Myosin was purified to a homogeneity from sea sponge, Halichondria okadai. The myosin consisted of 220 kDa heavy chain, 18 kDa calcium binding light chain and 21 kDa phosphorylatable light chain. Rotary shadowed images showed the two headed myosin (myosin II) with a 160 nm tail. The myosin was less soluble in a KCl solution as compared to rabbit skeletal myosin.
The K -stimulated and Ca2 -stimulated ATPase activities of sea sponge myosin were 0.46 and 0.07 μmol Pi min−1 mg−1, respectively. The Mg2 -activated myosin ATPase activity showed no significant enhancement by the addition of rabbit skeletal muscle actin despite that the light chain was phosphorylated by myosin light chain kinase from chicken gizzard. Sea sponge myosin 18 kDa light chain bound to Ca2 ion but was not phosphorylated like Physarum plasmodia myosin light chains.
INTRODUCTION
Myosin molecule constituting of two heavy chains and of four light chains (myosin II) is a major element of energy transduction and force development in muscle or nonmuscle tissues. Myosin has been isolated and characterized from various organisms [1, 2]. Sea sponge, a lower eukaryotic animal, has not distinct muscle tissues. In this paper, for the first time, we report isolation and characterization of myosin from sea sponge, Halichondria okadai. Sea sponge myosin 18 kDa light chain could bind Ca2+ ion, and 21 kDa light chain was phosphorylated by myosin light chain kinase (MLCK) from chicken gizzard. Physarum plasmodia myosin is reported to have two species of light chains; one is phosphorylated by endogenous kinase and another binds to Ca2+ ion resulting in the Ca2+ inhibitory regulation [3, 4]. Although the regulation of myosin ATPase activity was obscure, sea sponge myosin contained phosphorylatable regulatory light chain and Ca2+ -binding light chain like as Physarum myosin.
MATERIALS AND METHODS
Materials
Sea sponge was collected in the sea near the Marine Ecosystem Center, Chiba University, Kominato. Sea sponges were cut into small pieces by hands removing mussels, small stones etc. and the pieces were well washed with a cooled Mg2+- free artificial seawater. The washed sea sponges were added with a cooled salt solution consisting of 450 mM NaCl, 11 mM KCl, 2 mM NaHCO3, 25 mM Na2SO4, and 2 mM EDTA and homogenized for 10 sec at 10, 000 rpm three times in a Waring blender. The resultant suspension was filtered through a nylon gauze. The filtrate was centrifuged for 5 min at 500 rpm to sediment large debris, and then the supernatant was further subjected to centrifugation for 5 min at 5,000 rpm. The precipitated sea sponge cells were suspended in the above solution, added with glycerol to a final concentration of 50%, and stored at −20°C until use. The yield of sea sponge cells were approximately 30 g from 100 g live sea sponges.
Preparation of proteins
Actin was prepared from acetone powder of rabbit skeletal muscle and purified by Sephadex G-150 column chromatography. Myosin light chain kinase (MLCK) and smooth muscle myosin were purified from chicken gizzard according to Nakamura and Nonomura [5] and Ebashi [6], respectively. Calmodulin was purchased from Sigma. Protein concentrations were determined by the method of Bradford [7] using bovine serum albumin as a standard.
Phosphorylation of myosin light chains
Purified sea sponge myosin (1 mg/ml) was treated with MLCK, 5 μg/ml, for 30 min at 25°C in a mixture of 8 mM MgCl2, 1 mM CaCl2, 20 μg/ml calmodulin, and 20 mM Tris-HCl, pH 7.5. The reaction was stopped by the addition of EGTA to a final concentration of 10 mM. After dilution with 10 vol. of cold distilled water, the precipitate centrifuged for 10 min at 13,000 rpm was dissolved in a solution containing 0.6 M KCl, 1 mM NaHCO3 and 0.5 mM DTT (dithiothreitol). Clarified sample was used for ATPase measurements.
Autoradiography
Sea sponge myosin was incubated for 30 min at 25°C with 6 μCi/ml of [γ-32P] ATP (Amersham, 6000 Ci/mmol) in the above described MLCK solution. The reaction was terminated by the addition of 3 vol. of an SDS solution containing 10% SDS, 5% 2mercaptoethanol and 20 mM Tris-HCl, pH 6.8. After SDS gel electrophoresis, gels were stained with Coomassie Brilliant Blue, and dried on a Whatman No. 5 filter paper. Autoradiography was carried out at −80°C for 1 day with Kodak X-omat film and Cornex Lightening-plus intensifying screen (DUPONT).
ATPase measurements
The reaction mixture for ATPase assay was as following. K+ -EDTA-activated: 1 M KCl, 10 mM EDTA, 20 mM Tris-HCl, pH 7.5 and 1 mM ATP; Ca2+-activated: 0.1 M KCl, 3 mM CaCl2, 20 mM Tris-HCl, pH 7.5 and 1 mM ATP; Mg2+-activated: 30 mM KCl, 1 mM MgCl2, 20 mM Tris-HCl, pH 7.5, rabbit skeletal muscle F-actin, 1/3 by weight to myosin and 1 mM ATP. The reaction was started by adding ATP to a final concentration of 1 mM and the mixture was incubated for 5 min at 25°C. After terminating the reaction by adding malachite green solution, inorganic phosphate was assayed as described by Kanzawa et al. [8].
SDS gel electrophoresis
An SDS gel electrophoresis was performed according to Laemmli [9] using 15% (for myosin light chain) and 7.5–18% (for myosin purification step) polyacrylamide gels. Two dimensional gel electrophoresis was carried out as described by Mikawa et al. [10]. Ca2+ binding assay was performed as described by Maruyama and Nonomura [11] after two dimensional gel electrophoresis. Stained gels with Stains-all were photographed through a red filter.
Solubility test of myosin
Sponge myosin and rabbit skeletal muscle myosin dissolved in 0.6 M KCl were dialyzed overnight against various concentrations of KCl buffered with 10 mM potassium phosphate buffer, pH 7.0. The supernatant after centrifugation for 30 min at 15,000 × g was assayed for protein concentrations. The relative solubility is given as % protein concentration relative to the one before dialysis.
Electron microscopy
Samples in 50% glycerol was sprayed onto freshly cleaved mica. The samples were rotary shadowed with platinum and carbon at 7.5°C and then with carbon at 90°C on the specimen stage of a JEOL JFD-9000 freeze-fracture apparatus. The specimen was observed under a JEOL 100S electron microscope operated at 80 kV.
RESULTS
Purification of myosin
Stored sea sponge cells were washed with a low salt buffer (40 mM KCl, 20 mM Tris-HCl, pH 7.0, 0.5 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM benzamidine and 5 mM EGTA), and centrifuged for 5 min at 10,000 rpm (TOMY, No. 4 rotor). Precipitate was resuspended in the same buffer and homogenized in a teflon homogenizer. This procedure was repeated 3 times. All the procedures were carried out at 4°C.
The washed precipitate was extracted for 30 min with an ATP solution containing 10 mM ATP, 0.3 M KCl, 40 mM Tris-HCl, pH 7.0, 1 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 5 mM benzamidine, 0.2 mM CaCl2, leupeptin, 10 μg/ml and 0.01% casein. After centrifugation for 15 min at 13,000 rpm, the supernatant was filtrated through Whatman No. 3 filter paper and diluted with 10 vol. of 0.5 mM DTT solution. The precipitate collected by centrifugation for 10 min at 10,000 rpm was dissolved in 0.15 M potassium phosphate buffer, pH 7.5 containing 0.5 mM DTT and dialyzed against the same buffer (Fig. 1c).
Fig. 1
Purification of sea sponge myosin. a, whole SDS extract; b, extract with ATP-KCl solution; c, dissolved fraction with 0.15 M potassium phosphate buffer; d, eluate of DEAE Sephadex A-50 column chromatography; e, after HPLC. Numerals show molecular mass in kilodalton.
Clarified solution by centrifugation for 30 min at 13,000 rpm was subjected to a DEAE-Sephadex A-50 column (1.5 × 10 cm) equilibrated with 0.15 M potassium phosphate buffer, pH 7.5, and 0.5 mM DTT. Crude myosin was eluted with 0.3 M KCl, 0.17 M potassium phosphate buffer, pH 7.5, and 0.5 mM DTT. The eluted fraction containing crude myosin was dialyzed against 0.5 M KCl, 2 mM ATP, 3 mM MgCl2, 20 mM Tris-HCl, pH 7.0, and 0.5 mM DTT. Clarified dialyzate was concentrated by Amicon Centriprep 30 and subjected to TOSOH HPLC using a TSK gel G-4,000SW column. The main peak was collected. The yield was approximately 1 mg starting from 40 g of sea sponge cells.
Figure 1a is an SDS extract of dissociated sea sponge cells. Figire 1b is the first ATP-KCl extract and Figure 1d is the eluate of DEAE Sephadex A-50 column. Myosin was effectively concentrated in this step. HPLC gel chromatography largely removed contaminants. Sea sponge myosin consisted of 220 kDa heavy chain and two species of light chains (Fig. 1e).
Molecular shape of sea sponge myosin
Rotary shadowed image of sea sponge myosin was conventional two headed myosin (Fig. 2a). The myosin tail length was about 160 nm like that of rabbit skeletal muscle myosin (Fig. 2b). Both myosins were not morphologically distinguishable.
Solubility of sea sponge myosin
Sponge myosin was less soluble in KCl as compared to rabbit skeletal muscle myosin (Fig. 3). In the presence of 0.25 M KCl and 10 mM potassium phosphate buffer, pH 7.0, rabbit skeletal myosin was soluble by 80%, whereas 80% of sea sponge myosin was precipitated (Fig. 3). At 0.5 M KCl, sponge myosin was maximally soluble.
Sponge myosin regulation
Two dimensional gel electrophoresis revealed that the myosin has two species of light chains (Fig. 4a). Relative molecular masses of the myosin light chains compared with chicken gizzard smooth muscle myosin light chains were approximately 21 and 18 kDa, respectively (data not shown). Figure 4b shows that the sea sponge myosin light chain was phosphorylatable by chicken gizzard MLCK. Only the basic 21 kDa myosin light chain was phosphorylated. Chicken gizzard smooth muscle myosin and sea sponge myosin light chains were stained with cationic carbocyanine dye “Stains all” after the two dimensional gel electrophoresis. Gizzard myosin 21 and 17 kDa light chains were stained blue and red, respectively (data not shown). Because of photography through a red filter the gizzard 21 kDa light chain spot was enhanced and the gizzard 17 kDa light chain spot was faded out (Fig. 5d). It is evident that the 21 kDa light chain bound to Ca2+ ion. On the contrary, in sea sponge myosin, the 18 kDa light chain was a Ca2+ binding light chain (Fig. 5c).
Fig. 4
Phosphorylation of sea sponge myosin light chains by chicken gizzard myosin light chain kinase. a, two dimensional gel electrophoresis pattern; b, autoradiogram. S21 and S18, 21 kDa and 18 kDa light chains from sea sponge myosin. IEF, isoelectricfocusing; SDS-PAGE, SDS gel electrophoresis pattern.
Fig. 5
Ca2+ binding ability revealed by staining with Stains-all after two dimensional gel electrophoresis. a, b, Coomassie Brilliant Blue stain of sea sponge and chicken gizzard myosin light chain respectively. c, d, Stains-all stain of sea sponge and chicken gizzard myosin light chain respectively. c and d are photographed though a red filter after Stains-all staining. S21 and S18 are the same as Fig. 3. G21 and G17 are 21 kDa and 17 kDa chicken gizzard myosin light chain. IEF, isoelectricfocusing; SDS-PAGE, SDS gel electrophoresis pattern.
ATPase activity of myosin
The K+-EDTA activated and Ca2+-activated ATPase activities of sponge myosin at pH 7.5 and 25°C were 0.462 and 0.075 μmol Pi min−1 mg−1, respectively. Low salt Mg2+ -activated myosin ATPase activity was only faintly enhanced by rabbit skeletal F-actin from 0.03 to 0.037 μmol Pi min−1 mg−1. After the phosphorylation of myosin light chain by chicken gizzard MLCK, actin-activated Mg2+-ATPase activity was not elevated (Table 1).
Table 1
ATPase activity of sea sponge myosin
DISCUSSION
Sea sponge was the highest multicellular organism without distinct muscle tissues. The present study clearly revealed that sea sponge has conventional myosin II. The molecular mass of the heavy chain (220 kDa) is similar to those of other muscular or nonmuscular myosin II. The myosin was less soluble in KCl at pH 7.0 as compared to rabbit skeletal myosin, and formed bipolar filaments at low salt strength (data not shown). The solubility of sponge myosin in KCl was similar to that of sea anemone myosin [8]. It appears that myosin from animals of the seawater is less soluble in KCl solution as compared to myosins from terrestrial animals and myosins from animals of the freshwater (for crayfish myosin, see ref [12]).
Sea sponge myosin contained two species of light chains of 18 kDa which showed Ca2+ ion binding ability and of 21 kDa which was phosphorylated by MLCK, although the phosphorylatable myosin light chains of chicken gizzard showed Ca2+ binding ability (Fig 5d). In Physarum plasmodia, the myosin with phosphorylatable and Ca2+ binding light chains, the actin-activated myosin Mg2+-ATPase activity is inhibited by micromolar levels of Ca2+ and the Ca2+ binding light chain plays a key role in the inhibitory regulation of myosin ATPase [3]. However, calcium ions had no influence on Mg2+ ATPase activity of sea sponge myosin in the presence of actin (data not shown). It is to be noted that actin-enhanced ATPase activity is not observed with scallop myosin [13] and insect muscle myosin [14, 15]. Any significant enhancement in actin-activated Mg2+ ATPase activity of sponge myosin was not caused by phosphorylation with MLCK and also by direct Ca2+ binding. It is desirable to do the above mentioned experiments using actin isolated from sea sponges. Table 2 summarizes the regulation and the light chains of several invertebrate myosins.
Table 2
Myosin light chains of eukaryote myosin II
