Studies on the proteins from skeletal muscle of the bullfrog Rana catesbiana

.kKCHIVES

OF

BIOCHEMISTHY

Studies

on the

.\NU

113,

BIOPHYSICS

Proteins

20-33

from Rana

(1966)

Skeletal

Muscle

of the

Bullfrog

catesbiana I. Actin

R. M. DOLP’ Department

of Zoology,

University Received

of California, March

Berkeley,

California

30, 1965

Actin was isolated and characterized from the skeletal muscle of the bull frog, Rana catesbiana. Frog G-actin polymerizes rapidly with 0.1 M KCl, has an $0 = 3.1H, V app = 0.730 + ,006 and MW = 64,000 f 8000 as determined by ultracentrifugation. A preliminary amino acid analysis suggests that the pattern obtained from frog G-actin is similar to that reported for the rabbit protein. The protein binds 1 mole of nucleotide per 59,780 f 2000 gm. Sulfhydryl titrations with PMB and NEM indicate 2 moles of -SH per mole protein are reacted initially and 4 maximally. This latter value is 2 less than that observed for rabbit G-actin under the same conditions. The -SH groups which are readily accessible are not directly involved in polymerization. Bound Ca@ of frog G-actin exchanged with Ca, Mg, and Mn to the same degree whereas it was refractory to free Ba, K, and Na. In all cases, the nucleotide retained its mole/mole relationship and the protein was polymerized. The rate of exchange of CYATP bound to frog G-actin is influenced by the type of divalent cation present; i.e., exchange proceeded maximally with Mg, Ca, and “no cation” following in that order. The end point of exchange is the same, however. The labeled nucleotide (CY4ADP) of frog F-actin is refractory to both ADP and ATP. G-Actin nucleotide exchange is specific for ATP and possibly ADP. Nucleotide specificity is not a function of the cationic species. It is concluded that frog actin is like that of rabbit in the physical and chemical parameters analyzed except for a difference in the -SH groups; i.e., the frog has 4 maximally titrated wit,h PMB whereas the rabbit has 6.

In the animal kingdom, contractility is a universal phenomenon, and it appears that specialization of the contractile apparatus must have occurred early in evolution (1). The question of how a muscle works has been approached from many points of view, and, although a definitive explanation still lies in the future, we are now able to construct molecular models which can be test’ed (l-5).

Studies on the fine structure of muscle show that the basic unit of contract’ion is the myofibril, a unit consist’ing of thick and thin myofilamcnts. The arrangement of t,hese filaments is quite uniform in vertebrate striated muscle whereas variations are reported for vertebrate smooth and cardiac and also for all types of invertebrate muscles. Although these spatial modifications exist, it is t,aken as fact that the thick filaments universally cont’ain myosin and the t#hin filaments actin (and possibly tropomyosin) (6-8). The physiological and biophysical experiments concerning the energy metabolism mechanics, and thermodynamics (9-W, (13, 14) of the contractile process have been

1 This and the following paper were submit,ted in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This work was supported by a predoctoral fellowship to the author from the Nat,ional Institutes of Health and by grants USPHS GM-06025 and NSF BG-632 to R. C. Strohman. 20

K. CrlTESRlkVd

SKELETAL

confined almost exclusively to frog skeletal muscle as well as studies on the develop mental and synthetic aspect)s of the contract,ile prot(eins (15, 16). In addition, the cytological aspects of muscle differentiation have been described for amphibian material

(17). In contrast to the above have been those biochemicaal studies regarding the molecular characteristics of the contractile proteins themselves; although actomyosin and its components (actin and myosin) have been isolat>ed from many species of both invertebrates and veti,ebrat,es, detailed information is derived almost, entirely from mammalian prot tins, namely rabbit (18). The present study was undert,aken, then, with a view toward comparing the biochemicbal properties of amphibian skeletal muscle proteins with those of the rabbit primarily and with other data as it exists. If there is, in fact,, a universal model of contraction such a study might provide a clue as to which features of the contractile proteins are essential and which are evolutionary nuances. Similar reasoning has led to new insights about the stru&ure and function of several other proteins (19). In the work to be presented, the isolation and characterization of the contractile prot#eins from striated muscle of the bull frog Rana catesbianawill be described. This paper is concerned with some physical and chemical parameters of actin. MATERIALS

9X-1)

METHOl)S

Actill acetone powder m-as prepared from the leg muscles of fr’unu catesbiana and rabbit hy the method of Tsao and Bailey (20) and stored at -15°C in a desiccator up to 2 months wit,hout an? change in properties. The protein was extracted when needed by grilldillg the powder with 20 parts Coy-free douhle-distilled water for 30 minutes on ice. The mixture ~-as filtered through Whatman No. 1 paper with negative pressure, and the preparation was completed by the Mg polymerization method of Laki et ul. (21). The material prepared for sulfhydryl titrations contained 0.2 mill ascorbic acid at all stages. Zone electrophoresis was done at 4°C in a starch-gel medium according to the method of Carsten alrd Mommaerts (22) with some modifications; a 16” /C starch suspension (hydrolyzed,

MUSCLE

PROTEINS

I.

21

Connaught) was made with 0.012 k’ Tris,z pH 8.0. Fifty lambda of G-actin solution (8.12 mg per milliliter) was added to each well. Four-tenths rn,\l ATP and 0.2 mM ascorbic acid were added to the buffer at the start of the run. Current was applied for 4 and 19 hours, 8 V per centimeter. At the t,erminat,ion of the run, the gel was sliced with a nylon thread; one half of the gel was stained with Amido Black and the other half with Nigrosill. Analytical ultracentrifugation was performed in a Spinco model E at 20°C. iX2olecular weight was determined in the ultracentrifuge by the Ehrenberg modification (23) of the Brchibald method in which the boundary condition at the meniscus is used to measure the ratio S/D. $reas were calculated by tracing with a planimeter the boundaries of the curves on lo-fold enlargements. Apparent partial specific volume was measured in a lo-ml pycnometer at 25’C as ouL1ined by Schachman (24). Viscosity was determined in an Ostwald viscometer on 5-ml samples in a 25°C water bath. The solvent outflow time was about 70 seconds. Wit)h very small volumes (less than 0.1 ml) an Hellige viscometer No. 1200 was used with t,he appropriate solvent in the reference capillary; this measurement gave relative viscosity directly. The protein was hydrolyzed according to the method of Hirs ef al. (25) in 1.5 ml of 0 .V HC1 at 110°C for 20 and 70 hours. The hydrolyxates were dried dowtt by distillation on a Craig apparatus and redissolved in 2.5 ml of 0.2 111 citrate-ITAc buffer, pH 2.2. The amino acid analyses were carried out on an automatic Spinco amino acid analyzer. Protein colicelltrations were measured colorimet,rically by either of two methods, depending upon the concelrtratioll and accuracy desired. The biuret method standardized against a microKjeldahl procedure was used for concentrations of 4 my per milliliter and above, and the method of Lowry et al. (26) was used for more dilute solutiotls with egg albumin as a standard. 11 was found that eyuivalettt weights of lyophilized egg albu-~ 2 Abbreviat ioIls used: XTP, adetlosine triphosphate; PMB ,pu~a-hydroxvrnercuribellzo~tte; TEM, n-ethylmaleimide; E, molar extinction coefficient; Tris, trihydroxymethylan~iIlometharle; TCA. trichloroacetic acid; XaEl)TB, sodium ribonuethylencdiamitle t.etraacctate; RNase, clease; SH, sulfh)-dryl; SDS, sodium dodecyl su1fat.e; Al)]‘, adenosillediphosphate; ,4MP, adetlositlemollophospllate; (+TP, guanosine triphosphate; ITP, illosine triphosphate; CTP, cytosine triphosphate; UTP, uridine triphosphate; Cl’, creatine phosphate.

22

DOLP

min and frog actill gave identical absorption at i50 mp. Sulfhydryl groups were measured by the methods of Boyer (27) and of Alexander (28); PMB w-as recrystallized and the reaction, with a final PMB concentration of 0.1 m,V, was measured at, 250 mp and t = 7.6 X 103. SEM reactions at a final molarity of 2 In.11 were followed at 300 mr with e = 6.2 X 102. Fresh stock solutions of both reagents were prepared daily and the methods were checked against mercaptoethanol with less than a l$& error. Reactions proceeded at 20°C and were measured as difference spectra in a Beckman DB spectrophotometer using 4 cuvettes to provide the proper blanks simultaneously. Act,in was treated with Dowex-1 Cl- as described below to remove excess nucleotide which otherwise masked the absorption. Excess nucleotide was removed by stirring the preparation at 0°C for 10 minutes with one half its volume of centrifuged Dowex-1 Cl- slurry equilibratedwith2 rn.V Tris, pH 8.0 (29). The resin was removed either by filtration or centrifugation. To measure bound nucleotide, the protein was treated as described and the filtrate was deprot,einized with cold 5’9; TCA. The subsequent filtrate was analyzed at 260 rn* for nucleotide against the proper TCA blank, and the number of moles were calculated with E = 14,000. In some experiments, separation of the protein from free cation and nucleotide was accomplished simultaneously by passing the solution through a Sephadex G-50 Fine (bead form) column which was equilibrated with 2 rn:l4 Tris, pH 8.0, in the cold. A 3.5 X 44.cm column with a flow rat,e of 2.1 ml per minute handled up to 50 ml protein. Dialysis tubing was heated 4 hours at 60°C in 0.1 ilf NaEDTA (pH 7) and washed 1 week with dist,illed water circulating inside and out. Such treat,ment removed all cations which might interfere with the exchange studies to be described. C14ATP was purchased from Schwarz BioResearch, Inc. as the tetralithium salt and was converted to it,s disodium form by passage through a column of Dowex 50 X 4 (2o(t400 mesh) in the sodium cycle. The resulting ATP solution had a specific activity of about 20 mC per millimole. Cl” experiment,s were measured with a Packard Tri-Carb scintillation counter. Cad5 was purchased from Oak Ridge National Laboratory with an activity of 2-3 mC per milligram, and counts were detected with a thin-window, gas-flow Geiger counter by Tracer Lab, Inc. Water was glass double-distilled and all other chemicals were reagent grade.

RESIJLTS

AND

Preparative

I)TSCUSSION

Aspects

It

has been recognized t,hat when the poIvdcr extract of rabbit material was polymerized with 0.1 ~11 KCl, trotjomyosin was frequently incorporated into the polymerizing protei II. Although the tropomyosin content may be reduced by ext)raotion in t)he cold (SO),substit)ution of 0.6 mM hIgC& for KC1 as the polymerizing agent resulted in a I)reparation which was pure as judged by analytical ultracentrifugation in the absence and presence of EDTX (21). In view of t)his finding, the frog extract was polymerized with both salts, and the resulting material was assayedfor Ijurity by using analytic~al ultracentrifugation and starch-gel elec%rophoresisns the caritcria. The data presented in Fig. 1 indicate that the KC1 polynlerized material c*ontains at least, t,wo components, i.e., two bands appeared with electrophoresis, and EDTA treatment gave rise to a second peak in the ultracentrifugal pattern. In contrast’ are the results obtained with the Rig polymerized material. One band was observed in the starch gel; the slower one seenwit’h KC1 material, reportsed by Carsten (22) to be tropomyosin, was absent. In addition, EDTA did not produce a slower sediment’ing spiked peak, charact#eristicof tropomyosin (21). Therefore the hlg polymerization method of Laki et al. (21) was used throughout. Furthermore, in view of the above result,s, it may be concluded that the frog material exhibits the same behavior as does the rabbit with regard to actin and tropomyosin interaction. acetone

Physical

Parameters

Polymerization. Of the fibrous muscle proteins isolat’ed from rabbit, skeletal muscle, actin is unique in that the addition of salt, notably KCl, induces polymerization of the monomer (G-a&in) to the polymer (F-actin) (18). That. this phenomenon is also observed with frog actin is demonstrat,cd by the dat)a in Table I. The magnitude of the increase in specific viscosity (from 0.43 t’o 5.48) is quite startling and occurs v&h great rapidit,y. Similar saltlteffects are reported for

FIG. 1. Purity electrophoresis.

of frog

G-a&in

as judged

by analytical

Acetone

ultracentrifugation

powder

water

extract

and starch-gel

polymerized

with

Criterion 0.1 mb1 KCI, 2 mhf Tris, pH 8.0

Starch-gel electrophoresis (16yb) 0.012 M Tris, pH 8.0 0.4 mM ATP 0.2 mM ascorbic acid protein 8 mg/ml; 50 19 hours, 8 volts/cm 4°C. Analytical ultracentrifugation 59,780 rpm; 20°C Bar angle GO” 32 minutes at speed 0.5 mM ATP 2 mM Tris, pH 8.0 0.5 m&f ATP 2 mM Tris, pH 0.5 mM EIITA,

8.0 pH

7.0 23

0.6 mM MgClz, 2 mM Tris, pH 8.0

24

l)OJ,P

a&n extracted from rabbit, cardiac musc~lc (31) and for insect preparations (32, 33). Sedimentation coeficient as a function of protein concentration. A plot of t,he reriprocals of the calculated sedimentation coefficients at nine different protein conren trations in 0.5 mM ATP, 2 mM Tris, pH 8, is given in Fig. 2. The line, drawn by the method of least squares, extrapolates at c = 0 to an S,“O= 3.1s. This value is within t,he range of earlier report,ed values (18), between those recernly given for rabbit skeletal muscle [3.02S by Kay and 3.25s by Lewis et al. (35)] and slightly below that given for mammalian cardiac muscle [3.44S by Katz and Hall (31)]. In addition, Connell (36) reports an $0 = 3.3s for G-actin from cod. The similarity of these values suggests that, within the bound of sensitivit)y of this method, frog G-actin does not differ radically from that of other vertebrat,es studied. Partial specific volunze. The apparent partial specific volume of frog G-a&in at 25°C in 0.5 mM ATP, 2 mM Tris, pH 8.0, at 2.68 mg per milliliter was determined to be TSBLE POLYMERIZ.ITIOX

0.730 f 0.006. This average is slight.ly higher than the 0.715 reported for rabbit skeletal (34) and cardiac muscle (31). Molecular weight. I\Iolecular weights were determined by t,he Ehrenberg modificat#ion (23) of the Archibald method. A preliminary measurement was made with a 1%’ solution of bovine pancreatic ribonuclease in 0.1 J1 Tris, pH 7.5, to determine the error involved. The date in Table II show that. for RKase t’he MW was 12,302, which represents a 10 % decrease from t’he real value (13,700). The same experiment, for frog G-a&in in 0.5 m31 ATP and 2 mdl Tris, pH 8.0, gave 64,000 f 8000 as the NW. Comparison of this estimate with those published for rabbit skeletal and cardiac muscle shows that it, falls well within the range of 67,000-66,000 as measured by sedimentation and viscosity, and lghts catt,ering (31, 34, 35, 37). While additional measurements by independent methods are needed before establishing with certainty the real molecular weight, the value reported here gives a workable estimat’c. In addition, the S/D ratio remained const’ant with time, indicating a homogeneous preparation. This latter fact, coupled with a molecular weight of 64,000 f 8000 excludes for frog G-actin t)he possibility of the dimer structure proposed by Tsao (38) and Ooi (39).

1 OF FROG

&TIN

system

WJ

G-actin (3.36 mg/ml) 5 X lo- M ATP 5 X 10e3 M Tris, pII 8.0 As above plus 0.1 M KCl, 1 minute

Chemical Parameters

0.43 later

A~zino acid analysis. results of a preliminary

5.48

40 , .d

Table III give< the survey of the amino

.

s2o(s) -I;

4 mg. FIG. 2. l/szo as a function dium contains 0.5 mM ATP

of frog G-act&l and 2 mM Tris,

protein

6 /

8

ml.

concentration. pH 8.0. When

Speed = 59,780 q~“l; 20”~; me c = 0, s&, = 3.1~.

ZZ. CATESBIANA

SKELETAL

MUSCLE

TABLE MOLECULAR

WEIGHTS

DETERMINED OF THE

Protein

BY

Bovine pancreatic RNase Frog G-a&in

0.1

M Tris,

Cont.

pH

7.5

0.5 mM ATP 2 mM Tris, pH

8.0

TABLE ACID

COMPOSITION

OF HYDROLYZATES

Frog G-actin, Amino

(23)

EHRENBERG

(mg/ml)

Vapp used

MODIFICATION

Calculated

10

0.709

12,302

9

0.736

64,000=

MW

A protein concentration isimposed by the tendency

Error

107, based on MW = 13,760 ~k80OQ

of 1% and above of actin topolym-

III OF G-ACTIN

FROM

Frog G-actin, and Katz

this workc

25

METHOD

a This value is based on two runs at the same concentration. is desirable for this method but an upper limit around 0.9% erize at higher concentrations.

AMINO

I.

II THE

ARCHIBALD

Medium

PROTEINS

SKELETAL

Carsten (40)

MUSCLE Rabbit

G-actin, and Katz

Carsten (40)

acid

5.52 2.53 6.00 10.20 6.82 5.43 13.34 4.95 4.81 5.82 5.78 4.37 8.42 7.44 6.88 4.20

Lysine Histidine Arginine Aspartic acid Threoninea Serine” Glutamic acid Proline Glycine Alanine Valineb Methionine Isoleucineb Leucine Tyrosine Phenyalanine Total

22.6 9.8 20.6 46.0 34.1 31.0 54.4 25.8 38.5 39.2 29.6 17.6 38.5 34.1 22.8 15.2 479.8

2.3 1.0 2.1 4.7 3.5 3.2 5.6 2.6 3.9 4.0 3.0 1.8 3.9 3.5 2.3 1.6

26.8 10.3 24.1 48.6 38.4 34.5 58.1 24.7 38.8 42.3 25.5 17.3 37.3 37.2 20.2 15.9 500.0

2.6 1.0 2.3 4.7 3.7 3.4 5.6 2.4 3 .8 4.1 2.5 1.7 3.6 3.6 2.0 1.5

26.0 10.3 25.1 48.3 40.6 34.3 56.1 26.3 39.5 42.1 25.2 17.4 38.7 35.8 20.3 16.3 503.3

2.5 1.0 2.4 4.7 4.0 3.3 5.4 2.5 3.8 4.1 2.5 1.7 3.8 3.5 2.0 1.6

a Corrected for losses during hydrolysis. b Corrected for slow release during hydrolysis. c Data based on preliminary runs only. d Histidine

analyzed

residues

taken

as 1 to facilitate

comparison.

(One

preparation

hydrolyzed

20 and

70 hours,

in duplicate).

acid composition of hydrolyzat’es of frog G-actin. Also given are dat#a obtained recently by Carsten and Katz (40) for rabbit and frog actin. Alt’hough the values obtained in the present work for the number of amino acid residues per 60,000 grams protein are, in most cases, slightly lower than those reported by Carsten and Katz for both the frog and rabbit material, they do corroborate the evidence presented by the latter workers t,hat, the amino acid composition of actin

isolated from these two sources does not appear to reflect a species’ variation. This fact is best illustrated by a comparison of the member of residues relative t,o histidine for each of the analyses listed (Table III). Such an expression compensates for the smaller total yield obtained in this work. It can be seen that the amount of valine detected in this study is higher t,han that obt’ained by Carsten and Katz for either the rabbit, or frog protein (29.6 residues per

26

DOLP

60,000 grams as opposed to the values of 25.5 and 26.2). This discrepancy is probably due to the fact that in this study the valine was corrected for slow release during hydrolysis by extrapolation to infinite time from a zero order plot. Grams protein per mole bound nucleotide. In 1950 it was demonstrated that rabbit G-actin contains bound nucleotide in the form of ATP (41, 42). This relationship was then quantified by Szent-Gyorgyi (43) and Mommaerts (44), who concluded that 57,000-60,000 gm G-actin (or 1 mole) contained 1 mole of bound ATP, a value which has been subsequently confirmed (4549). A similar experiment was made on 5 different preparations of the frog material and a value of 59,750 f 2000 obtained which is in close agreement with that for the mammalian protein. -SH titrations. In view of the possible role of -SH groups both in the polymerization of G to F-a&in (50, 51) and in maintenance of the integrity of the monomer form through possible involvement with ATP binding (48), it was of interest to assay the frog material with respect to -SH groups. Due to the variability of the methods concerned, the experiments were first performed on rabbit G-actin in the same manner as with t,he frog material and these dat’a used as a point of comparison. A molecular weight of 60,000 is assumed for all calculations and values represent moles -SH per mole protein. The upper curves in Fig. 3 represent the t,ime course of the PMB titrations. With rabbit G-actin, it is observed that initially two -SH groups react and maximally 6. After 15 minutes, 4 of the groups are titrated and the total reaction begins to level off at about 40 minutes (20°C). The maximal value of 6 -SH groups is confirmed by the observation that incubation of the protein with PMB and 0.1 M SDS (added in that order) for either 20 minutes or 3 hours reveals 5.9 -SH groups (Table IV). NEM, a reagent known to react with only the most accessible -SH groups, titrated 1.9 of the -SH groups of rabbit G-actin, as shown in Table IV. This same protein polymerized upon the addition of 0.1 Jf KCl, i.e., the specific viscosity increased from 0.4 to 4.2, indicating that the 2 -SH groups blocked with NEM are not directly involved in the polymerization process. All of the

20

40 time

in

60 minutes

FIG. 3. Rate curves for --SH titrations of rabbit and frog G-actin. G-actin at 0.5 mg/ml; 2 mM Tris, pH 8.0. Upper curves, 0.1 mM PMB; lower curves, G-actin reacted with NEM for x hour, dialyzed against buffer overnight, and then treated with 0.1 mM PMB. Vertical lines through every 4th point represent the deviation from the average of the values recorded. The actin was prepared in 0.2 mM ascorbic acid to ensure constancy of results. Before assay, excess nucleotide was removed with Dowex-1 Cl- as described in Methods. Closed circles, rabbit actin; closed triangles, frog actin.

foregoing results are in direct accord with those obtained by Katz and Mommaerts (52). When the rabbit material was previously treated with NEM and then titrated with PMB (Fig. 3, Table IV) the maximal value titrated is 3, one less than would be expected. This discrepancy, perhaps due to an oxidation during dialysis, is not important to the main point of the argument developed below. The data for identical experiments with frog G-actin taken from three different acetone powder preparations are recorded in Fig. 3 and Table IV. PMB initially reacts with 2.2 -SH groups but, in contrast to the rabbit, only doubles this value maximally, i.e., the total number of -SH groups is 4.0. The third group is titrated in about 10 minutes and the reaction levels off at about 30 minutes. The deviation recorded indicates that at no time does the value approach that of the rabbit protein. In addition, titration with PMB in the presence of SDS gave a value of 3.8 moles -SH per mole protein. NEM reacted with 2.2 -SH groups and NEM substituted frog G-action when titrated with PMB gave 2 -SH groups maximally as would be expected (Fig. 3).

Clearly

then,

frog G-actin

differs

from

R. CATESBZANA

SKELETAL

MUSCLE

TABLE -SH

TITRATIONS

PROTEINS

IV

OF RABBIT

AND

FROG

G-ACTIN Rabbit

Reagent

A. Moles SH/mole protein (assuming MW = 60,000) 0.1 mM PMB 2 mM NEM NEM (unreacted removed by dialysis) plus 0.1 mM PMB 0.1 mM PMB plus 0.1 M SDS B. Viscosity (q.*) 2 mM NEM 2 mM NEM plus 0.1 M KCl, 35 hour

that of rabbit with respect to its sulfhydryl group titration under the conditions of these experiments. The problem then arises of how to explain this difference. It appears that there are two general possibilities: (a) that frog actin does indeed contain 2 more potential -SH groups, perhaps in the form of a S-S bridge; or (b) that rabbit actin possessestwo “extra” -SH groups which are not basic requirements for the contractile process. The first alternative was considered earlier (53) for rabbit actin to explain the difference between methylmercury nitrate and PMB titrations (33). This notion was denied, however, by Carsten (54) who found no rystine residues in an amino acid analysis of S-carboxymethyl-actin. Very recently, she reported a similar analysis of actin from several species, among them the frog (40). No cystine residues and 6.1 S - carboxymethyl - cysteine residues per 60,000 gm were detected. These values were qualified by the opinion that due to bhe method of ca,lculation, it seemsquite possible t,hat the preparations actually differed more t,han the list indicated. Thus neither the possibility of a S-S bridge nor t,he t’otal number of -SH groups being 4 in frog G-a&in can he excluded 011 the basis of present evidence. If t,hc second alt,ernative mentioned above is, in fact, the explanation, then a good system is at hand for st’udying several aspects of a&n structure and function. Wit,h regard to actin structure, it would be interesting t,o st,udy which 2 SH groups are “missing,” so t’o speak, and consequently

27

I.

Frog

Initial

Final

Initia!

Final

1.99

5.8 1.9 3.0

2.2

4.02 2.19 2.1

0.44

0.87

5.9 0.4 4.2

3.8 0.9 4.4

those which are essential for monomer integrity. An understanding of the role of SH groups in polymerization might also be further enhanced by a comparison of the two protein systems. Lastly, the role of actin SH groups in the interaction with myosin and consequently, the contractile process might be further elucidated through such a comparative study. Cation exchange. It has long been known that, rabbit actin preparat’ions contain divalent cations, namely calcium (50). More recently it has been established that the calcium (and only calcium) is bound to the protein mole per mole, that it,s presence is necessary to maintain the integrity of the actin molecule, and that in the monomer this divalent cation can be exchanged wit,h others without impairment of protein function (29,41, 46-48, 55-57). Early speculation that perhaps the cation served as a bridge between the protein and bound nucleotide has been substantiat’ed by recent, experiments (46, 48, 55). Due to the importance of this facet in rabbit actin structure, it was of interest to determine the sit#uation with frog material. Before discussing the experiments, mention must be made that excess Ca45 and ATP were simultaneously removed from the protein solution by passage through a Sephadex G-50 column as described in Methods; thereby eliminating the 2.step procedure involved with use of Dower; 1 Cland Dowex 50. That the desired separation is obtained is demonstrated by the elution pattern given in Figure 4. It can be seen that the first peak has an 0D260 ,/ODnao ,,,fi of 0.70 contains all the material react,ing with Lowry reagent and has

28 cpmC-1

500

1000

1500

2000

O.D.,,,f-----I

.zoo

.400

,600

BOO

,500

1.000

1.500

2.00

0.D.26o

,,,&

4

0

FIG. 4. Separation of excess ATP and Cad5 from frog G-actin on a Sephadex (bead form) column. Column equilibrated and eluted with 2 mM Tris, pH 8.0; mensions, 1 X 26.3 cm.; 2 ml protein applied (4 mg/ml); flow rate, 0.55 ml/min;

2500 cpm; in addition, the second peak has an OD 260mll/OD2mnqr of G.70 and 400 cpm. Therefore actin containing bound Ca”S is separated from free Ca45 aud ATP which move together through the column. Actin so treated is able to polymerize as judged by the increase ill specific viscosity from 0.85 to 2.60 upon the addit.ion of 0.1 M KCL.

In general, the experiments reported here concern the abilit’y of Ca45 bound to frog G-a&in to exchange with other cations and the subsequent affect of such an exchange, if any, upon the bound nucleobide and polymerizabi1it.y of the protein. The specific det,ails of t,he experimental procedure are given in Table V. It might be pointed out, t,hat the direction in which exchange was measured here (i.e., hot chased by cold) gives a true measure of act’ual exchange and excludes measurement of cations which might be nonspecifically adsorbed. The data in Table V show that bound Ca45 of frog G-a&n can exchange with Ca, Mg, and ;\ln and about to the same extent in each case (40.548.2 %). No exchange was observed with t,he divalent Ba nor the monovalents K and Na. The addition of Cd, Hg (mercuric), and Fe (ferrous) precipitated the prot’ein and therefore the exchange was not determined. It is interesting to note that with the exchanged cations, Ca and Mg, nucleotide was bound mole for mole and the prot,ein polymerized with 0.1 M KCl. In

G-50 Fine column di4°C.

the case of Mn, exchange occurred, the prot,ein polymerized but the value for the bound nucleotide is low (0.76) ; in view of ot’her evidence, it seems unlikely that the protein would polymerize without bound nucleotide (58) and thus t,he value for t,he nucleotide may be considered erroneous. As would be expected with those cations which did not exchange (Ba, K, Na), the nucleotide remained bound and the protein polymerized indicating that t’hese cations, although perhaps adsorbed to the protein, do not alter it,s behavior. The slightly reduced value for bound nucleotide in the presence of cold Ba might be due to the extremely low solubilit,y product’ of BaATE’ which is formed during the dissociation equilibrium of the protein and nucleotide elucidated by Asakura (29). Those cations which will exchange with Ca in frog G-aetin display the same behavior with respect to the rabbit (56, 57). It is difficult to compare the ext’ent to which this exchange takes place because the rabbit data are reported as percent,age exchange relative to that of Ca. The actual cpm are not’ given and consequently it is not known whether t’he Ca exchange does, in fact, represent’ 50% of that which occurred absolutely. It is reported, however, that within a few minutes, the Ca in rabbit a&n exchanges completely (56). The dat,a in

R. C/lTESBI/W/l

SKELETAL TABLE

EXCHANGE

Cold ;;ti&chase

None Ch Mg ml Ba K Sa Cd 11g+2 Fe+3

(HZO)

701 417 388 373 706 G71 G35 Protein Protein Protein

29

I.

V G-ACTING

Bound nucleotide

% Activity exchanged 0 40.5 44.7 48.2 0 4.5 9.4 precipitated precipitated precipitated

PROTEINS

Ca45 IN FROG

OF BOUND

Radioactivity avg. cpm/ mg prot.

MUSCLE

mole/mole prot. (60,000 grn)

Spectrum Spectrum Spectrum

1.10 1.00 1.03 0.76 0.86 0.95 0.99 not that not that not that

Viscosity

-

hp-?sp)

0.1 .$I KC1

0.30 0.20 0.30 0.20 0.30 0.25 0.30

1.80 1.20 1.00 1.00 0.95 0.90 1.10

of a nucleotide of a nucleotide of a nucleotide

a The experiments were performed in the following manner at 4°C. The F-a&in pellets obtained by ultracentrifugation of the Mg polymerized acetone powder extract were homogenized in 0.4 mdE Cads, 0.5 mM ATP, and 2 mM Tris, pH 8, and dialyzed against the same for 18 hours. The dialyzate was then filtered through the Sephadex G-50 column. 5-ml aliquots were incubated 1 hour with the chloride salts of the cations listed above at a final conceutration of 1 mM. Following this chase with cold cation, the mixtures were dialyzed in EDTA-treated tubing against 100 ml of buffer for 18 hours. The dialyzate was then assa,yed for radioactivity, protein, bound nucleotide, and viscosity. Protein concentrations were all about 3 mg/ml (0.053 mM). That Ca45 released by exchange did pass through the dialysis tubing was indicated by the presence of counts in the medium. yti activity exchanged = (cpm without chase) - (cpm with chase)/(cpm without chase) X 100.

Table V for frog G-actin indicate t’hnt after 1 hour only 40.2 % of t.he Ca has exchanged. This difference may lie in technique or in the nature of the prot,ein. The first allternative contains two possibilities: (a) the presence of F-a&n which is known to be refractory t’o cation exchange (-55) ; and (b) t’he temperature of incubation (t’he cation molarity was the same in experiments being compared). The former condition is doubted due to the low viscosit’y of t’he preparation although Nagy and Jencks (58) suggested that perhaps Mg polymerized actin does not completely depolymerize. In these experiments, react)ions were carried out at 4°C rather than the 25°C used for t,he rabbit exchanges. Kinetics must be studied before it can be known whether the end point of exchange is the same for both rabbit and frog G-actin. The second alternative mentioned above is related to the latter, i.e., perhaps the absolute molar ratio of bound divalent cation differs in the two species prot,eins. This seems less plausible in view of the similarity of nucleotide ratios yet remains a possibility which cannot be discarded on the evidence at hand.

Nucleotide exchange and specificity. Studies concerning the function and specificity of the nucleot’ide bound to rabbit actin have shown that Ohis portion is necessary for all the characteristic properties of actin (59) and that t,here is a strict requirement for a triphosphate chain, an adenine-like configuration in the 1, 2, and 3 positsionof the ring and the ribose moiety (48, 60, 61). In addition it is only with the G-form of the protein t’hat nucleotide exchange is possible, F-actin being refrachory to both ATP and ADP (62). It has also been observed with rabbit G-actin that the rate of exchange of ATP in t,he presence of hlg is greater than with Ca (48). Similar experiments with frog a,ctin are reported here. First, the exchange rates of ATP with C14ATP G-actin were measured with Ca, Mg, and “no cation.” The latter phrase does not indicate the absenceof any cations in the reaction mixt’ure but rather t,hat one is not present in preponderance. Experimental details are given in the legend of Fig. 5. The rate curves for frog G-actin indicate that exchange does, in fact, occur wit’h the entire ATP molecule (Fig. 5).

30

DOLP

s

L--;--;---,---;-

-;----AD.J

40

00 time

in

I20

ATP

F-actin

,

160

minutes

FIG. 5. Rate curves for the nucleotide exchange of frog G- and F-actin. The protein was prepared by homogenizing the F-a&n pellets obtained by ultracentrifugation in 0.5 mM Cl4ATP and 2 mM Tris, pH 8.0. Excess nucleotide was removed by Dowex-1 Cl-. Upper curves: G-actin-C14ATP. Separate aliquots of the protein were preincubated with 0.55 mM CaCl,, MgC12, or water representing “no cation” for 10 minutes. ATP was added to 0.05 mM and at various times l-ml aliquots were removed, treated with Dowex-1 Cl- and counted for radioactivity. Lower curves: F-actin-C14ADP. Labeled F-actin-CJ4ADP was obtained by 0.1 M KC1 polymerization of G-actin-C14ATP prepared as above. One portion was incubated with 0.06 mM ATP and a second with 0.06 mM ADP. At various times l-ml aliquots were removed, dialyzed 36 hoursagainst0.1 M KCl, 2 miM Tris, pH 8.0, andcounted for radioactivity. All incubation, 4°C in 2 mill Tris, pH 8.0; proteirl-2 mg/ml (0.035 mM). 70 activity exchanged = [(cpmta) - (cpmt=,)]/(cpmt=o) X 100.

Secondly, it appears that the equilibrium is approached most rapidly in the presence of Mg, with Ca and “no cation” following in that order; at 20 minutes of incubation, 57.8 7c of the activit’y exchanged with Mg, 49.9 ‘% with Ca, and 38.4 % with “no cation.” Although more detailed studies are needed of the early part of the reaction, a first order plot of these dat’a confirm t’he trends indicated by Fig. 5. Finally it may be concluded that the end point or extent of ATP exchange is the same with Ca and hlg (59.1 and 58.0 % at 120 minut’es) and slightly lower with “no cation” (48.0 %). Similar cation effects upon the rate and extent of ATP exchange were observed by Strohman (48) with rabbit material, i.e., the approach to equilibrium was faster with Mg than with Ca whereas the final value of exchange was the same. It must be noted, however, that the ret,ardat’ion by Ca observed for the rabbit material is much more pronounced than that reported here; in addition, without added cation, the rate for

the frog ATP exchange was lowest whereas for the rabbit it was highest. It is difficult to assess the significance of this last observation due to lack of information about’ the actual cation content of the reaction. The remaining curves of Fig. 5 represent exchange experiments with frog F-actinC14ADP when chased wit’h ATP and ADP. In neither case did the free nucleotide replace that bound to the fibrous form of actin, indicating that during the polymerization of frog actin the nucleotide is somehow masked in a manner long reported in the case of rabbit (41, 62, 63). The specific&y of the nucleotide bound to frog G-a&in was also studied by measuring the extent to which bound CY4ATP exchanged when “chased” with an assortment of other nucleotides and related compounds in the presence of Ca, XIg, or “no cation.” Figure 6 gives the details and results of such an experiment. It can be seen that exchange was greatest with ATP (82.6 %) and that ITP is able to replace the bound nucleotide

R. C,4TESB1&J14

Adenosine

compounds

SKELETAL

MUSCLE

AMP

GTP

added

to

incubating

ITP

PROTEINS

CTP

UTP

I.

31

CP

mixture

FIG. 6. Specificity of the nucleotide bound to frog G-actin. G-actin-Cl4ATP prepared as in Fig. 5 and divided into three aliquots: one with 0.5 mM CaCl2, the second with 0.5 mM and the third with “no cation” (water). Each of the three mixtures was divided W&l,, into nine portions and incubated for 1 hour with 0.1 mM (pH 7.0) solutions of the compounds listed above. Nucleotide released by exchange was removed by Dowex-1 Cl-’ and the filtrate was count,ed for radioactivity. All incubations, 4°C in 2 mM Tris, pH 8.0; protein = 2 mg/ml (0.035 mM) y0 activit,y exchanged = [(cpmA20) - (Cpk)]/(CpmHtO) X 100.

to a limited extent (32.0% radioactivit’y exchanged). All other compounds except ADP showed a very low activit’y in this respect. A similar ADP effect has been reported with rabbit material (48, 61, 62) and has been attributed to the action of myokinase and another enzymatically catalyzed ATP-ADP exchange reaction observed in the actin preparat’ions (61). Whether or not this explanation applies to the frog material cannot be concluded at this point. Although time curves were not run with each nucleotide, the above data indicate either that only ATP (and ITP) can be bound or that the protein has a much great,er affinity for these two nucleotides than for the others. Comparison of t’he %j radioact#ivity exchanged in Figs. 5 and 6 indicates that the end point of ATP exchange is dependent upon the concentration of free nucleotide, i.e., in 1 hour at 0.06 mM ATP, 58 % of the activity exchanged whereas at 0.1 m)V ATP

83% exchanged. Such a concentration dependence corroborates and extends Asakura’s (29) notion of an equilibrium dissociation to frog G-a&in. The data in Fig. 6 also show that the nucleotides which exchanged with the labeled ATP were not effected to any great extent by the type of cation which was present. This observation suggeststhat the specificity of t’he nucleotide which can be bound is determined by an interaction of the protein and that nucleotide, and not’ by the cat,ion present. Although the cation is essential for nucleotide binding, its influence may be exerted through the degree of stability which it confers upon t>hecomplex. ACKNOWLEDGMENTS The author wishes to extend a special t’hanks to Dr. It. C. Strohman for his patience and helpful suggestions throughout the course of this work. I am also grateful to Dr. R. D. Cole for sharing his time and for many kindnesses, including the

32

DOLP

use of facilities for starch-gel amino acid analysis.

elect,rophoresis

and

REFERENCES 1. PERRI-, $. V., in “Comparative Biochemistry” (RI. Florkin and H. Mason, eds.), Vol. II, pp. 245-340. Academic Press, New York (1960). 2. D.\v-IES, R. E., Satu?‘e, 199, 1068 (1963). 3. PODOLSKY, R. J. Naval Med. Res. Inst. Res. Repf. 16, 417-432 (1958). 4. HOEVE, C. 9. J., WILLIS, Y. A., AND M,\RTIN, I>. J. Biochemistry, 2, 282 (1963). 5. HUXLEY-, H. E., .IND HANSON, J., in “The Structure and Function of Muscle” (G. H. Bourne, ed.), 1’01. 1, pp. 183-227. Academic Press, New York (1960). 6. BENNETT, H. S., in “The St,ructure and Function of Muscle” (G. II. Bourne, ed.), Vol. 1, pp. 1377181. Academic Press, New York (1960). 7. HUXLEY, H. E., J. Mol. Biol. 7, 281 (1963). 8. HANSON, J., .ZND LOWY, J., J. Mol. Biol. 6, 46 (1963). 9. CAIN, D. F., INFANTE, .4. A., .\ND I~AVIES, R. E., A\rature 196, 214 (1962). 10. CAIN, D. F., KUSHMERICK, M. J., BND DAVIES, R. E. Biochim. Biophys. Acta 74, 735 (1963). 11. MOMMAERTS, W. F. H., SEHAYDARIAN, K., AND M.~RECHAL, G., Biochim. Biophys. Acta. 57, 1 (1962). 12. C,\RLSON, F. I>., AND SIGER, A., J. Gen. PhysioL 44, 33 (1960). 13. BsMSEY, R. W., in “The Structure and Function of IMuscle” (G. IT. Bourne, ed.), Vol. 2, pp. 303-358. Academic Press, New York (1960). 14. PODOLSKY, R. J., in “The Structure and Function of Muscle” (G. H. Bourne, ed.), i’ol. 2, pp. 359-385. Academic Press, New York (1960). 15. N;\ss, M. K., Develop. Biol. 4, 289 (1962). 16. STROHMAN, R. C., CERWINSKY, E., AND HOLMES, D. W., Exp. Cell Res. 35,617, (1964). 17. HAY, E. D., 2. Zellforsch. 69, 6 (1963). 18. SZENT-GYORGYI, A. D., in “The Structure and Function of Muscle” (G. H. Bourne, ed.), Vol. 2, pp. l-54. Academic Press, New York (1960). 19. ANFINSEN, C. B., “The Molecular Basis of Evolution.” Wiley, New York (1959). 20. BAILEY, K., in “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. 2B, p. 980. Academic Press, New York (1954). 21. LAKI, K., MBRUYAMA, L., END KOMINZ, I>. R., $rch. Biochem. Biophys. 98, 323 (1962). 22. CIRSTEN, M. E., AND MOMMAERTS, W. F. H., Biochemistry 2, 28 (1963).

23. EHRENBERG, A., Acta Chem. Stand. 11, 1257 (1957). 24. S~H~~HM~N, H. K., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Tel. 4, pp. 32-103. Academic Press, New York (1957). 25. HIRS, C. H. W., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 211, 941 (1954). 26. LOWI~Y, 0. II., ROSEBRO~G~, N. J., F~RR, A. I,., AND I~ANIIALL, B. J., J. Biol. Chem. 192, 265 (1951). 27. BOYER, P. I)., J. Bm. Chem. Sot. 76, 4331 (1954). 28. ALEXANDER, N. M., ;I&. Chem. 30, 1292 (1958). 29. AS.4KuR.4, S., Arch. Biochem. Biophys. 92, 140 (1961). 30. DRSBIKO\VSKI, W., .~ND GEKGELY, J., J. Biol. Chem. 237, 3412 (1962). 31. KATZ, A. M., ANL) HALL, E. J., Circulation Kes. 13, 187 (1963). 32. MARUY:\MI, K., J. Insect Physiol. 3,271 (1959). 33. KOMINZ, I). R., MARUY.iM.4, K., LEVENBOOK, I,., AND LEWIS, M., Biochim. Biophys. *lcta 63, 106 (1962). 34. Kay, C. M., Biochim. Biophys. Acta 43, 259 (1960). 35. LEWIS, M. S., M.4RUYAM.4, K., CARROLL, W. R., KOMINZ, I>. R., AND LAKI, K., Biochemistry 2, 34 (1963). 36. CONNELL, J. J., Biochem. J. 70, 81 (1958). 37. MOMM.IERTS, W. F. H. M., J. Biol. Chem. 198, 445 (1952). 38. Ts,zo, T. C., Biochim. Biophys. Acta 11, 227 (1953). 39. 001, T. M., J. Biochem. 60, 128 (1961). 40. CBRSTEN, M. E. 14~~ KII~Z, A. M., Biochim. Biophys. Acta 90, 534 (1964). G., Biochim. 41. STRAUB, F. B., AND FEUER, Biophys. Acta 4, 455 (1950). 42. LAKI, K., BOWEN, W. J., AND CLARK, A., J. Gen. Physiol. 33, 437 (1950). 43. SZENT-GYORGYI, A. D., Arch. Biochem. Biophys. 31, 97 (1951). 44. MOMMAEKTS, W. F. H. M., J. Biol. Chem. 198, 469 (1952). 45. ULBRECHT, J., GRUBHOFER, N., J~ISLE, F., AND W.ILTEK, H., Biochim. Biophys. hta 46, 443 (1960). 46. TONOMUR.~, Y., AND YOSHIMURA, J., J. Biothem. 60, 79 (1961). 47. CHRAMBACH, A., BARONY, M., END FINKELMAN, F., Arch. Biochem. Riophys. 93, 456 (1961). 48. STHOHMAN, R. C., AND S~~MORODIN, A. J., J. Biol. Chem. 237, 363 (1962). 49. TSUBOI, K. K., AND HAYASHI, T., Arch. Biothem. Biophys. 100, 313 (1963).

R. C’-4TESBIA,VA

SKELETAL

50. FEUER, G., MOLNAR, F., PETTKO, E., AXD STRAUB, F. B., Hung. Acta Physiol. 1, 150 (1948). 51. KUSCHINSLT, C., AND TURBA, F., Biochinr. Biophys. Acta 6, 426 (1951). 52. KATZ, A. M.: AND MOM~XAERTS, W. F. H. M., Biochim. Biophys. Acta 66, 82 (1962). 53. LAKI, K., AND HTANDAERT, J.: Arch. Biochem. Biophys. 86, 16 (1960). 54. CARSTEN, M. E., Biochemistry 2, 32 (19G3). 55. BAR.IXY, RI.,FINKELYAN, F., AND THERATTILAXTONY, T., Arch. Biochem. Biophys. 96, 28 (1962). 56. B.m.m;r, G., AXD CHlL4xBAC!H, A., J. Gen. Physiol. 45, 589A (1962).

MUSCLE

PROTEINS

I.

33

57. I~RABIK~W~~I, W ., AND STRZELECKA-GOLASZE\VGKI, J., Biochim. Biophys. Acta 71, 486 (1903). 58. NAGY, P., AXD JENCICS, Biochemistry 1, 987 (1962). 59. BARANY, M., NAGY, B., FINKELMSN, F., AND CHRU~BACH, A., J. Biol. Chem. 236, 2917 (1961). 00. MARTONOSI, A., AND GOUYEA, M. 9., J. Biol. Chem. 236, 1345 (1961). til. MARTONOSI, A., Biochim. Biophys. Acta 57, 163 (1902). 62. MARTONOSI, A., Gouvm, M. A., AND GERGELY, J., J. Biol. Chem. 235,170O (1960). 63. STKOHMIN, R. C., Biochim. Biophys. Acta 32, 436 (1959).