Effects of freezing on particulate enzymes of rat liver.

The fact that freezing caused marked changes in oxidative activity of tissues was of comparatively little interest until it became apparent that most if not all the enzymatic activities of cells could be reproduced under suitable conditions in cell-free systems. Isolated soluble enzymes were usually unaffect,ed or even stabilized by freezing, and damaging effects of freezing on other enzymes could be attributed to destruction of cells. Thus Lynen (I), in a study of frozen yeast cells, ascribed the effects of freezing to dilution of the enzymes and cofactors resulting from breaking of cell membranes. He also showed that diphosphopyridine nucleotide (DPN) restored activity of alcohol dehydrogenase after freezing. In a comparable study on animal tissues (a), he also ascribed the effects of freezing to breaking of cells. De Robertis and Nowinski (3, 4) showed that respiration and succinate oxidation not only of frozen liver slices, but also of frozen liver homogenates, were greatly reduced. Lynen (5), using kidney homogenates, obtained the same results and concluded that there were effects of freezing other than destruction of cell membranes. The sensitivity of particulate or mitochondrial enzyme systems to freezing has been a common observation. Loomis (6) reported a stabilization of such activity by fast freezing in the presence of substrate and sucrose. Huennekens and Green (7) showed that added DPN would restore some of the activity of frozen washed homogenates. Repeated slow freezing and thawing have been used in methods of preparation of pyruvic and a-ketoglutaric dehydrogenase (8, 9). Williams (lo), after freezing mitochondrial preparations, found an increased requirement for adenosinetriphosphate (ATP) in choline oxidation. The particulate oxidative enzymes of rat liver are largely inactivated by freezing. A detailed study of these enzymes was therefore undertaken, with the results described in this report.