Studies on the Electrophoretic Mobility of Troponin in the Presence of Ca 2+,Mg 2+,and (Mg-ATP) 2− 1

Abstract

The regulation of actomyosin contraction by low concentrations of Ca²⁺ (∼10^–5 M) involves the participation of troponin, tropomyosin, Mg²⁺ and ATP³ (1–5). The precise mechanism of action of the “Ca sensitizing factor” (troponin, tropomyosin) is unknown. Under certain experimental conditions however, the binding of 10^–6–10^–5 M Ca²⁺ to the myofibrils initiated contraction (6). Fuchs and Briggs (5) have shown conclusively that troponin is the only known myofibrillar protein which bound Ca²⁺ if the concentration of this ion was 10^–5 M. The removal of Ca²⁺ from a myofibrillar binding site and/or its replacement by Mg²⁺ was shown to occur in the relaxed state (2). Kaminer found that the “Ca sensitizing factor” required the presence of equimolar quantities of ATP and Mg²⁺ (5 mM) to produce maximal effect on actomyosin (4). In view of all these results, it appeared to us that troponin may be the only known site on the myofibrils suitable for a Ca²⁺–Mg²⁺ exchange during the contractile event. For this reason we have investigated in a rather qualitative fashion whether or not troponin is able to bind Mg²⁺.

If a divalent metal is bound to an ionized carboxyl group of a protein, it will change the charge of this group from (COO)^– to (COOMe)⁺. For this reason it is likely that the net charge of this protein will also change. In the subsequently described experiments we have studied the net surface charge alterations on troponin in the presence and absence of Ca²⁺, Mg²⁺, ATP^4–, and (Mg–ATP)^2–. The electrophoretic mobility was used as a qualitative or perhaps semiquantitative indicator of the surface charge on this protein.

Materials and Methods. The sodium salt of ATP was purchased from Sigma Co.; all other reagents were of reagent grade. Troponin was prepared and tested as described by Ebashi (3). Electrophoretic experiments were carried out in a microelectrophoretic apparatus supplied by Arthur Thomas Co. as described in an earlier paper (7).