786 



7. MERCURIALS 



ible with cysteine. Muscle 3-PGDH behaves similarly but, after charcoal 

 treatment, reacts with 3 equivalents of p-MPS. If the enzyme is ultracen- 

 trifuged it carries down most of the NAD but the inhibited enzyme does 

 not, most of the NAD being free in the medium. The correlations between 

 these various events are shown in Fig. 7-12. The change in spectral absorp- 

 tion at 340 ji/m associated with NAD binding disappears progressively with 

 added p-MPS, again indicating coenzyme release. Velick was careful not to 

 assume that these results necessarily point to a binding of the NAD by 

 the SH groups, but stated that the p-MPS can sterically or electrostatically 



1.5 



p-MPS BOUND 



0.5 , 



"0 I 2 



EOUIVS p-MPS ADDED 



Fig. 7-12. Dissociation of NAD from 3-phosphogly- 

 ceraldehyde dehydrogenase by . p-MPS, and the si- 

 multaneous loss of activity. (From Velick, 1953.) 



interfere with the coenzyme binding if the SH groups are close enough to 

 the active center. Fluorometric titration of the 3-PGDH- (NADH)3 complex 

 from rabbit muscle with p-MB demonstrates NADH dissociation, but the 

 kinetics indicate that each p-MB bound weakens the coenzyme binding at 

 the other sites, so that perhaps structural changes in the protein occur 

 (Velick, 1958). The same number of equivalents of p-MB is required to 

 release the NADH from 3-PGDH- (NADH )i, 3-PGDH- (NADH)2, and 3- 

 PGDH-(NADH)3. If the displacement were due to direct competition with 

 the NADH for SH groups, one would expect the p-MB to attack the unoc- 

 cupied SH groups first, with no release of NADH, but this is not the case, 

 the release beginning immediately, as if the reaction of any SH group al- 

 tered the enzyme structure throughout. These structural changes, if they 

 occur, must be readily reversible. 



Results with a few other enzymes will be discussed briefly. Reports on 

 coenzyme displacement from lactate dehydrogenase have not been entirely 

 consistent. Apparently the state of the enzyme and particularly the source 

 are important factors. Kaplan and Ciotti (1954) found that p-MB releases 

 NAD from the liver enzyme, this being associated with a fall in absorption 

 at 300 m//, but Chance (1954) could detect no release from heart lactate 



