the presence of 0.1 mM-CaCl, and 0.2 mM-ATP (pH 7.8), 25°C. S, 0.075 mg subtilisin/ml; C, 0.125 mg chymotrypsin/ml; P, 0.033 mg Pronase/ml; T, 0475 mg trypsin/ml.

difference in the susceptibilities of G- and of F-actin to enzymatic digestion suggests that G-actin undergoes a general change in conformation upon polymerization int,o F-actin. The observed difference in the susceptibilities of the t’wo states of actin to proteolysis could be due to the presence or absence of 0.1 M-KCl. A comparison of the rates of hydrolysis of synthetic substrates, hemoglobin and denatured actin by trypsin, chymotrypsin, Pronase and subtilisin under the two different conditions (Table 1) shows that the presence of 0.1 M-KC1 decreased the activity of these enzymes up to 40’7;. This effect of salt clearly cannot account for the 400% to SOOT1 difference repeatedly observed between the rates of digestion of G-actin and of F-actin. The greater digestibility exhibited by G actin might be due to a higher ratcb of autolvsis of the various enzymes under G-actin conditions than in the presence of 0.1 M-KCl. Experiments were therefore conducted to determine only the autolysis rates by separately incubating the enzymes in the presence or absence of O-1 nz-KC1 before adding synthetic substrate and determining their activities. During the incubation times employed, which correspond to the period of time that was used to determine the initial steady-state digestion rates shown in Figure 1, no significant change in the activities of the enzymes was observed. Another possible explanation

Ejject of KC1 on enzyme activity [Enzyme] @%/ml)

Hydrolysis rate (pequiv/min) No KC1 added 0.1 ~-Kc1 added

Substratt’

Chymotrypsin

0@03 0.125 0.125

ATEE hemoglobin denatured actin

21x 0.152 0.353

205 0.137 0.350

Pronase

0.06i

0.033 0.033

BAEE hemoglobin denatured actin

737 0.27 I w407

712 0.153 0.352

Subtilisin

0.236 0.075 0.075

TAME hemoglobin denatured actin

310 0.412 0.444

350 (I.302 0.313

Trypsin

0.010 0.075 0.075

BAEE hemoglobin denatured actin

236 0~180 0.457

240 0.165 0.442

Rates of digestion were determined, as described in Materials and Methods, in the presence of 0.1 mM-CaCl, and 0.2 mM-ATP at pH 7.8, 25°C. Each entry represents an average of multiple determinations. The titrant was 0.1 N-NaOH for synthetic substrate digestions and 0.01 3.NaOH for protein digestions. Actin was denatured by heating G-actin to 50°C in 0.1 N-KOH for 1 h followed by neutralization to pH 7.8 with HCl and removal of the salt formed by extensive dialysis. The concentration of protein substrates was 1 mg/ml. ATEE, N-acetyl-L-tyrosine ethylester; BAEE, N-benzoyl-L-arginine ethylester. HCl; TAME, P-tosyl-L-arginine methyl ester.

for the consistently lower digestion rate of F-a&in is the high viscosity of an F-actin solution which could reduce the measured rate of digestion and create an inhomogeneous solution by preventing adequate mixing from occurring. To exclude this possibility, trypsin was incubated in the presence of 1 mg F-actin/ml for various times, and then N-benzoyl-L-arginine ethylesteraHC1 was added and the activity of trypsin, still in the presence of F-actin, determined from the rate of hydrolysis of N-benzoyl-L-arginine ethylester.HCl. Only a 9% loss in activity was observed after a 30-minute incubation, and this loss was probably due to autolysis. Thus, the low rate of digestion of F-actin polymers is not due to an inhomogeneous solution or to any other apparent loss in enzyme activity resulting from the presence of F-actin polymers. The decrease in digestibility of a G-actin solution when made 0.1 M in KC1 must thus mean t,hat the number of sites available to enzymatic attack is reduced. Such a reduction could occur if G-a&in undergoes a change in conformation upon polymerization, and this possibility is supported by previous reports of conformational changes during the G- to F-actin transformation (Higashi & Oosawa, 1965; West, 1970; Stone et aZ., 1970; Murphy, 1971). (b) Studies with partiaEly-polymerized Since there is always some G-a&in in equilibrium 1962), the observed low rate of digestion of presence of a few digestible G-actin molecules in population. To ascertain whether this could be the actin in equilibrium with the polymeric form was

actin

with F-actin (Oosawa & Kasai, F-actin may be due to the a relatively indigestible F-actin case, the fraction of monomeric varied by preparing equilibrium

EVIDENCE

FOR

AN

F-ATE-ACTIN

ix3

hlONOMER

samples of partially polymerized actin solutions and estimating the F-actin content of such solutions by determining their specific viscosities. In the range of 0 to 100 mM-KC1 concentration, the almost total transformation of G-actin monomers to F-actin polymers occurred in the region of 9 to 20 mrvr-KG1 (Fig. 2(a)). The rates of digestion of these partially polymerized actin solutions were then measured using Pronase and subtilisin, which had previously shown readily measurable rates with both G- and F actin (Fig. 1). Figure 2(b), sh ows that increasing the fraction of F-actin

[KC11 (mtd)

050. .

0

(b)

I 05 Specific viscosity

I. I.0

FIG. 2. (a) Viscosity of partially polymerized actin solutions. The specific viscosities of 1 mg actin/ml solutions which had been standing for 24 h to reach equilibrium were determined st 25°C in the presence of 0.1 mMn-Cd&, 0.2 mM-ATP (pH 7.8) and various concentrations of KCl. (b) Proteolytic digestion of partially polymerized actin solutions. The above solutions of 1 mg actin/ml at various extents of polymerization (as determined by specific viscosity) were subjected to proteolysis under the conditions of Fig. 2(a). (0) 0.075 mg subtilisin/ml; (0) 0.033 mg Pronase/ml.

in 1 mg actin/ml samples, as indicated by the increasing specific viscosity, results in a linear decrease in the rate of digestion. If the unpolymerized actin in equilibrium with F-actin in these partially polymerized actin samples is assumed to be G-actin, then it can be concluded from this experiment that the F-actin molecule is essentially indigestible compared to the G-a&in molecule.

7x4

S. A. HI(IH (c) Studies

with

Ah’l)

,I. E.

low concentrations

ESTES of actin

Another possible int,erpretat)ion of the difference in digestibilit,y between G- and F-actin is that there are susceptible digestion sites on G-act,in monomers which become occluded upon polymer formation without any change in the conformation of the monomer molecule. To examine this possibility, the viscosity and digestibi1it.y of actin were measured below the critical concentration of actin, where actin, even in the presence of 0.1 M-KC& does not polymerize (Oosawa et al.. 1959; Ooi, 1960: Asakura et al., 1960; Oosawa $ Kasai, 1962). The specific viscosity of solutions having various actin concentrations without added KC1 show a low value throughout. the actin concentration range used (Fig. 3(a)). Th e viscosity measurementZs of actin

(b)

[Actn]

(mg/ml)

FIG. 3. (a) Determination of the critical actin concentration. The specific viscosities of solutions of various concentrations of aetin containing 0.1 mm-CaC1, and 0.2 mu-ATP (pH 7.8) at 25°C were obtained with Ostwald viscometers in the presence (solid symbols) or absence (open symbols) of 0.1 M-KCI. (b) Proteolytic digestion of actin in the region of critical actin concentration. Solutions of various concentrations of actin containing 0.1 mM-CaCl, and 0.2 mmATP (pH 7.8), at 25°C were subjected to digestion in the presence (solid symbols) or absence (open symbols) of 0.1 M-KU. (O), ( l ) 0.017 mg Pronaselml; ( q ), ( n ) 0.04 mg subtilisin/ml.

samples which had been made 0.1 M in KC1 24 hours previously, however, have this low value up to an actin concentration of O-04 to 0.05 mglml (the critical actin concentration) whereupon they increase as the polymer content of the sample increases. When these same actin solutions were subjected to proteolytic digestion with subtilisin and with Pronase, the G-actin samples with no added KC1 had significantly higher digestion rates than the actin samples in 0.1 M-KC1 at every actin concentration examined (Fig. 3(b)). The difference in the actin concentration-dependence

EVIDENCE

POR

AN

F-ATP-A(‘TIN

MONOMER

TM

of t,he digestion rates obtained in the absence of KC1 reflect an apparent difference in the kinetic properties of Pronase and of subtilisin under these conditions (open symbols in Fig. 3(b)). It is clear from these data that in the presence of 0.1 M-KCl, the low rates of digestion of actin occur even below t,he critical actin concentration, and thus the observed difference in digestibility between G- and F-actin cannot b(l due to the occlusion of specific susceptible sites on t’he monomer when it binds to a polymer. At, actin concentrations below the critical concentration, the significant difference between the digestibilities of monomeric actin in the presence or in the absence of 0.1 M-KC1 (Fig. 3(b)) suggests that there is a difference in the nature of the substrate present under the two conditions. Such a difference is also seen in other experiments not shown here, where the rates of digestion of 0.02 mg actin/ml samples by subtilisin and Pronase were found to increase linearly with increasing enzyme concentration in the absence of salt, but remain low and essentially independent of enzyme concentration in t)he presence of 0.1 M-KCl. Since the presence of 0.1 M-KC1 does not, alter t,he activity of the enzymes employed enough to account for the difference in the digestion rates, it must be the conformation of the actin monomer which is changed by the addition of salt. Apparently, then, monomeric actin can exist as two species: as normal G-actin in t,he absence of salt, and in the presence of 0.1 M-KU at sub-critical concentrations as a monomer with a conformation different from G-act,in and more similar to that of the subunits in F-actin polymers. The preceding data with actin at low concentrations could also be explained if t,hr experimental samples were very dilute solutions of small polymers or actin nuclei (trimers or tetramers) which have the same viscosity as a monomeric species when measured with the Ostwald viscometers used. In an attempt to physically detect the presence of such small polymers, both the shear dependence and the specific viscosity of solutions of actin were determined with an Ubbelohde shear &corn&r. The result)s of these determinations at actin concentrations of 0.02, 0+4 and 0.08 mg/ml under four different shear forces can be compared in Table 2 for G-actin diluted to these concentrations in the absence of KCl, for G-actin diluted to t hew concentrations, made 0.1 M in KC1 and allowed to st’and for 24 hours, and TABLE

,”

Spec@c viscosities qf actin solutions at low actin concentration 0.08 mg aotin/ml No 0.1 MKC’1 KC1 Dilntrcl &Lddd added F-a&n

The aperific- viscosities of actin solutions containing 0.1 mwCaC1, and (I.2 mM-ATP (pH 7.8), at 25”(‘, were determined with an Ubbelohdr viscometer at 4 different shear forces. No KC1 added: G-actin diluted to concentration shown. 0.1 M-Kc’1 added: G-aotin diluted to the concentration shown, made 0.1 M in KCI, and allowed to stand for 24 h. Diluted F-a&in: fully polymerized F-a&in diluted to conrentration shown with 0.1 M-KU immediately before viscosit?; measurements.

7X6

S. A.

HTC’H

ANI)


ESTES

for F-actin which was fully polymerized at a concent’rat’ion of I mg/ml and then dilut,ed with 0.1 ~-Kc1 just prior to the viscosity measurements. The dilut,ed F-act)in polymer solutions show a shear dependence at all three actin concentrations. while no shear dependence was observed with the G-actin samples or with the actin samples at sub-critical concentrations in the presence of 0.1 M-KCl. In some cases the outflow time of the solution was not significantly different’ from the outflow time of the solvent, and thus a viscometric characterizat’ion could not be determined. However, t#hevalues of the specific viscosities of the F-actin samples diluted to sub-critical concentrations were always markedly higher than the corresponding solutions of G-actin diluted in the absence of KC1 or of diluted G-actin made 0.1 M in KCI. The apparent’ discrepancies between the specific viscosity values in Table 2 and those presented in Figure 3(a) arise due to the difference in the shear forces developed in the Ubbelohdr shear viscometer and in the Ostwald viscomeber. But even though some of the experimental values are low, these data support the previous conclusion that actin at sub-critical concentrations in 0.1 &r-KC1 is not, in the form of small polymers or probably not nuclei but rather exists in a monomeric state. To gain further evidence t’hat a true monomeric species exists in the presence of 0.1 M-KC1 at sub-critical actin concentrations, the effect of adding such a solution on the rate of polymerizatJion of G-actin was examined. The addition of actin nuclei or short polymers to a polymerizing F-actin solution reportedly enhances the rate of polymerization (Kasai et al., 1962; Asakura et aZ., 1963). Such enhancement is seen in Figure 4, where the initial rate of polymerization of G-actin after the addition of a small amount of F-actin in 0.1 M-KC1 is greater than that of G-actin with only KC1 added. The addition of an equal amount of actin at a sub-critical concentration (0.02 mg/ml) in O-1 M-KC1 causes little change in the initial rate of polymerization (Fig. 4). While this result again sugges& that) actin at sub-crit,ical concentrations in the presence of 0.1 M-KC1 is not in a small polymer or nucleated st’at’r but rather in

Time (min) FIG. 4. Initial rate of polymerization of G-ATP-actin. The polymerization of solutions of G-ATP-actin was induced by the following additions: (0) 1.4 ml of 0.1 M-KCl; (0) 1.4 ml of 0.02 mg actin/ml in 0.1 M-KCl; (A) 1.4 ml of a fully polymerized F-actin solution which was diluted with 0.1 M-KC1 to a concentration of 0.02 mg/ml immediately before being added. Each of these volumes was added to 5.6 ml of a 0.625 mg G-actin/ml solution to give final conditions of 0.5 mg a&in/ml, 20 mM-KC& 0.1 mM-call, and 0.2 mmATP (pH 7.8), at 25°C.

EVIDENCE

FOR

AN

F-ATP-ACTIN

IXi

MOh-OMER

a monomeric state, the lack of an increase in the rate of polymerization of G-actin could also be explained if the actin sample at sub-critical concentration was someho% denatured. To demonstrate that this monomeric species was not denatured and could form polymers, actin at 0.02 mg/ml in 0.1 M-KC1 was concentrated, and it,s viscosity was found to be nearly the same as that’ of a solution of 0.02 mg G-actin/ml which was concentrated and then made 0.1 M in KCI. The charact’eristics nf the act)in monomer species in 0.1 M-KC1 at sub-crit’ical concentrations do not, a,ppea~ to b(> due to a denatured form of the molecule. (d) Boun,d wcleotide

determination

While t#he above studies give some indication of the characteristics of actin at sub-critical concentrations, the case for the presumed monomeric actin species in the presence of 0.1 M-KC1 would be stronger if the identity of the nucleotide bound to this monomeric species was known. Nucleotide analysis of 1 mg G-actin/tnl solutions containing a fourfold stoichiometric excess of free ATP show t$hat ATP is t,hr predominant, nucleotidc present) (Table 3). Similar solut’ions of G-actin wwc

TABLE 3 Bound nucleotide analysis Sample G-actin F-actin Diluted in

0.1

( 1 mg/ml) (I mg/ml) actin (0.02 mg/ml) M-I(c1

of actin

ATP content (%I

ADP content COh)

99.8 71.X

?“.O

0 “0

2.1

(I

!I$.5

Expected “6 ADI’

Nnrleotide analysis was performed as described in Materials and Methods on the following samples: 1 mg G-actin/ml in the absence of KCI, 1 mg F-a&n/ml in WI wKC1, and a similar amount of G-actin diluted to 0.02 mg/ml and then made 0.1 M in KCI. Prior to the expwimrnt, the artin used was found to have I mole of bound adenine nurlrotide per mole of artin. Aftct preparation for the experiment, all actin solutions contained a fourfold stoichiometric vxwss of ATP. The ATP content of each experimental solution was therefore determined from t,h(s ratio of the amount of ATP in each actin sample to the amount of ATP in an idtmtical volume of dialysis solution treated in the same manner as the actin sample. The ADP content was takm as the difference between the ATP content of the G-a&in sample and the ATP content of thr particular actin sample. The expected y. ADP was calculated assuming that the hound nurlrot,id~~ of G-a&in is lOOq$ ATP, that the bound nucleotide of F-wtin is 1000;, :I III’. and that the dillltr~tl wtin species in WI wKC1 contains only hound ATI’.

made 0.1 M-KC1 and analyzed as F-actin samples after 24 hours of polymerization. Under these experimental conditions, the ATP content’ of the F-acbin solutions would be expected to decrease approximately 200,b since only the nucleotide bound t’o G-actin in the presence of a fourfold stoichiometric excess of ATP would be dephosphorylated to ADP. The observed difference in the nucleotide content of t,he G- and F-actin samples is 22% and is thus in excellent agreement with the expected value. The analysis of the nucleotide bound to the actin monomer species at, subcritical concentration was performed on an amount of G-actin which had been diluted to 0.02 mg/ml and allowed to stand for 24 hours after being made 0.1 M

S5. A.

iXX

l
i\NI)

J.

15. ESTES

in KCI. The difference in ATP content between G-actin and the diluted actin sample, also in the presence of a fourfold stoichiometric excess of ATP, is a 2.1% decrease in ATP content. Assuming that this increase in ADP content could only have been produced by the dephosphorylation of the actin-bound ATP, and that the increase would have a value of 22% if full polymerization had occurred, the 2.1% increase actually means that 9% of the nucleotide bound to the sub-critical monomeric actin species is ADP and therefore that 91% of it is ATP. Thus the identification of the nucleotide bound to actin atj sub-critical concentrations in 0.1 ~-Kc1 as BTP confirms the previous results which suggest that actin is in a monomeric state. Furthermore, it indicates that the conformational change occurring in G-BTP-actin upon the addition of salt does not by itself cause the hydrolysis of the actin-bound ATP, but rather that the hydrolysis of ATP must take place during the formation of nuclei and polymers from “F-ATP-actin monomers”. (e) Proteolytic

digestion of G-ADP-actin

The data in Figure 3(b) show the digestibility of actin in 0.1 ~-Kc1 to be relatively low and independent of whether the protein is in a monomeric state containing bound ATP or in a polymeric state containing bound ADP. This suggests that the phosphorylated state of the actin-bound nucleotide does not markedly effect the surface conformation of the substrate actin molecules. Further verification of this finding was obtained by comparing the digestibilities of G-ATP-actin and of G-ADPactin with the digestibilities of the F-ADP-actins they form. In Figure 5, both the G-ATP- and G-ADP-actin samples with no added KC1 are shown to have relatively high and essentially identical digestion rates with both Pronase and subtilisin. Upon addition of KC1 to 0.1 M, the rates of digestion of both F-actin samples were characteristically lower than the G-actin samples, with the F-actin from G-ADP-actin showing a slightly higher digestion rate which is probably due to the presence of a small amount of highly digestible denatured actin formed during the preparation

0

I

3

5

Time (min)

FIG. 5. Digestibility of G-ADI?-actin, G-ATEactin and F-ADP-actin. The rates of digestion of 1 mg actin/ml samples by 0475 mg subtilisin/ml (--) and 0.035 mg Pronase/ml (------) were measured in 0.1 mix-CaCl,, 0.2 mmATP (pH 7.8), at 25”C, and the presence (solid symbols) or absence (open symbols) of 0.1 M-KU. For comparison, the subtilisin digestion of denatured actin (- x - x -), prepared by first adding 0.1 N-KOH and then an equivalent vol of 0.1 x-HCl to actin, is shown. (o), (0) G-ADP-actin; (a), ( n ) F-ADP-actin from G-ADP-actin; (a), (V ) G-ATP-actin; (A), (v ) F-ADP-actin from G-ATP-actin.

EVIDENCE

FOR

AN

F-ATP-ACTIN

58R

MONOMER

of G-ADP-actin. Therefore, based on the susceptibility to proteolytic digestion technique used in this study, the conformation of actin is not dependent on whether it is binding ATP or ADP. Apparently then, it is the addition of salt alone to G-actin that induces a change in the tertiary struct,ure of the monomer molecule t)o a much more indigestible conformation.

4. Discussion Compared to the rates of digestion of G-act’in. the significantly lower rates of digestion of F-actin by all of the enzymes employed in this study show that the susceptibility of actin to proteolysis changes upon polymerization (Fig. 1). This increased resistance to enzymatic digestion could not be attributed t,o factors affectmingthe proteolytic enzymes (Table 1) and thus supports previous reports that G-actin undergoes a conformational change when polymerizing. Since the enzymes employed vary greatly in their specificities, the conformational change appears to occur generally throughout the entire G-actin molecule. Results of analyses of the proteolytic digestion products, currently in progress, should provide information about the extent of the change. The reduced digestibility in the presence of 0.1 M-KC1 was shown to be characteristic of actin whether it. is in the polymeric form or in a monomeric state below the critical concentration (Fig. 3(a) and (b)). This implies that t’he conformation of the protein in these two physical stat,es is the same or very similar. Evidence that actin is in a monomeric state in 0.1 M-KC1 at sub-critical concentrations and not nucleat,ed or polymerized, is provided by the apparent lack of shear dependence in its viscometric characteristics (Table 2) its failure to enhance t,he rate of polymerization of G-a&in (Fig. 4), and its possession of ATP as a bound nucleotide (Table 3). Therefore, there must, exist a distinct species of actin in the presence of 0.1 M-KU which has digestion properties similar to F-ADPactin. Lt, is this species that we have called F-ATP-actin monomer. A mechanism of polymerization incorporating this newly-detected species of actin in a series of reversible steps is presented in Figure 6 as a modification of the polymerization model suggested by Oosawa and his colleagues (Kasai et aZ., 1962). In the proposed scheme, the addition of 0.1 M-KC1 induces a rapid change in the conformation of G-ATP-actin to form F-ATP-actin monomers prior to the formation of nuclei. It is assumed here t,hat G-actin molecules cannot form nuclei or be added onto a growing polymer until they undergo this conformational change. As in the earlier mechanism (Kasai et al.. 1962), the rate-limiting step in the polymerization reaction is the formation of nuclei. Accompanying bot,h nuclei formation and the addition of F-a&in monomers onto growing polymers, the actin-bound ATP is

G-ATP-Actin monomers

E’Ic.

F-ATP-Actin monomers

6. Modified

mechanism

F-ADP-Actin nuclei

for the polymerization

F-ADP-Actin polymer

of CT-actin to F-a&n.

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S. .I.

NIC’H

;\Kl)

.I.

I<. ESTES

hydrolyzed to ADP. While the proposed model shows the nucleotide bound t,o t.hck monomer unit’s at the ends of the nuclei and polymers as ADP, both the work presentcd here and t’hat done by others has not &ablished this to be the case. Implicit, in this condensation polymerization mechanism is the existence of a critical concentration of monomer actin molecules. below which polymerization will not occur (Oosawa et al.? 1959; Kasai & Oosawa, 1962). The presence of the newl! discovered species of monomeric actin suggests that the type of actin monomer which must reach a critical concentration is not G-actin but F-a&in monomer. The lorates of digestion of monomeric actin in the presence of 0.1 M-KU indicate that, G-actin is probably not present in detectable amounts, and thus the observed critical actin concentration of approximately 0.05 mg/ml must be the concentration of F-actin monomers (Fig. 3(a) and (b)). In the presence of lower concentrations of KCl, the critical actin concentration increases (Oosawa et al., 1959; Asakura & Oosawa, 1960; Kasai et al., 1962), and under these conditions the rates of digestion of equilibrium solutions of partially polymerized actin also increase (Fig. 2(b)). This indicates that the monomer population making up the critical actin concentration at t’hese lower KC1 concentrations contains not only F-actin monomers, but also some digestible G-actin monomers. Such a possibility could occur if the concentration of salt present determines bot,h the size of the monomer population in equilibrium with the polymeric state, and the ratio of F-actin monomers to G-actin in the monomer population. This interpretation of the effect of salt on the various populations present) in an equilibrium actin solution explains t’he linear decrease in digestibility that is experiment,ally observed when increasing concentrations of salt are added to G-actin (Fig. 2(b)). An essential feature of the above model is the key role the presence of neutral salt plays in determining the conformation of actin, and thereby regulating the polymerization process. After the salt-induced conformational change has converted the G-actin monomers into F-actin monomers, the formation of nuclei and polymers may occur spontaneously. It is generally acknowledged that the type and concentration of neutral salt affect the rate and extent of polymer formation, and an a,lt,eration of the polymerization rate constants by salt may still occur, but the results of this study suggest that a major effect of salt is on the rate and extent of F-a&in monomer formation. It is also generally recognized that the actin-bound nucleotide affects the conformation of actin, but the essentially identical rates of digestion of G-ATP- and G-ADP-actin (Fig. 5) would suggest that the two forms of G-actin have the same tertiary structure. This result appears to contradict earlier ultraviolet difference spectra measurements which indicate that the substitution of ADP for &4TP in G-actin causes a conformational change (Higashi & Oosawa, 1965; West et aZ., 1967; West. 1970). However, a variety of changes in protein st,ructure, including internal changes as well as surface effects, will give rise t,o ultraviolet difference spectra, and it is quite possible that the portion of the structure influencing ultraviolet absorption varies with the tvpe of nucleotide bound, while the surface conformation of actin, as seen by a proteolytic enzyme, remain+ constant whether ATP or BDP is the bound nucleotide. The low identical rates of digestion of F-ATPactin monomers and F-ADP-actin polymers (Fig. 3(b)) further suggest that tho surface conformation of actin is not significant,ly affected by the phosphorylated state of the bound nucleotide. Hence the presence or absence of neutral salt appears to be the most significant factor in determining the conformation of monomeric actin.

EVIDENCE

F’OK

AN

F-A’l’F-A(“rIX

MOI1’OMElC

;!I I

The existence of a salt-induced change in the conformation of G-actin to form F-actin monomer means there are at least two conformation changes associated with actin polymerization. The occurrence of a separate conformational change in t,he actin monomer when it joins an F-actin nuclei or a polymer has been previously concluded from both ultraviolet spectral studies (Higashi & Oosawa, 1965) and eleetron paramagnetic resonance studies (Stone et al.? 1970) of the polymerization process. This conclusion is further supported by recent preliminary experiments examining the effect of applied hydrostatic pressure on actin polymerization, which also indicate that, the polymerization step itself is accompanied by a large volume change ot activation. possibly resulting from a change in F-actin monomer conformation (EstfK 1974.1975). Support, by grants acknowledged.

from

t,hc Heart

Association

of Eastern

N~aw York

is gratcfnll>,

REFERENCES Asai, H. & Tawada, K. (1966). J. Afolol. Biol. 20, 403-417. Asakura, S. (1961). Biochim. Biophys. Acta, 52, 65-75. Asakura, 8. & Oosawa, F. (1960). Arch. Biochem. Biophys. 87, 2480.-2488. Asakura, S., Kasai, M. & Oosawa, F. (1960). J. PoEy. Sci. 44, 35-50. Asaktxra, S., Taniguchi, M. & Oosawa, F. (1963). J. *l//02. Biol. 7, 55-69. Circwlar OR-1 7 (1961). P-L Biochemicals, Milwaukee, Wise. Cohn, W. E. & Carter, C. E. (1950). J. Amer. Chem. Sot. 72, 4273-4275. Cooke, R. & Murdoch, L. (1973). Biochemistry, 12, 3927-3932. Drabikowski, W. & Gergely, J. (1962). J. Biol. Chem. 237, 3412-3417. Elliott,, (:. F., Lowy, J. & Millman, B. (1967). J. Mol. BioZ. 25, 31--45. Est’es, tJ. E. (1974). Fed. Proc. Fed. Amer. Sot. Ezp. BioZ. 33, 1522 (abstr). Estes, ,J. E. (1975). Biophys. Sot. Abstr., 19th Ann. Mtg, p. 34a (abst,r). Eskrs, J. E. & Moos, C. (1969). Arch. Biochem. Biophys. 132, 388-396. (:rant. R. J., Cohen, L. B., Clark, E. E. & Hayashi, T. (1964). Biochem. Biophys. Rex. Commun. 16, 314-318. Higashi, S. $ Oosawa, F. (1965). J. A’foZ. BioZ. 12, 843-865. Huxley, H. E. & Brown, W. (1967). ,J. Mol. BioZ. 30, 383-434. Kasai, M., Asakura, S. & Oosawn, F. (1962). Biochim. Biophys. Acta, 57, 22-31. of Muscle Contraction (Gergely, .J., ed.), pp. 135-13i, Laki, K. (1964). In Biochemistry Little, Brown and Company, Boston, Massachusott’s. Lchrer, S. S. & Kerwar, (2. (1972). Biochemistry, 11, 1211-1217. Lewis, M. S., Maruyama, K., Carrol, W. R., Kominz, D. R. & Laki, K. (1963). Biochemistry. 2. 34 -39. Markus, G. (1965). Proc. Nat. Acad. Sci., (T.S.A. 54, 653-258. Martonosi. A., Gouvea, M. a. & Gergely, J. (1960). J. BioZ. Chem. 235, 1700-1703. Maruyama, K. & Gergely, J. (1962). J. BioZ. Chem. 237, 1095-1099. Matssubara, H., Kasper, C. B., Brown, D. M. $ Smit’h, E. L. (1965). J. BioZ. Chem. 240, 1125-1130. Mihalyi, E. (1953). J. BioZ. Chem. 201, 197-209. Mihalyi, E. (1972). Application of Proteolytic Enzymes to Proteins Structure Studies, CRC Press, Cleveland, Ohio. Momma&s, W. F. H. M. (1952). J. BioZ. Chem. 198: 445-458. Moos, C., Eisenberg, E. & Estes, J. E. (1967). Biochim. Biophys. Acta, 147, 536L545. Murphy, A. (1971). Biochemistry, 10, 3723~-3728. Nagy, R. & Jencks, W. P. (1962). Biochemistry, 1, 987L996. Nagy, B. & Jencks, W. P. (1965). J. Amer. Chem. Sot. 87, 2480-2488. Offer, (i., Baker, H. & Baker, L. (1972). J. Mol. BioZ. 66, 435.-444. Ooi, T. (1960). J. Phys. Chem. 64. 984-988.

ix!

S. A RT (‘ H A N I) ,J 14:. 13AT R 6

OOSil~\Vil,F. pr KRSRi, M. (1962). .I. .llo/. l~iul. 4, lo- PI. Oosa\v~, F., Asakwa, S., Hot,ta, K., Inrai, N. K: Ooi, ‘I’. (1959). J. f’ol,y. Sri. 37, 323-340. I’cY*hiTc~, .I. F. nt,-(iyorgyi, A. (2. & Prior, G. (1966). .I. ~\lol. Viol. 15, 515 538. Riophys. Actu, 160, 420-434. ‘I’suhoi, I(. K. (1968). Biochina. ‘l’suhoi, K. & Hnyushi, T. (1963). Arch. Hiochem. Biophys. 100, 313-322. Tsuboi, K. K. & Price, T. D. (1959). Arch. Riochen~. Riwphys. 81, 223-237. Wchw, K. & Oshom, M. (1969). .J. Rid. Chew. 244, 4406~ 4412. LVest., J. J. (1970). Hiochemiutry, 9, 3847~ 3853. \Vwt,, J. .J., Nagy, IS. & (iwgely, J. (1967). Kochem. Biophys. Res. Common. 29, 611-616.