PYRIDINE NUCLEOTIDES IN MITOCHONDRIA 221 



we found \'ery similar results with glutamate as substrate whether lack of 

 Pj or ADP was responsible for the inhibition of respiration (see Table VIII). 

 In the controlled state, the degree of oxidation of the pyridine nucleotides 

 is probably largely controlled by equilibria catalyzed by the DPN-specific 

 dehydrogenases, such as 



glutamate + DPN + = a-ketoglutarate + NH+ + DPNH 



Adding dinitrophenol to a mitochondrial suspension in the presence of 

 glutamate and absence of P, causes about a 3-fold increase in the rate of 

 Oo uptake [23]. This is presumably due to activation of DPNH oxidation, 

 with a consequent increase in the rate of oxidation of glutamate to a-keto- 

 glutarate. Further oxidation of the a-ketoglutarate cannot occur at an 



TABLE VIII 



diphosphopyridlne nucleotide compounds of r.at-l1ver mitochondria in 



Controlled State 



Glutamate substrate. The P-deficient medium was the same as that in Fig. 

 2. The ADP-deficient medium contained 15 mM KCl, 5 mM MgCl,, 30 mM 

 nicotinamide, 40 mM tris(hydroxymethyl)aminomethane — HCl buffer, pH 7-4, 

 40 mM potassium phosphate, pH 7 -4. 2 mM EDTA, o • i mM ADP, 0-12 M sucrose, 

 30 mM glutamate. Single experiment (fluorimetric). Values in fimoles/g. protein. 

 Unpublished experiment of M. Bailie. 



State of mitochondria DPN + DPNH " Extra DPN "* 



Fresh 1-51 2-30 0-54 



In Pj-deficient medium 1-72 i-8q 0'74 



In ADP-deficient medium i-8.S 1-83 0-64 



* Determined as in Table \\. 



appreciable rate, because P, is necessary for the substrate-linked phos- 

 phorylation step of a-ketoglutarate oxidation even in the presence of 

 dinitrophenol [2:;, 26]. The marked decrease in the DPNH concentration 

 shown in Fig. 2 is to be expected. 



In other experiments, not shown in Fig. 2, respiration was fully 

 activated (sevenfold) by adding P, instead of dinitrophenol. This not only 

 activates DPNH oxidation, but also DPN ^ reduction by a-ketoglutarate 

 and by malate (the oxidation of malate to oxaloacetate is involved in the 

 oxidation of glutamate bv mitochondrial preparations [27]. In fact, the 

 sevenfold stimulation of the respiratory rate was accompanied by very 

 little change in the degree of oxidation of the diphosphopyridine nucleotide 

 (means of four experiments : J DPN *, +0-35 /u.mole/g. protein ; J DPNH, 

 — 0-21 |Limole/g. protein). Thus, under the conditions of our experiments, 

 the diphosphopyridine nucleotide of rat-liver mitochondria oxidizing 

 glutamate in the presence of Pj and ADP is about 50" o reduced. This is 



