METABOLIC CONTROL OF STRUCTURAL STATES OF MITOCHONDRL\ 87 



instances, kinetics of scattering changes were measured simultaneously 

 with the utihzation of oxygen by means of the vibrating platinum electrode. 

 The electrode was placed in the cuvette and employed in a manner similar 

 to that described bv Chance and Williams [8]. Mitochondria were isolated 

 in sucrose-Versene by the technique of Cleland and Slater [9] and sus- 

 pended in a medium of o-i m sucrose fortified with 0-025 ^^ "tris" 

 buffer at pH 7-5. An increase in light-scattering indicates shrinking. 

 Addition of reducing equivalents in the form of glutamate gave a rapid 

 shrinkage which terminated in a steady state after a few seconds. As 

 electron transport bv tightly coupled mitochondria requires phosphate, 

 very little respiration of glutamate was recorded at this time; this is 

 denoted bv the figure o-oi which refers to the calculated rate of oxygen 

 utilized in /xM sec. Adding phosphate augmented respiration sevenfold, 

 and also initiated swelling which continued over several minutes before 

 reaching a steadv state. The reverse experiment of adding phosphate in the 

 absence of substrate does not result in swelling. Thus phosphate is unable 

 to induce swelling in the absence of reducing equivalents interacting 

 with the electron transport chain. The dependence of swelling on electron 

 transport has been widely reported on by Chappell and Greville [10] 

 and Hunter et al. [11]. Initiation of phosphorylation in the mitochondrial 

 suspension bv the addition of ADP results in acceleration of respiration 

 and a rapid shrinkage. When ADP is converted into x-lTF, respiration 

 declines, and the scattering state returns to that found prior to the brief 

 cycle of phosphorylation. Synthesis of ATP under these conditions 

 apparentlv has not given rise to a net change in mitochondrial volume. 

 When Dr. Chance and I [12] first observed this rapid reversal of swelling 

 by ADP, and the relative lack of eff'ectiveness of ATP under these con- 

 ditions, we suggested that energy-linked intermediates may be more 

 efi^ective than ATP itself. Implications for the role of intermediates in 

 the control of this phenomenon have been reported by others, for example, 

 Ernster [13] and Lehninger and associates [5], employing diflPerent systems. 

 Again returning to the experiment, after some lapse of time, the dissolved 

 O2 of the system is exhausted and at this point a reversal of swelling occurs 

 which, if allowed to continue in this record, would have reached the level 

 seen in the presence of ADP. This effect has been termed autonomic 

 reversible swelling by Beechey and Holton [14]. The experiment suggests 

 how fluctuations in the concentration of the reactants of the respiratory 

 system are capable of controlling the state of mitochondrial volume 

 [i:^, 16], as the ability of these substances to elicit changes in volume 

 follows closely their effects upon the respiratory chain on a concentration 

 basis. Thus the half-maximal value for activation of respiration by ADP 

 and phosphate is about 50 |UM and i mM respectively [17] and they have a 

 half-maximal effect on shrinkage or swelling at the same concentrations. 



