Sadashi Hatano

Isolation and characterization of plasmodium actin

Abstract 1. 1. Methods have been developed for the isolation of an actin-like protein from a myxomycete plasmodium (plasmodium actin). 2. 2. Purified plasmodium actin exists as a monomer (G-actin) in salt-free solution. On the addition of salt, such as 0.1 M KCl, G-actin is polymerized to a fibrous polymer, F-actin. 3. 3. The molecular characteristics of plasmodium G-actin appear to be similar to those of muscle G-actin. The sedimentation constant ( s ° 20, w ) is 3.7, and the mol. wt. determined by the Archibald method is about 57000. G-actin combines 1 molecule of ATP per molecule of protein. 4. 4. The amino acid compositions of plasmodium actin and muscle actin are strikingly similar.

Isolation, purification and characterization of myosin B from myxomycete plasmodium

Abstract 1. 1. Methods have been developed for the isolation of myosin B-like protein (plasmodium myosin B) from a myxomycete plasmodium. 2. 2. Purified plasmodium myosin B has an ATP sensitivity and an ATPase activity as high as those of myosin B from rabbit striated muscle. 3. 3. Mg 2+ increases the rate of superprecipitation of myosin B, while Ca 2+ inhibits its extent. Superprecipitation never takes place in the presence of EDTA. When Mg 2+ is added together with EDTA or ethylene glycol bis (β- aminoethyl ether )-N,N′- tetraacetate , strong and rapid superprecipitation occurs. 4. 4. Electronmicrographs of myosin B show that myosin B is a filament having a diameter of about 100 A. Globular particles of myosin A attach to the surface of the filament. When ATP is added to it, (natural) F-actin appears (75 A in diameter). Natural F-actin has the double-stranded helical structure, half a pitch of which is 350 to 420 A. 5. 5. Myosin A-like protein (plasmodium myosin A) has been isolated by ultracentrifugation of myosin B solution in the presence of ATP and Mg 2+ . Plasmodium myosin A can combine with plasmodium F-actin and muscle F-actin to form actomyosin-like complexes. Some physicochemical properties of purified plasmodium myosin A are reported.

Polymerization of plasmodium actin

Abstract 1. (1)|On the addition of monovalent cations, plasmodium G-actin is polymerized to a fibrous polymer, termed plasmodium F-actin. The F-actin has a high viscosity, and shows a positive flow birefringence. During polymerization, 1 mole of inorganic phosphate is liberated from 1 mole of G-actin. Thus: G-actin-ATP ⇄ ATP 0.1 M KCl F-actin-ADP + P i Kinetic and structural analyses shows the F-actin to be a microfilament, 60 A in diameter, having a two-stranded helical structure with a half pitch of about 290 A containing about 13 spherical units. 2. (2)|Addition of divalent cations forms another kind of polymer, termed Mg-polymer, which has a relatively low viscosity. During the polymerization, 1 mole of inorganic phosphate is liberated from 1 mole of G-actin, suggesting the reaction: G-actin-ATP ⇄ ATP 2 mM MgCl 2 Mg-polymer-ADP + P i Moreover, this polymer itself seems to have ATP-splitting activity. Mg-polymer appears as a globular aggregate in electron micrographs, with a diameter of about 100–600 A. 3. (3)|F-Actin and Mg-polymer are depolymerized to G-actin by removing salts from their solutions in the presence of ATP and cysteine, for example, by dialysis. The resultant G-actin may be polymerized again to one of the two polymer forms. Moreover, it is suggested that F-actin and Mg-polymer can transform into each other directly, not passing through the G-actin state. 4. (4)|Plasmodium G-actin can be copolymerized with rabbit striated muscle G-actin on the addition of monovalent cations or divalent cations.

Purification and characterization of myosin A from the myxomycete plasmodium

Abstract 1. 1. Methods have been developed for the isolation of myosin A-like protein (plasmodium myosin A) from a myxomycete plasmodium. 2. 2. Plasmodium myosin A is soluble in 0.5 M KCl solution at pH 7.0. Its molecular characteristics are examined and compared with those of myosin A from rabbit striated muscle. The sedimentation coefficient ( s 20, w ) is 6.05 S which is the same order as that of muscle myosin A. However, the viscosity ([ η ] = 1.60 dl/g) is only about two-thirds of that of muscle myosin A. 3. 3. Generally speaking, amino acid composition of plasmodium myosin A is very similar to that of muscle myosin A, but cysteine is not detected in plasmodium myosin A as far as we have examined. 4. 4. ATPase activity of plasmodium myosin A is as high as that of muscle myosin A, and it is activated by Ca 2+ and inhibited by Mg 2+ . Plasmodium actin as well as muscle actin activates ATPase of plasmodium myosin A. ATPase activity of plasmodium myosin A is always inhibited by EDTA and p -chloromercuribenzoate, even at low concentrations, whereas ATPase of muscle myosin A is greatly activated at such low concentrations of EDTA and p -chloromercuribenzoate. 5. 5. Plasmodium myosin A is completely soluble and forms only small aggregates with sedimentation coefficients which are about 9 S at low salt concentrations, including the physiological salt concentration of plasmodium (0.03 M KCl) at pH 7.0.

Inositol phospholipid — Induced suppression of F-actin-gelating activity of smooth muscle filamin

Filamin, a high molecular weight actin-binding protein, cross-links actin filaments and produces a gel composed of F-actin. The effects of polyphosphoinositides on the gelating activity of smooth muscle filamin were examined by measuring the low shear viscosity of the F-actin solutions containing filamin incubated with phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), or phosphatidylinositol 4,5-bisphosphate (PIP2). Micelles of these inositol phospholipids bound to filamin inhibited the ability to form a gel of F-actin. The inhibiting activity of each phospholipid was in the following order, PIP2 greater than PIP greater than PI. The F-actin binding assay of filamin revealed that the inhibition of F-actin-gelation resulted in the loss of the F-actin-binding activity of filamin. Thus, polyphosphoinositides may play important roles in regulating the gelating activity of filamin.

Extraction of native tropomyosin-like substances from myxomycete plasmodium and the cross reaction between plasmodium F-actin and muscle native tropomyosin

Abstract 1. 1.|Native tropomyosin-like substances were extracted from plasmodium of the myxomycete Physarum polycephalum, in the presence of which superprecipitation of muscle actomyosin was regulated by Ca2+. 2. 2.|The superprecipitation of actomyosin synthesized from plasmodium F-actin and muscle myosin A was regulated by Ca2+ in the presence of native muscle tropomyosin. 3. 3.|It was shown by sedimentation analysis and measurements of flow birefringence and viscosity that muscle tropomyosin was bound to plasmodium F-actin.

ATPase activity of plasmodium actin polymer formed in the presence of Mg2

Abstract 1. In the presence of Mg2+, plasmodium actin forms a polymer different from F-actin. This polymer, termed Mg-polymer, has an ATPase activity. Its specific activity is about one-hundredth of the activity of myosin from muscle or plasmodium and of the same order as that of muscle F-actin under sonic vibration. 2. The formation of Mg-polymer and its ATPase activity are not due to modification of the protein in the preparation procedure. The ATPase activity is not due to contamination in the preparation but is closely related to the amount of Mg-polymer formed. 3. The ATPase activity of Mg-polymer is markedly inhibited by high concentration of ATP (about 1 mM) in the presence of KCl; but it is not inhibited in the absence of KCl. 4 Mg-polymer releases its bound [14C]ADP in a cold ATP solution. The half time of the release is about 10 min at 22°, which is of the same order as the rate of ATP splitting. This is understandable if both the ATP splitting and the ADP exchange are associated with the same cyclic process of intrapolymer reaction occurring everywhere along the Mg-polymer. 5. Copolymers of plasmodium and muscle G-actins are formed in the presence of Mg2+. The ATPase activity of copolymers decreases rapidly with increasing content of muscle actin; it is suggested that interaction of neighboring plasmodium actin molecules in the polymer is required for the ATPase activity.

Reversible superprecipitation and bundle formation of plasmodium actomyosin.

Synthetic actomyosin from plasmodium was found to undergo reversible superprecipitation upon addition of ATP. According to electronmicroscopic investigation upon clearing, short myosin filaments of about 0.2 micron in length appeared predominantly coexisting with actin filaments, and after superprecipitation, bundles of actin filaments were formed where short myosin filaments or myosin molecules were bound to the side of the bundle, making a whisk-like structure. The turbidity and the ATPase activity of actomyosin were measured at various ATP concentrations clamped by using an ATP-regenerating system. The turbidity was high below 1 . 10(-6) M ATP, corresponding to the state of superprecipitation, and with increasing ATP concentration it dropped in the range of 1 . 10(-6)--1 . 10(-5) M ATP. On the other hand, the ATPase activity was low below 1 . 10(-6) M ATP and increased above 1 . 10(-5) M after the turbidity dropped. Characteristic features of superprecipitation of plasmodium actomyosin observed here were discussed in relation to the mechanism of motility in vivo.

Actin-Binding Proteins in Cell Motility

Publisher Summary This chapter focuses on the actin-binding proteins (ABP) in cell motility. Many actin-binding proteins (ABP) have been isolated from nonmuscle cells and are classified into several groups based on specific properties which are characterized in vitro . These ABPs play key roles in the dynamic behavior of the actin cytoskeleton in nonmuscle cells. Two types of actin-myosin-based cell motility have been studied. 1. Streaming of the cytoplasmic sol on the inner surface of the cortical gel layer. The typical cytoplasmic streaming is seen in internodal cells of Characeae. The sol continuously slides on bundles of actin filaments located on the cortical gel layer. These bundles of actin filaments are stable structures and show no appreciable structural changes during cytoplasmic streaming. 2. Passive flow of the cytoplasmic sol within the cortical gel layer. This type of cytoplasmic streaming is seen in plasmodium of an acellular slime mold, Physarum polycephalum , and in a giant ameba, Chaos chaos . The contractile apparatuses are constructed in cell regions when they contract, and disintegrate after the contraction. Therefore it is a complex problem to determine where and when the contractile apparatuses are constructed, in addition to how they work in these cells. The ABPs are the key to solving these problems. The chapter discusses passive cytoplasmic streaming in Physarum plasmodium , and also includes ameboid movements of other cells.

Purification of myxamoebal fragmin, and switching of myxamoebal fragmin to plasmodial fragmin during differentiation ofPhysarum polycephalum

SummaryWe have isolated and purified an activity from amoebae ofPhysarum polycephalum that reduces the flow birefringence of a solution of F-actin in a Ca2+-dependent manner. The purified activity from 100 g of amoebae consisted of 1 mg of a 40000 mol. wt protein. DNase I-affinity chromatography demonstrated that the protein binds toPhysarum actin in a Ca2+-dependent manner, and the binding is not reversed by excess EGTA. Viscometric measurement indicated that the protein (i) accelerates polymerization of G-actin, and (ii) severs F-actin, in a Ca2+-dependent manner. Thus, the protein appeared functionally similar to the fragmin previously isolated fromPhysarum plasmodia (plasmodial fragmin). However, the two proteins had slightly different mobilities on urea-SDS-PAGE, and antibodies raised against the two proteins scarcely cross-reacted with each other. Hence, we conclude that the two proteins are closely related to but are different from each other, and we have named the novel protein ‘myxamoebal fragmin’. Immunoblot analysis indicated that myxamoebal and plasmodial fragmins are specifically present in amoebae and plasmodia, respectively. Results of immunofluorescence staining suggest that the synthesis of plasmodial fragmin is switched on coordinately with the synthesis of the heavy chain of plasmodial myosin and other plasmodium-specific contractile proteins during the apogamic differentiation of amoebae to plasmodia.