In the period between 1945 and 1948 studies were carried out on the oxidation of long-chain fatty acids by particulate enzyme preparations derived from liver and heart. It was shown that a two-carbon intermediate of the oxidation state of acetate which arose during the oxidation of fatty acids or of pyruvate could condense to form acetoacetate or, in the presence of oxal-acetate, to form citrate and the subsequent intermediates of the Krebs citric acid cycle (Lehninger, A. L. J. Biol.Chem., 164:291,1945; 165:131, 1946). These experiments defined the manner of integration of the Krebs citric acid cycle with fatty acid oxidation, particularly in the liver. At that time, of course, the identity of this active acetate intermediate was not yet known as acetyl coenzyme A.

Studies on the oxidation of long-chain fatty acids (Kennedy, E. P., and Lehninger, A. L. J. Biol. Chem., 179:957,1949; 183:275, 1950) revealed that the fatty acid oxidase system of mitochondria could oxidize to completion not only the naturally occurring saturated acids, such as palmitic and stearic, but also a wide variety of unsaturated fatty acids. Both the natural cis- and the "unnatural" trans acids, such as elaidic acid, were found to be oxidized readily, as was vaccenic acid. The long-chain fatty acids were shown to be almost entirely oxidized via the Krebs cycle, in contrast to shorter-chain fatty acids, such as butyrate, which give rise to considerable amounts of acetoacetate. Thus the integration of fatty acid oxidation with the Krebs cycle depends to some extent on chain length. It was also learned that oxidation of fatty acids could be "sparked" or activated by the oxidation of reduced diphosphopyridine nucleotide in a self-perpetuating manner (Kennedy, E. P., and Lehninger, A. L. J. Biol. Chem., 190:361, 1951). Once the oxidation is initiated, it continues long after the DPNH primer is oxidized to completion.

Identification Of The Role Of Mitochondria In Biological Oxidations

The particulate preparations capable of oxidizing fatty acids and pyruvate referred to above were for some time suspected to be mitochondria. However, when mitochondria were isolated by early methods involving differential centrifugation from saline homogenates, the so-called mitochondrial fraction was found to be totally inert, whereas the nuclear fraction contained the enzymatic activity. This seemed rather unexpected. In 1948 Hogeboom, Schneider, and Palade published their now well-known method for isolating mitochondria in sucrose media. It was found that mitochondria isolated by the new sucrose method, which are relatively intact morphologically, could oxidize fatty acids and Krebs cycle intermediates and were also able to carry out oxidative phosphorylation (Kennedy, E. P., and Lehninger, A. L. J. Biol. Chem., 179:957, 1949; Lehninger, A. L., and Kennedy, E. P. J. Biol. Chem., 172:847, 1948). These experiments thus provided the first direct evidence that mitochondria are the site not only of terminal electron transport, but also of the entire complex of enzymes involved in the Krebs cycle, in fatty acid oxidation, and in oxidative phosphorylation. This study was the first of a now long series on the relation between structure and function of mitochondria.

Oxidative Phosphorylation

Studies on the mechanism of oxidative phosphorylation were initiated in 1948 (Friedkin, M., and Lehninger, A. L. J. Biol. Chem., 178:611,1949). The immediate objective was to determine whether or not phosphorylation occurred along the electron transport chain, as thermodynamic considerations suggested. Actually, what little experimental evidence existed at this time appeared to deny this possibility. It was found that when reduced DPN was allowed to be oxidized by isolated mitochondria in the presence of inorganic phosphate labeled with P32, a rapid incorporation of the P32 into the terminal phosphate of ATP occurred, without any net uptake of phosphate (Friedkin, M., and Lehninger, A. L. J. Biol. Chem., 178:6II, 1949). This was the first indication that phosphorylation was associated with the oxidation of DPNH. Later experiments using the β-hydroxybutyrate dehydrogenase as a generating system for DPNH established that at least two and probably three phosphorylations were associated with the respiratory chain (Lehninger, A. L. J. Biol. Chem., 178:625, 1949)- Net uptake of phosphate by this system was easily observable. Conclusive proof of the participation of the respiratory chain was provided by subsequent work (Lehninger, A. L. J. Biol. Chem., 190:345, 1951). The oxidation of chemically reduced pure DPNH by isolated mitochondria was found to be accompanied by net phosphorylation with a P: O ratio of about 2. This was subsequently raised to 2.6. These experiments therefore proved the occurrence of three phosphorylation sites along the respiratory chain.

Other studies revealed the respiration-linked incorporation of P32-labeled phosphate into phospholipids, ribonucleic acid, and phosphoprotein in isolated mitochondria (Friedkin, M., and Lehninger, A. L. J. Biol. Chem., 177:775, 1949). These observations provided the basis for the later experiments by E. P. Kennedy on phospholipid synthesis in extracts of mitochondria (p. 97).

In other studies, the relationship of myokinasc to oxidative phosphorylation was clarified (Barkulis, S. S., and Lehninger, A. L. J. Biol. Chem., 190-339. J951)- Oxidative phosphorylation coupled to the oxidation of pyruvate to acetoacetate was also demonstrated (Barkulis, S. S., and Lehninger, A. L. J. Biol. Chem., 193:597, 1951).