Light Chains of Myosins from White, Red, and Cardiac Muscles

Abstract

Purified preparations of rabbit skeletal white, red, and cardiac muscle myosin (WM, RM, and CM) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences in both the molecular weights and number of light chains in these myosins were found. WM has three distinct light-chain components (LCiw, LC2W, LC3W) having molecular weights of 25,500, 17,400, and 15,100, respectively (29), extensively used for the separation of various proteins and the determination of their molecular weights, we decided to apply this method to a reinvestigation of the light chains of rabbit WM, RM, and CM. Our results clearly show that both quantitative and qualitative differences exist between light chains in the different types of muscle; these will be discussed in terms of the isozymic nature of myosin. MATERIALS AND METHODS Preparation of myosinRabbit soleus, semitendinosus, crureus, and intertransversarius (red muscles); the outer layers of vastus lateralis and adductor magnus (white muscles); and heart ventricle (cardiac muscle) were used for the preparation of myosin (8). Myosin was further purified by column chromatography on DEAE-Sephadex A-50, essentially by the methods of Richards et al. (30). 150-200 mg of myosin, dissolved in a solution containing 0.04 M Na4P207 (pH 7.5), 0.1 mM EDTA, and 0.1 mM dithiothreitol (buffer I), were applied to a column (2.5 X 35 cm) of DEAE-Sephadex A-50 equilibrated with the same buffer. The column was then washed with 2-3 column volumes of buffer I. Under these conditions myosin remained on the column, but other proteins (5-10% of the input) were eluted. A linear gradient of 0-0.5 AI NaCl in buffer I was then applied. Myosin was eluted as a sharp single peak at 0.13-0.15 M NaCl and concentrated by precipitation either at low ionic strength after dilution or with (NH4)2SO4 at 50% saturation. Myosin was finally dissolved in a solution containing 0.01 M sodium phosphate (pH 7.0), 0.5 M NaCl, 0.1 mM EDTA, and 0.1 mM dithiothreitol (buffer II). For the determination of ATPase activities of column-purified myosin, K4P207 and KCl were used in the elution buffers for chromatography and myosin was finally dissolved in a solution containing 0.5 M KCl, 1 mM EDTA, and 10 mM Ntris(hydroxymethyl) methyl-2-aminoethane sulfonic acid (TES), pH 7.0.Typical

Differences in maximum velocity of shortening along single muscle fibres of the frog.

“…Much interest has been directed recently to the substructure of myosin and its possible role in determining the kinetics of the actin-myosin interaction. Myosins from 'fast' and 'slow' mammalian muscles can be distinguished on the basis of their light chain composition (see Gazith, Himmelsfarb & Harrington, 1970;Weeds & Lowey, 1971;Sarkar, Sreter & Gergely, 1971;Weeds, Hall & Spurway, 1975;Weeds, 1976). Furthermore, changes of the light chain pattern have been found to modulate both the maximum speed of shortening of skinned muscle fibres (Moss, Giulian & Greaser, 1983) and the actin-activated ATPase activity of homogeneous myosin subfragment-1 preparations in vitro (Wagner & Weeds, 1977).…”

Effect of cross-reinnervation on physiological parameters and on properties of myosin and sarcoplasmic reticulum of fast and slow muscles of the rabbit.

“…Protein concentration was determined by a microbiuret method (Itzhaki and Gill, 1964). Na dodecyl-SO4 gel electrophoresis was carried out as reported earlier (Sarkar et al, 1971) except that 100-mm gels were used in 115-120 X 5-mm glass tubes.…”

“…Myosin was extracted from the sedimented myofibrils with 5 vol of cold KCIpotassium phosphate, pH 6.5 (0.3 M KC1, 0.1 M KH2PO4, 5 mM EDTA, and 5 mM ATP) for 10 rain and further purified essentially as described earlier (Sarkar et al, 1971).…”

John Gergely (1919–2013): a pillar in the muscle protein field

“…Major contributions were also made to our understanding of tissue and fiber type differences in myosin light chains (Sarkar et al 1971) and in myosin during muscle development (Sréter et al 1975). In addition, John also collaborated with Noriaki Ikemoto on several papers related to the structure and function of the sarcoplasmic reticulum (see, for example, Ikemoto et al 1974).…”