Yoh Okamoto

The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil α-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified1 Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP)2. Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents3. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings4,5 that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate6 and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.

Fluorescent tracer method for protein SH groups. II

Abstract An improved fluorescent tracer technique for protein SH groups is described using a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The direct measurement with a scanning fluorometer of the fluorophore-labeled proteins separated by SDS polyacrylamide gel electrophoresis achieved a simple, precise, and sensitive determination of the SH group of the order of picomoles per band. In addition, the direct recording of peaks enabled us to analyze more complex protein systems, compared with our previously reported method using an extraction step from gel slices. As an application, we compared the reactivity of the SH groups of proteins in glycerinated muscle fibers under two conditions, in rigor and in contraction.

Domain Structure of Physarum Myosin Heavy Chain

Physarum myosin is inhibited by Ca2+. Specifically its actin-activated ATPase activity (Kohama and Kendrick-Jones 1986) and its movement along actin-cables (Kohama and Shimmen 1985) are inhibited by physiological levels of Ca2+. The involvement of the Ca-inhibition in regulating plasmodial motility is suggested by the Ca-dependent contraction of demembranated plasmodial cells (Yoshimoto et al. 1981, Yoshimoto and Kamiya 1984, Achenbach and Wohlfarth-Bottermann 1986, 1986 a).