[33] Preparation and assay of phosphorylating submitochondrial particles: Particles from rat liver prepared by drastic sonication

Sonication of rat liver mitochondria in a hypotonic medium for up to 60 minutes yields particles which carry out oxidative phosphorylation (P:O ratio approximately 1.0), the 2,4-dinitrophenol-stimulated ATPase (DNP-ATPase) reaction, and the ATP-Pi and ATP-ADP exchange reactions. The particles are obtained in good yield and they possess a specific respiratory activity comparable to that of other submitochondrial preparations. These preparations are noteworthy because of their stability to freezing and thawing, the peculiar behavior of the partial reactions, and the fact that they show very pronounced reverse acceptor control of respiration. The chapter describes the assay for oxidative phosphorylation, assay for ATPase activity, assay for ATP-Pi exchange, and assay for ATP-ADP exchange. The correlation between the ability to stimulate respiration in the absence of ADP and Pi and to stimulate ATPase activity is not seen in the sonic particles. EDTA stimulates respiration but inhibits ATPase activity completely. ADP inhibits respiration but not ATPase activity, whereas the effect of Mg++ is just the opposite of that of ADP. The action of uncoupling agents is similarly anomalous; dicoumarol, DNP, and gramicidin all inhibit acceptor-less respiration. DNP stimulates ATPase activity, whereas dicoumarol and gramicidin actually inhibit ATPase activity.