102 III. OXIDATION AND METABOLISM 



tive intermediate in acetoacetate formation and in citrate synthesis is a 

 two-carbon compound. ^^^ Lehninger^ later postulated that the two-carbon 

 unit in question is acetyl-CoA as isolated by Lynen et al.^*- In outlining the 

 requirements of the fatty acid oxidase system from mitochondria, Leh- 

 ninger^^^ recorded, in 1952, that Mg++, ATP, an optimal concentration of 

 electrolytes or of non-electrolytes such as sucrose, and a priming metab- 

 olite, possibly a &ebs citric acid cycle intermediate, which is necessary for 

 producing oxidative phosphorylation, are the essential components. Leh- 

 ninger suggests that an asymmetric acetoacetate may be formed from the 

 fatty acid, probably due to the formation of more than one molecular 

 species of active acetyl from the fatty acid chain. Lehninger' reviewed the 

 subject of enzymic oxidation of fatty acids through 1951, and Lynen^^* 

 summarized the role of CoA in fatty acid oxidation. 



a'. Conditions Affecting Stability: Although the system described by 

 Lehninger was considerably more stable than that of Munoz and Leloir^^ 

 it was destroyed when attempts were made to purify it further. It was 

 stable for some hours at 0°C., but decomposed within twenty minutes 

 when incubated in the absence of sul)strate and ATP. The optimum pH 

 was found to be 7.4. Lehninger^ noted that oxidation of fatty acids by this 

 enzyme was strongly inhibited by fluoride, calcium ions, 2,4-dinitrophenol, 

 methylene blue, arsenate, arsenite, as well as by certain phenyl-substituted 

 fatty acids. High concentrations of fatty acids, especially of the long-chain 

 members, were shown to inhibit the activity of the enzyme, presumably by 

 their surface effects. ^-^^^ Optimum concentrations of acids in the substrate 

 were as follows: butyrate, 0.01 AI; octanoate, 0.001 M; and palmitate, 

 0.00025 M. 



W . The Nature of the Particulate Matter: Potter ^^^ was of the opinion 

 that the activity of the fatty acid oxidase system was dependent upon the 

 presence of unbroken cells in the particulate preparations, especially be- 

 cause of the sensitivity of the system to changes of tonicity. ^'^^ However, 

 Lehninger* did report that the oxidase system could withstand exposure of 

 the particles to distilled water for short periods, but it was necessary to re- 

 store approximate isotonicity to obtain oxidation. Moreover, no intact 

 liver cells could be observed microscopically. 



In later work, the liver homogenates were fractionated according to the 



"2 F. Lynen, E. Reichert, and L. Rueff, Ann., 574, 1-32 (1951). 



1^3 A. L. Lehninger, Biochem. J., 51, xv-xvi (1952). 



1" F. Lynen, Harvey Lectures, 48, 210-244 (1952-1953). 



i« V. R. Potter, J. Biol. Chem., 163, 437-446 (1946). 



i« A. L. Lehninger and E. P. Kennedy, /. Biol. Chem., 173, 753-771 (1948). 



