of the Temperature of Extraction

Tropomyosin Content in Actin*

IV. DRAFIIKO~SKI~ ASD J. GERGELY

on the

Prom the Department of Jluscle Research, Institute of Biological and Medical Sciences, Retina Foundation, the Cardiac Biochemistry Research Laboratory, Massachusetts General Hospital, and the Department of

Xedicine, Harvard University, Boston, .lfassachusetts

(Received for publication, iipril 2.5, 1962)

In the course of our studies 011 the rapid depolymcrization of F-actin (l), a publication by Grubhofcr and \Veber (2) came to our attention in which the authors reported on work with actin prepared according to Ulbrecht et al. (3). The latter procedure differs from the one routinely rmployrd in our laboratory in two points: (a) the muscle residue, after removal of myosin with the Cuba-Straub solution which is generally used (4), is washed with 0.01 u ?;aHC&, whereas we use 0.05 Y SaHC&, and (h) 1Jlbrecht et al. estractetl the acetone-dried muscle residue (ace- tone powder) at 0” whrrcas we, following an earlier procedure (4), have been extracting nctin at room temperature. Since ae found (1) that the properties both of G-a&in and of rapidly depolymerizrd F-a&in varied awording to which of the two methods was used, we became interested in the effect of the conditions of estraction from muscle acetone powder on the properties of actin. The viscosity of both G-a&in and rapidly depolymerized F-actin, prepared according to the usual method, was higher than that of actin prrpared with the use of the pro- cedure of Ulbrecht et al. (3). Moreover, the ultracentrifu- gal pattern of rapidly dtpolymerized F-actin obtained from the usual G-actin showed an additional sharp, faster moving bound- ary which was cssrntially absent when actin was prepared ac- cording to Ulbrecht et al.

Evidence to be presented in this paper shows that of the fac- tors mentioned above the temperature is the decisive one. The reduced viscosity of G-actin purified from extracts obtained at 23-24” has a considerably greater concentration dependence than that of an actin preparation from 0” estract. This phe- nomenon made us suspect the involvement of tropomyosin in view of the earlier results of Laki and Cairns (5) pointing to the presence of tropomyosin in alkali-denatured actin preparations, particularly when Martonosi (6, 7) in our laboratory found that one of the boundaries (y) appearing in the ultracentrifuge pattern of actin in 0.6 to 0.8 mhI big contains tropomyosin.

We can show, utilizing both these findings, that the above differences in viscosity depend on the presence of varying amounts of tropomyosin which is csswtially absent in 0” ex-

tracts. This c*onclusion is further corroborated by experiments in which tropomyosin was added to a&n extracted at 0’ or to

* This work was supported by grants from the National Henrt Institute (II-5040), the Muscular Dystrophy -4ssociations of America, Inc., the I,ife lnsur~nce Medical Hescrwch Fund, and the American Heart Association.

t l’ermancnt address, Nerwki Institute of ISxperimentnl Biol- ogy, Wnrsaw, Poland.

actin freed of tropomyosin by sedimentation at (90,000 X g in 0.6 tu 0.8 ml1 Jig (6, 7). Problems relating to the interaction of nctin and tropomgosin arc discussed in this paper; the prop- erties of ral)idly drpolymerizcd F-a&in prrpared from actin extracted under various conditions will be the subject of a subse- quent paper.

I:SPERIhIENT.4I, PROCEDURE

Actin was extracted at various temperatures lvith 20 volumes of water for 30 minutes from acetone-dried muscle residue pre- pared, unlrss otherwise stated, with the USC of the procedure of Feucr el al. (4), but omitting the washing with Na2C03 + Na- IICOI (8, 9). Unless the temperature of extraction is stated, “low temperature” will mean O-2”, and room temperature 23-24”. Crude extracts were polymerized for 2 hours at rnnm temperature in 0.1 M KC1 and 1 rnhl MgClt, and F-a&in was sedimented in the preparative ultracentrifuge (10). G-&tin was obtained from F-&in pellets by dcpolymerization by dialysis with in- Wnal and external stirring (II), against 0.2 mM ATP + 2 mM Tris, pH 8.0, for 40 hours and clarification by centrifugation for 30 minutrs at 18,006 X g. This preparation will be referred to as purified actin. It was transformed to F-a&in by pnlymeriza- tion in 0.1 u KC1 + 1 mM hIgC&. Tropomyosin was prepared according to Hailcy (12).

Viscosity measurements were carried out on G- and F-a&in in Ostwald viscomctcrs with outflow times of apprnsimately 80 and 30 seconds, respectively, at 27’.

Double refraction of flow was measured, unless otherwise stated, at a velocity gradient of 12 set-1 with an Edsall type of instrument made by the Rae Instrument Company. Ultra- centrifugal sedimentation patterns were obtained in a Spinco model I? analytical ultracentrifuge. Protein determinations were made with the use of a biuret reaction.

RESULTS

Table I contains wmparative data on crude actin preparations extracted from acetone-dricul ponder at low and at room tem- perature. More protein is extracted per gram of acetone powder at 24” than at O-2”. The percentage of polymerizable actin, as judged by ultmccntrifugation and double refraction of flow, is, however, approximately the same at the two temperatures, suggesting, prima facie, no gross difference in the composition of the two extracts.1

’ The extraction of acetone powder with 0.2 rnlr sdenosinc tri- phosphate instrud of water had no influence on yield or viscosity.

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November 1962 W. Drabikowski and J. Gergely

However, when purified G-a&in was investigated it appeared that, within the temperature range used, the higher the temper- ature of extraction the higher the reduced viscosity at a given concentration of G-a&in and the greater its comb&ration de- pendcnce (Fig. 1). If G-a&in extracted at room temperature is subjected to another cycle of purification, viz. polymerizat.ion with KC1 and Mg, sedimentation in the ultracentrifuge, and depolymerization by dialysis, the reduced viscosity remains high and retains the strong concentration dependence. So double refraction of flow was observed in G-a&in solutions and the analytical centrifugal patterns of G-a&in extracted at cold or at room temperature were thr same (Fig. 2:1). On addition of KI, in a final concentration of 0.8 M, the viscosity of G-actin. regardless of the temperature of extra&ion, dropped to values even lower than those reported by Kay (13) (‘l’ablc II). In contrast with the observations on G-a&in the viscosity and double refraction of flow of F-actin show practically no depmd- ence on the temperature of the extraction (Fig. 3).

In view of the facts mentioned in the introduction, we per- formed extractions at 0” and 24’ of acetone powders prepared according to IJlbrecht et al. (2), including a washing with 0.01 M xaIICOs, rather than 0.05 M SaHC&, before thr acetone trcat- ment. Again the viscosity and concentration drpcndence of the reduced viscosity of G-a&in were much higher in the case of room temperature extract than in the cast of cold extract. It appears that the conditions of washing the muscle residue were of little consequence compared with the tcmpcrature of estraction. (This is further borne out by data which are pre- sented later, in Table V.)

In view of the recent evidence pointing to the presence of tropomyosin even in purified actin preparations obtainrd by the conventional method from room temperature extracts of acrtone- dried powders (S-7), it seemed to us that the differences between actin preparations estracted at low and at room temperature might be due to the differences in the tropomyosin content. In fact, ultracentrifugal sedimentation patterns of partially polymerized, 0.6 to 0.8 mM Mg, low temperature actin (Figs. 2B to 20) do not show the characteristic hypersharp y boundary characteristic of preparations containing tropomyosin. This boundary is found in room temperature preparations (6, 7).

These results then suggest that the higher viscosity, with its higher concentration dependence, of G-actin prepared at room temperature is due to the presence of tropomyosin which sedi- ments in the same boundary as either the G or, under conditions of full polymerization, the F form of actin. It would seem that the viscometric properties of G-a&in depend on the tropomyosin- a&in ratio; the following experiments deal with conditions in which one would expect higher or lower tropomyosin-actin ratios than those obtained in room temperature extracts.

Preparative ultracentrifugation at 20,000 x g for 3 hours of a partially polymerized actin solution in 0.6 to 0.8 mhr MgClz results in the separation of a tropomyosin-rich supernatant solution and an essentially tropomyosin-free F-a&in (refcrrcd to below as hlg-purified actin) pellet (6, 7). In order to study the effect of decreasing the tropomyosin content of room tem- perature actin we examined the reduced viscosity of G-actin obtained from the pellet. It appeared low, viz. comparable with the viscosity of G-actin extracted at 0” (Fig. 1). When big- purifird actin was recombined with the 20,000 X g supernatant solution that had previously been dialyzed against ATI’, the viscosity wsus concentration plot was the same as that of the original room temperature actin.

TABLE I

h’fect of temperature on total and sedimentable protein content of actin. extracts

Acetone powders prepared according t,o the standard method (4) were cstracted with distilled water at. the temperature in- dicated. Sedimentable protein is that which 2 hours after addi- tion of 0.1 M KCI + 1 IBM M&l2 is removed by centrifugation at fJO,OOO X g for 3 hours.

._.. -_- Temperature of extraction

- . _---

.4cetone

c%. ~-

1 2 3 4 5 G

* F,xtr

-

1

-

O-2” U-24’

/ Oude ! Sedimentable protein extract

fmdar

20.5 22.0 19.7 21.0 23.3 26.0

w/g acc1om pnwdcr

I -

-

Crude extract

29.8 27.0 25.3 26.6 27.0 40.8*

Sedimentable protein

I -_

5i : 17.3 55 1 14.8 48 12.2 01 16.2 GO 16.2 5G* 22.V

,action at 28’.

lfl-

t6-

7

QI a3 &IO0 ML

a4 a

Fro. 1. Reduced viscosity of G-actin extracted at various temperatures, A, actitt extracted at 23-24”; 0, 28”; V, 34”; A, 0, V, actin extracted at O-2”. Each symbol shape indicates a dif- ferent lot of acetone powder. X, actin purified from room temp- erature actin after centrifugation in 0.8 rnM MgCl, (Mg-purified actin). For details see text.

After addition of 0.1 M KC1 to the undialyzed, tropomyosin- rich supernatant solution: 62’s of the protein left behind was still sedimentable at 90,600 X g and G-actin could again be ob- tained from the pellet. The reduced viscosity of such a G-a&in, although lower than that of the supernatant fluid itself freed of Mg ions, was still much higher than that of G-actin estracted at room temperature and showed a greater concentration depend- ence (Fig. 4).

;\ssuming that extraction of the acetone powder in the cold preferentially removes actin rather than tropomyosin, a second

2 This supernatant solution was essentially a mixture of Mar- tonosi’s 7 component and F-act.in. Since our purpose was t.he isolat.ion of tropomyosin-enriched actin, no special effort was made t,o remove the supernatant solmion without small admix- tures of P-uctin. Hence the viscous propert,ies of the supernatant fluid are not directly comparable with those of t.he more rigor- ously prepared y component (6,7).

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3414 Tropomyosin in &in Vol. 237, So. 11

extraction at room temperature should also lead to an enrich- ment in tropomyosin. Indeed, the effects of the temperature of extraction on the properties of actin arc accentuated if a room temperature extraction follows a cold extraction of the acetone powder. Although after polymerization the second crude ex- tract had a considerably lower double refraction of flow and viscosity than the first, when both were examined at the same protein concentration, the ratio of sedimentable protein to total protein in the two extracts was not very different (Table 111). However, only 8% of the protein in a G-a&in preparation iso- lated in the usual way from the second extract was sedimentable in 0.7 to 0.8 mM Mg, in contrast to approximately 40% in the case of standard room temperature actin. The former had a much higher reduced viscosity and showed a considerably greater concentration dependence than even G-actin obtained by direct estraction at room temperature (Fig. 5).

Both of the above experiments strongly suggest that the higher the tropomyosin to actin ratio in a G-a&in solution the higher the reduced viscosity in a salt-free medium and the greater its concentration dependence.

It is well known that salt-free solutions of tropomyosin are

FIG. 2‘4, ultracentrifugal sedimentat.ion of G-actin. Upper curve, actin extracted at 2”; protein concentration, 4.70 mg per ml. Lower curve, actin extracted at 24”. Protein concentration, 4.55 mg per ml; 2 mM Tris, pH 8.0; speed, 56,100 r.p.m.; tempera- ture: initial, 8.5”, final, 10.5”; bar angle, 45”. Picture taken 89 minutes after-reaching speed.

FIGS. 2B to 20, ultracentrifugal sedimentation of partially polymerized G-actin solutions. Upper curve, actin extracted at 2”. Leer curue, actin extracted at 34”. Protein concentration, 3.8 mg per ml; 2 mM Tris and 0.9 mM MgCl2; speed, 56,100 r.p.m.; temperature: initial, 4”, final 4.5”; bar angle, 45”. Pictures taken (R) 13, (C) 21, (D) 69 minutes after reaching speed.

Fro. 2B, ultracentrifugal sedimentation of partially polymerized G-actin without and with addition of tropomyosin. Upper curve, G-actin extracted at 2” and purified twice by polymerization in 0.1 XI KC1 and depolymerization by dialysis; protein concentra- tion, 4.38 mg per ml; 2 mM Tris, pH 8.0, and 0.8 mu MgC12. Lower curre, the same actin, but after the first purification cycle tropo- myosin was added so that it amounted to 1770 of the total prot.ein. The mixture was once more polymerized in 0.1 M KCI and de- polymerized by dialysis; protein concentration, 4.48 mg per ml; 2 rnhf Tris, pH 8.0, and 0.8 rnxf MgCIt; speed, 56,109 r.p.m.; tem- perature: initial 6.2”, final 9.0”; bar angle, 45”. Picture taken 133 minutes after reaching speed.

TABLE II

Effect of KI on reduced viscosity of G-actin

ACE+XE nowder lot

Temperature of Concentration of ‘)rsduesd

extraction

I orotein

1 _ 1 No salt added I

m-/ml A 2O 3.12 0.21

34 3.12 1.46

B 2 3.80 0.16 30 4.30 1.77

0.8 Y KI

0.05 0.10

0.11 0.12

2

t OJ 02 0.3 04 I

C,G/lOOML 5

FIG. 3. Reduced viscosity of F-a&n extracted at various temperatures, 0, V, 0, actin extracted at 2’; 0, V, W, actin extracted at room t.emperature. Each symbol shape indicates a different lot of acetone powder.

5 I

4-

Y $3'

2

I/

01 c,G%o ML

a3 cl4

FIG. 4. Effect of actin impoverishment on reduced viscosity. Actin extracted at 20” was polymerized in 0.8 xnM MgClt and sub- sequently centrifuged for 3 hours at 90,090 X g. One portion of the supernatant solution was dialyzed for 24 hours against 2 rnM Tris, pH 8.0, and 0.2 mM ATP. To another portion of the super- natant solution 0.1 M KC1 was added and after 2 hours it was cen- trifuged at 90,000 X 0 for 3 hours and G-actin was prepared from the pellets in the usual way. X, original G-actin; 0, supernatant solution after dialysis; and 0, G-actin prepared from supernatant solut.ion after dialysis.

rather viscous (12). Adding tropomyosin in a 1:6 to 1:5 ratio, the ratio found in “purified” G-actin preparations, to 1Ig-purified or cold-extracted G-actin increases the viscosity and produces a considerable concentration dependence (Fig. 6),3 If the mixture of the two proteins was polymerized in 0.1 M KC1 and 1 mM lMgC12, G-actin reisolated in the usual way showed a reduced viscosity comparable with that of G-actin extracted at room temperature after a second purification cycle (Fig. 6). In 0.8 mM Mg a sharp peak, essentially absent in the original G-actin extracted at low temperature, appeared in the analytical ultra- centrifugal pattern (Fig. 2E). This is similar to the reappear- ance of the sharp peak on adding tropomyosin to Mg-purified actin (6, 7).

If the specific viscosity of the G-actin-tropomyosin mixture is compared with the individual specific viscosities of tropomyosin

8 The addition of 25y0 of tropomyosin to G-actin produces practically no double refraction of flow at lower velocity gradients. This explains the fact that even with room temperature extraction only traces of double refraction of flow were occasionally found at the highest velocity gradients (1006 set-I).

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November 1962 W. Drabikowski and J. Gergely 3415

TABLE III

Effect of pteezttaction at .P on subsequent e&action at 2P of an acetone powder

Acetone powder was extracted with 26 volumes of water for 39 minutes at 2” (first extract), then the residue was again treated with 10 volumes of water at 26” for 30 minutes (second extract). Both extracts were polymerized in 0.1 M KC1 + 1 mM MgClt. After 2 hours they were centrifuged at 90,094l X g for 3 hours.

mg protein/g “/p protein m-/g

ac&M powder s* crude acetans cxtroct pow&r

1st extract 19.5 68 13.3 6.72 63’ 2nd extract 31.7 54 17.0 3.25 42”

* Protein concentration, 0.15 g per 109 ml. t DRF, double refraction of flow. Protein concentration, 0.2

mg per ml.

After pdymeritation

al 0.2 a3 0.4 C, GAOOML

Fxa. 5. Effect of preextraction of acetone powder at 2” on reduced viscosity of G-actin extracted at 24”, 0, purified G-actin extracted at 2”; 0, purified G-actin extracted at 24” after ex- traction at 2”; V, for comparison, G-actin extracted directly at room temperature.

x

a 0

x

/

8 x

*

x

-- I

0.1 d2 0.3 0.4 C,G/lOO ML

Fm. 6. Effect of addition of tropomyosin on reduced viscosity of G-actin, X, mixture of tropomyosin and Mg-purified G-actin (tropomyosin, 18.0% of total protein); W, G-actin prepared from a mixture of tropomyoain (177c of total protein) and cold-ex- tracted actin; V, original cold-extracted actin; V, for comparison, room temperature actin; both the original cold-extracted actin and the room temperature actin underwent a second cycle of purification. Absciesa, total protein concentration.

ACTI N CONC., G/l00 ML

FIG. 7. Specific viscosity of a mixture of tropomyoain and G- actin, V, mixture of tropomyosin and actin as described in the legend of Fig. 6; 0, Mg-purified G-actin; X , for comparison, the specific viscosity of tropomyosin in 2 mad Tris, pH 8, shown in concentrations at which it is present in the mixtures for which the actin content is indicated on the abscissa.

TABLE IV

Redwzed viscosity of Iropomyosin-actin mizturee affer polymerization

Actin and the mixture of tropomyosin and actin were poly- merized in 0.1 Y KC1 + 1 my MgClz (for details see legend to Fig. 8).

I m/c

Total protein concentration F-b.in F-din + tropmyosin

m/ml

0.315 10.2 0.240 7.5 0.178 5.9 0.185 6.0 0.100 5.9

10.8 8.2 6.3 5.4 5.0

and actin at the appropriate concentrations the specific viscosity of the mixture is essentially the sum of the contributions of the two components (Fig. 7). Comparison of the reduced viscosity of the above actin-tropomyosin mixture after polymerization with that of Mg-purified F-actin, at the same total protein con- centration, shows practically no difference (Table IV). This is in agreement with the observations on the reduced viscosity of F-a&in extracted at cold and room temperature (see Fig. 3). If, however, the spec$ic viscosity is plotted against actin con- centration it appears that at higher concentrations the viscosity of the F-actin-tropomyosin mixture greatly exceeds the sum of the contributions of the two aomponenta (Fig. 8). At lower concentrations this cWerence tends to disappear, This ob- servation may be attributable to a concentration dependent interaction between tropomyosin and actin in 0.1 M KCI.

According to L&i and Cairns (5) alkali-denatured actin is readily separated from tropomyosin at 20% saturation of am- monium sulfate. Table V shows that the percentage of protein remaining in the supernatant solution under these conditions is considerably decreased when actin is extracted at low tempera- ture. The tropomyosin content does not depend on which of the two methods (3, 4) discussed in this paper is used for the preparation of the acetone powder. As far as fractionation with

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3416 Tropomyosin in A&n Vol. 237, No. 11

3-

2-

ii! F

I-

FIQ. 8. Specific viscosity of a polymerized mixture of tropomyo- sin and actin. To tropomyosin, Mg-purified G-actin and a mix- ture of both in proportion as described in the legend of Fig. 6, 0.1 nr KCL + 1 rnr,r MgClr were added and after 2 hours the vis- cosity was measured. A, mixture of tropomyosin and actin (actin content indicated on the abscissa); 0, F-actin; 0, tropo- myosin in concentrations at which it is present in the mixtures.

TABLE V

Protein remaining in solution at 20% saturation with (NH,)rSO,

To samples of actin preincubated for 15 minutes in 0.1 M NarCOI at room temperature solid (NHd)nSOc was added to 20y0 satura- tion (0.82 u). After centrifugation at 28,000 X g for 30 minutes the protein remaining in the supernatant solution was precipi- tated by adding (NH,)tSOk, to 70% saturation (2.87 M). Protein determinations were made on the precipitates dissolved in water.

Acetone powder, 1st No.

1 2 3 3 4 5t 6$

Cold actin

%

2.1 5.2 2.8

10.3* 3.8 3.8 2.8

Room temperature actin

%

10.5 10.0 15.6

10.9

* Tropomyosin (8.2% of total protein) added to G-actin. t Actin from acetone powder prepared according to Ulbrecht

et al. (3). $ Mg-purified actin.

ammonium sulfate is concerned low temperature actin and Mg- purified actin are indistinguishable.

DISCUSSION

The electrophoretic heterogeneity of crude actin extracts and the presence of material unable to combine with myosin (14) clearly showed the presence of impurities. Tsao and Bailey (9) were the first to point out that one of these impurities was tropomyosin, and that the higher the pH at which the muscle residue was washed before drying with acetone, the more tropo- myosin there was in the extract. Although the ultracentrifugal purification of actin introduced by Mommaerts (10) removed grossly discernible impurities, recent evidence points to the presence of tropomyosin (5-7) even in purified actin preparations.

The data here presented show that if the extraction of the acetone powder is performed at 0” the tropomyosin content of

actin is decreased to approximately the same extent as by the purification by partial polymerization with Mg (6, 7). Extrac- tion in the cold is the simplest way of obtaining actin essentially free of tropomyosin.

It is interesting that cold extraction has in the past been in- troduced by several authors without their either realizing, or stressing, the significance of low temperature. Thus, as men- tioned in the introduction, Ulbrecht et al. (3) extracted at 0” but recommended other modi6cations as well. In fact, Grubhofer and Weber (2), utilizing this procedure, considered only the avoidance of alkaline pH essential, although this precaution has usually been taken. The procedure of Tsao and Bailey (9) involves the extraction of actin with 30% acetone from a butanol-treated muscle mince at 0”, although their emphasis is on the use of acetone in order to suppress the extraction of tropo- myosin; indeed this type of preparation lacks the tropomyosin- rich y-peak (6,7), as would be expected also in view of the pres- ent results on aqueous extraction at 0”. It appears that this method has not gamed general use because of low yield and the ease with which denatured actin is obtained. Finally, at the time of writing we became aware of the fact that a recent de- scription by Momma&s (15) of the method of preparing actin mentions extraction with an ATP solution in the cold, although no rationale is given for the choice of temperature.’

It seems that the striking difference between the behavior of the viscosity of room temperature-extracted G-actin and that of cold-extracted a&in6 can be accounted for in terms of the known high viscosity and the concentration dependence of the viscosity of tropomyosin.

In contrast, the reduced viscosity of F-actin is much less de- pendent on the concentration; this is also clear from a recalcula- tion of the data of Oosawa et al. (16) and the presence of 10 to 26% tropomyosin (see Table IV) does not substantially change the r,~r/c versus c plot.

It appears that as the temperature of extraction is increased from 0” to 34” there is a corresponding increase of the viscosity and its concentration dependence. Fractionation of alkali- denatured actin with ammonium sulfate (5) shows that the amount of tropomyosin increases with the temperature of ex- traction. The ultracentrifugal sedimentation pattern of cold- extracted actin in 0.7 mnr MgClr lacks the characteristic sharp boundary of tropomyosin-containing preparations, which has been found by Martonosi (6, 7) who carried out the extraction at room temperature. Fig. 2 also shows that when the y-peak is missing, the next one, /3 (6, 7) also disappears; both reappear with the addition of tropomyosin.

Fractionation with ammonium sulfate in 0.1 M NarCOs shows that extraction at room temperature of an acetone powder pre- viously extracted in the cold yields a solution containing more

4 Dr. Mommaerts has told us recently t.hat in the work described in the 1952 publication (lo), too, the extraction of actin was car- ried out at 0”; since this was not stated in the paper many workers who adopted the ultracentrifugal purification recommended by Mommaerts continued to employ the original procedure of ex- traction at room temperature introduced by Straub. This has been the procedure until now in this laboratory; according to a personal communication from Dr. F. Oosawa their extractions were carried out at approximately 18”.

6 Dr. F. Oosawa et al. (personal communication) have, in view of the results obtained in our laboratory, reexamined the electrical birefringence of G-actin extracted in the cold or purified by partial Mg-polymerization. They found that the birefringence was considerably decreased in the first caee, and completely absent in the second.

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tropomyosin than one directly obtained at room temperature. This second extract is also more viscous with a stronger concen- tration dependence. The properties of room temperature actin could also be accentuated by purifying actin from the tropo- myosin-rich supernatant solution obtained after centrifugation at 90,000 X g in 0.7 mM MgCl*. Conversely, tropomyosin-free G-actin obtained from the pellet of this centrifugal experiment (6,7) behaved like cold-extracted actin, i.e. its reduced viscosity was almost independent of the concentration (Fig. 1).

The intrinsic viscosity of both Mg-purified G-actin and that extracted at low temperature is less than 0.10. Viscosities of our room temperature preparations were not appreciably higher than those of the cold extracts. The above value is lower than that previously obtained in 0.6 M KI, 0.14 (13), and 0.2 (17). Under certain conditions Tsao (17) obtained even higher values (0.5) and considered them evidence for the existence of dimers.

It is rather puzzling that the reduced viscosity data on G-actin presented by Grubhofer and Weber (2) are similar to those we found for G-actin extracted at room temperature, viz. they show fairly strong concentration dependence despite the fact that the former authors used an extraction at 0”.

That actin preparations (room temperature extraction) now known to contain tropomyosin show only one peak in the ultra- centrifugal diagrams suggests, but does not prove, an interaction between G-a&in and tropomyosin. The latter would normally exist in the form of aggregates at low ionic strength. The vis- cosity data here presented do not furnish evidence for an intcr- action since the viscosity of G-actin-tropomyosin mixtures is essentially the sum of the contributions of the two components. The rather low viscosity of G-actin-tropomyosin mixtures found by Martonosi (6, 7) might have been due to the presence of small amounts of salt leading to the lowering of the viscosity of tropomyosin.

According to Laki and Cairns (5) repeated ultracentrifugal purification of actin does not lead to the decrease of the tropo- myosin content. We confirmed this observation and found moreover that, even when the ratio of tropomyosin to actin was increased after removal of the major part of actin (see Table III), the percentage of sedimentable protein at 90,000 x g was roughly the same as in the case of low temperature actin.

These observations suggest the presence of interactions, per- haps only in a dynamic sense, between F-actin and tropomyosin, as does the higher viscosity of solutions containing tropomyosin. A similar increase was also found by Martonosi (6, 7) with actin purified after partial polymerization to which various amounts of tropomyosin were added. As pointed out in connection with Fig. 8 these interactions appear to be concentration dependent.

It might be worthwhile to point out here that, owing to the rather high initial viscosity of tropomyosin at low ionic strength, the interaction between actin and tropomyosin after polymeriza- tion may be obscured by a drop in viscosity, on adding salts, resulting from the disaggregation of tropomyosin. This is particularly noticeable in tropomyosin to actin ratios 2 1. Thus, it is not always possible to recognize the actin-tropomyosin interaction from an increase in A 7 on polymerization, as hap- pened to be the case in Martonosi’s experiments (6,7).

The degree of increase in viscosity of F-actin in the presence of tropomyosin shows some variability depending on factors which are not yet clear. A study of the details of this inter- action may give valuable information regarding the hitherto elusive role of tropomyosin in the structure and function of striated muscle.

Work is now in progress in our laboratory directed at a de- tailed study of tropomyosin-free actin. Clearly many problems relating to actin will have to be reexamined: the existence of a critical concentration (16), the process of rapid depolymerization (1, 2), determinations of molecular weight, size, shape (17-19), the stoichiometry of phosphate liberation during polymerization, and finer details of the interaction with myosin, to name only a few, since results obtained to date may have been seriously affected by the presence of the hidden tropomyosin contamina- tion.

SUMMARY

The tropomyosin content of ultracentrifugally purified actin preparations is considerably reduced if the extraction of the acetone powder is performed at O-2”. The reduced viscosity of actin preparations purified according to the usual procedure shows strong concentration dependence. This is absent in actin isolated from cold extracts, or in actin separated from tropomyosin in 0.6 to 0.8 mM Mg by ultracentrifugation. The viscosity pattern of room temperature-extracted actin could be reproduced by adding tropomyosin to actin free from, or con- taining small amounts of, tropomyosin. Whereas the viscosity of G-actin appears to be due to the independent contributions of actin and tropomyosin, deviations from additivity found in F- actin suggest concentration-dependent interactions between the two proteins. The evaluation of the effect of the presence of tropomyosin on hitherto described properties of actin will re- quire further investigation.

Acknowledgments-The authors are grateful to Miss .4. Castro for skillful technical assistance, to Miss A. Polis for ultracen- trifugal analysis, and to Dr. A. Martonosi for much valuable discussion and for the preparation of tropomyosin.

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W. Drabikowski and J. GergelyActin

The Effect of the Temperature of Extraction on the Tropomyosin Content in

1962, 237:3412-3417.J. Biol. Chem. 

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CORRECTIONS

In the preliminary communication by Subir K. Bose, Howard Gest, and John G. Ormerod on page PC13, Vol. 236, No. 3, March lQG1, for the concentrations of ferricyanide given in figures and text, read molarity, rather than millimolarity.

In the paper by W. Drabikowski and J. Gergely on page3417, Vol. 237, No. 11, November 1962, Reference 10 should read: W. F. H. M. Mommaerts, J. Biol. Chem., 188, 559 (1951).

In the paper by J. Hurwitz, J. J. Furth, M. Anders, and A. Evans on page 3755, Vol. 237, No. 12, December 1962, in the legend of Table IV, the incorporation factors for the thymus DNA-primed reaction should read: a = 0.278, u = 0.291, g = 0.213, c = 0.218.

In the preliminary communication by Robert L. Heinrikson and E. Goldwasser on page PC486, Vol. 238, No. 1, January 1963, the first sentence of the “Summary” should read: The data we have presented in this communication demonstrate that the formation of +uridine monophosphate by Tetrahymena preparations involves the condensation of uracil with ribose 5-phosphate or some ribose derivative other than 5-phosphoribosylpyrophosphate.

1913