localization of the actin-binding sites of acantharnoeba myosin ib

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263. No. 1, Issue of January 5, pp. 427-435,1988 Printed in U. S. A.

Localization of the Actin-binding Sites of Acantharnoeba Myosin IB and Effect of Limited Proteolysis on Its Actin-activated Mg2+-ATPase Activity*

(Received for publication, June 5, 1987)

Hanna Brzeska, Thomas J. Lynch, and Edward D. Korn From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NHderminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-ter- minal68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH,-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH2,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Md+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80- kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NHa terminus, and the second actin- binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.

Acantharnoeba myosins 1A and IB differ from conventional myosins in having only one, relatively small, heavy chain and one light chain: 140 and 17 kDa for I A and 125 and 27 kDa for IB, by SDS-PAGE’ (1-3). Both are incapable of self- association into bipolar filaments and both are monomeric under physiological conditions (4). In spite of these physical differences, myosins IA and IB are enzymatically and functionally similar to other myosins. They have the characteristic (NH:,EDTA)-ATPase, (K+,EDTA)-ATPase, Ca2+-ATPase, and M$+-ATPase activities, bind to F-actin, and their M$+-

* The costs of publication of this article were defrayed in part. by the payment of page charges. This article must therefore be hereby marked ‘‘aduertisemnt” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; EGTA, [ethylenebis(oxy- ethylenenitri1o)ltetraacetic acid.

ATPase activities increase about 30-40 times in the presence of F-actin (1-3) when a single serine in the heavy chain is phosphorylated (5, 6). And, although unable to form bipolar filaments, monomeric myosins IA and IB can cross-link actin filaments (7, 8) and support superprecipitation of F-actin dependent on the hydrolysis of ATP (7). Myosins IA and IB also support translocation of latex beads (4) and unidentified Acanthumeba organelles (9) along actin cables in vitro.

We would like to identify all of the active sites of myosins IA and IB and to understand their functional interactions. Similarly to muscle myosin subfragment 1 (10,11), the heavy chain of myosin IB is active in the absence of the light chain (12) and, therefore, we have restricted our initial studies to the domain structure of the heavy chain. Albanesi et al. (13) showed that trypsin cleaves the heavy chain of myosin IB into a 62-kDa peptide, which contains the ATP-binding and regulatory phosphorylation sites, and a 68-kDa peptide. By analogy to muscle myosins, in which the ATP-binding site is near the NHz terminus (14-16), it was assumed in that study that the 62-kDa peptide originated from the NH2 terminus of the myosin IB heavy chain. This assumption gains support from the recent finding that the ATP-binding site of the myosin IA heavy chain occurs within 27 kDa of its NH, terminus and its regulatory phosphorylation site lies between 38 and 58 kDa from the NH, terminus (17). The very great similarities between the physical properties, enzymatic activities, and functional capabilities of myosins IA and IB make it reasonable to assume that their sequences and functional domains are similar, even though they are coded for by different genes (18).

Little is known about the location of the actin-binding sites in myosins IA or IB. From the unusual kinetics of the actin dependence of the M$+-ATPase activities of myosins IA and IB and their abilities to cross-link F-actin filaments, it was proposed (19) that their single heavy chains contain two actin- binding sites: one which interacts with the catalytic site and, as for other myosins, would be responsible for activation of M$+-ATPase activity, and a second actin-binding site not directly associated with catalysis. Direct evidence for this hypothesis was obtained by chymotryptic cleavage of myosin IA heavy chain into an NHz-terminal, 112-kDa peptide with actin-activated M$+-ATPase activity and a COOH-terminal 27-kDa peptide (rich in glycine, proline, and alanine residues) that also bound to F-actin but had no catalytic activity (8).

In this paper, we report the results of studies on the influ- ence of F-actin on the tryptic digestion of myosin IB heavy chain and on the actin-activated M$+-ATPase activities of the proteolytic fragments. The new data for myosin IB are fully consistent with the earlier data for myosin IA and allow us to locate more specifically the position(s) of the actin- binding site(s) involved with catalysis and relate the domain

427

428 Actin-binding Sites of Acanthamoeba Myosin IB

structures of myosins IA and IB t o the domain structure of muscle myosin.

MATERIALS AND METHODS

Purification of Protein+"canthumeba myosin IB was purified as described previously (2,3,8). Myosin heavy chain kinase was purified according to Hammer et al. (6). Rabbit skeletal muscle actin was purified according to Spudich and Watt (20) followed by gel filtration on Sephadex G-200. The actin was polymerized by the addition of MgClz to 2 mM, pelleted at 100,000 X g, drained, and stored in ice for no more than 10 days. The actin used for the viscosity measurements was a generous gift from Lois Greene (National Heart, Lung, and Blood Institute) and was not gel-filtered.

ATPase Assays-ATPase activities were measured by the release of 32Pi from [Y-~~PIATP (1) after incubation for 5-15 min at 30 "C. (K+,EDTA)-ATPase was assayed in 500 mM KCI, 2 mM EDTA, 2mM ATP, and 15 mM imidazole chloride, pH 7.5. (NIX+,EDTA)-ATPase was measured in 400 mM NH,CI, 35 mM EDTA, 2 mM ATP, and 25 mM Tris.HC1, pH 7.5. MP-ATPase, in the presence or absence of F-actin, was assayed in 2 mM MgCI,, 2 mM ATP, 1 mM EGTA, and 15 mM imidazole chloride, pH 7.5.

Tryptic Digestion of Myosin-Myosin IB (0.5 p h f ) was digested at 20 "C with trypsin (4 pg/ml, except as noted) in buffer T (10 mM KCI, 5% glycerol, 5 mM MgCl,, and 20 mM Tris. HCI, pH 7.5) in the presence or absence of F-actin (5 PM). Digestions were stopped by adding 250 mM phenylmethylsulfonyl fluoride in ethanol to a final concentration of 5 mM or soybean trypsin inhibitor in a 1:l weight ratio to trypsin. The number and the molecular weights of the proteolytic fragments were determined by SDS-PAGE according to Laemmli (21) in gels containing 12% polyacrylamide. Gels were stained with Coomassie Blue (22).

Active-site Labeling of Myosin ZB and Its Fragments-Myosin IB (0.5-1 mg/ml) was phosphorylated by myosin I heavy chain kinase (10 pg/ml) at 30 "C for 10 min in 5 mM Tris.HC1, pH 7.5, containing 50 mM KCI, 5 mM MgC12, 0.2 mM ATP, 0.5 mM dithiothreitol, and 25% glycerol. For fluorographic purposes, [ Y - ~ ~ P ] A T P was added up to 0.05 mCi/ml. Proteolytic myosin fragments (0.25-0.5 pg/ml) were phosphorylated by incubating at 30 "C for 30 min with heavy chain kinase (10 pg/ml) in buffer T with ATP added to a final concentration of 0.2 mM. Myosin IB or its digestion mixture was labeled with [5,6-3H]UTP according to Maruta and Korn (23). Proteolytic frag- menta labeled with 32P or 3H were visualized by fluorography according to Laskey (24) after separation by SDS-PAGE.

Protein Concentration-Actin concentration was calculated from the optical density at 290 nm assuming an extinction coefficient of 0.617 mg" . ml . cm". Myosin I concentration was determined by the Bradford assay (25) using bovine serum albumin as standard. The (K+,EDTA)-ATPase activity of myosin IB with no impurities detect- able by SDS-PAGE was 9 pmol min" mg". No preparation was used which had a specific activity less than 6 amol min" mg-'. The concentrations of the 80- and 62-kDa tryptic peptides were determined by their (NH:,EDTA)-ATPase activities, assuming their specific activities were the same as the specific activity of native myosin IB.

Actin Binding Assay-Binding assays were performed on myosin IB, the 80-kDa peptide derived from its heavy chain in the presence of F-actin, and the 62-kDa peptide produced by redigestion of the 80- kDa peptide in the presence of 5 mM ATP. To ensure equivalent conditions, myosin IB and the 80-kDa peptide (after addition of soybean trypsin inhibitor) were incubated with 5 mM ATP under conditions identical to the digestion of the 80- and the 62-kDa peptides (see "Results"). Residual ATP in all samples was then removed by incubation at 25 "C for 20 min in the presence of hexokinase (0.01 mg/ml) and glucose (20 mM). The protein samples were then diluted 10 times to final concentrations in the binding assay of 0.025-0.05 p~ (according to their (NHf,EDTA)-ATPase activities). The binding assays were performed in buffer T with F-actin (15 p M ) and bovine serum albumin (1 mg/ml) with or without addition of 7 mM ATP. The solutions were centrifuged for 30 min at 30 psi (165,000 X g ) in a Beckman Airfuge at room temperature. The amounts of myosin IB and the peptides bound to F-actin were calculated from the differences between the (NH;,EDTA)-ATPase activities of the original solutions and the supernatants after centrifugation.

carried out by the rolling ball method described by MacLean-Fletcher Viscosity Measurements-Low-shear viscosity measurements were

and Pollard (26) with the microcapillary set at an angle of 30" from the horizontal. Myosin LB or digestion mixtures were mixed with F-

actin in buffer T, vortexed, and drawn into a 100-pl microcapillary. The filled microcapillary was incubated for 60 min at a horizontal position before measuring the viscosity.

Reagents-ATP, L-1-tosylamide-2-phenylethyl chloromethyl ke- tone-trypsin, soybean trypsin inhibitor, and phenylmethylsulfonyl fluoride were from Sigma. [ Y - ~ ~ P ] A T P was from New England Nu- clear and [5,6-3H]UTP from ICN. Hexokinase (400 units/mg) was from Sigma. Electrophoresis reagents and molecular weight standards were from Bio-Rad. All other materials were reagent grade.

RESULTS

Effects of F-actin on Tryptic Digestion of Phosphorylated Myosin ZB-The conditions of digestion in these experiments (see "Materials and Methods") were chosen to maximize the interaction of myosin IB with F-actin; glycerol and salt concentrations were kept at the minimum level necessary to avoid precipitation of myosin in the absence of F-actin and the myosin/actin ratio was 1/10, so that both of the actin-binding sites of myosin IB would be bound to F-actin (8,19). Although these conditions were quite different from those used by Albanesi et al. (13), a similar pattern of digestion was obtained in the absence of F-actin. Initially, two major peptides of 68 and 62 kDa were formed, reaching peak concentrations at about 10-20 min (Fig. lA, lanes 5 and 6) and disappearing by 60 min (Fig. lA, lane 7). (The 38-kDa peptide seen in Fig. lA was a degradation product of the 49-kDa peptide that contam- inated this particular preparation of myosin IB (Fig. lA, lane 1 ) and was not present in digests of myosin preparations that did not contain the 49-kDa contaminant.) The phosphorylation site was located on the 62-kDa peptide and on the 57- kDa and smaller peptides that were derived from it by longer digestion (Fig. 1B).'

In the presence of F-actin, tryptic digestion of myosin IB heavy chain had a very different time course (Fig. IC). Ini- tially, a 92-kDa peptide was formed (Fig. lC, lanes 2-6); it was then cleaved to an 80-kDa peptide (Fig. lC, lunes 4-7) which was resistant to further proteolysis. Both the 92- and 80-kDa peptides contained the phosphorylation site (Fig. 1D). As the myosin IB heavy chain is only 125 kDa, i t is apparent from these results that F-actin protects the 62/68 site against tryptic cleavage while probably promoting cleavage at the sites producing the 92- and 80-kDa peptides.

The small amounts of 68- and 62-kDa peptides formed early in the digestion of myosin IB in the presence of F-actin (Fig. IC, lanes 2-5) were probably derived from small amounts of denatured myosin IB that did not interact normally with F- actin; the amount increased with the age of the myosin and was inversely related to the (K+,EDTA)-ATPase activity of the myosin. Whether or not F-actin was present, the 27-kDa light chain of myosin IB was digested within 1 h to small peptides not detected by Coomassie Blue (Fig. 1, A and C). After incubation for 1 h, the digestion mixture consisted almost exclusively of the 80-kDa peptide, actin, and a 36-kDa peptide that was derived from actin, as shown by fluorograms of digestion mixtures in which the actin was labeled with [3H] N-ethylmaleimide (data not shown). Thus, that portion of the myosin IB heavy chain that was not recovered as the 80-kDa peptide was apparently digested, under these conditions, into small fragments (e10 kDa) that were not resolved on the electrophoretic gels.

* These results and the deduced amino acid sequence of the myosin IB heavy chain (27,28) are inconsistent with the previous preliminary localization of the site to which UTP is cross-linked (13). The earlier conclusion depended on the formation of a 60-kDa tryptic fragment that appeared to be labeled by UTP but not phosphorylated. We have not observed such a fragment in the present studies.

Actin-binding Sites of Acanthamoeba Myosin IB 429

Location of the ATP-binding Sites in the T v p t i c Peptides- Tryptic digestions, in the presence and absence of F-actin, of myosin IB photoaffinity-labeled at the ATP-binding site with [‘HIUTP (23) produced the same peptides as those shown in Fig. 1 for phosphorylated myosin IB (Coomassie Blue-stained gels, not shown). The 62-kDa peptide formed in the absence of F-actin contained the ATP-binding site (Fig. 2A, lanes 2- 5) ; this site was also present in the 57-kDa and smaller peptides that appeared with longer digestion times (Fig. 2A, lanes 4-7). The same 62-kDa peptide, and its 57-kDa degradation product, was also photoaffinity-labeled with [‘HIUTP when the labeling reaction was carried out after, rather than

1

200- 116 - 97 - 66-

45 -

31 -

21.5 -

14.4 - A

MYOSIN I6 ALONE

2 3 4 5 6 7 1 2 3 4 5 6 7

”- - - HC

-68 - 62 - 57

-49

-38

- LC

Coornassie =P

before, tryptic digestion (Fig. 2B). As shown in Fig. 2C, both the 92- and 80-kDa peptides formed by tryptic digestion of myosin IB in the presence of F-actin also contained the site that can be photoaffinity-labeled with [‘HIUTP. In this case, labeling was carried out only after digestion to avoid the possibility that UTP would have dissociated the actomyosin IB complex which would have prevented the formation of the 92- and 80-kDa peptides. The presence of both the phosphorylation and nucleotide-binding sites on the 92- and 80-kDa peptides, as well as the time sequence of their appearances, is consistent with the 92-kDa peptide being the precursor of the 80-kDa peptide.

MYOSIN I6 + ACTIN

1 2 3 4 5 6 7 1 2 3 4 5 6 7

m””” - ~ . -

” I

- HC

b ma- ,%

- ACTIN

-36

- LC

C Coornassie =P

FIG. 1. Effect of F-actin on tryptic digestion of myosin IB heavy chain. Myosin IB, phosphorylated with [32P]phosphate, was incubated with trypsin in the absence ( A , B ) and presence (C, D) of F-actin as described under “Materials and Methods.” Aliquots of 100 pl were removed for SDS-PAGE, and the digestions were stopped by addition of phenylmethylsulfonyl fluoride, at 0, 1, 2, 5, 10, 20, and 60 min (lanes 1-7). Coomassie Blue-stained gels ( A , C) and fluorograms ( B , D) of the same gels are shown. The positions of standard mass markers are at the left in kDa: muscle myosin heavy chain, &galactosidase, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme (top to bottom). The positions of the myosin IB heavy ( H C ) and light ( L C ) chains and actin and the major proteolytic fragments of interest (identified by mass in kDa) are shown at the right of the panels.

MYOSIN IB ALONE MYOSIN IB + ACTIN 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3 4 5 6

r- . ”

A

- 62 - 57

C

- ACTIN

FIG. 2. Identification of tryptic peptides of myosin IB containing the ATP-binding site. The ATP- binding site was photoaffinity-labeled with [3H]UTP before ( A ) or after (B , C) digestion with trypsin in the absence (A , B ) or presence (C) of F-actin. Digestion conditions were identical to those in Fig. 1. Aliquots of 50 pI were removed for SDS-PAGE at 0,2,5,10,20,40, and 60 min (A , lanes 1-7) or 0,5, 10,30, and 60 min ( B and C, lanes 1-5). Panel C, lane 6 is a sample of F-actin digested for 60 min in the absence of myosin. The Coomassie Blue-stained gels were identical to those in Fig. 1 so only the fluorograms are shown. The position of the myosin IB heavy chain (HC) and actin are indicated.

430 Actin-binding Sites of Acanthumoeba Myosin IB

Effect of ATP on Tryptic Digestion-In the absence of F- actin, the presence of ATP had no significant effect on the digestion pattern of the phosphorylated myosin IB heavy chain (Fig. 3, A and B ) . In contrast, the presence of ATP abolished the effect of F-actin on the tryptic digestion pattern; in the presence of ATP, the 92- and 80-kDa peptides were no longer formed and the 68- and 62-kDa peptides appeared instead, just as if actin had not been present (Fig. 3, C and D). Fluorograms of these gels (not shown) were consistent with the reversal of the effect of F-actin by ATP, which suggests that the change in the digestion pattern by F-actin in the absence of ATP was due to its specific interaction with the ATP-sensitive actin-binding site involved in catalysis.

Localization of the 80-kDa Peptide in the Heavy Chain- The origin of the 80-kDa peptide was established by first digesting phosphorylated myosin IB to the 80-kDa peptide in the presence of F-actin and absence of ATP and then contin- uing the digestion after the addition of ATP to dissociate the F-actin and allow cleavage to occur at the now unprotected 62/68 site. If the 80-kDa peptide represented the NH2-terminal end of the heavy chain, its further tryptic digestion when dissociated from F-actin should produce the same 62-kDa NH2-terminal peptide obtained by the digestion of myosin IB in the absence of actin; if the 80-kDa peptide represented the COOH-terminal end of the heavy chain it should subsequently be converted to the COOH-terminal 68-kDa peptide, and, if the 80-kDa peptide were derived from the middle of the 125- kDa heavy chain, it would subsequently be cleaved to two peptides smaller than 62- and 68-kDa, respectively.

Fig. 4, A and E, shows the time course of digestion of the myosin IB heavy chain to the 80-kDa peptide in the presence of F-actin and absence of ATP for 60 min. At that time, one portion of the incubation mixture was allowed to incubate for a further 60 min to demonstrate the stability of the 80-kDa peptide when bound to F-actin (Fig. 4, B and F ) . ATP was added to a second portion of the 60-min digest to dissociate the SO-kDa peptide from F-actin and the incubation continued for another 60 min. In this case (Fig. 4, C and G), the 80-kDa peptide was degraded to a peptide with identical mobility to the 62-kDa peptide formed directly from myosin IB in the absence of F-actin (Fig. 4, D and H). No 68-kDa peptide was formed from the 80-kDa peptide (Fig. 4C). These results

- ACTIN + ACTIN

- ATP + ATP - ATP + ATP

M 1 2 3 4 1 2 3 4 1 2 3 4 A 1 2 3 4

I)- " - . HC

80

62 68

. 57

m- ACTIN

A B

FIG. 3. Effect of ATP on tryptic digestion of myosin IB in the presence and absence of F-actin. Myosin IB, phosphorylated with [R2P]phosphate, was digested with 20 pg/ml t m s i n in the absence or presence of F-actin and in the absence or presence of 5 mM ATP as indicated. Aliquots of 100 pl were removed for SDS- PAGE at 1,2,5, and 10 min (lanes 1-4 ) . The lane marked M at the left of p a n e l A contained undigested myosin IB and the lane marked A, at the right of p a n e l C, contained F-actin incubated for 10 min with trypsin in the absence of myosin IB. The positions of the myosin IB heavy chain (HC), actin, and identified tryptic peptides are shown on the right. Fluorograms (not shown) identified all fragments that contained the phosphorylation site.

1 2 3 4 5 1 2 1 2 3 4 5 6 1 2 - ".- - H C

" "" -80 " - 68 " '62 - 57 ~ ""_

C.B. . . - m - - - ACTIN

D

1 2 3 4 5 1 2 1 2 3 4 5 6 1 2

E F G H FIG. 4. Tryptic digestion of the 80-kDa peptide to the 62-

kDa peptide. Myosin IB, phosphorylated with [32P]phosphate, was digested in the presence of F-actin and absence of ATP, as in Fig. 1, and aliquots of 100 pl were removed at 0, 2, 10, 30, and 60 min (A and E, lanes 1-5) to monitor the formation of the 80-kDa peptide by SDS-PAGE. At 60 min, the sample was divided into two portions. One was incubated without change for an additional 60 min and aliquots were removed at 90 and 120 min for SDS-PAGE ( B and F, lanes 1 and 2). The other had ATP added to 5 mM at 60 min and again at 90 min to dissociate the 80-kDa peptide from the F-actin and aliquots were removed for SDS-PAGE at 62, 65, 70, 80, 90, and 120 min ( C and D, lanes 1-6). In a control, myosin IB was digested with trypsin in the absence of F-actin and ATP and aliquots were removed at 2 and 10 min for SDS-PAGE (D and H, lanes 1 and 2). All aliquots were 100 pl. Coomassie Blue-stained gels (C.B.) and fluorograms ("P) of the same gels are shown.

indicate that the 80-kDa peptide formed by tryptic digestion in the presence of F-actin was derived from the NH2-terminal end of the heavy chain. This interpretation was confirmed by showing that the 62-kDa peptide derived from the 80-kDa peptide contained the phosphorylation site (Fig. 4G), just as did the 62-kDa peptide formed directly from the heavy chain (Fig. 4H). Of course, these results do not rule out the possibility that a small segment at the NH2 terminus of the native heavy chain was missing from the 80-kDa peptide. It should be noted that an 18-kDa peptide, which would have been formed had the 62-kDa peptide been produced from the 80- kDa peptide by a single cleavage, was not detected; the COOH terminus of the 80-kDa peptide was probably digested to fragments smaller than 10 kDa.

Catalytic Activities of the 80- and 62-kDa Peptides-To obtain preparations containing essentially only the 80- or 62- kDa peptide, phosphorylated myosin IB was incubated with trypsin, initially in the presence of F-actin and no ATP and then with added ATP, as just described. Aliquots were removed to monitor, by SDS-PAGE, the formation of the 80- kDa peptide in the absence of ATP and its conversion to the 62-kDa peptide after addition of ATP (Fig. 5, top). Other aliquots were assayed for Mg2"ATPase (in the presence of 30.8 PM F-actin) and (NH:,EDTA)-ATPase activities (Fig. 5, bottom).

After 45 and 60 min of digestion in the absence of ATP, the 80-kDa peptide accounted for about 78% of the original heavy chain (on a molar basis as quantified by scanning the Coomassie Blue-stained gels) and essentially no 62-kDa peptide was present; 94% of the (NH:,EDTA)-ATPase and 64%

Actin-binding Sites of Acanthamoeba Myosin IB 431

TIME, min 0 -60 * 120

"" - HC

-80 - 62

I +ATP I

c \

:i a 3 4 0 k

20

0 0 20 40 60 80 100 120

DIGESTION TIME, rnin

FIG. 5. Effect of tryptic digestion on the ATPase activities of myosin IB. Phosphorylated myosin IB was incubated with trypsin in the presence of F-actin and absence of ATP for 60 min, as described in the legend to Fig. 1. Aliquots were removed at the times indicated in the lower panel, soybean trypsin inhibitor was added to stop proteolysis, and the samples were assayed for (NH;,EDTA)-ATPase (A) and actin-activated Me-ATPase (0) activities (lowerpanel) and formation of the 80-kDa peptide by SDS-PAGE (upper panel). Only the upper portion of the Coomassie Blue-stained gel is shown. At 60 min, ATP was added to 5 mM and the incubation was continued for another 60 min with equal aliquots assayed at the indicated times for both ATPase activities and for conversion of the 80-kDa peptide to the 62-kDa peptide by SDS-PAGE. In the (NH:,EDTA)-ATPase assays, the concentration of undigested myosin IB was 5 nM. In the actin-activated Me-ATPase assays, the concentration of myosin IB before i ts digestion was 80 nM and the concentration of F-actin was 30.8 p ~ . Because all of the samples contained F-actin, the actin- activated MP-ATPase activities could not be corrected for the activities in the absence of actin.

of the actin-activated MgZ+-ATPase activities of the original myosin IB were still present at this stage. After digestion for another 45 and 60 min in the presence of added ATP, the 80- kDa peptide had almost completely disappeared and 32% (on a molar basis as quantified by scanning the Coomassie Blue- stained gels) of the original heavy chain was accounted for as the 62-kDa peptide. This material retained 39% of the original (NH:,EDTA)-ATPase activity (i.e. it had 100% of the specific activity of the undigested myosin IB) but only 2.3% of the original actin-activated Mg2"ATPase activity (i.e. it had only about 7% of the specific activity of undigested myosin IB). Therefore, it seems that the 80-kDa peptide retained appre- ciable actin-activated Mg2"ATPase activity (see next section), whereas the 62-kDa peptide had very little actin-activated Mg2"ATPase activity, even though both retained essentially all of the (NH:,EDTA)-ATPase activity of the undigested myosin IB. The difference between the actin-activated Mg2"ATPase activities of the 80- and 62-kDa peptides cannot be attributed to the loss of the light chain, as it had

already been lost during the formation of the 80-kDa peptide. Thus, we conclude that removal of the COOH-terminal 45- kDa of the myosin IB heavy chain has little if any effect on the actin-activated Mg2"ATPase activity at high concentrations of F-actin. In contrast, some or all of the COOH- terminal 18-kDa segment that is removed from the 80-kDa peptide to produce the 62-kDa peptide is important for actin- activated Mg2"ATPase activity, even though the catalytic site per se remains fully active.

One possible explanation of these results is that loss of the 18-kDa segment between 62 and 80 kDa from the NH2 terminus removes a site essential for binding of F-actin. How- ever, as shown in Table I, both the 62- and 80-kDa peptides bound to F-actin in the ATP-sensitive manner expected for the actin-binding site associated with actin-activated ATP hydrolysis.

Actin-activated Mg2+-ATPase Actiuity of 80-kDa Peptide- The Mg2"ATPase activity of myosin IB has an unusual triphasic response (activation, inhibition, re-activation) to an increase in F-actin concentration which can be explained by the presence of two actin-binding sites (8, 19, 29, 30). This triphasic response was retained by intact myosin IB after a control incubation for 60 min under digestion conditions in the absence of trypsin, although the magnitude of the initial activation phase was reduced (Fig. 6A). On the other hand, the Mg2'-ATPase activity of the 80-kDa peptide formed by tryptic digestion of phosphorylated myosin IB showed simple, hyperbolic dependence on the F-actin concentration (Fig. 6B) , consistent with the presence of only a single actin-binding site in this fragment. The actin-activated Mg2'-ATPase activity of the 80-kDa peptide was still regulated by phosphorylation (Fig. 6C). On Hanes plots, the data in Fig. 6, B and C, fell on the same straight line which gave values of 10 s" for V,, and 30 PM for K A T p - (the actin concentration required for half-maximal activity). This Vmax value is about 56% that of native myosin IB (3, 19) and the K A T p - value is the same as for the second activation phase of native myosin IB (3,191.

Effect of 80-kDa Peptide on Viscosity of F-actin-We previously showed by measurements of low-shear viscosity (7) and electron microscopy (7, 8) that one of the consequences of the presence of two actin-binding sites in the myosin IB heavy chain is that native myosin IB can cross-link actin filaments. For example, Fig. 7 shows the increase in low-shear viscosity of 20 PM F-actin with increasing concentrations of myosin IB. Gels formed at myosin concentrations greater

TABLE I Binding of myosin IB and the 80- and 62-kDa peptides to F-actin in

the presence and absence of ATP The 80- and 62-kDa peptides were obtained by tryptic digestion of

myosin IB heavy chain as described in the legend to Fig. 5. Myosin IB or the tryptic peptides (0.025-0.05 p ~ ) were mixed with F-actin (15 pM) in the absence or presence of ATP (7 p ~ ) ; the F-actin was sedimented and the amount unbound was quantified by the (NH:,EDTA)-ATPase activity remaining in the supernatant. For details see "Materials and Methods." The binding of myosin IB in the presence of ATP was weaker than previously published (3, 19), presumably because of differences in conditions under which the assays were performed. Myosin IB bound more strongly than either the 80- or 62-kDa peptide, presumably because only the undigested myosin IB contained the second, ATP-insensitive F-actin-binding site.

Protein Bound

-ATP +ATP %

Myosin IB 100 54 80 kDa 98 13 62 kDa 98 18

432 Actin-binding Sites of Acanthumoeba Myosin IB

I I I I I I A 16

I 1 I I I I J 0 5 10 15 20 25 30

[ACTIN], )IM

8 I I I I I I ” Phosphorylated Before Digestion B -

fi L-

2 Unphosphorylated - I I I r ; A

0 0 5 10 15 20 25 30

[ACTIN], )IM

FIG. 6. Mgg+-ATPase activities of myosin IB and the 80- kDa peptide as functions of F-actin concentration. A, activities of native phosphorylated myosin IB (0) and phosphorylated myosin IB incubated for 60 min in the presence of F-actin under digestion conditions but without trypsin (0). B, ATPase activity of the 80-kDa peptide obtained by tryptic digestion of phosphorylated myosin IB in the presence of F-actin. C, ATPase activities of the 80-kDa peptide obtained by tryptic digestion of unphosphorylated myosin IB in the presence of F-actin with (0) and without (0) phosphorylation of the 80-kDa peptide before assaying its ATPase activity. Myosin IB and the 80-kDa peptide were present a t 40 nM. Because F-actin was present during the tryptic digestion, its lowest concentration in the Mg*-ATPase assay was 0.4 PM. The 80-kDa peptide preparation was shown to be free of undigested myosin IB by SDS-PAGE.

than 0.6 MM. In contrast, the 80-kDa peptide had no significant effect on the low-shear viscosity at the highest concentrations that could be used, indicating that a second F-actin- binding site had been removed with the COOH-terminal 45-

kDa segment. These results are analogous to those obtained previously with myosin IA in which the second F-actin- binding site is in the COOH-terminal27 kDa (8).

DISCUSSION

In the absence of F-actin, tryptic digestion of the myosin IB heavy chain produced an NH2-terminal 62-kDa peptide that contains an ATP-binding site, the regulatory phosphorylation site and an actin-binding site, and a COOH-terminal 68-kDa peptide. In the presence of F-actin and absence of ATP, this 62/68 site is protected from tryptic cleavage and an NH2-terminal 80-kDa peptide is produced from an unstable 92-kDa precursor. The deduced origins of these peptides from the myosin IB heavy chain are illustrated in Fig. 8. Protection of the 62/68 cleavage site by F-actin suggests that an actin-

GEL

600

\ : s c V g 400

v, >

200 I

0 0 0.2 0.4 0.6 0.8 1.0 1.;

[MYOSIN I61 or 180 kDa1, pM

,

FIG. 7. Increase in viscosity of F-actin as a function of myosin IB or 80-kDa peptide concentration. The increase in low-shear viscosity of 20 PM F-actin is shown in the presence of increasing quantities of native myosin IB or the 80-kDa peptide formed by tryptic digestion of myosin IB in the presence of F-actin as described previously. The absence of undigested myosin IB in the 80-kDa preparation was established by SDS-PAGE. Other specifics are described under “Materials and Methods.”

MYOSIN IB

ATP PO. ACTIN 11 u

ACTIN I

t-“ S3 kDa ____1

I-23 kOe-Ylkh+2€ k D a i MUSCLE MYOSIN 1

ATP ACTIN FILAMENT

FIG. 8. Schematic comparison of the structure of the heavy chains of myosin IB and skeletal muscle myosin. Arrows indicate the positions of trypsin-sensitive sites. The heavy arrows are the tryptic sites protected by F-actin which were used to align the two polypeptides. The general positions of some of the ATP- and actin- binding sites, the phosphorylation site of myosin IB, and the filament- forming portion of muscle myosin are indicated. For a more detailed description see the text. S-I, subfragment 1; S-2, subfragment 2; LMM, light meromyosin. Heavy meromyosin (not depicted) com- prises S-1 plus S-2.

Actin-binding Sites of Acanthumoeba Myosin I B 433

binding site (ACTIN I in Fig. 8) is located in this region of the heavy chain.

The 80-kDa peptide has high actin-activated Me-ATPase activity, indicating that this peptide retains all regions of the actin-binding site that are essential for activation of the ATPase activity. Although the 62-kDa peptide has a functional catalytic site and binds to F-actin, it has little actin- activated Mg2"ATPase activity. The loss of activity that occurs when the 80-kDa peptide is digested to the 62-kDa peptide suggests that the COOH-terminal 18-kDa segment of the 80-kDa peptide is important for actin-activated M$+- ATPase activity, Possibly, this 18-kDa region contributed to the actin-binding site.

This interpretation of the data depends on the assumption that the ATPase activities of the digestion mixtures, the components of which were not purified, were due solely to the 80- and 62-kDa peptides. It is possible that these two peptides were associated with smaller fragments, derived from other regions of the heavy chain, that influenced their enzymatic activities, but we think this is unlikely. In these experiments, the COOH-terminal domain was extensively digested to fragments no larger than -10 kDa. When myosin IB was cleaved by trypsin in the absence of F-actin to an NHz-terminal 62- kDa peptide and a COOH-terminal 68-kDa peptide (Fig. 1, Ref. 13) actin-activated Me-ATPase activity was also lost (13). If the 68-kDa COOH-terminal peptide is unable to activate the 62-kDa peptide, it seems improbable that fragments of <10 kDa were responsible for the activity of the 80- kDa peptide.

The existence and location of a second actin-binding site, one not directly involved in the activation of the M e - ATPase activity but which was predicted by kinetic analyses (19), can be deduced from the properties of the 80-kDa peptide. Intact myosin IB cross-links actin filaments (7), whereas the 80-kDa peptide did not. Also, the actin-activated M$+- ATPase activity of native myosin IB is positively cooperative at high ratios of myosin to actin (19), whereas the M$+- ATPase activity of the 80-kDa peptide showed simple hyperbolic dependence on the concentration of F-actin. These two observations suggest that the COOH-terminal45-kDa region of the myosin IB heavy chain includes a second actin-binding site (Actin II in Fig. 8) which is necessary for cross-linking actin filaments and, thereby, inducing cooperativity. This interpretation of the data is consistent with the direct dem- onstration of a second actin-binding site in the COOH-terminal 27-kDa segment of the myosin IA heavy chain (8). However, it has not yet been possible to demonstrate directly the presence of a second actin-binding site in myosin IB because the entire 45-kDa COOH-terminal region is digested to small fragments under all conditions investigated.

The homology between the domain structure of myosin IA and IB also extends to the localization of the actin-binding site responsible for activation of their M$+-ATPase activities (ACTIN I in Fig. 8). Similar to the behavior of myosin IB, a tryptic cleavage site 64 kDa from the NH, terminus of the myosin IA heavy chain is also protected by F-actin leading to the formation of an NHn-terminal 80-kDa tryptic fragment.3 Previous studies have localized the ATP-binding site of the myosin IA heavy chain to within 27 kDa of the NH, terminus (13,17). From the deduced amino acid sequence of myosin IB (Ref. 27, see below), this site is probably 12 kDa from the NH2 terminus. It also seems likely that the regulatory phosphorylation site in the NHz-terminal 62 kDa of myosin IB will be at a similar position as the phosphorylation site of

H. Brzeska, T. J. Lynch, and E. D. Korn, unpublished observations.

myosin IA that has been shown to lie between 38 and 58 kDa from the NH2 terminus (13, 17). The probable locations of these functional sites are shown in Fig. 8.

The location of some functionally important regions of the NHz-terminal 80 kDa of the myosin IB heavy chain appears to be very similar to that of the more extensively studied NH2-terminal 93 kDa of skeletal muscle myosin, subfragment 1 (Fig. 8). Subfragment 1 is cleaved by trypsin at sites 23 and 73 kDa from the NH2 terminus (31-33). Although myosin IB heavy chain is not cleaved readily a t a position corresponding to the 23-kDa site of subfragment 1, the 62-kDa site in myosin IB (and a 64-kDa site in myosin IA3) are similar to the 73- kDa tryptic site of subfragment 1: both are protected against tryptic cleavage by F-actin (this paper and Refs. 34-36). F- actin also protects smooth muscle myosin against proteolytic cleavage at a similar position about 68 kDa from the NH, terminus (37). F-actin binds to and can be covalently cross- linked to both the 50- and 20-kDa segments of subfragment 1 (38-47). These data suggest that the actin-binding sites responsible for the activation of the Mg+-ATPase activities of Acanthamoeba myosins I, skeletal muscle subfragment 1, and smooth muscle myosin are all located in the vicinity, and possibly on both sides, of this common tryptic cleavage site. With this tryptic site as the point of reference (Fig. 8), the COOH-terminal 18-kDa segment of the 80-kDa peptide of myosin IB corresponds to the reactive thiol region of subfragment 1 (48). This region is thought to be involved in the binding of subfragment 1 to F-actin (47, 49-51) and it seems to be necessary for the actin-activated Mg2"ATPase activity of myosin IB. However, a recent report suggests that this portion of subfragment 1 is not essential for its actin-activated M$+-ATPase activity (52).

There is convincing evidence that at least part of the ATP- binding site of subfragment 1 is located within the 23-kDa domain (14-16), in agreement with the data for myosin IA and IB; the 50-kDa domain of subfragment 1 has also been shown to be involved in ATP binding (53-56). Also, the region that lies between the F-actin-binding site and the ATP- binding site in the 23-kDa domain of subfragment 1 is important in the transmission of information between these two sites (57-59) probably through ligand-induced conformational changes (57,60, 61). It is, therefore, of some interest that the phosphorylation that regulates the actin-activated M r - ATPase activity of myosin IB occurs within the corresponding region.

As the work described in this paper was nearing completion, the complete amino acid sequence of the myosin IB heavy chain was deduced by Jung et al. (27) from the nucleotide sequence of its genomic DNA. The sequence data are completely consistent with, and allow further extension of, the domain assignments and comparisons between myosin IB and skeletal muscle myosin. The sequence of the NHz-terminal 76 kDa of myosin IB is 36% identical to the corresponding region of muscle myosin (55% if conservative substitutions are con- sidered), except for the first 80 amino acids which are missing in myosin IB. A few specific regions are of interest in relation to the data in this paper. First, -Lys-Lys-Lys- sequences apparently allow tryptic cleavage of subfragment 1 at the 231 50 and 50/20 sites. The myosin IB heavy chain has this same sequence at the 62/68 cleavage site (60/67 according to the sequence analysis), which corresponds to the 50/20 site in subfragment 1, but not at the position which would correspond to the 23/50 site, thus explaining the absence of cleavage there. Second, although myosin IB contains neither of the 2 cysteine residues that characterize the active thiol region of subfragment 1, this region of myosin IB is otherwise one of the most highly similar to muscle myosin (27). Finally, the

434 Actin-binding Sites of Acanthamoeba Myosin IB

ATP-binding site of myosin IB identified by photoaffinity labeling by UTP can probably be further localized by the sequence data to a site 12 kDa from the NHz terminus because the sequence there (-Thr-Glu-Ala-Ser-Lys-Lys- (27)) is very similar to the sequence of the peptide of Acanthamoeba myosin I1 (-Thr-Glu-Asn-Thr-Lys-Lys-) that is similarly photoaffinity-labeled (28). The region surrounding this sequence is among the more highly conserved between Acanthamoeba and muscle myosins (27). These sequence homologies are reflected in the similar functional properties of the NHz-terminal subfragments of myosin I (this paper and Refs. 8, 17) and of subfragment I.

The sequence homology between muscle myosins and Acan- thamoeba myosins IA and IB does not extend beyond the domains corresponding to subfragment 1, which is the region solely responsible for the actin-activated M$+-ATPase activities of all myosins. The remaining portion of muscle myosins contains the tail that forms the a-helical, coiled-coil rod through which the molecules self-associate into bipolar, thick filaments. In marked contrast, the remaining 51 kDa of myosin 1B (specifically the COOH-terminal 23 kDa) is greatly enriched in glycine, proline, and alanine (27), as is the COOH- terminal 27-kDa peptide of myosin IA that also contains the second actin-binding site (8). It is interesting that the amino acid compositions (but not the sequence) of the COOH- terminal domains of myosins IA and IB that contain the second F-actin-binding site bear a general similarity to the amino acid composition of the NH2-terminal domain of the A1 light chain of muscle myosin (62) to which F-actin also binds (63).

The common features of subfragment 1 and the NH2- terminal 80-kDa of Acanthamoeba myosin IA and IB are probably all that are needed for “motile” activity, as evidenced by the abilities of myosin IA and IB (4,9) and subfragment 1 (64) to move particles along actin cables. The bipolar filaments of muscle myosins and the second F-actin-binding sites of Acanthamoeba myosin IA and IB may then provide alter- nate mechanisms by which myosins can move one actin filament relative to another. Perhaps monomeric myosins with two F-actin-binding sites in one molecule can function more efficiently than conventional myosin bipolar filaments within the subplasma membrane, cortical networks of non- muscle cells where the actin filaments are not as precisely organized geometrically as in muscle. It is also possible that the unusual COOH-terminal domains of myosin IA and IB may be able to bind to cellular structures other than microfil- aments.

Acknowledgments-We thank Drs. Goeh Jung and John Hammer 111 for providing us with the sequence of Acanthamoeba myosin IB and Laura Hall for editorial assistance.

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