118 Britton Chance 



after addition of ethanol, there is a further downward sweep 

 of the spectrophotometric trace, indicating further reduction 

 of cytochrome b. The increased absorbancy at 430 mfx is 

 suggestive of a transition in the mitochondria of state 3 to 

 state 4. There is a corresponding decrease of respiratory rate 

 from 2 • 1 to 1 • 5 (xM Og/sec. This is not a very large decrease, 

 and is in agreement with other considerations that indicate 

 that the yeast cell may not represent a "tightly coupled" 

 system. The possibility that respiration was slackened due to 

 lack of substrate is disproved by the fact that cytochrome 

 becomes more reduced, indicating, if anything, an increased 

 substrate concentration. In view of the foregoing discussions, 

 this effect could be attributed to a number of control sub- 

 stances: a decrease of ADP or phosphate concentration, an 

 increase of magnesium concentration, or a decrease of any 

 physiological uncoupling substance such as calcium, mito- 

 chrome, lipid, etc. 



Evidence for the type of substance exerting respiratory 

 control over the mitochondria of the yeast cell is afforded by 

 both the spectroscopic and the respiratory responses of the 

 cell suspension to glucose addition (Fig. 19). The rapid up- 

 ward deflexion of the spectrophotometric trace to the level 

 obtained a few seconds after adding ethanol indicates that 

 whatever control substance was depleted a minute after the 

 addition of ethanol has been restored. This is supported by the 

 fact that the respiration increases to a value somewhat above 

 that obtained shortly after ethanol addition (2-3 [xm Og/sec). 



It is clear, from the known pathway of glucose metabolism 

 of the yeast cell and from the great rapidity with which its 

 respiration is accelerated upon glucose addition, that ADP 

 produced in glucose phosphorylation has reactivated the 

 respiratory chain. An increase of substrate concentration is 

 eliminated by the spectroscopic response, indicating an 

 oxidation of cytochrome b. This type of spectroscopic change 

 could only be caused by a decrease of substrate concentration. 

 An increase of phosphate concentration is unlikely, since the 

 primary reaction involves the formation of ADP, phosphate 



