INHIBITION OF ENZYMES 



355 



of iV-ethylmaleimide; e. g., 5.5 moles of iV-ethylmaleimide/10 g^ actomyosin 

 at pH 7.5 cause activation up to 20 min but this is then followed by depres- 

 sion. It is interesting that ATP protects the ATPase activity against 

 A'-ethylmaleimide, whereas ITP increases the loss of ITPase acvitity, 

 this indicating that ATP and ITP interact differently with myosin. The 

 pyrophosphate-binding site is protected from A-ethylmaleimide by py- 

 rophosphate, ADP, and 2,4-dinitrophenol, but the ATPase site is not 

 protected (Martonosi and Meyer, 1964). Using A^-ethylmaleimide-C^*, Se- 

 kine et al. (1962) studied the kinetics of the reaction of myosin SH groups. 



60 80 100 120 



Fig. 3-4. Reactions of iV-ethylmaleimide with the SH groups 



of myosin at pH 7. The figures on the curves give the amounts 



of iV-ethylmaleimide present, the volume being 2 ml. Myosin 



SH == 2 /anoles. (From Sekine et al., 1962.) 



Native myosin reacts fairly slowly, whereas after denaturation by guan- 

 idine the rate is high (Fig. 3-4). The ATPase activity is completely lost 

 when two SH groups per subunit of myosin are reacted. iV-Ethylmaleimide 

 is more selective than the mercurials, although it reacts slower, in that 

 with mercurials half titration leads to only 70-75% inhibition. The myosin 

 treated with the labeled A^-ethylmaleimide was hydrolyzed and the pep- 

 tides examined chromatographically, and it was demonstrated that the 

 cystein residues are attacked. 



The ATPase activity associated with a fraction from ox brain (supposed 

 to be in the cell membranes and involved with ion transport) is inhibited 

 by A^-ethylmaleimide, but the Mg++-activated and Mg++— Na+— K+-acti- 

 vated components respond somewhat differently (Skou, 1963). The former 



