Oxidative phosphorylation by mitochondrial suspensions after freezing and storage at low temperatures.

There is much evidence to suggest that the structural and functional integrity of many biochemical materials is unaltered by freezing if the rate of cooling is rapid and the storage temperature low (2-6). Isolated enzymes and mixtures of enzymes and their substrates, after rapid freezing at low temperatures, often retain their activities and in some cases show enhanced activity upon thawing (7-Q). Attempts to preserve complex, integrated systems, as represented by mammalian cells, by freezing and storage at low temperatures have been disappointing (10, 11). Skin, ovary, testis, pituitary, and adrenal, however, have been reported to give functional grafts or growths in tissue culture after being frozen slowly at -79” and -190” and stored at these temperatures (10-12). The effects of freezing and low temperature storage on the biochemical activities of isolated cell particulates have been investigated in a preliminary manner. Oxidative phosphorylation by homogenates of rat liver was reported to be completely destroyed by freezing and thawing (13, 14). Hunter et al. (15) have stressed the importance of using freshly prepared mitochondrial suspensions for demonstrating oxidative phosphorylation. The qualitative results obtained by Loomis (16), however, suggest the possibility of preserving the above capacities in suspensions of mitochondria after freezing and storage at low temperatures. The present report deals with the quantitative aspects of oxidative phosphorylation by mitochondria after freezing at various rates and temperatures, storage in the frozen state at various temperatures, and repeated freeze-thaw cycles.