modia, another motile stage in the. Physarum life cycle. Amoebal myosin contained heavy chains. (M,. -220,000), phosphorylatable light chains (Mr 18,000),.
Vol. 261, No. 17, Issue of June 15, pp. 8022-8027,1986 Printed in U.S.A.
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1986 by The American Societyof Biological Chemists, Inc.
Isolation and Characterizationof Myosin from Amoebae of Physarum polycephalum” (Received for publication, February 6,1986)
Kazuhiro KohamaSP, Hiromi Takano-Ohmurofl, Takeshi Tanakall ,Yoshikazu Yamaguchi**, and Tomoko KohamaS From the $Department of Pharmology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, the lITokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113, Japan, the IlResearch Division, Saitama Red Cross Blood Center, Yorw-shi 338, Japan, and the **BiomedicalLaboratories, Kawagoe-Shi 350, Japan
Myosin was isolatedfromamoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. (M, Amoebal myosin contained heavy chains -220,000), phosphorylatable light chains(Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electronmicrographs after rotary shadow casting.In thepresence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity < K-EDTA-ATPase activity < Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2’. Amoebal myosin was indistinguishable fromplasmodial myosin in ATPase activities and molecular shape. However, the heavychainandphosphorylatable light chainsof amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamidegelelectrophoresis,peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosinswere found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity inPhysarum myosins by micromolar levels of Ca2+.
(Acanthamoeba myosin I) coexists with a double-headed longtailed “conventional myosin” (Acanthumoeba myosin 11) (410). Mini-myosin analogous to Acanthumoeba myosin I was also obtained from Dictyostelium amoeba (11)in addition to conventional Dictyostelium myosin (12-15). The biochemical properties of mini-myosins and consensus myosins are also quite different; mini-myosins have high K-EDTA-ATPase activity but low Ca-ATPase activity (4, 5, l l ) , whereas the Ca-ATPase activity of conventional myosins is higher than the K-EDTA-ATPase activity ( 5 , 6, 9, 12-15). Phosphorylation of mini-myosin (Acanthamoeba myosin I) enhances actinactivated ATPase activity ( 5 , 16, 17), whereas the converse is true for conventional myosin (Acanthumoeba myosin 11, Dictyostelium myosin) (13, 15, 18, 19). Physarum plasmodial myosin is double-headed and long-tailed (20, 21), and its CaATPase activity is higher than itsK-EDTA-ATPase activity (21, 22). These observations indicate that plasmodial myosin shares conventional myosin properties with Acanthamoeba myosin I1 and Dictyostelium myosin. Ca2+-sensitivemyosin has been prepared from Acanthumoeba by Collins and Korn (18,19); the actin-activated ATPase activity of dephosphorylated Acanthamoeba myosin I1 is higher in Ca2+than in EGTA.’ We prepared Ca2+-sensitive myosin from Physarum plasmodium (21-27). Unlike Acanthamoeba myosin 11, the actin-activated ATPase activity of our plasmodial myosin (p-myosin) is higher in the presence of EGTA. Ca2+inhibited the activity half-maximally at micromolar levels (21-24, 27). ca‘+-binding to the M , 14,000 light chain is thought to be involved in this Ca2+-sensitivity (22, 25, 27). Amoebal myosin (a-myosin) was first isolated by Ohta and The Physarum lifecycle contains two motile stages, i.e. amoeba and plasmodium, and actomyosin systems have been his colleagues (28). However, they could not find any differdemonstrated in each (Ref. 1for amoeba, Ref. 2 for plasmo- ences between amoebal and plasmodial myosins. This paper dia). The mode of motility is quite different in each case; describes the purification and characterization of a-myosin, uninucleate amoeba show slow amoeboid movement while with reference to myosins from Acanthamoeba and Dictyospolynucleate plasmodia show rapid cytoplasmic streaming. telium amoeba mentioned above. Evidence is presentedwhich We obtained indications of stage-specific myosins (myosin suggests that: (i) a-myosin shows the two-headed long-tailed isoforms) by extracting crude actomyosin from both stages shape of conventional myosins; (ii) a-myosin contains the same M , 14,000 Ca2+-binding light chain as p-myosin, ac(3). Kornandhis colleagues (4-8, 10, 11) demonstrated the counting for the observation that Ca2+ inhibits the actinexistence of myosin isoforms in lower eukaryotes using Acan- activated ATPase activity of a-myosin at micromolar levels; thamoeba, where a single-headed short-tailed “mini-myosin’’ and (iii) a-myosin and p-myosin contain heavy and phosphorylatable M, 18,000 light chains which differ in primary stmcture.
*This work was supported in part by grants from the Yamada Science Foundation and the Aino Hospital Foundation and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Thecosts of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed.
The abbreviations used are: EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; p-myosin, Physarum plasmodial myosin; a-myosin, Physarum amoebal myosin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; IEF, isoelectric focusing; E64C, N-[N-(~-trans-3-carboxyoxirane-2-carbonyl)-~-leucyl]-3-methylbutylamine.
8022
Physarum polycephalum Amoeba of from Myosin Parts of this study were previously reported inpreliminary forms (27, 29).
8023 to pH 7.5 by adding 1 N HCl and then centrifuged a t 25,000 X g for
15 min. The supernatant was concentrated by ultrafiltration using an Amicon PM-10 membrane. The concentrate was clarified by centrifugation a t 25,000 X g for 20 min, mixed with solid ammonium EXPERIMENTAL PROCEDURES sulfate to 34% saturation, and centrifuged a t 25,000 X g for 20 min. Materials-Heterothallic amoebae of strain TM-4, which was The supernatant was mixed with solid ammonium sulfate to 68% cloned from spores of plasmodial strain Ng-1, were grown on agar saturation and centrifuged at 25,000 X g for 20 min. Fractions plates by the method of Uyeda and Furuya (30). Plasmodia (Ng-1) enriched in M, 18,000 light chain and in M, 14,000 light chain were were grown on rolled oats by modifying the method (31) of Camp recovered in the precipitate and supernatant,respectively. (32). Amoebae and plasmodia were harvested in 20 mM NaCl conActin was prepared from rabbit (36) or chicken (37) skeletal muscle. taining 15 mM phosphate buffer at pH6.5, pelleted by the centrifugaAntibodies-Proteins used as antigens, i.e. a-myosin heavy chain, tion a t 1,000 X g for 3 min, and thensuspended in thesame solution. p-myosin heavy chain, M, 18,000 light chain, and M, 14,000 light The suspension was recentrifuged a t 10,000 X g for 10 min, weighed, chain were obtained by preparative SDS-polyacrylamide gel electroand immediately subjected to theprotein preparation procedures. phoresis of a-myosin, p-myosin, plasmodial M, 18,000 light chainPreparation of Proteins-All procedures were performed a t 0-4 "C enriched fraction, and plasmodial M, 14,000 light chain-enriched using double distilled water. fraction, respectively. They were concentrated by dialyzing first Preparation of Amoebal Native Actomyosin-Procedures were de- against solid sucrose, then against 1 mM NaHC03 containing 0.1% rived from those described by Kohama and Takano-Ohmuro (3). The SDS, and mixed with adjuvant complete H37Ra (Difco). principles were essentially the same as those described by Kohama A monoclonal antibody againstp-myocin heavy chain was produced (22,33) for the preparation of plasmodial native actomyosin. by hybridoma formation (38) between spleen cells of BALB/c mice The amoebal pellet was homogenized 4 times with a Polytron for immunized with p-myosin heavy chain and a nonsecreting myeloma 30 s at maximum speed in 2 volumes of high salt buffer (0.5 M NaCl, cell line P3-X63-Ag8-Ul as described previously (39). Polyclonal 10 mM EGTA, 0.1 mM DTT, and protease inhibitors, i.e.0.1 mM antibodies against a-myosin heavy chain, plasmodial M, 18,000 light phenylmethylsulfonyl fluoride from Sigma, 0.1 mM E64C from Taisho chain, orplasmodial M, 14,000light chain were prepared by injecting Pharmaceutical Co., and 75 kallikrein-inactivating units/ml of Tra- individual antigens into guinea pigs. sylol from Bayer). The homogenate was adjusted to pH7.8 by slowly These monoclonal and polyclonal antibodies were used for immuadding 1 N NaOH and centrifuged a t 10,000 X g for 30 min. The nostaining as described previously (39). We used lmI-protein A or precipitate was mixed again with high salt buffer (1 volume of the '251-anti-mouse Ig (New England Nuclear) and peroxidase-labeled amoebal pellet), homogenized with a Polytron for 30 s a t maximum anti-guinea pig IgG (Cappel) or peroxidase-labeled anti-mouse IgG speed, and the pH was adjusted to 7.8 by adding 1 N NaOH. The (Cappel) to detect antibodies. supernatant and homogenate were centrifuged a t 50,000 X g for 40 Biochemical Methods-Protein concentration was determined by min. The supernatants(high salt extract) (Fig. 1, lune a ) were pooled, the method of Lowry et al. (40) using bovine serum albumin as a p H adjusted to 6.7 by slowly adding 1 N HC1, and dialyzed against 9 standard. volumes of 1mM NaHC03 containing 10 mM 2-mercaptoethanol and SDS-PAGE was performed by modifying the method of Laemmli 0.05 mM phenylmethylsulfonyl fluoride. The dialysate was centri(41) using an 8%upper separation gel to analyze myosin heavy chains fuged a t 10,000 x g for 10 minto precipitate crude native actomyosin and a 16% lower separation gel to analyze actin and myosin light (Fig. 1, lune c). chains. The following molecular weight standards were used phosCrude native actomyosin was mixed with one-fifth of the amoebal phorylase b (M, 94,000), albumin (Mr67,000), ovalbumin (Mr43,000), pellet volume of 0.5 M NaCl buffer (0.5 M NaCl, 0.1 mM DTT, 10 mM trypsininhibitor (M, 20,000), a-lactalbumin (M, 14,000),myosin EGTA, and 2 mM NaHC03, final pH = about 7.5) and gently homog- heavy chain of rabbit skeletal myosin (Mr200,000). SDS-PAGE gels enized by pipetting the mixture. The homogenate was centrifuged a t were stained with Coomassie Brilliant Blue. The molar stoichiometry 25,000 X g for 20 min. The supernatantwas mixed with 5 volumes of of heavy and light chains was determined by extractingstained water, allowed to stand for 1h, and centrifuged a t 10,000 x g for 10 protein bands in 1%SDS containing 5 mM phosphate buffer (pH 7.0) min. The precipitate was subjected to the a-myosin purification followed by densitometry a t 550 nm (42). Two-dimensional gel elecprocedure described below or was used as native actomyosin after trophoresis, a combination of SDS-PAGE and isoelectric focusing dissolving in 0.5 M NaCl containing 0.1 mM DTT and dialyzing (IEF), was performed by the method of O'Farrell(43) as modified by against 2 mM NaHC03 containing 0.1 mM DTT (Fig. 1, lane d). Mikawa et al. (44). Two-dimensional electrophoresis gel was stained Purification of Amoebal Myosin-Procedures were derived from using,highlssensitive silver stain (45). those described by Kohama (34) for the purification of plasmodial a-Chyaotryptic peptide maps of amoebal and plasmodial heavy myosin. chains isolated using SDS-PAGE were performed by modifying the Native actomyosin was dissolved in 100 mM ATP containing 10 method of Cleveland et al. (46). Peptide maps of M, 14,000 and M, mM DTT (pH 6.7) (one-tenth of the amoebal pellet volume) and 18,000light chainsof a-myosin and p-myosin were prepared by tryptic clarified by centrifugation at 25,000 X g for 20 min. The dissolved digestion of M, 14,000 and M, 18,000 light chains isolated from IEF native actomyosin (Fig. 1,lane e ) was diluted with water, mixed with gels? stock NaHC03 and EGTA to give final concentrations of20 mM 45CaZ+ binding to the M, 14,000 light chainwas demonstrated using ATP, 2 mM DTT, 5 mM NaHC03, and1mM EGTA, and thenmixed Western blotting (47) and SDS-PAGE of myosin as described by with stock magnesium acetate to give a final concentration of 0.1 M. Maruyama et al. (48). The final pH was about 6.3. After stirring for 20 min, the mixture ATPase activities were measured by the pH stat method at pH was centrifuged a t 25,000 X g for 20 min. The supernatantwas placed 7.50, 25 "C (22, 49). Measurement of the relationship between Ca2+ in dialysis membrane tubing (Spectrapor, with a M, 3,500 cut-off) concentration and actin-activated Mg-ATPase activity was initiated and concentrated byburying the dialysis tubing inpolyethylene glycol in 0.1 mM EGTA (Ca" C 10 nM) in a 10-ml assay pot. By successive powder (Sigma, average molecular weight = 20,000). The concen- addition of CaClZaliquots to the same assay pot, ATPase activities trated solution was clarified by centrifugation a t 100,000 X g for 20 at higher Ca2+concentrations could be determined, giving a precise min. The clarified concentrate (Fig. 1, lune f) was mixed with an measure of relative activity. The Ca2+concentration was calculated equal volume of water and allowed to stand for 3 h to precipitate using Ca/EGTA buffers with apparent binding constants of 2.5 X lo7 amoebal myosin. The precipitate was dissolved in 0.6 M NaCl conat pH7.5 (50, 51). taining 0.1 mM DTT and 2 mM NaHC03 and clarified by the centrifElectron Microscopy-a-Myosin monomer in 50%glycerol containugation at 25,000 X g for 20 min. The supernatantwas dialyzed first ing 0.5 M ammonium acetate was sprayed on freshly cleaved mica, against 0.6 M NaCl containing 0.1 mM DTT and 2 mM NaHC03 subjected to rotary shadow casting ina JFD-7000 (JEOL) as described extensively, then against 1mM NaHC03 containing 0.1 mM DTT and by Onishi and Wakabayashi. (52) and examined in a JEM1200EX used as a-myosin (Fig. 1, lane g). (JEOL) or anAkashi 002A operated at 80 kV. Preparation of Other Proteins-Plasmodial high-salt extract was prepared by the method described for amoebal high-salt extract, from RESULTS which p-myosin was prepared (33, 34). Light chains were isolated Subunit Compositiom"DS-PAGE reveals that purified afrom p-myosin by modifying the method described for isolating skelmyosin preparations contain three major polypeptide bands: etal light chains (35).p-Myosin was dissolved in 0.4 M NaCl containing 0.1 M Na2C03(final pH was about 10.5), heated a t 55 "C for 10 min, and mixed with 9 volumes of water. The mixture was adjusted T. .Mikawa, unuublished method.
Amoeba of Physarum polycephalum
Myosin from
8024
M, 220,000 heavy chain andM , 18,000 and 14,000 light chains (Figs. 1 and 3). The molar ratio (heavychain:M, 18,000 light chain:M, 14,000 light chain) is 1:0.9:0.8, which probably represents a 1.O:l.O:l.O molar stoichiometry. On two-dimensional gels, the M, 18,000 light chains of amyosin and p-myosin have minor subspots corresponding to species of different charge (Fig. 2), suggesting that the light chains are phosphorylatable. Ca"-binding activity of M, 14,000 light chainsof a-myosin and p-myosin is suggested by the detection of radioactivity on M, 14,000 light chain bands when the Western blots of 3). SDS-PAGE gels were incubated with 45Ca2+ (Fig. Electron Microscopy of a-Myosin Monomers-Electron microscopy of single a-myosin molecules visualized by rotary shadow casting (Fig. 4) reveals that a-myosin consists of a fibrous tail, 184.8 f 1.37 nm (n = 30) in length, withtwo The two-headed globular headsabout 13 nmindiameter. structure suggests that a-myosin contains two heavy chains and, therefore, two pairs of light chains. The totalmolecular weight of a-myosin should, therefore,be about 500,000.
200
83
-
-Ha
a
b
c
d
FIG. 3. CaZ+binding to M, 14,000 light chains. a-Myosin (a, c) and p-myosin ( b , d ) were subjected to SDS-PAGE followed by
Western blotting( a ,b). Blots were incubated in a solution containing 60 mM KC], 5 mM MgC12, 10 mM imidazole HCI, pH 6.8, and "CaC12 and were then subjected t o autoradiography (c, d ) (48). Hu/p, heavy chains of a-myosin and p-myosin; h / p , phosphorylatable M , 18,000 light chains of a-myosin and p-myosin; L14, Ca2+-bindingM , 14,000 light chains shared to a-myosin and p-myosin.
94-
67-
-A
T"" 2014-
...
1 '
a
-L18a d-14
b
c
e
d
f
9
FIG.1. SDS-PAGE of proteins from different steps in the a-myosin preparation procedure. SDS-PAGE was prepared using an 8.0% upper separation gel to analyze heavy chains and a 16.0% lower separation gel to analyze actin and light chains. Amoeba1 high salt extract ( a ) was dialyzed against low salt and centrifuged. The supernatant and precipitate, i.e. crude native actomyosin, are shown in b and c, respectively. Native actomyosin ( d ) purified from crude native actomyosin was dissolved in ATP ( e ) ,mixed with magnesium acetate,and centrifuged. Thesupernatant was concentratedand recentrifuged. The supernatant ( f) was mixed with a n equal volume of water to precipitate a-myosin (g). The numbers represent molecular weight in x103 daltons. Ha, heavy chain of a-myosin; A , actin; LI8a, phosphorylatable ( M , 18,000) light chain of a-myosin; L14, Ca'+-binding ( M , 14,000) light chain.
H + L OH-
Lu
A A+P P FIG.2. Two-dimensional gel of a-myosin (A), p-myosin ( P ) , and their mixture (A+P) using IEF in the first dimension and SDS-PAGE in the second dimension. The gels are oriented from the acidic side (H+) (left) to the basic side(OH-) (right). Minor subspots accompanying M , 18,000 light chains of a-myosin and pmyosin are indicated by arrowheads.
FIG.4. Electron microscopy of single a-myosin molecules. a-Myosin molecules were subjected to rotary shadowing with platinum using the method of Onishi and Wakabayashi (52). The micrograph was taken using an Akashi operated a t 80 kV. The bar represents 100 nm.
Enzymatic Activities-The ATPaseactivity of a-myosin was stimulated by Ca2+ (158.0 nmol min" mg" myosin) and EDTA (80.4 nmol min" mg" myosin) and inhibitedby Mg2' (28.9 nmol min" mg" myosin) when measured under high salt conditions (Table I).3 Under low salt conditions, ATPase activity in the presence ofM$+ was reduced to 15.3 nmol min" mg" myosin. The presence of0.1 mM EGTA (Ca" C 10 nM) or 50 pM Ca'+ did not affect this low-salt MgATPase activity (Table I). Mg-ATPase, Ca-ATPase, and K-EDTA-ATPase activities of pmyosin under the comparable assay conditions are 30.9, 506.5, and 101.5 nmol min" mg" protein, respectively (21,22). Itremains to be established whether the difference in Ca-ATPase activity between amyosin and p-myosinisdue to a difference in myosin primary structure or to modification of myosin during preparation.
8025
Myosin fromAmoeba of Physarumpolycephalum TABLE I
Amoeba
ATPase activities of amoebal native actomyosin and myosin ~
~
~
"
~
~
~~~~
~
~
~
~~
~
~
Ha
ATPase activity at 25 "C. pH 7.5 ~
~
~~~~
Native actomyosin " "
~
~-
nmol
0.5 M KC1 + 1 mM EDTA 31.0 0.5 M KC1 + 1 mM Ca'+ e 0.5 M KC1 + 1 mM M + 0.1 mM EGTA KC1 30 mM + 1 mM M e + 0.1 mM EGTA KC1 30 mM + 1 mM M$+ 50 P M Ca2+
ATP min" mg" protein 80.4
50.3 8.9
158.0 28.9
22.1
15.3
6.9
+
~~
a-Myosin ~~~~
~
15.3 .
~-
""-. 0 50 100 500 loo0
0
50 100 500 loo0
ns a l/ne
FIG.6. a-Chymotryptic peptide map of myosin heavychains using the method of Cleveland et al. (46). Left (Amoeba) and rizht (Plasmodium) panels refer t o a-myosin and p-myosin, respectively. The numbers indicate the amounts of n-chymotrypsin used for digesting myosin heavy chains. Ha, a-myosinheavy chain; Hp, pmyosin heavy chain; *, n-chymotrypsin.
FIG. 5. Inhibitory effect of Caz+on actin-activated ATPase activity of a-myosin.a-Myosin was mixed with skeletal muscle Factin, and ATPase activity was assayed in 30 mM KC], 0.5 mM MgATP, 1 mM M$+,0.1 mM EGTA-Ca buffer at pH 7.50, 25 'C. The 100% ATPase activities (in 0.1 mM EGTA) were 130.7 & 11.6 nmol min" mg" myosin (mean f S.E., n = 3). Bars represent standard error (n = 3).
However, theactin-activatedMg-ATPaseactivity of amyosin in the presence of 0.1 mM EGTA (130.7 f 11.6 nmol min-l mg" myosin, n = 3) was higher than in the presence of 50 PM Ca'+ (39.3 f 3.5 nmol min-l mg" myosin, n = 3) (Fig. 5). The Ca" concentration giving half-maximal inhibition was about 1 PM (Fig. 5). This inhibitoryeffect of Ca'+ on actin-activated Mg-ATPase activity was comparable with that of p-myosin (21-25). Difference in Primary Structure between Heavy Chains of a-Myosin and p-Myosin-We failed to detect any obvious differences in myosin monomer shape (compare Fig. 4 with Refs. 20 and 21), subunit composition in SDS-PAGE (Fig. 3, Ref. 28), or ATPase activities (compare Table I and Fig. 5 with Refs. 21 and 22). Is a-myosin identical with p-myosin? a-Myosin was directly compared withp-myosin. In SDS-PAGE, the mobility of a-myosin heavy chain was slightly lower than that of p-myosin (not shown), confirming results obtained with native actomyosins from amoeba and plasmodium (3). a-Chymotryptic peptide maps of a-myosin heavy chain are distinct from those of p-myosin (Fig. 6), confirming the result obtained with native actomyosin from amoeba and plasmodium (3). Monoclonal antibody toamyosin heavy chain reacted with the heavy chain band in amyosin Western blots but not p-myosin in Western blots(Fig. 8). The heavy chain band in a-myosin Western blots did not react with antibody to the p-myosinheavy chain (Fig. 8). These observations indicate that a-myosin heavy chainis different in the primary structure top-myosin heavy chain. Difference i n the Primary Structure between M , 18,000 Light
Chains of a-Myosin andp-Myosin-The M , 18,000 light chain of a-myosin migrated differently to that of p-myosin in twodimensional gels (Fig. 2). The tryptic peptide map of a-myosin light chains is distinguishable from that of p-myosinlight chain (Fig. 7). Antibody to p-myosin light chaindid not react with a-myosin light chain in Western blots (Fig. 8). These observations indicate that the primary structure of the M, 18,000 light chain of a-myosin differs from that of the M , 18,000 light chainof p-myosin. Identity of M , 14,000 LightChainsofa-Myosinand pMyosin-In two-dimensional gels, the migration of the M , 14,000 light chain of a-myosinwasidentical to that. of pmyosin (Fig. 2). Tryptic peptide maps of the M , 14,000 light chain of a-myosin were indistinguishable from those of the M , 14,000 light chainof p-myosin (Fig. 7). Moreover, antibody to the M , 14,000 light chain of p-myosin reacted with both amoebal and plasmodial M , 14,000 light chain bands(Fig. 8). In Western blots, the M , 14,000 light chain of a-myosin bound Ca'+ to the same extent as that of p-myosin (Fig. 3), suggesting that the light chain of a-myosin hinds Ca" to the same extent as that of p-myosin. These observations strongly suggest that a-myosin shares a common M , 14,000 light chain with p-myosin, which further supportsourhypothesisthat Ca'+ inhibitsactin-activated ATPase activity inp-myosin by binding to the M , 14,000 myosin light chain (21-27,531. DISCUSSION
The present study reports that amoebal and plasmodial forms of Physarurn possess stage-specific myosin isoforms. aMyosin and p-myosin differ inthe primary structures of heavy andphosphorylatable light chains. The Ca'+-binding light chain is common to both a-myosin andp-myosin. These two myosins are similar in shape (double-headed and long-tailed, Fig. 4 and Refs. 20 and 21) and myosin ATPase activity (MgATPase < K-EDTA-ATPase < Ca-ATPase, TableI and Refs. 21 and 22), indicating that they can be classified as conventional myosinsanalogous to Acanthamoeba myosin I1 and Dictyostelium myosin ( 5 , 6, 9, 12-15). In addition to conventional myosin, Acantharnoeba and Dictyostelium amoeba possess mini-myosin isoforms which are totally different in the
Myosin from Amoeba of Physarum polycephalum
8026
18 kd chain light Plasmodium Plasmodium Amoeba Amoeba
14 kd chain light
- -.-
"
I(,
0
0.5
2
0
0.5
0
2
0.5
2
0
0.5 2 w/ml
FIG.7. Tryptic peptide map of light chains ofa-myosin and p-myosin. Left panel, phosphorylatable M, 18,000 light chain (18-kDa light chain)of a-myosin (Amoeba) andp-myosin (Plasmodium) were excised from IEF gels and digested in 0, 0.5, or 2 pg/ml of trypsin solution and then subjected to SDS-PAGE. Right panel, Ca2+bindinc M. 14.000 light chains (14-kDa light chain) were excised from IEF gels and digested in trypsin solution. For others; see the legend to left panel. '.
lasmodium
Protein
Amoeba -
Amoeba
n
Plasmodium
0
. - b !
Ha-
HP-
L18aL14 -
L18pL 14-
a b c d e
Anti-Hp Anti-Ha
0
FIG.9. Dot-blot analysis of high salt extracts from amoeba (upperpanel)and plasmodium (lowerpanel).High salt extracts were dot-blotted on nitrocellulose and subjected to immunostaining with antibodiesagainsta-myosin heavy chain (Anti-Ha) andpmyosin heavy chain (Anti-Hp).Protein refers to protein stainedwith Amido Black. c
0
a b c d e
FIG.8. Immunoblots of a-myosin and p-myosin subunits. Antibodies were produced againstheavy chains of a-myosin ( H a )and p-myosin ( H p ) and phosphorylatable M, 18,000 (L18p) and Ca'+binding M,14,000 (L14) light chainsof p-myosin. a-Myosin (Amoeba, left panel) and p-myosin (Plasmodium, right panel)were subjected to Western blotting (47) followed by immunostaining. a, protein staining; b, immunostaining with antibody to a-myosin heavy chain; c, with antibody to p-myosin heavy chain; d, with antibody to Ca2+binding M. 14,000 light chain ( L f 4 ) ;e, with antibody to the phosphorylatable M, 18,000 light chain ( L f 8 p )of p-myosin. molecular shape and ATPase activity. It remains tobe demonstratedwhetherornot mini-myosinisoforms exist in amoeba and/or plasmodiaof Physarum. Although the myosin isoforms of Acanthamoeba coexist in Physarum the same cell (7), there are no indications that myosin isoforms co-exist in amoebae or plasmodia; dot blots of amoeba1 high salt extract reacted with antibody a-myosin to heavy chain but not with antibody top-myosin heavy chain. Similarly dot blots of plasmodial high salt extract reacted only with antibody top-myosin heavy chain (Fig. 9). Conventional myosins prepared from Acanthamoeba (18, 191, Dictyostelium amoebae (13-15), and Physarum plasmodia (22,23, 54) exist in the phosphorylatedform. Dephosphorylation of conventional myosin heavy chains of Acanthamoeba (55, 56, cf. Ref. 57) and Dictyostelium amoebae (14, 15) enhance ATPase activity andassembly of these myosins. However, dephosphorylation of Physarum p-myosin tends to reduce ATPase activity andassembly of p-myosin (23, 54, 58). Thus, it would be interesting to examine the effect of phos-
phorylation state on the ATPase activity andassembly of aa minorsubspot myosin. The M , 18,000 lightchainhas corresponding to a species of different charge (Fig. 2), suggesting that a-myosin is partly phosphorylated. a-Myosin ATPase activity in native actomyosin preparations can be monitored only under high-salt conditions. KEDTA-ATPase and Ca-ATPase activities of a-myosin were 2.5- and 5.7-fold greater than Mg-ATPase activity, respectively (Table I). Comparable results were obtained by comparing for the ATPase activities of purified a-myosin (Table I), suggesting that thea-myosin preparation is as"native" as in the native actomyosin preparation. The inhibitory effect of Ca2+ on Mg-ATPase activity observed with actomyosin reconstituted from a-myosin and skeletal muscle actin is as high as in native actomyosin (TableI). This indicates that amyosin is sensitive to Ca2+ and suggests that the Ca2+ inhibition of a-myosin is not an artifact produced during preparation by processes such as additional phosphorylation and the oxidation of sulfhydryl residues. We started this study anticipating that the ATPase activities of a-myosin mightbe quite different to those of p-myosin, since the mode of motility is quite different in the two cell types. However, we failed to detect any distinctdifferences in ATPase activities between a-myosin and p-myosin. Preliminary immunofluorescence studies suggest that the organization of actin and myosin in amoeba is quite different to that found in plasmodium (cf. Ref. 53).4 Thus, the cellular organization of contractile proteins, such as actin and myosin, may play a key role in producing stage-specific movements.
' T. Q. P. Uyeda and K. Kohama, unpublished data.
Myosin from
Amoeba of Physarum polycephalum
Acknowledgments-We thank Prof. Y. Nonomura for his discussions, interest, and support. Our thanks are also due to Prof. S. Ebashi for his discussion and interest and to Dr. Wakabayashi for instruction in rotary shadow casting.
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