l6o BIOCHIMICA ET BIOPHYSICA ACTA VOL. 4 (1950) 



THE COMBINATION OF DIPHOSPHOPYRIDINE NUCLEOTIDE WITH 

 GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE 



by 



CARL F. CORI, SIDNEY F. VELICK, and GERTY T. CORI 

 Department of Biological Chemistry, Washington University, School of Medicine, St. Louis, 



Missouri {U.S.A.) 



It has been shown in a previous paper^ that glyceraldehyde phosphate dehydrogenase 

 from rabbit muscle contains one mole of diphosphopyridine nucleotide (DPN) per 

 50000 g of protein. This ratio did not change after prolonged dialysis against distilled 

 water or after repeated recrystallizations from ammonium sulphate solutions. When an 

 aqueous solution of the enzyme was treated with activated charcoal (norit) and filtered, 

 DPN was removed. Addition of an excess of DPN and ammonium sulphate to the DPN- 

 free enzyme solution resulted in the formation of crystals which contained the original 

 ratio of DPN to protein. From these and other observations it was concluded that the 

 enzyme contained firmly bound DPN. The fact that DPN could be removed with norit 

 made it clear that the union between enzyme and coenzyme was not through a covalent 

 bond. 



Earlier work^ had indicated that the dissociation constant of the enzyme with 

 DPN, as estimated from the concentration of DPN at which the reaction with glycer- 

 aldehyde phosphate occurred at half maximal velocity, was of the order of 4- io~^ M/ml. 

 This agreed with a value obtained by Warburg and Christian^ with yeast enzyme 

 and free glyceraldehyde as substrate. According to existing criteria the constant so 

 obtained is sufficiently large to permit easy separation of enzyme and coenzyme by 

 dialysis or recrystallization. The fact that such a separation was not observed suggests 

 either that the enzyme combines with DPN at two sites, one of which binds DPN more 

 firmly than the other, or that the conclusions drawn from the kinetic measurements or 

 from dialysis and recrystaUization are not valid. 



In the present paper experiments are described in which some aspects of the two- 

 site hypothesis are tested. In order to make reactions of bound DPN measurable in a 

 I cm cell at 340 m^a in the Beckman spectrophotometer, it is necessary to use enzyme 

 concentrations of 2 to 4 mg per ml which are about 1000 times greater than those 

 necessary to give good rates with added DPN and glyceraldehyde phosphate. Accord- 

 ingly the reaction with glyceraldehyde phosphate is too rapid for convenient study, 

 unless one works at a pn far from the optimum. When glyceraldehyde is used as sub- 

 strate, however, the reaction rate is conveniently measurable over a wide range of 

 conditions, the slower reaction being due, as will be shown, to a low affinity of glycer- 

 aldehyde for the enzyme. 

 References p. i6g. 



