32 1. lODOACETATE AND lODOACET AMIDE 



other explanations. It is possible, although perhaps not likely, that iodo- 

 acetate replaces some of the mercurial from the SH groups; since glu- 

 tathione reactivates quite rapidly, the mercurial cannot be very firmly at- 

 tached. Also the mercurial might not have reacted with all the SH groups 

 on the enzyme, although enough to inhibit the activity, so that iodoacetate 

 by reacting with the remaining groups could alter the enzyme so that it 

 had lost some activity even when the mercurial was removed. Finally, it 

 may be that excess iodoacetate present reacted with the glutathione, there- 

 by reducing its ability to reactivate the enzyme. Hirade and Hayashi (1953) 

 found disappearance of SH groups and inhibition by iodoacetamide to pro- 

 ceed in parallel fashion with rat liver enzyme, but for some reason obtained 

 only 21% inhibition and 11% of the SH groups reacted with 170 m.M inhi- 

 bitor; they do not state how long the reaction ran. 



Ribonuclease 



Zittle (1946) found that pancreatic ribonuclease is inhibited slowly and 

 moderately by iodoacetate and iodoacetamide, and felt that this probably 

 indicated the presence of SH groups. However, he was troubled by the fact 

 that iodoacetate inhibits faster and better than iodoacetamide, contrary to 

 the usual experience with other enzymes, but felt that other explanations 

 must be tested before assuming that groups other than SH are reacted. 

 We now know that ribonuclease possesses no SH groups. A number of 

 workers have found iodoacetate to have little or no action on ribonucleases: 

 2-10 mM on pancreatic enzyme (Davis and Allen, 1955; Rabinovitch and 

 Barron, 1955; Dickman et al., 1956), 100 mM on Bothrops venom enzyme 

 (Taborda et al., 1952 a,b), up to 30 mM on the tobacco leaf enzyme (Frisch- 

 Niggemeyer and Reddi, 1957), and up to 48 mM on Bacillus subtilis en- 

 zyme (Watanabe and Yamafuji, 1961). Indeed, several workers have ob- 

 served a stimulation of the activity: thus the ribonuclease in Taka-diastase 

 is stimulated 20% by 10 mM iodoacetate (Naoi-Tada et al, 1959). The 

 enzyme from ascites tumor cells is first stimulated at low concentrations, 

 reaching a peak near 0.001 mM, and is then depressed (Fig. 1-1) (Hilz and 

 Klempien, 1959). Other SH reagents may also stimulate. Koth (1956, 1958) 

 believed the stimulation might arise from the inactivation by the SH reagent 

 of a naturally occurring inhibitor present in unpurified preparations. It 

 appears in any event that, under the usual conditions, iodoacetate has very 

 little effect on ribonuclease, and probably would not inhibit it in cellular 

 preparations at the concentrations used to block glycolysis. 



The interest in the effects of iodoacetate on ribonuclease come from studies 

 done not under the usual physiological conditions but at relatively high 

 iodoacetate concentrations and over a wide range of pH. When 30 mg of 

 ribonuclease and 30 mg of iodoacetic acid in 10 ml are allowed to react at 

 40o, and amino acid analyses of the enzyme are then made, it is found that 



