PYRIDINE NUCLEOTIDES IN MITOCHONDRIA 219 



series of eight such experiments with rat-Hver mitochondria. On the 

 average, there was httle change in the DPX ^ content while the DPNH 

 declined. As a consequence, the (DPN++DPNH) content declined in 

 every experiment, by an average of 0-73 jumoleg. protein. The amount 

 of total diphosphopyridine nucleotide which disappeared varied con- 

 siderably from experiment to experiment — between o • 2 and i • 5 /xmoles/g. 

 protein. It exceeded 0-4 ^nmole g. protein in six of the eight experiments. 



Time (min) | Time {mini 



Clu + ADP(-P,) +DNP Clu + ADP(-P,) + DNP 



Fig. 2. DPN + and DPNH contents of rat-liver mitochondria after incubation 

 with glutamate in the absence of P; and after adding dinitrophenol. 



A. A set of eight experiments, in which the fluorimetric enzymic assay was 

 used. The first series of vertical lines show the range of values for the DPN +, 

 the DPNH and the DPN - + DPNH contents of the fresh mitochondria. The 

 points show the individual values. At zero time the mitochondrial suspension was 

 added to a mixture containing 13-3 mM KCl, i -8 mM EDTA, 13 5 mM glucose, 

 4-5 mM MgCl.2, 9 mM L-glutamate, o-8 mM ADP, 38-7 mM nicotinamide, 0-15 M 

 sucrose (derived from the mitochondrial suspension) and hexokinase. The final 

 concentration of mitochondrial protein was about 12 mg./ml. After 15 min. in a 

 manometer flask the suspensions were analyzed for DPN ~ and DPNH. The values 

 are shown on the second set of vertical lines. Duplicate flasks were treated in the 

 same way, until at 15 min. o- i-i mM dinitrophenol was added from the side-arm. 

 After a further 5 min. the DPN + and DPNH contents of these suspensions were 

 determined. The values are shown in the third set of vertical lines. The full lines 

 connecting these vertical lines show the changes in the mean values of DPN +, 

 DPNH and DPN ^ + DPNH. The discontinuous lines show the changes in 

 (DPN * + DPNH) in the individual experiments. 



B. A set of four experiments, in which the spectrophotometric enzymic method 

 was used. Conditions and reaction mixture as in A, except that a concentration of 

 about 18 mg. mitochondrial protein /ml. was used. The full lines connect the mean 

 values of DPN + and DPNH. The discontinuous lines show the change of DPN + 

 + DPNH in each experiment. One of the DPN + values after dinitrophenol is 

 probably a mistake. The dotted line is drawn to the mean obtained by ignoring 

 this value. 



