Fragmentation of actin by thrombin-like snake venom proteases

34

Biochimica et Biophysica Acta, 577 (1979) 34--43

© Elsevier/North-Holland Biomedical Press

BBA38130 FRAGMENTATION OF ACTIN BY THROMBIN-LIKE SNAKE VENOM PROTEASES

L. MUSZBEK and M. HAUCK a Department of Clinical Chemistry and a Department of Biochemistry, University School of Medicine, 4012 Debrecen, Pf. 40 (Hungary)

(Received August 25th, 1978) Key words: Actin fragmentation; Protease; Thrombin-like enzyme; Venom

Summary The effect of thrombin-like snake venom proteases (Ancrod of A g k i s t r o d o n r h o d o s t o m a and Batroxobins of B o t h r o p s m o o j e n i and B o t h r o p s marajoensis) on skeletal muscle actin was studied and compared to the thrombic cleavage of this protein. Only EDTA-pretreated G- and F-actin were split b y thrombin and Ancrod, while Batroxobins hydrolyzed native G-actin, too. The time course of digestion was followed b y sodium dodecyl sulfate polyacrylamide gel electrophoresis. A split p r o d u c t of 37 500 daltons appeared first which was cleaved further resulting in three lower molecular weight fragments. The sodium dodecyl sulfate gel pattern of thrombic fragmentation was well distinguishable from those caused b y Ancrod and Batroxobins. The first split products of Batroxobin digestion -- a smaller peptide and the 37 500 dalton fragment -- were isolated and by estimating their N:, and C-terminal end groups and amino acid compositions the peptide bond hydrolyzed first was located in the primary structure of actin. It was established that while thrombin split off two actinopeptides (at Arg(28)-Ala(29) and Arg(39)-His(40)) from the N-terminal end of the molecule only Arg(39)-His(40) was cleaved b y Batroxobins.

Introduction Several snake venoms contain thrombin-like proteolytic enzyme. Ancrod isolated from the venom of A g k i s t r o d o n r h o d o s t o m a and Batroxobins of the subspecies of B o t h r o p s a t r o x are of particular interest because of their therapeutical use as defibrinating agents. These enzymes are very specific proteases and share t h e fibrinogen clotting property of thrombin (see recent reviews by Nolan et al. [1], Stocker and Barlow [2] and Aronson [3]}. However, in con-

35 trast to thrombin only fibrinopeptide A but not B is removed by them from fibrinogen [4--7], they are without effect on other clotting factors [1--3] -with the exception of factor XIII in some cases [8--10] -- and lack the platelet activating capability of thrombin [11--13]. Besides fibrinogen and, in certain cases, factor XIII no protein substrate has been found so far for these protease$.

In a previous paper we have shown that thrombin can split skeletal muscle actin [14] and the peptide bonds hydrolyzed by thrombin have been located in the primary structure of the molecule [15]. The present study is an attempt to explore if actin can be a substrate for Ancrod and Batroxobins of Bothrops moojeni and Bothrops marajoensis, as well. Materials and Methods G-Actin from rabbit skeletal muscle was prepared according to Spudich and Watt [16] (final buffer solution: 2 mM Tris-HCl/0.5 mM ATP/0.5 mM mercaptoethanol/0.2 mM CaCI2) and, if indicated, was transformed to F actin by 0.1 M KC1. Purified Ancrod (Arwin, Knoll AG), Batroxobin moojeni and Batroxobin marajoensis (from Pentapharm) were the kind gifts of Dr. Z.S. Latallo, Institute Badan Jadrowych, Warsaw. For thrombic digestion U.S. standard thrombin (human, Lot H-l} was used. The following reference proteins were used for molecular weight determinations: horse heart cytochrome c, rabbit skeletal muscle aldolase, bovine liver catalase (commercial preparations of Serva), tubulin and modulator protein of cyclic nucleotide metabolism from bovine brain, rabbit skeletal muscle troponin I (prepared in our Laboratory). Phenylmethylsulfonyl fluoride-treated carboxypeptidase A and carboxypeptidase B were purchased from Worthington. Precoated polyamide sheets were the products of Chin Trading Co., Taiwan. For digestion, thrombin (14 NIH U/ml) or one of the following thrombinlike enzymes: Ancrod (28 Twyford U/ml),Batroxobin moojeni (15 U/ml), Batroxobin marajoensis (8 U/ml}, was added to 2.6 mg/ml G- or F-actin. The snake venom proteases were dialyzed prior to digestion against 5 mM Tris-HC1, pH 8.0. Some of the actin samples were preincubated with 1 mM EDTA (pH 8.0) for 1 h. The digestion was carried out at 37°C and at various intervals aliquots were removed and added to equal volumes of denaturing solution containing 6% sodium dodecyl sulfate (SDS) and mercaptoethanol, 8 M urea and 0.08 M phosphate buffer, pH 7.1. SDS polyacrylamide gel electrophoresis analysis of the samples and the determination of the molecular weight of the larger fragments were performed according to Weber and Osborn [17]. A lower molecular weight fragment split off by Batroxobins was detected and separated from the higher molecular weight fragment by gel filtration. In this case 6.5 ml EDTA pretreated G actin (3.5 mg/ml) was incubated with 37.5 U Batroxobin moojeni or 20 U Batroxobin mara]oensis at 37°C for 14 hours, then applied to a Sephadex G50 (superfine, Pharmacia} column (2.5 X 52 cm) equilibrated by 1.5 mM phosphate buffer, pH 8.0.4-ml fractions were collected and A was measured at 280, 260 and 220 nm. The peak fractions were examined by SDS polyacrylamide gel electrophoresis and submitted to further chemical analysis.

36

For amino acid analysis samples containing the isolated fragments were hydrolyzed in 6.0 N HC1 under vacuo at l l 0 ° C f o r 24 or 72 h and the amino acid compositions were determined on a Model LYZ 75 (Chinoin, Budapest) amino acid analyzer. The N-terminal amino acid residues were identified by the dansyl method with a slight modification [15] of the procedure described by Gray [18]. The carboxypeptidase method was used for the determination of the C-terminal end groups [19]. The amino acids released by carboxypeptidase A or B were identified by thin-layer ion exchange chromatography on Fixion 50 X 8 (Chinoin) chromatoplates [20]. The concentration of actin was measured by its ultraviolet adsorbance at 280 nm [21]. Results In the absence of EDTA neither G- nor F-actin were split by Ancrod (Fig. l b , d). In case of F actin even after EDTA pretreatment only a faint band representing actin split product could be observed on the SDS gel (Fig. lc). At the same time EDTA treated G-actin lost its resistance to Ancrod {Fig. le--j) and at advanced stages of digestion besides actin 4 extra bands appeared with molecular weights of 37 500 (Ka) 27 000 (Lal), 22 500 (La2) and 11 000 (Ma). The time course of digestion indicates that fragment Ka and a lower molecular weight peptide undetectable on the gels are the first split products, then Ka is cleaved further resulting in Lal and Ma. Finally, Lal is converted to La2 by the removal of a smaller peptide. In the case of Batroxobin moojeni the general pattern of splitting is similar

i~ ii i~ !~ iii ~

iiiiiiiii!ii

Fig. 1. F r a g m e n t a t i o n o f actin b y A n e r o d . A: actin; Ka, L a l , La2 and Ma: actin f r a g m e n t s . 2 . 6 m g / m l Gor F-actin w i t h or w i t h o u t 1 m M E D T A w a s i n c u b a t e d w i t h 2 8 T w y f o r d U / m l A n c r o d at 3 7 ° C . (a): c o n trol G-actin ( w i t h o u t e n z y m e ) , (b): F-actin, (c): E D T A p r e t r e a t e d F-actin, (d): G-actin, (e--j): E D T A p r e t r e a t e d G-actin i n c u b a t e d for 1 5 rain ( e ) , 3 0 m i n ( f ) , 1 h ( g ) , 2 h ( h ) , 4 h ( i ) , a n d 1 6 h ( a - - d , j ) . 2 3 p g p r o t e i n w a s applied o n t o t h e gels.

37

l i ~ ii !!iii~:/~: i

ii!ii i ~i

All qllllP

a

b C

d:

¢i

!f g

F i g . 2. F r a g m e n t a t i o n o f a c t i n b y B a t r o x o b i n moojeni. K m o , L m o l , L m o 2 , M m o : f r a g m e n t s p r o d u c e d b y Batroxobin moojeni digestion. 2.6 mg/ml G- or F-actin with or without 1 mM EDTA were incubated with 15 U/ml Batroxobin moojeni at 37°C Other experimental conditions were identical to those described i n t h e l e g e n d t o F i g . 1.

.............ii~i~iiI

M

F i g . 3. F r a g m e n t a t i o n o f a c t i n b y B a t r o x o b i n maraJoensis. K m a , L m a l , L m a 2 , M m a : s p l i t p r o d u c t s o f a c t i n f r a g m e n t e d b y B a t r o x o b i n marajoensia. 2 . 6 m g / m l G o r F a c t i n w i t h o r w i t h o u t 1 m M E D T A w a s i n c u b a t e d w i t h 8 U/m1 B a t r o x o b i n marajoensis a t 3 7 ° C . O t h e r e x p e r i m e n t a l conditions were identical t o t h o s e d e s c r i b e d i n t h e l e g e n d t o F i g . 1.

38 and the molecular weight of the fragments are identical to those of produced by Ancrod (Fig. 2). Comparing to Ancrod, however, there are also considerable differences. At the used concentration the first splitting of EDTA-treated G - a c t i n - resulting in fragment K m o - is faster and after 16 h the actin band completely disappeared. At the same time the splitting of Kmo was slower than that of Ka, consequently, Kmo accumulated. Lmol is represented only by a faint band hardly seen on the photo, apparently once formed it is immediately converted into Lmo 2. It is to be noted that Batroxobin moojeni split native G-actin, too. Comparing the effect of Batroxobin moojeni and Batroxobin marajoensis on actin only slight differences could be detected. Batroxobin marajoensis in half of the concentration was as effective as Batroxobin moojeni and in this case the accumulation of fragment Kma is even more definite (Fig. 3). As is revealed on the SDS gels of Fig. 4 the molecular weights of the split products of Ancrod and Batroxobin digestion differ from those of thrombic fragments. Fragment K t m o v e s somewhat slower while M t migrates definitely

Fig. 4. C o m p a r i s o n o f t h e S D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s p a t t e r n o f a c t i n fragmentation caused b y t h r o m b i n and thrombin-like snake v e n o m proteases. EDTA pretreated G-actin digested for 4 ( t l , m a ) o r 1 6 h ( t 2 , t o o , a) b y t h r o m b i n ( t l , t 2 ) , B a t r o x o b i n marajoensis ( m a ) , B a t r o x o b i n m o o j e n i ( m o ) o r A n c r o d (a). A: a c t i n , K t , Lt, Mt: thrombic split p r o d u c t s ; K a , L a l ' L a 2 , Ma: fragments produced b y Ancrod.

39

A

J

A

2,0 /,5

4 O.

LO

I' I

O,5

•

0

2o

AI •

m

iml m,m ,-iKma

:.

Jo 40

Q

50

¢ /f

b

g

FRACTION NUMBER Fig. 5. T h e d e t e c t i o n a n d i s o l a t i o n o f a c t i n f r a ~ ' n e n t s of B a t r o x o b i n m a r a j o e n s i s d i g e s t i o n b y gel f i l t r a t i o n o n a S e p h a d e x G 50 c o l u m n . T h e a b s o r b a n c e o f t h e f r a c t i o n s w a s m e a s u r e d a t 2 2 0 n m ( o . . . . . o), 2 6 0 n m (o . . . . . . o) a n d 2 8 0 n m (÷ , J ). T h e SDS p o l y a c r y l a m i d e gels r e p r e s e n t : a c t i n b e f o r e (a; 50 Dg p r o t e i n ) a n d a f t e r (b; 20 # g p r o t e i n ) f r a g m e n t a t i o n , f r a g m e n t e l u t e d in f r a c t i o n s 2 6 - - 3 0 of p e a k A (c--g). A l i q u o t s of f r a c t i o n s w e r e d e n a t u r e d as d e s c r i b e d in t h e M e t h o d s a n d 25 ILl o f t h e d e n a t u r e d s a m p l e s w e r e a p p l i e d o n t o t h e gels. N o p r o t e i n h a n d c o u l d b e d e t e c t e d in f r a c t i o n s ( 3 5 - - 4 0 ) o f p e a k B ( n o t s h o w n in t h e F i g u r e ) . L o w m o l e c u l a r w e i g h t n o n p r o t e i n s u b s t a n c e s e l u t e d in t h e t o t a l s o l v e n t v o l u m e a p p e a r e d in f r a c t i o n s 5 7 - - 6 4 ( o m i t t e d f r o m t h e figure).

faster than the corresponding fragments produced by snake venom proteases. The electrophoretic mobility of Lt comes to between Lal and La2. In order to reveal the formation of lower molecular weight split product(s) (actinopeptide(s)) by Batroxobins at the earlier stage of digestion and isolate them the digestion mixtures were gel filtrated on a Sephadex G 50 column. After a series of preliminary experiments the actin concentration, the actin: enzyme ratios and the incubation time were fixed at a point where actin was completely converted into fragment Kmo (or Kma) but no further splitting of this fragment occurred (see Fig. 5, gel b). In addition to the peak (A) representing fragment Kma (Fig. 5 gels c--g) a further peptide containing peak (B) could

TABLE I T H E E N D G R O U P S O F T H E S P L I T P R O D U C T S P R O D U C E D BY B A T R O X O B I N S N-terminal

C-terminal

F r a g m e n t K m a and K m o (peak A) A c t i n o p e p t i d e m a a n d m o ( p e a k B)

His _ a

Arg

Aetin

AcAsp b

Phe

a N o free a m i n o g r o u p c o u l d b e d e t e c t e d b y t h e d a n s y l m e t h o d . b Alving a n d L a k t [ 2 2 ] .

Phe

4O

T A B L E II AMINO ACID COMPOSITION OF THE SPLIT PRODUCTS PRODUCED BY BATROXOBIN MARAJOENSIS DIGESTION

Amino acid

a Fragment Kma

Actinopeptide a Expected b

Analyzed (hydrolysis for) 24 h

Lys His Arg Cys Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe

1 _ d 3 1 6 2 2 2 3 5 5 4 _ d 1 2 -- d 2

Trp c

-- d

Expected b

72 h

1.1 ~0.1 2.8 1.1 5.8 1.8 1.7 2.2 3.4 4.6 4.4 4.0 ~0.1 1.2 2.4 ~0.1 2.3

1.2 (0.1 3.0 0.6 5.8 1.5 1.3 2.3 3.2 4.7 4.6 4.1 ~0.1 1.3 2.7 (0.1 2.5 --

18 9 15 4 28 25 20 37 16 23 24 17 15 29 24 16 11 4

Analyzed ( h y d r o l y s i s for) 24 h

72 h

17.0 8.4 14.2 4.8 26,0 24,3 20,2 40.2 16.6 24.0 22.6 16.5 15.8 23.7 23.9 19.2 13.3

16.9 9.2 14.6 3.3 29.6 24.0 16.5 39.1 18.0 22.8 24.8 17.8 12.5 27.9 23,8 17.3 12.9 +

a T h e a n a l y z e d s a m p l e s r e p r e s e n t the r e s p e c t i v e e l u t i o n p e a k ( a c t i n o p e p t i d e : p e a k B, f r a g m e n t K m a : p e a k A). b C a l c u l a t e d f r o m the s e q u e n c e data of actin [ 2 3 - - 2 5 ] , c T h e a b s e n c e or p r e s e n c e o f t r y p t o p h a n residues w a s revealed b y f l u o r e s c e n c e spectral analysis. T h e s a m e results w e r e o b t a i n e d using the f r a g m e n t s o f Batroxobin moojeni d i g e s t i o n . d L a c k o f an a m i n o acid in the primary s t r u c t u r e .

be detected at 220 nm revealing the existence of one, and only one, actinopeptide. If the split products of thrombic digestion were analyzed in the same conditions t w o actinopeptides with molecular weights of 3 0 0 0 and 1500 could be separated [15]. Although only experiments with Batroxobin marajoensis are shown in Fig. 5 the very same results were obtained with Batroxobin moojeni. The end group analysis of fragment Kma and Kmo and actinopeptide of peak B (Table I) and the absence of tyrosine and tryptophan residues in the actinopeptide (Table II) strongly indicate that as a result of the hydrolysis of an ArgHis peptide bond a smaller molecular weight peptide is removed from the N-terminal end of the molecule. The estimated amino acid composition of the isolated split products agrees well with the values calculated on the basis of the presumption that A r g ( 3 9 ) - H i s ( 4 0 ) - the only Arg-His bond in the primary structure of actin [23--25] -- is cleaved by Batroxobins (Table II). Discussion It is well known that actin is highly resistant to proteolytic enzymes. The fact that in certain conditions thrombin and thrombin-like enzymes which are

41 considered very specific proteases can split actin is therefore of considerable interest. It has been shown earlier that if Ca 2÷ (around 10 -4 M) is included in the buffers during the preparation procedure (as it was in the present experiments) neither the filamentous polymeric F nor the globular monomeric G form of skeletal muscle and platelet actin are attractive for thrombin [14,26]. The same is true for Ancrod b u t n o t for Batroxobins. In the latter cases 'native' G actin also undergoes fragmentation. The finding that Arg(39)-His(40) is a c o m m o n place of splitting for Batroxobins and thrombin (see Fig. 6) and presumably for Ancrod, too, suggests that this peptide bond is exposed on the surface of G-actin and the lack of splitting in case of thrombin and Ancrod is probably due to the nonaccessibility of certain other structures necessary for the binding of the enzymes. As some of the tryptic fragments of actin retain their original functional features [27], the present findings raise the possibility that future isolation and structural-functional characterization of fragments of 'native' G-actin produced by Batroxobins may provide important information on the role of certain structural entities of the molecule. Actin contains 1 mol of tightly b o u n d divalent cation (Mg 2÷ or Ca 2+) which is easily exchangeable in G- b u t practically unexchangeable in F-actin. EDTA quickly removes the divalent cations from G- b u t n o t from F-actin and as a result nucleotide (ATP) binding becomes weaker and G-actin gradually becomes denatured [28--30]. As Ca :÷ or Mg 2÷ has no direct effect on the activity of thrombin-like enzymes this probably means that structures originally buried became exposed and these changes in the secondary structure of G-actin made the binding and the proteolytic action of Ancrod possible and considerably enhanced the effect of Batroxobins. The poor accessibility of divalent cation for EDTA in F-actin explains w h y digestibility was only slightly increased in this case. As mentioned above, Ancrod and Batroxobins are considered highly specific thrombin-like proteolytic enzymes with the c o m m o n feature of removing fibrinopeptide A from fibrinogen, i.e., in the case of human fibrinogen splitting a (A) chains at Arg(16)-Gly(17). These enzymes leave/3 (B) and 7 chains intact even after prolonged incubation, although further proteolysis of the a chains takes place at a slow rate [31--33]. Batroxobins, like thrombin, hydrolyze Arg(19)-Val(20), t o o [31]. As it became evident on an isolated N-terminal segment of the ~ (A) chains of h u m a n fibrinogen, Arg(23)-His(24) (but not Arg(19)--Val(20)) is one of the peptide bonds split b y Ancrod [31]. Reports concerning the effect of Ancrod [10,32--35] and Batroxobins [8--10] on factor XIII are quite contradictory and no peptide bond cleaved by these enzymes has been identified, in the primary structure of the molecule, so far. EDTA treated actin was found to be a new protein substrate for all three snake venom enzymes. The SDS polyacrylamide gel electrophoretic analysis of fragmentation revealed that at least three peptide bonds are cleaved b y these proteases and the peptide bonds split b y Ancrod and Batroxobins are probably identical though the rate of cleavage of the individual bonds show considerable differences. Compared to thrombic fragmentation the pattern of cleavage is different, i.e., the location of one of the susceptible peptide bonds, at the least, differs. The isolation and chemical characterization of the split products also

42 (A) lO AcAsp-Glu- Asp-Glu- Thr- Thr- Ala- Leu-Val- Cys2o ( A s p - A s n ) G l y - Ser- G l y - L e u - V a l - L y s - Ala- G l y 3o P h e - A l a - G l y - A s p - A s p - A l a - Pro- A r g - Ala- Val4o P h e - P r o - Ser- Ile- Val- G l y - A r g - P r o - A r g - H i s -

(B) F i b r i n o g e n o~(A) c h a i n : - G l y ~ - ~ A r g - G l y ~

2o : Val-

Skeletal muscle actin:

: His-

-Ile-

GI

rg

Fig. 6. (A) Primary structure o f a c t i n o p e p t i d e s released by t h r o m b i n and Batroxobins. (B) Comparison of the amino acid s e q u e n c e s preceeding two t h r o m b i n and Batroxobin susceptible p e p t i d e b o n d s in fibrinogen (~A) chain a n d aetin. The p e p t i d e b o n d s split by t h r o m b i n ( -) and by Batxoxobins ( . . . . . . ) are i n d i c a t e d . T h e s e q u e n c e data o f Elzinga et ai. [23] with the correction of Vandekerckhove and Weber [25] were used for rabbit skeletal muscle actin and those of Blomb~ick [36] for h u m a n fibrinogen ~ (A) chain fragment.

revealed that while thrombin cleaves Arg(28)-Ala(29) and Arg(39)-His(40), removing actinopeptide I and II from the N-terminal end of the molecule [15], the first place of Batroxobin splitting is Arg(39)-His(40) but Arg(28)-Ala(29) remains untouched (Fig. 6A). Thus, using the terminology applied to thrombic splitting the single peptide removed by Batroxobins could be termed as actinopeptide (I + II). Looking for similarities in the cleavage of fibrinogen and actin by thrombin and Batroxobins two interesting analogies should be pointed out: 1, both fibrinopeptide B and actinopeptide I are highly acidic peptides with blocked N-terminal residue and both are released by thrombin but neither of them by Batroxobins. (Batroxobins remove actinopeptide I only together with the nonacidic actinopeptide II.) 2, Comparing the primary structure of actinopeptide (I + II) to that of fibrinopeptide A no significant analogy is evident. However, if instead of Arg(16)-Gly(17) one starts with the comparison three residues further, i.e., at Arg(19)-Val(20) -- a peptide bond of a (A) chain also susceptible to thrombin - - a definite analogy in the sequence of five amino acid residues preceeding the hydrolyzed bonds is apparent (Fig. 6B). The data available at the present moment are not sufficient to draw a conclusion on the structural basis of similarities and differences in substrate specificity of thrombin and Batroxobins. Comparative studies on further protein substrates with known primary structure are needed to elucidate this problem. Acknowledgements We are indebted to Dr. Z.S. Latallo and Prof. Maria Kopec (Institute Badan Jadrowych, Warsaw, Poland) for stimulating discussion and helpful advice. The excellent technical assistance of Ms. Zsuzsa Peth6 is gratefully acknowledged.

43 This work was supported by the Scientific Research Council, Ministry of Health, Hungary (Grant No. 2-52-0306-01-1/M). References 1 Nolan, C., Hall, L..S. and Barlow, G.H. (1976) in Proteolytic E n z y m e s (L6r&nd, L., ed.), pp. 205--213, Methods in E n z y m o l o g y , Vol. 45, Academic Press, N.Y. 2 Stoeker, K. and Barlow, G.H. (1976) in Proteolyt i c E n z y m e s (L6rfind, L., ed.), pp. 214--223, Methods in E n z y m o l o g y , Vol. 45, Academic Press, N.Y. 3 Aronson, D.L. (1976) Thrombos. Haemostas. 36, 9--13 4 Holleman, W.H. and Coen, L.J. (1970) Biochim. Biophys. A c t a 200° 587--589 5 Ewart, M.R., Hatton, M.W.C., Basford, J.M. and Dodge, K.S. (1970) Biochem. J. 116,603---609 6 Blomb~'ck, B., Blomb&ck, M. and Nilsson, J.M. (1957) Thromb. Diath. Haemorrh. 1, 76--84 7 Stocker, K. and Straub, P.W. (1970) Thromb. Diath. Haemorrh. 24, 248--255 8 Kopec, M., Latallo, Z.S., Stahl, M. and Wegrzynowich, Z. (1969) Biochim. Biophys. Acta 181, 437--445 9 McDonagh, J. and McDonagh, R.P. (1975) Br. J. Hacmatol. 3 0 , 4 6 5 - - 4 7 9 10 Walter, M., N y m a n , D., KJmjnc, V. and Duckert, F. (1977) Thrombos. Haemostas. 38, 438--446 11 Brown, C.H., Bell, W.R., Shreiner, D.P. and Jackson, D.P. (1972) J. Lab. Clin. Med. 79, 758--769 12 Niewiarowski, S., Regoeczi, E., Stewart, G.J., Senyi, A.F. and Mustard, J.F, (1972) J. Clin. Invest. 51, 685--701 13 Tangen, O., Wik, O.K. and Berman, H.J. (1973) Microvasc. Res. 6, 342--347 14 Muszbek, L. and Laid, K. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2208--2211 15 Muszbek, L., Gladner, J.A. and Laid, K. (1975) Arch. Biochem. Biophys. 167, 99--103 16 Spudich, F.A. and Watt, S. (1971) J. Biol. Chem. 246, 4866--4871 17 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 18 Gray, R.W. (1972) in E n z y m e Structure, Part B (Hits, C.H.W. and Timasheff, S.N., eds.), pp. 121--138, Methods in E n z y m o l o g y , Vol. 25, Academic Press, N.Y. 19 Ambler, R.P. (1972) in E n z y m e Structure, Part B (Hits, C.H.W. and Timasheff, S.N., eds.), pp. 143--154, Methods in E n z y m o l o g y , Vol. 25, Academic Press, N.Y. 20 Sajg6, M. and D~v~nyi, T. (1972) Acta Biochim. Biophys. Acad. Sci. Hung. 7, 233--236 21 Houk, W.T. and Ue, K. (1974) Anal. Bioehem. 62, 66--74 22 AWing, R.E. and Laid, K. (1966) Biochemistry 5, 2 5 9 7 - - 2 6 0 1 23 Elzinga, M., Collins, J.H., Kuehl, W.M. and Adelstein, R.S. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2687--2691 24 Elzinga, M. and Lu, R.C. (1976) in Contractile Systems in Nonmuscle Tissues (Perry, J.V., M a r ~ e t h , A. and Adelstein, R.S., eds.), pp. 29--37, Elsevier, North Holland, Biomedical Press, A ms t e rda m 25 Vandekerckhove, J. and Weber, K. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1 1 0 6 - - 1 1 1 0 26 Muszbek, L., F~siis, L., (31veti, E. and SzabS, T. (1976) Biochim. Biophys. A c t a 4 2 1 , 1 7 1 - - 1 7 7 27 Jaco bso n, G.R. and Rosenbusch, J.P. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2742--2746 28 T o nomu ra, Y. and Yoshimura, J. (1961) J. Biochem. 50, 79--80 29 B~r~ny, M., F i n k e l m a n , F. and Therattil-Antony, T. (1962) Arch. Biochem. Biophys. 167, 230--237 30 West, J.J. (1971) Biochemistry 10, 3 5 4 7 - - 3 5 5 3 31 Hessel, B. and Blomb~ck, M. (1971) FEBS Lett. 18, 3 18--320 32 Mattock, P. and Esnouf, M.P. (1971) Nat. New Biol. 2 3 3 , 2 7 7 - - 2 7 9 33 Gaffney, P.J. and Brasher, M. (1974) Nature 251, 53--54 34 Barlow, G.H., HoUeman, W.H. and LSr~nd, L. (1970) Res. Commun. Chem. Path. Pharmacol. 1~ 39--53 35 Pizzo, S.V., Schwartz, M.L., Hill, R.L. and McKee, P.A. (1972) J. Clin. Invest. 51, 2 8 4 1 - - 2 8 5 0 36 Blomb&ck, B. (1969) Br. J. Haematol. 17, 145--157