Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing.

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria. The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25 degrees C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation. The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32 degrees C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid. The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.