Modification of Cardiac and Smooth Muscle Myosins with 2, 4, 6-Trinitrobenzenesulfonate

Evidence for Differences in Structure around the Active Sites of Cardiac, Smooth, and Skeletal Muscle Myosin ATPase

Abstract

Myosins purified from cardiac (porcine heart) and smooth (chicken gizzard) muscles were modified with 2, 4, 6-trinitrobenzenesulfonate (TNBS) and the effects on the kinetic properties of myosin ATPase [EC 3.6.1.3] were studied. The following results were obtained.
1. About 0.5mol of TNBS per mol of myosin head was incorporated rapidly, irrespective of the presence of PP₁ (2mM), into both types of myosin studied.
2. The size of the initial burst of P₁ liberation for both myosins was found to be 0.5-0.6 mol/mol head.
3. The rapid incorporation of TNBS into cardiac muscle myosin was accompanied by a rapid decrease in the size of the initial P₁ burst, and it was completely lost after modification for 20 min. However, smooth muscle myosin retained its P1 burst.
4. The EDTA (K⁺)-ATPase activity of both myosins modified in the presence or absence of PP₁ decreased sharply with incorporation of TNBS.
5. Superprecipitation and ATPase activity of reconstituted actomyosin from cardiac myosin and skeletal F-actin decreased only after 10min of modification with TNBS in the absence of PP₁.
6. The spectra of TNP bound to myosins from cardiac and smooth muscles were unchanged by the addition of PP₁.
The above findings are compared with those previously obtained for skeletal muscle myosin [Miyanishi, T., Inoue, A., & Tonomura, Y. (1979) J. Biochem. 85, 747-753], and the structural and functional differences among the myosins derived from skeletal, cardiac, and smooth muscles are discussed.