June M. Fessenden

[35] A-particles1 and P-particles2

Publisher Summary A partial resolution of oxidative phosphorylation in submitochondrial particles can be achieved by a variety of physical, chemical, and enzymatic degradation procedures. Such apo-particles catalyze oxidation of DPNH or succinate with little accompanying ATP formation. The oxidation can be coupled to phosphorylation by the addition of specific proteins (coupling factors) isolated from mitochondria. Among the most useful and reproducible preparations of apo-particles are the A-particles (ammonia particles) and P-particles, which are stimulated by coupling factor 1, coupling factor 2, coupling factor 3 and coupling factor 4 (F1, F2, Fs, and F4, respectively). This chapter investigates the preparation of A-particles and P-particles. Deficient particles are used that catalyze oxidation but negligible phosphorylation. On addition of multiple coupling factors to these particles, a marked stimulation of phosphorylation is observed. The preparation of phosphatide suspension is also described in the chapter. For the preparation of P- particles, one gram of heavy layer beef heart mitochondria is mixed with 0.4 ml of 0.5M EDTA, pH 7.4, 10 ml of 1.0M sucrose, and 8 ml of asolectin in a final volume of 40 ml. This suspension is exposed to sonic oscillation in 20-ml batches in a Raytheon (250 watts, 10 kc) sonic oscillator for 2 minutes.

[84b] Coupling factor 3 (F3)☆

Publisher Summary This chapter discusses the coupling factor 3 (F 3 ). F 3 is measured with A-particles by the stimulation of the ATP-linked reduction of DPN by succinate in the presence of F 1 or by the increase of the P:O ratio in the presence of F 1 and F 2 . F 3 , 1-50 μl of the enzyme solution is used for assay. If necessary, the enzyme may be diluted in 50 mM Tris-S04, pH 8, but the final concentration should not be below 3-5 mg of protein per milliliter. Protein is determined spectrophotometrically at 280 mμ, assuming that 1 mg/ml gives a density reading of 1.0. The purification procedure includes the steps such as preparation of crude extract, chromatography on DEAE-cellulose, and ammonium sulfate fractionation. In the frozen state, F 3 (DEAE-cellulose eluate or Sup II) is stable for several months, but loses 80% of the activity in 10 minutes at 40°. Sup II is stable above 5 mg/ml but loses 40-60% of its activity when diluted to 1 mg/ml.

[84a] Coupling factor 2 (F2)☆

Publisher Summary This chapter discusses coupling factor 2 (F 2 ). A-particles catalyze oxidations, but require coupling factors for phosphorylation. In the presence of excess F 1 and F 3 or F 4 , the stimulation of phosphorylation by F 2 is used as an assay of F 2 activity. The purification procedure includes the various steps that are described in detail in the chapter. For the preparation of mitochondrial acetone powder, heavy layer of beef heart mitochondria is added to reagentgrade acetone at –10o and blended in an explosion-proof waring blender for 30 seconds, then rapidly added to acetone at –10o. In extraction, the acetone powder is resuspended and homogenized in 80 ml of 0.1 M potassium phosphate pH 7.4, previously flushed with nitrogen. In adsorption on calcium phosphate gel, calcium phosphate gel is added to the glycine extract, homogenized, and kept at 0o with gentle stirring for ten minutes. In the last step, Chromatography of P-100 gel, in a cold room, a column of P-100 gel, with a spacer gel of P-60 gel is set up and equilibrated with 0.3 M potassium phosphate pH 7.4.

[107] Antibody against F1

Publisher Summary This chapter discusses the antibody against F12. Mitochondrial ATPase (F1), which has been obtained in homogeneous form, stimulates oxidative phosphorylation in Fl-deficient submitochondrial particles. Such particles usually contain residual ATPase, sometimes in considerable quantities, and energy-producing reactions can be readily demonstrated under suitable conditions even without added coupling factors. The incorporation of 32Pi into ATP, catalyzed by submitochondrial particles is inhibited by an antiserum against F1. The degree of inhibition can be used in titration experiments to evaluate the potency of the antiserum. The Purification of γ-globulins from antiserum is described. All steps are carried out at 23°, at room temperature unless specified. As a control, a sample of normal serum is carried through the same procedure. The antibody from both chicken and mice that has been examined in the chapter is specific for F1. Using double diffusion analysis, a single precipitin band appears with F1, but nothing appears with any of the other coupling factors. The ATPase activity of F1 and oxidative phosphorylation in submitochondrial particles is inhibited by the antibody.