PROTEIN-PROTEIN INTERACTIONS OF PROTEOLYTIC FRAGMENTS OF ACTIN
PETER JOHNSON a, PATRICIA J. WESTER b and ROBERT S. HIKIDA c
a Department of Chemistry and College of Osteopathic Medicine, b Department of Chemistry, and c Department of Zoology and Microbiology, Ohio University, Athens, OH, 45701 (U.S.A.}
Proteolytic fragments of actin, prepared by removal of up to sixty-eight residues from the N-terminal end of the molecule, can form filamentous struc- tures after denaturation in urea solution. The filaments have a diameter similar to F-actin filaments and interact with myosin and tropomyosin. A frag- ment comprising residues I to 207 of the actin sequence did not form fila- ments or interact with myosin after the urea treatment.
The actin molecule of the thin filament of striated muscle plays a central role in the contractile process, and it is now believed that the actin monomer contains a variety of protein binding sites; for other actin molecules, for myosin, for t ropomyosin, and for t roponin I [1,2,3]. Because of the im- portance of such sites in the contractile process, tbere is considerable interest in locating these sites in the actin amino acid sequence, so that when the tertiary structure of the molecule is known [4], a functional model of actin can be proposed. The two principal approaches which have been used to identify such sites have been studies of the effects of chemical modifications [1] and proteolytic fragmentations [ 5,6] on the interaction properties of actin. Reports of limited proteolysis of actin and of a procedure for the re- naturat ion of actin after denaturation in urea [ 7] have led us to investigate the properties of proteolytic fragments of rabbit skeletal muscle actin with re- spect to their abilities to participate in biologically significant protein-protein interactions.
Tryptic and chymotrypt ic fragments of actin (comprising residues 69 to
254
T A B L E I
AM IN O ACID C O M P O S I T I O N OF T H E A C T I N F R A G M E N T O B T A I N E D BY S. A U R E U S V8 P R O T E A S E D I G E S T I O N
A m i n o acid Residues pe r m o l Res idues in actin of fragment* sequence f rom
pos i t ions 1 to 207* *
Asx 23 21 Thr 16 16 Ser 7 8 Glx 22 20 Pro 13 I I GIy 20 18 Ala 18 17 Val 12 15 Cys n.d. § 1 Met 8 8 nc 11 11 Leu 15 17 Tyr 9 9 Phe 6 6 His §§ 7§§ Lys 9 8 TrP 2 2 Arg 10 11
* D e t e r m i n e d by analysis on a B e c k m a n 119CL a m i n o acid ana lyze r after acid hydrolys i s [ I 0 ] . **~From Elzinga et al. [ 11 ] .
Not determined. §§ Inc lud ing a single res idue of NT-methylhis t id inc .
374 and 68 to 374, respectively) were prepared as described previously [5], ex- cept that the digests were terminated by the addition of diisopropylfluorophos- phate (5 mM final concentra t ion)and further incubation for 1 h at 25°C. The fragments were then purified by chromatography at 4°C on a 2 × 100 cm Sephadex G-200 column equilibrated in the digestion buffer. Digestion of actin in 0.2 mM ATP, 0.2 mM dithiothreitol, 10 mM ammonium bicarbonate pH 7.8 [8] with S taphy lococcus aureus V8 protease (Miles) was performed at a molar substrate: enzyme ratio of 100. After 2 h at 23°C, the digestion was ter- minated by diisopropylfluorophosphate t reatment and fractionated on Sephadex G-200 as described previously, resulting in the isolation of a poly- peptide of approximately 25 000 daltons as determined by electrophoresis in the presence of sodium dodecyl sulfate [9]. From its molecular weight, its amino acid composition (Table I), the absence of a free N-terminal on dansyla- tion [ 12 ], and the known proteolytic specificity of S. aureus protease [ 8 ], this fragment appeared to comprise residues 1 to 207 of the actin sequence [11].
The purified fragments and native actin were denatured [ 7] wi thout alkylation by incubation in 4 M urea, 0.1 M ~-mercaptoethanol, 10 mM Tris, pH 9.0 for 2 h at 0°C, followed by dialysis at 40C against 0.2 mM dithio- threitol, 5 mM Tris, pH 8.5. The dialyzed samples were then adjusted to pH 5.5 with HC1 and the precipitates were redissolved and dialyzed against 0.2 mM dithiothreitol, 2 mM Tris, pH 8.5. After the addition of NaH2PO4 (20 mM final concentration), the samples were adjusted to pH 6.5 with HC1 and the solutions stored at 4°C for six days.
Examination of these samples in the electron microscope revealed the presence of filamentous structures of identical appearance in both the tryptic (Fig. 1A) and chymotrypt ic (not shown) fragment preparations and in the un-
255
Fig. I . F i l amen t s of the tryptic fragment of aet in. A. The fragment ( res idues 69 to 374) was p r o d u c e d by l imi ted p ro teo lys i s w i th t ryps in , a nd a f t e r pur i f i ca t ion , was d e n a t u r e d an d r e n a t u r e d as desc r ibed in the t ex t . B. Nat ive ( n o n - d e n a t u r e d ) F-act in f i l amen t s obse rved u n d e r ident ica l cond i t ions . Th e p repa ra - t ions were sp read o n formvar-coa ted~ carbon-s tab i l i zed c o p p e r grids an d nega t ive ly-s ta ined wi th 2% u r a n y l ace ta te . Magni f ica t ion × 100 800 .
digested actin preparation, whereas no filamentous structures were observed in preparations of the S. aureus protease digest of actin. Although the filaments obtained from the typtic and chymotryptic fragment preparations were shorter in length than those from undigested actin, the filaments had diam- eters within the range of 5 to 7 nm, values which are close to that for the native F-actin filament (Fig. 1B). In contrast to these results with renatured actin fragments, when fragment preparations were directly exposed to the pH 6.5 treatment without prior denaturation, or were dialyzed directly against 80 mM KC1, 5 mM MgC12, 0.2 mM dithiothreitol, 50 mM Tris, pH 8.0, no filaments were observed in the samples.
The abilities of the filament preparations to interact with rabbit skeletal muscle myosin were measured by activation of the Mg2+-activated ATPase of myosin [13]. As shown in Table II, it was found that the undigested re- natured actin and both the tryptic and chymotryptic filament preparations activated the Mg2+-activated ATPase of myosin to significant extents, indica-
256
T A B L E II
A C T I V A T I O N OF Mg2+-ACTIVATED M Y O S I N ATPase BY R E N A T U R E D A C T I N F I L A M E N T P R E P A R A T I O N S
Digest ion and r e n a t u r a t i o n p r o c e d u r e s are desc r ibed in the text . Prote in c o n c e n t r a t i o n s of the prepara- t ions were d e t e r m i n e d b y the Coomass ie Blue p r o c e d u r e [ 14] , using a s t anda rd curve c o n s t r u c t e d wi th a G-act in solut ion, the c o n c e n t r a t i o n of wh ich had b e e n accurate ly d e t e r m i n e d by a m i n o acid analysis. Resul ts are expressed as a pe rcen t age o f the ac t ivat ion e f f e c t in co ntro l samples of a c t o m y o s i n p r e p a r e d f r o m n o n - d e n a t u r e d nat ive F-act in (act ivi ty o f c o n t r o l a c t o m y o s i n was 35/~g P/5 rain p e r m g prote in ) . Assays were p e r f o r m e d in 2.5 m M ATP, 2.5 m M MgCl2, 25 m M Tris-HC1, p H 7.6 using 0.5 m g o f rabbit skeletal muscle m y o s i n and 0.2 m g of act in p r e p a r a t i o n pe r 2 ml [ 1 3 ] . Contro l s to m e a s u r e e n d o g e n o u s free p h o s p h a t e in bu f fe r s a nd prote in preparat ions were p e r f o r m e d for each e x p e r i m e n t .
Ac t in preparat ion Relat ive ac t iva t ion e f fec t o n Mg2+-activated m y o s i n ATPase (%)
Undiges ted , r e n a t u r e d 79 actin f i laments
R e n a t u r e d t r yp t i c 80 f i l amen t s
R e n a t u r e d c h y m o t r y p t i e 57 f i l aments
tive of specific interaction between the filament preparations and myosin. No activating effect on the Mg2+-activated ATPase of myosin was shown by the fragment produced by S. aureus protease digestion, indicating that interaction between this non-filamentous fragment and myosin did not occur. The abil- ities of the filament preparations to interact with rabbit skeletal muscle tropo- myosin were determined by ultracentrifugation and electrophoretic analysis [15], and it was found that the filament preparations possessed tropomyosin- binding abilities equivalent to those of control samples.
The significance of this s tudy is the demonstrat ion that renatured frag- ments of actin can interact to form filamentous structures which are morphol- ogically similar to F-actin filaments and which interact with myosin and tropo- myosin. The results suggest that the N-terminal sixty-eight residues of actin are not essential for actin-actin, actin-myosin and actin-tropomyosin interac- tion. This conclusion contrasts with results from earlier chemical modification studies on His-40 [16] and Tyr-53 [17] which implicated these residues in actin-actin interaction. An explanation for this discrepancy could be that the substi tuent groups introduced onto these residues interact with other regions of the actin molecule which are directly involved in actin-actin interaction. On the basis of this indirect effect of the presence of the N-terminal region on actin-actin interaction, normal actin-actin interaction in the absence of this N-terminal region would still be possible. The present studies also indicate that a region in the C-terminal half of the actin sequence may be involved in actin- actin and actin-myosin interactions. Further studies will be necessary to de- lineate more precisely the sequences in this region which are involved in these biologically important interactions.
This work was supported by a grant to P. Johnson from the Muscular Dyst rophy Association.
2 5 7
References
1 Mannherz, H.G. and Goody, R.S. (1976) Annu. Rev. Biochem. 45, 427--465 2 Wakabayashi, T., Huxley, H.E., Amos, L.A. and Klug, A. (1975) J. Mol. Biol. 93, 477--497 3 Taylor, E.W. (1972) Annu. Rev. Biochem. 41, 577--616 4 Dos Remedios, C.G. and Barden, J.A. (1977) Bioehem. Biophys. Res. Commun. 77, 1339--1346 5 Jacobson, G.R. and Rosenbu$ch, J.P. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2742--2746 6 Drabikowski, W., Lehrer, S.S., Nagy, B. and Gergely, J. (1977) Arch. Biochem. Biophys. 181,
359--361 7 Mihashi, K. (1972) Biochim. Biophys. Acta 267 ,409- -421 8 Drapeau, G.R. (1976) in Methods in Enzyrnology (Lorand, L., ed.), Vol. 45, pp. 469--475,
Academic Press, New York 9 Weber, K. and Osborn, M. (1969)J . Biol. Chem. 244, 4406--4412
10 Liu, T.-Y. and Chang, Y.H. (1971) J. Biol. Chem. 246, 2842--2848 11 Elzinga, M., Collins, J.H., Kuehl, W.M. and Adelstein, R.S. (1973) Proc. Natl. Acad. Sci. U.S. 70,
2687--2691 12 Gray, W.R. (1967) in Methods in Enzymology (Hits, C.H,W., ed.), Vol. 11, pp. 139--151, Academic
Press, New York 13 Schaub, M.C. and Perry, S.V. (1969) Biochem. J. 1 1 5 , 9 9 3 - - 1 0 0 4 14 Spector, T. (1978) Anal. Biochem. 86, 142--146 15 Johnson, P. and Blazyk, J.M. (1978) Biochem. Biophys. Res. Commun. 82, 1013--1018 16 Hegyi, G., Premecz, G., Sain, B. and Muhlrad, A. (1974) Eur. J. Bioehem. 44, 7--12 17 Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K.E. (1976) Eur. J. Biochem. 64,
![Actin cytoskeleton and cell motility - Indico [Home] ?· Actin cytoskeleton and cell motility Julie…](https://static.documents.pub/doc/80x56/5bcc339f09d3f232618dcbfd/actin-cytoskeleton-and-cell-motility-indico-home-actin-cytoskeleton-and.jpg)
