MITOCHONDRIAL LIPIDS AND THEIR FUNCTIONS IN THE RESPIRATORY CHAIN 1 83 



shaking with an organic solvent was due principally to small amounts 

 of the solvent retained in the enzvme suspension acting as a physical 

 inhibitor. Removal of this residual solvent by physical means, e.g. dis- 

 persion with a surface-active agent, gave complete restoration of enzymic 

 activities. These findings, which have since been confirmed by others [8, 

 9], make it necessary to be extremely cautious in evaluating the results of 

 extraction-reactivation experiments. Thus inactivation of an enzvme svstem 

 due to an inhibition by the solvent must be clearly distinguished from 

 inactivation brought about by the removal of lipid essential for some 

 structural or functional role. 



In a study of the effect of removal of lipid on enzyme activities, pig 

 heart-muscle preparations were extracted with organic solvents by Nason's 



TABLE III 



KXTRACTION OF A PlG HeART-MuSCLE PREPARATION WITH DlETHVL EtHER 



Number 



of 



extractions 



Succinic 



oxidase 



{% original 



activity) 



Succinic- 

 cytochrome c 

 reductase 

 ( "1, original 

 activity) 



L hicjuinone 



extracted 



(",, total 



extractable 



ubiquinone) 



Lipid 

 extracted 

 (",, total 



lipid) 



100 



106 

 75 

 63 

 50 



100 

 73 

 63 

 61 



54 



50 

 78 

 79 



96 



o 

 I 



7 

 II 



16 



I ml. preparation (35 mg. proteinnil.) extracted successively with 5 ml. 

 peroxide-free diethyl ether for 1-5 min. Extracts washed with water, dried and 

 solvent evaporated. Lipid residue weighed. Lipid dissolved in 40-60 light petrol- 

 eum and ubiquinone separated and determined as described by Pumphrey and 

 Redfearn [i]. Enzyme activity determined by the methods described by Redfearn 

 it al. [11]. 



technique and the residual soh ent remo\ed by incubating the suspension 

 in a Warburg manometer until solvent evolution had ceased. In this way, 

 the effects of extraction could be studied without the additional complica- 

 tion of the inhibitory effects of the solvent itself. In experiments using 40- 

 60 light petroleum as the solvent it was found that one or two extractions 

 produced marked increases in the succinic oxidase and cytochrome oxidase 

 activities (Table II). The amount of total lipid removed from the particles 

 appeared to be small and less than 50",, of the total extractable ubiquinone 

 was removed even after forty successive extractions. 



With diethyl ether, the endogenous ubiquinone of heart-muscle 

 preparations could be extracted much more effectively. The results of an 

 experiment are shown in Table III. 96",, of the total extractable ubiquinone 

 was removed after eight extractions with ether; the succinic oxidase and 



