Alteration of Mycobacterium phlei membrane structure by freezing and thawing; reversal by heating

Abstract

Membrane vesicles from Mycobacterium phlei which contain the electron transport chain, when subjected to freezing to −70 °C followed by slow thawing, exhibited a decreased level of phosphorylation coupled to the oxidation of substrates. This loss in oxidative phosphorylation was restored following heat treatment (50 °C for 10 min) of the membranes. Freeze treatment (−70 °C for 10 min) of membrane vesicles also resulted in a decrease in membrane bound coupling factor-latent ATPase activity. The soluble coupling factor(s) or cryoprotective agents (i.e., glycerol or dimethyl sulfoxide) were found to protect the membrane vesicles from the effects of freezing. Membrane vesicles depleted of particulate bound coupling factor were sensitive to exposure to low temperatures; however, complete protection was afforded by the addition of coupling factor. In addition, prolonged sonication of electron transport particles resulted in lowered P O ratios, and heat treatment of these sonicated particles restored P O ratio. Therefore, it appears that the effects of heat treatment and freeze treatment on membrane vesicles are reversible. The steady state level of reduced cytochrome b was considerably higher (40%) in heat-treated electron transport particles as compared to untreated particles (28.5%); electron transport particles subjected to freeze treatment showed a lower steady state level of cytochrome b (16.6%) as compared to electron transport particles. The steady state level of cytochrome b in freeze-treated particles returned to the original level (27.5%) for electron transport particles when subjected to heat treatment. Nevertheless, the total amount of enzymatically reducible cytochrome b was the same for all membranes after subjection to the various types of treatment. In contrast to cytochrome b, the reduced steady state levels of cytochrome c, and a + a 3 were not altered by heat or freeze treatment.