The sulfhydryl groups of actin

82

BIOCHIMICA ET BIOPHYSICA ACTA BBA 3787

THE SULFHYDRYL

GROUPS OF ACTIN

ARNOLD M. KATZ AND W. F. t{. M. MOMMAERTS

The Department of Medicine and the Los Angeles County Heart Association Cardiovascular Research Laboratory, The University of California, Los Angeles, Calif. (U.S.A.) (Received May 7th, 1962)

SUMMARY

I. Actin has been found to contain 6 SH groups per mole when titrated with p-mercuribenzoate. Concentrated urea solutions do not increase this number, but precautions against oxidation of the protein must be carried out to prevent loss of SH groups. 2. Titration with N-ethylmaleimide yields 2 SH groups per mole of G-actin and F-actin, denaturation in urea or sodium lauryl sulfate increases the number to 6. 3. A modification of the amperometric silver titration yielded 6 SH groups per mole of G-actin. Only 4 SH groups reacted in the case of F-actin when titrations were carried out b~fore this protein depolymerized. The number of SH groups rose to 6 when F-actin was depolymerized b y Ag+. 4. The following classification of SH groups for actin is proposed: (a) Two accessible groups which react with N-ethylmaleimide and are rapidly blocked by p-mercuribenzoate and silver. (b) Two intermediate reacting groups which can be blocked b y silver or p-mercuribenzoate without loss of polymerizability. (c) Two slowly reacting groups whose integrity appears necessary for the G - F transformation. 5. The possibility that there is a disulfide bridge in actin is excluded b y comparison of the present result with existing amino acid analyses. 6. The role of SH groups in the polymerization of actin is discussed in the light of evidence now available. While no final conclusion can be reached, it now appears that two SH groups per mole are required to hold actin in a configuration favorable for polymerization and for ATP binding.

INTRODUCTION

The role of sulfhydryl groups in the architecture of the actin molecule and in the polymerization reaction has gradually become accepted. It is of interest, however, to recall the development of this concept, the more so since we shall arrive at different quantitative conclusions, thus questioning the correctness of some of the earlier points of evidence. The first indication of the importance of sulfhydryl groups for actin was obtained Abbreviations: PMB: p-mercuribenzoate; NEM: N-ethylmaleimide.

Biochim. Biophys. Acta, 65 (I962) 82-92

SULFHYDRYL GROUPS OF ACTIN

83

b y one of us in 1947 when it was observed that PMB* prevented the polymerization of actin ~. Three interpretations of this result seemed possible at that time (compare a discussion by PERLZWEm2 on the case of urease): that sulfhydryl groups were essential for the reactivity of actin itself; that the polymerization was catalyzed by a sulfhydryl enzyme ; or that polymerization was dependent on a low molecular weight mereaptan such as glutathione. Since it was also found at that time that actin lost its polymerizability after dialysis (an observation explained later as due to the removal of ATP 8, 4), the latter interpretation especially seemed possible, and further work was postponed until purification of the protein would permit a distinction between these possible mechanisms. A further indication for the role of sulfhydryl groups came when FEUER et al. 5 reported that actin lost its ability to polymerize when treated with mild oxidizing agents (although these authors held that other oxidizable groups were involved). KUSCHINSKY AND TURBAG then reported that several sulfhydryl reagents inhibited the polymerization of actin and concluded that actin itself contains essential SH groups. This conclusion'has subsequently become accepted. After actin had been purified 7-9 and appeared to behave as a pure protein 8 ~0 without indications of a dependence upon an enzyme or a mercaptan cofactor for its polymerization, this problem was reinvestigated. Preliminary results indicated that there were 4 moles of SH in G-actin and 3 moles per monomer in F-actin, and it seemed at that time that polymerization might be due to the engagement of I SH group in a bond formation similar to that encountered in ttl'.~ s~mthesis of acyl coacetylase H. Subsequent reports in the literature'% ~2 1.~agreed on the presence of 4 5 SH groups per m,~)le of G-aetin, but suggested the disappearance of 2 rather than I in the polymerization reaction~2, ia. In the work of this laboratory, however, all of tlle analytical procedures employed showed anomalies and no final conclusion as to the number of SH groups was drawn. As a result of further efforts, \ve have now found that the sulfhydryl groups in G-actin number 6 per molecule**, of which two appear involved in polymerization. In the present paper we shall present our measurements in detail and discus~ the possible role of the sulfhydryl groups in the light of work that has been done with actin as well as with other proteins. METHOI)OLOGY

Preparation of actin Actin was prepared in pure form from rabbit back and leg muscle with the standard methods of this laboratoryT, 8. Several variations were made from time to time which were of no major importance for the present results. The method of BARANY19 is now preferred for the preparation of the tissue powder. Extraction was carried out at o °. In the preparation of actin from the powder, two polymerizations were carried out with complete depolymerization in between. Prolonged dialysis was aw)ided by homogenization of F-actin in salt-free solutions in a teflon homogenizer 2°. In later experiments, o.ooi M Tris nitrate was added to stabilize the pH of solutions of G-actin. Various methods were tried to avoid oxidation during the purification The PMB used in t h a t work was a generous gift b y Dr. L. HELLERMAN of J o h n s H o p k i n s University. *~ In this work we shall accept 60000 as tile molecular weight of actin; valid estimations of this q u a n t i t y have ranged from 560o0 (see ref. 16) t h r o u g h 58000 (see refs. 8, IO) and 6160o (see ref. t7) to 625oo (see ref. i8).

Biochim. Biophys. Acta, 05 (I962) 82-92

84

A . M . KATZ, W. F. H. M. MOMMAERTS

procedure; treatment of solutions with helium* was discarded as impractical in view of the repeated handling of the solutions and we returned to the procedure of STRAUB3 to add 2" lO -4 M ascorbate to all actin solutions. Polymerization was induced b y adding o.Io M KC1 or 0.05 M KNO 3 when chloride ions would interfere. The protein content of actin solutions was determined by the Kjeldahl procedure on the basis of a nitrogen content of 16.1%*,~1. After 5 different actin preparations had been standardized against bovine serum albumin (Armour lot No. U I 7 8 I I ), the biuret colorimetric method was used, it having been found that actin gave a color reading equal to that of 0.85 times its concentration of albumin (the latter not corrected to dry weight).

p-Mercuribenzoate titration The reagent was obtained commercially and purified b y two precipitations from NaOH. Solutions were prepared b y dissolving PMB at .pH 9-1o and adjusting the p H to 8.0-8.5 with HC1. PMB solutions were kept no longer than one week. The final concentration was determined from the absorbancy at 232 mtz using 1.69- lO 4 as the molar extinction coefficient ~2. Determinations of SH groups were carried out using the modification 23 of the method of BOYERz~. The increase in absorbancy at 255 m ~ of the PM]3 mercaptan complex was determined in a Hilger spectrophotometer. In the course of work with this method it was found that cyanide and NEM also react with PMB, causing a spectral shift similar to that given b y mercaptans.

N-ethylmaleimide titration The commercial reagent solution (Schwarz Bioresearch Inc., Lot NEM 5904) was prepared weekly in o.I M potassium phosphate or Tris nitrate buffer at p H 6.8 and was employed in the spectrophotometric method of ALEXANDER ~4, using the Hilger spectrophotometer.

Amperometric silver titration The method of BENESCH AND BENESCH ~5,~8, and later that of BENESCH, LARDY AND BENESCH 22 was used. However, anomalous behavior of the electrodes prevented precise measurement s of the slowly reacting SH groups using the published methods. A modification of this technique gave utilizable results as will be described in the experimental section. Silver nitrate standards were prepared from crystals of better than 99.98% purity.

Urea denaturation Commercial urea was deionized b y stirring for I h at 50 ° with a mixed-bed ionexchange resin in alcohol-water (4o:6o), then recrystallized from 85% alcohol. All urea solutions were prepared immediately before use ~.

Other precautions Since we have found that the sulfhydryl groups of G-actin are subject to oxidation, all titrations were carried out by adding actin, which had been kept on ice, to * The nitrogen value of 14.6 % or 15.2 % reported earlier b y one of us ~,s and the a b n o r m a l l y low value of the refractive index increment s are p r o b a b l y in error since no a b n o r m a l composition is suggested b y the amino acid analysis of actin.

Biochim. Biophys. Acta, 65 (1962) 82-92

SULFHYDRYL GROUPS OF ACTIN

85

solutions c o n t a i n i n g t h e S H reagent. T u b e s c o n t a i n i n g s t o c k sohltions of actin were k e p t sealed a n d on ice; stirring a n d foaming of the s t o c k solutions was avoided. U n d e r these conditions, in the presence of ascorbic acid, solutions of G-actin could be k e p t for several d a y s w i t h o u t significant loss of S H groups.

RESULTS

Reaction of actin with P M B The reaction was carried out in o.I M Tris chloride or o.o2 M Tris n i t r a t e buffer a t p H 8.2. The former solution led to the p o l y m e r i z a t i o n of actin in the absence of PMB, the l a t t e r d i d not. There was no difference in the result of the t i t r a t i o n of S H groups in these two buffers. P r e l i m i n a r y e x p e r i m e n t s revealed t h a t the reaction of PMB w i t h actin was more r a p i d at higher p H in the range from 7.o to 9.o, a b e h a v i o r p r e v i o u s l y n o t e d for t h e reaction of s a l y r g a n w i t h actin la. The r e a c t i o n of PMB w i t h G-actin s t a r t e d r a p i d l y a n d reached a n end value in a b o u t I h at p H 8.2 (Fig. i). A p p r o x i m a t e l y o n e - t h i r d of the S H groups h a d r e a c t e d in the first minute. A more d e t a i l e d r a t e s t u d y of this reaction, p a r t i c u l a r l y at lower p H where t h e r e a c t i o n is r e t a r d e d , m a y well c o n t r i b u t e further d a t a as to the r e a c t i v i t y of the i n d i v i d u a l S H groups. However, this was n o t u n d e r t a k e n as the t h e o r e t i c a l t r e a t m e n t of several overlapping, a n d p r o b a b l y m u t u a l l y influencing, kinetic phases was not obvious. •

•

0.3-

/ °'/

E oJ

0,2Z

/

/

/

o ~ e - - "

/--

/t

/"

o co <

z w

O,I -

o

~0 TIME

IN

I00

MINUTES

Fig. i. Rate curve for the reaction of PMB with G-actin in 0.02 M Tris nitrate at pH 8.2. After i min approximately one third of the SH groups had reacted and the reaction was complete after approximately I h. The t i t r a t i o n of freshly p r e p a r e d G-actin gave a value of 6 s u l f h y d r y l groups p e r mole, regardless of the m e t h o d of p r e p a r a t i o n (Table I). W h e n actin solutions prep a r e d w i t h o u t ascorbic acid were allowed to s t a n d at r o o m t e m p e r a t u r e in c o n t a c t w i t h air, t h e s u l f h y d r y l t i t e r diminished, often to less t h a n 5 groups/mole, b u t this was variable, a n d possibly d e p e n d e n t on trace metals. The e n d p o i n t of the reaction w i t h F - a c t i n was also 6 S H groups per mole of p r o t e i n m o n o m e r , a n d the a t t a i n m e n t of the

Biochim. Biophys..4cta, 65 (1962) 82--9z

86

A.M.

K A T Z , W . F. H . M. M O M M A E R T S

endpoint was associated with complete depolymerization as judged b y viscometric measurements and observation of flow birefringence. The reaction rate of PMB with F-actin could not be measured spectrophotometrically because the turbidity of the F-actin, which is considerable at 255 mtz, decreased coincident with the depolymerization 1°. I t was thus not possible to determine whether, early in the reaction, there was a difference between the number of SH groups in G- and F-actin. TABLE

I

THE NUMBER OF S H GROUPS PER MOLE AS DETERMINED BY" P M B TITRATIONS OF ACTIN PREPARED BY SEVERAL METHODS A e t i n prepared i n oxygen-free 2 • z o -~ M A T P F i n a l dialysis F i n a l dialysis F i ~ a l dialysis against against 0.2 % against oxygenz .z o -4 M free 2 .zo ~ M ascorbic a c i d a n d a l b u m i n a n d ATP 2.zo ~.MATP ~'r°-~MATP

Average

A e t i n prepared i n e . t o -~ M ascorbic acid a n d 2 . i o - * M A T P

G-actin

F-actin

G-actin i n 5 M urea

6.5 5.7 6.0 6.0

5.6 6.2

6.6 5.9

6.2 5 .8 5.4 6.3 5.5

7.0 6.0

5.8 5.5

6.0

5.9

6.2

5.8

6.5

5.6

Performance of the PMB titration in concentrated urea did not increase the number of titratable SH groups, but the endpoint was reached within I min.

Reaction of actin with N E M In accord with earlier findings 9,14 it was found that a total of 2 SH groups per mole was found to react with NEM (Table II). The result was the same for F-actin and for G-actin, and the reaction did not prevent polymerization of the monomer or cause breakdown of the polymer. When NEM-substituted actin was subsequently titrated with PMB, 4 additional SH groups were found to react. As shown in Fig. 2, the rapidly reacting groups were blocked by NEM since the PMB reaction of the NEM-substituted actin lacked the initial rapid reaction. Similarly, pretreatment with NEM diminished the initial rate of the silver reaction (see below). The number of groups reacting with NEM after denaturation with 5 M urea or with o.I M sodium lauryl sulfate was found to be 6 per mole of actin (Table II). Reaction of actin with complex silver ions Earlier experiments by one of us, using the amperometric titration with the silver ammonium ion ~8, were performed before the autoxidizability of actin was recognized. Endpoints ranging from 4 to 6 SH per mole of actin were determined after arbitrary times, e.g. 30 min, at which time tile drift had slowed. I t has now been demonstrated that silver currents will decrease in the polarograph in the absence of protein, a finding probably related to silver-plating of the platinum electrode and reaction of the silver with the saturated KC1 of the salt bridge. Replacement of the KC1 in the salt bridge Biochim.

Biophys.

Acta,

65 (1962) 8 2 - 9 2

SULFHYDRYL GROUPS OF ACTIN

~7

with KN03 slowed the drift but did not abolish it. Washing the platinum electrode with fuming nitric acid or cysteine in EDTA did not abolish the anomalies. It was finally concluded that reliable estimates of slowly reacting SH groups by this method are subject to significant error due to uncontrollable decrease of current in the presence of excess silver ion. TABLE II NEll

TITRATIONS

OF

ACTIN

PREPARED

IN

2" IO

4 ~//

ASCORBIC

ACID

AND 2' 10 .4 2~I A T P S u l f h y d r y l c o n t e n t s are e x p r e s s e d as mol e s pe r 6oooo g a c t i n. Number of S H groups per mole of actin

Conditions

2.O

P o l y m e r i z e d , in o.o666 M N a H 2 P O 4 (pH 7.o)

2.1

D e p o l y m e r i z e d , in o.o2 M Tris n i t r a t e (pH 7.o)

6.3

D e n a t u r e d , in 6.o M u r e a

6.4

D e n a t u r e d , in 5.2 M ur e a

6.[

D e n a t u r e d , in o.t M s o d i u m l a u r y l s u l f a t e

A determination of the number of sulfhydryl groups by the silver-Tris titration ~7 was found to be possible if the reaction was carried out with various amounts of silver in separate samples, and the silver excess, if any, measured amperometrically after completion of the reaction. This method avoided prolonged measurement on the polarograph which, for reasons discussed above, was undesirable. The terminal values for F- and G-actin were 6.2 and 6.5 SH groups per 6oooo g of protein (Fig. 3 and Table III). The values tended to be higher by this method than with either PMB or NEM. Similar high values with silver titration have been noted in the case of 0.15

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f , /

~

./

Z

i/x Z

/x

/

X'

(3

¢XI

o

--

,;

2'0 TIME

IN

5'0 MINUTES

Fig. 2. R a t e c u r v e s for t h e i n i t i a l r e a c t i o n of P M B w i t h G - a c t i n ( O - - O ) , a n d G - a c t i n p r e v i o u s l y r e a c t e d w i t h N E M ( × --- × ) in o.o2 3 I Tris n i t r a t e buffer a t p H 7.8. The i n i t i a l r a p i d r e a c t i o n of P M B w i t h a c t i n was a b o l i s h e d b y p r e t r e a t m e n t w i t h N E M .

Bioch~m Biophys..4cla, 65 (1962) 82 92

88

A . M . KATZ, W. F. H. M. MOMMAERTS

hemoglobin 29. In the initial stages, G-actin reacted more rapidly with silver-Tris ions than did F-actin. 30 min after the reaction was started, two more groups had reacted in G-actin than in the polymer (Fig. 4)-

/

+1,0.

/

zor'° 1 O 0 LU _J

F

o - - o - - • - - o/~-.-~

+0,5,

F-

./

G

/o /

O.

/o

>,

i

°j ° --o--o--o--o--o~

/

__o

o/

.~ i.b ml SILVER NITRATE Fig. 3. Silver t i t r a t i o n s of G-actin ( O - - O ) a n d F - a c t i n ( O - - O ) p r e p a r e d from t h e s a m e e x t r a c t i o n . A c t i n a n d v a r y i n g a m o u n t s of A g N O 3 were allowed to r e a c t in Tris n i t r a t e buffer o v e r n i g h t a n d read in t h e p o l a r o g r a p h t h e n e x t day. G - a c t i n w a s r e a c t e d in 0.o2 M Tris buffer, F - a c t i n was r e a c t e d in o. I M Tris buffer. T h e c o n c e n t r a t i o n of s i l v e r w h i c h g a v e t h e first increase in c u r r e n t was t a k e n as t h e e n d p o i n t , w h i c h in t h e s e e x p e r i m e n t s was 6.6 SH per mole.

MOLES OF SILVER PER MOLE OF A C T I N Fig. 4. Silver t i t r a t i o n s of G - a c t i n ( 0 - - 0 ) and F - a c t i n ( • - - • ) from t h e s a m e p r e p a r a t i o n a f t e r 0.5 h. I t c a n be seen from t h e s e c u r v e s t h a t 2 more SH groups per mole have reacted in the case of G - a c t i n t h a n in t h e F - a c t i n w h e n t h e latter has not depolymerized.

G o ~ °

b Fig. 5. R a t e c u r v e s for t h e r e a c t i o n of silver nitrate with G-actin (©--O), F-actin (×--×) a n d G - a c t i n w h i c h h a d p r e v i o u s l y been r e a c t e d with NEM (•--•). A t zero t i m e t h e a c t i n s o l u t i o n w a s a d d e d to a n a p p r o x i m a t e l y twofold excess of silv er ion per SH group, a n d t h e course of r e a c t i o n followed as a fall in current. These d a t a c a n n o t be quantified, b u t d e m o n s t r a t e t h a t N E M t r e a t m e n t c a u s e d t h e loss of r a p i d l y r e a c t i n g SH g r o u p s while p o l y m e r i z a t i o n c a u s e d t h e loss of slowly r e a c t i n g SH groups.

o.O-

o~

~ ~

9.o ~ X -------x op •/ xx.X-x ex'x ~ , , /

u. uJ

° "''~

F

ox/°

='~" mr_m

°"-----

° ~ ° ~ ° 1

=._.---|~:

x--.-----'e ~

''''-

,.m, o 2<> o o

TIME IN MINUTES Much time was expended in attempting to evaluate the number of instantly reacting groups of G- and F-actin, both with the silver-ammonium and silver-Tris methods. It was not possible to do this quantitatively for reasons mentioned previously. Such results as could be obtained when actin was added to solutions containing a slight excess of silver were consistent with the thesis that there are two more SH groups Biochim. Biophys. Acta, 65 (1962) 82-92

SULFHYDRYL

GROUPS

OF

ACTIN

89

available in G-actin than in F-actin, and that these are not the fast-reacting groups blocked by NEM (Fig. 5). It was found that the addition of sufficient silver nitrate to block 5 sulfhydryl groups would prevent polymerization of G-actin on subsequent addition of salt. However, 4 moles of silver per mole of actin left intact the ability to polymerize. Similar results were obtained with PMB (Table IV). Furthermore, addition of more than 4 moles of silver per mole of F-actin caused depolymerization. This depolymerization required approx. 24 h at pH 8.o with slight excess of silver. TABLE

ill

SILVER T I T R A T I O N S OF ACTIN P R E P A R E D IN 2 - I O 4 j~6r ASCORB[C ACID AND 2" IO - 4 M A T P SOLUTIONS

S u l f h y d r y l c o n t e n t s are e x p r e s s e d as m o l e s per 60 000 g of actin. Expt. I 2 3 4

Average

TABLE

G-actin

F actin

6.8 6.1 6.3 6.8

6.8 5.1

6.5

6.2

6.8

IV

T H E N U M B E R OF ' ~ N O N - E N S E N T I A L " S U L F H Y D R Y L GROUPS IN ACTIN I n t h e case of G - a c t i n this is t h e n u m b e r of S H groups w h i c h c a n be r e a c t e d w i t h o u t p r e v e n t i n g p o l y m e r i z a t i o n o n a d d i t i o n of salt. In t h e case of F - a c t i n this is t h e n u m b e r of S H g r o u p s w h i c h c a n lie b l o c k e d w i t h o u t c a u s i n g d e p o l y m e r i z a t i o n . The p r e s e n c e or a b s e n c e of p o l y m e r i z a t i o n w a s d e t e r m i n e d b y o b s e r v i n g flow birefringence of t h e a c t i n solutions.

Expt.

G-actin

F-actin

Sulfhydryl

I

4-5

4

Ag ÷

2 3 4

3 4 4

4 4 --

Ag + Ag + PMB

reagent

DISCUSSION

Actin has been found to contain 6.o (see ref. 3o) or 6.5 (see ref. 15) half-cysteine residues per mole, calculated on the basis of a molecular weight of 6o ooo. Since it had been found that an aetornyosin preparation from rabbit skeletal muscle contained both SH groups and disulfide bridges al, it seemed, when earlier investigations indicated a total of 4 SH groups per molecule, that one intra- or interehain disulfide bridge was contained in actin. Recent work has indicated that this is not the case ; POGLAZOVAND BAEVa2 reported 5 SH per mole (calculated on the basis of a molecular weight of 6o ooo) by amperometric titration with HgC12, and 6 SH per mole with silver titration. STROHMAN AND SAMORODIN33 have reported 8 SH per mole*. Our present finding is * T h i s w o r k w a s n o t specifically directed t o w a r d t h e d e t e r m i n a t i o n o f t h e n u m b e r of S H groups. b e l i e v e t h a t t h e findings w i t h P M B c a n b e e x p l a i n e d b y t h e use of a s p e c t r o p h o t o m e t r i c c o n v e r s i o n f a c t o r d e r i v e d f r o m t h e r e a c t i o n of P : V I B w i t h s e r u m a l b u m i n , w h i c h w e find to b e v a r i a b l e b u t o f t e n a b o u t 3 o % i n e x c e s s of t h a t for actin; a n d t h a t t h e results w i t h N E M m a y b e due to d e c o m p o s i t i o n of this r e a g e n t at a l k a l i n e p H . We

B i o c h i m . B i o p h y s . A c t a , 6 5 ( [ 9 6 2 ) ~$2--92

9°

A. M. KATZ, W. F. H. M. MOMMAERTS

that there are 6 SH groups per mole of actin and, in the light of the amino acid analysis, the possibility of a disulfide bridge in actin is therefore excluded. These results do not exclude the possibility that there m a y be 7 SH groups per mole of actin, particularly if the higher estimates fo~ molecular weight are valid. If the molecular weight of actin is 120000 as has been suggested, the correct number of SH groups per mole m a y be 12 or 13. Of the 6 SH groups two can be described as fast-reacting. This is the number that reacts with NEM, and which reacts within I min with PMB at p H 8.2. That these two pairs of SH groups are the same is indicated by the finding that previous reaction with NEM abolished the fast reaction with PMB. They also appear to be the two SH groups that react more rapidly with silver, although this could not be rigorously demonstrated. Two of the SH groups appear to be necessary for polymerization of actin, since blocking 4 groups neither prevented the formation of F-actin, as has been noted previously34, 85, nor caused depolymerization. The two groups which appear to participate in polymerization are not the rapidly reacting groups since they would have been the first to react with PMB and would have been blocked by NEM. It is suggested that they are the least reactive ones, since one can add 4 equivalents of either silver or PMB before polymerization is inhibited. The reported observation that HgCI~ does not cause depolymerization 3~ m a y be in error because this reagent did not react with all of the SH groups under the conditions of the experiments, and because depolymerization m a y have required more time than was allowed in the experiments. The precipitation reaction of actin below p H 6. 5 was observed with actin that had been fully reacted with PMB ; such actin will not polymerize, but instead precipitate, with o.oi M MgC1v These phenomena m a y be regarded as isoelectric precipitations, which are not dependent on SH groups or other specific reactivities. The fact that when the two least reactive sulfhydryl groups are blocked, polymerization of G-actin is prevented and depolymerization of F-actin occurs, as well as the finding that 2 sulfhydryl groups are less accessible in F-actin than in G-actin TM,13, 86, indicates that these groups participate in polymerization. The mechanism of this involvement is not obvious, and several possible roles for the sulfhydryl groups exist. These two groups could participate in the formation of a bond between the peptide chains of actin monomers in the polymer as was suggested b y B~Rf, NYTM who postulated a hydrogen bond between SH and N H 2 groups. However, the polymerization of actin appears to require both bound nucleotide and bound calcium as well as magnesium ions, making simple hydrogen bonding between peptide chains less likely. A second possibility is that sufhydryl groups act to bind certain cofactors to actin which are necessary for the a c t i n - a c t i n binding. If polymerization is linked, as has long been believed3,~, 37 with the breakdown of I mole of ATP, the SH group m a y be part of an ATP-splitting center in the actin molecule m. The recent finding that actin can polymerize without ATP breakdown2°, 39, 5o weakens this possibility, but does not rule out such a role for SH groups. A possible type of linkage of nucleotide to actin is suggested b y the work of MARTONOSI AND GOUVEA34 who found that ATP and I T P actin can polymerize while UTP, CTP and GTP actin cannot, in accordance with work of STROHMAN AND SAMORODIN33 and in partial accordance with work of our group (unpublished). These findings raise the possibility of a bond to actin through the purine ring of the nucleotide. Tyrosine and histidine have also been implicated Biochim. Biophys. Acta, 65 (1962) 82-92

SULFHYDRYL GROUPS OF ACTIN

91

in tile polymerization process 40. Magnesium ion has long been recognized to be necessary for polymerization 3 and more recently EDTA has been shown to prevent the binding of ATP to actin 14,aa, 34, 41. It has been shown 4~,4a that one mole of calcium is bound to actin, and a preliminary report suggests that removal of this ion causes a slow loss of polymerizability4a. The role of SH groups in the binding of nucleotide suggested by one of us la has recently been demonstrated 14,aa-a5 by effecting the release of bound ATP with sulfhydryl-blocking reagents. The time course of this reaction, however, raises a third possible role for the SH groups: that these groups hold the peptide chains of actin in a configuration favorable for ATP-binding and for polymerization. This is indicated in a preliminary report where ATP was slowly released from actin after all the sulfhydryl groups are reacted with organic mercurials 3a. Such a result can also be explained on the basis of two-point binding of ATP to actin as has been suggested by STROHMANAND SAMORODIN33. Indications that SH groups might act to maintain the stability of a configuration of the actin molecule are found in the work of FEUER et al. 5 where it was briefly noted that oxidation of actin increased the susceptibility of the protein to heat denaturation, and in the work of STRAUBAXD FEUERa where ascorbic acid slowed the rate of inactivation of G-actin by dialysis. The possible participation of ATP in this stabilization is suggested by the observation that this nucleotide protects aetin from inactivation, not only by organic mercurials, but by formaldehyde and urea la,44. The finding, first described by LAKI, BOWEN AND CLARK4, and more recently confirmed 14,zo,ag,4s, that nucleotide-free actin is not instantly inactivated, also suggests that the inactivation is the result of labilization of the actin molecule, rather than the loss of the nucleotide per se. The role of SH groups in maintaining a favorable configuration for protein activity is not unknown. MADSEN AND COR146 have demonstrated that the reversible inactivation of phosphorylase a by PMB is due to dissociation of this enzyme into subunits when the SH groups are blocked. This work supports the concept of a structural or configurational action of sulfllydryl groups as contrasted to the participation of these groups in an "active center". There is a final possibility, that the sulfhydryl groups play no direct role in the reactions of actin and that the observed effects are due solely to steric changes produced by the introduction of the blocking agents. The importance of such steric effects has been observed in the case of oxytocin where 0-methylation of the tyrosine residue caused almost complete inactivation of the rat uterine-contracting activity of the hormonO 7, while synthesis of an analog where tyrosine was replaced with phenylalanine yielded an active hormonO 8, 49. It is hoped that studies now in progress on the role of sulfhydryl groups in the interaction of actin with various cations will reveal more clearly the role of sulfhydryl groups in the polymerization reaction.

ACKNOWLEDGEMENTS

This investigation was supported by Research Grant H-3o6 7 of the National Heart Institute, National Institutes of Health, Bethesda, Md. (U.S.A.) and a grant of the Life Insurance Medical Research Fund. We are indebted to Mrs. S. SURANOWlTZtor her many forms of assistance and to Dr. M. CARSTEN for her helpful suggestions. One of us (A.M.K.) is an advanced Research Fellow of the American Heart Association. Biochim. Biophys. Acta, 65 (~962) 8:z-9z

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A . M . KATZ, W. F. H. M. MOMMAERTS

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Biochim. Biophys. Acta, 65 (1962) 82-92