288 W, W. Wainio 



to the membrane of the particle (Siekevitz and Watson, 1956). The insoluble 

 nature of the particle permitted Battelli and Stern (1912) to separate it from 

 the cell. Keilin and Hartree (1938b) modified the method of preparation so 

 that the grossly impure enzyme could be obtained in a high yield from heart 

 muscle where it occurs in a high concentration. 



It has been suggested by many investigators that the insolubility of the 

 enzyme may be due to its association with the lipids of the mitochondrion. 

 In fact it has been further suggested that the oxidase may be a lipoprotein 

 complex, because approximately 35% of the mitochondrion is lipid (Bensley, 

 1937) and because bile salts are needed to solubilize the enzyme (Yakushiji 

 andOkunuki, 1941; Straub, 1941). 



The soluble preparations that are being studied today are those that are 

 made with either cholate or deoxycholate. It has been clearly shown that the 

 solubility of this insoluble enzyme depends on the continued presence of a 

 solubilizing agent. Smith and Stotz (1954) reported that the solubihty of their 

 preparation is dependent on the presence of both the cholate, and the 

 ammonium sulphate used in the purification. Kremzner and Wainio (1955) 

 found that a complete removal of the deoxycholate from the preparation 

 '2-3' by ion exchange resins led to a precipitation of the enzyme, but not to its 

 denaturation. 



Among the soluble preparations of cytochrome c oxidase available for 

 study is the preparation of Yakushiji and Okunuki (1941) as modified by 

 Okunuki et al. (1958). However, these authors do not report a lipid analysis. 

 The preparation '2-3' of Eichel, Wainio, Person and Cooperstein (1950) was 

 analysed by Kremzner (1956), who found 45% of total lipid. A modified 

 preparation '2-3', made by first washing the insoluble heart muscle particles 

 with 20% methanol, was still active and contained only 19% of total lipid. 

 The lipids in both preparations were predominantly phosphatidylcholine, 

 and phosphatidylethanolamine. This finding has been verified and extended 

 by Marinetti, Scaramuzzino and Stotz (1957), who reported that the soluble 

 preparation of Smith and Stotz (1954) contains 14-7% of phospholipid 

 (primarily phosphatidylcholine and phosphatidylethanolamine), 12-8% of 

 neutral fat, 1-02% of free cholesterol and 3-12% of unidentified lipids. 

 Green (1958) states that the soluble preparation of Mackler and Penn (1959) 

 contains about 15% of lipid. 



Marinetti, Erbland, Kochen and Stotz (1958) have more recently extended 

 their chromatographic analysis of the phosphatides and have found again 

 that phosphatidylcholine (33-4 % of the total hpid P) and phosphatidylethanol- 

 amine (19-6 % of the total) were the principal phosphatides. There was 8-2 % 

 of phosphatidylserine, 13-6% of a component tentatively identified as poly- 

 glycerophosphatide, 5-1% of unidentified phosphatides, and traces of lyso- 

 lecithin and lysocephalin. Two compounds distinguished the lipids of the 

 oxidase preparation from those of a purified cytochrome b — c^ preparation 



