INHIBITION OF ENZYMES 345 



residue. When the enzyme and xV-ethylmaleimide are incubated alone, 

 no inhibition occurs, but when NADPH is added, inhibition occurs pro- 

 gressively; when iV-ethylmaleimide and NADPH are added together, in- 

 hibition is rapid and marked (80-90% in 10 min from 0.02 mM) (Mapson 

 and Isherwood, 1963). There is no reaction of .V-ethylmaleimide with the 

 NADPH but with an enzyme group evoked by the NADPH. 



iY-Ethylmaleimide has been used with varying success to titrate enzyme 

 SH groups. The first work of this type was by Krimsky and Racker (1954) 

 in efforts to demonstrate the presence and function of glutathione in glyc- 

 eraldehyde-3-P. dehydrogenase. Tryptic digestion of the enzyme releases 

 glutathione but if the enzyme is pretreated with iV-ethylmaleimide the 

 yield of glutathione is very low, indicating that the bound glutathione 

 has reacted with the reagent. Thetin-homocysteine methylferase is in- 

 hibited progressively by A'-ethylmaleimide and simultaneously there is a 

 reduction in the number of free SH groups (Durell and Cantoni, 1959). 

 The activity decreases more rapidly than the free SH groui)s, however, in 

 contrast to the results with p-MB, and this could be due either to a sec- 

 ondary structural alteration of the enzyme or to the fact that all the SH 

 groups are not involved in the enzyme activity. Microsomal cytochrome c 

 reductase reacts rapidly with A-ethylmaleimide and is completely inactivat- 

 ed when two SH groups have disappeared, a third SH group reacting more 

 slowly (P. Strittmatter, 1959). One of the two reactive SH groups can be 

 protected by NADH. Ficin also reacts completely with A"-ethylmaleimide 

 at pH 6.8, one SH group being accessible in the native enzyme and two 

 SH groups in the denatured enzyme (Liener, 1961). Sweet potato /^-amylase 

 has four SH groups which react readily with A'-ethylmaleimide at pH 8 

 but do not at pH 4.5 (Speck et al., 1961). Reaction of one SH group leads 

 to 50% inhibition, so that one critical SH group may be involved, but the 

 inhibition never becomes complete even with reaction of all the SH groups. 

 They followed the reaction by disappearance of A'-ethylmaleimide and also 

 by determination of *S-cysteinosuccinate in the hydrolyzates of the enzyme, 

 and since these data always checked it seems likely that only SH groups 

 are attacked. The some seven SH groups of lactate dehydrogenase, however, 

 do not react at all with A^-ethylmaleimide (Pfleiderer et al., 1958). Sum- 

 marizing the titration work, one must conclude that A'-ethylmaleimide has 

 proved itself to be a rather effective agent, especially when used in con- 

 junction with other SH reagents. 



Effects on ATPase and Actomyosin 



The SH groups of actin are 40-50% titrated with A'-ethylmaleimide but 

 the reactivity is somewhat less than with myosin (Tsao and Bailey, 1953). 

 If actin is treated with 2.5 mM A'-ethylmaleimide for 20 min the ability 

 to undergo the G-actin — >■ F-actin transformation is not imi)aired, although 



