VOL. 4 (1950) DPN AND GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE 



167 



TABLE VI 



REACTION OF "BOUND" DPNH WITH LACTIC DEHYDROGENASE SYSTEM 



The DPN in 24 mg of glyceraldehyde phosphate dehydrogenase was first reduced by addition of 

 glyceraldehyde phosphate and arsenate. One ahquot (A) was precipitated directly with ammonium 

 sulphate, while another aliquot (B) was first exposed to 0.024 M iodoacetate before being precipitated 

 with ammonium sulphate. The precipitates were separated by centrifugation, dissolved in cysteine- 

 pyrophosphate buffer, pH 8.3, and pyruvate (i-io-^ M/ml) was added. The reaction was started by 

 the addition of a catalytic amount of lactic dehydrogenase. To reaction mixture (C) free DPNH was 

 added in place of glyceraldehyde phosphate dehydrogenase containing bound DPNH. 



in rate was not observed leaves one with two alternatives. Either DPNH is more highly- 

 dissociated than has been assumed or lactic dehydrogenase can react with bound 

 DPNH. The latter alternative would involve collisions between protein molecules which, 

 from a kinetic standpoint, is not incompatible with a rapid rate of reaction. 



In a final experiment we tried to see whether the lactic dehydrogenase system could 

 reduce the DPN bound to the enzyme. The reaction mixture contained 4 mg of glycer- 

 aldehyde phosphate dehydrogenase per ml as a source of DPN, sodium lactate, cyanide 

 (to trap the pyruvate formed) and a catalytic amount of lactic dehydrogenase. The 

 bound DPN was reduced at a good rate as soon as the lactic dehydrogenase was added. 

 The considerations mentioned above when the reverse reaction was discussed apply 

 here as well. 



IODOACETATE 



Iodoacetate (0.004 M) completely inhibited the reduction of enzyme DPN by sub- 

 strate. An enzyme solution of about 8 mg of protein per ml was prepared with pyro- 

 phosphate buffer at pn 8.4 containing no cysteine. Five minutes at 25° was allowed for 

 reaction with iodoacetate before glyceraldehyde was added. A suitable control without 

 iodoacetate was run simultaneously. This was necessary because the enzyme loses 

 activity quite rapidly in the absence of cysteine. No enzymatic activity could be detected 

 in the presence of iodoacetate. Whether or not a differentiation of two catalytic sites 

 is possible by means of addition of smaller concentrations of iodiacetate has not been 

 tried. 



In experiment B, Table V, iodoacetate was added after the DPN bound to the 

 enzyme had been reduced. The object was to see whether the inactivation of the enzyme 

 by iodoacetate would influence the rate of reaction of bound DPNH with the lactic 

 dehydrogenase system. As shown in Table V no difference could be detected. 



This paper is presented as a token of esteem for the numerous scientific contributions 

 of Otto Meyerhof. 



References p. i6g. 



