Exchange and Localization of 3H and 18O from Water and Substrates by Mitochondria

Abstract

Abstract Mitochondria that have catalyzed oxidative phosphorylation at 37° in 3H-labeled water, and which are then chilled and subjected to gel filtration near 0°, retain small amounts of tritium. Retention is decreased by lowering the temperature of incubation below 37°. At 37° most of the response is complete in 1 min of incubation, and after 5 min of incubation only about 3 µatoms of hydrogen from media water are retained per mg of protein. The extent of tritium retention is not modified by inhibitors or uncouplers of oxidative phosphorylation. Comparison of 3H and 18O retention in a less sensitive experiment shows that the tritium retention is not accompanied by an equivalent retention of 18O from H18OH. 3H released from 2-3H-glutamate and 18O released from 18O-Pi during oxidative phosphorylation are not demonstrably retained by mitochondria. The results indicate that the tritium retention from media water may represent proton exchange with mitochondrial structures, probably mitochondrial proteins, and that the exchange is largely independent from oxidation or phosphorylation processes. The data give no support for possible transient localization of hydrogen from substrates or oxygen from Pi in mitochondrial water pools during oxidative phosphorylation, or for protein conformation changes associated with phosphorylation.