“Survival” of Mitochondria in Vitro

Abstract

Isolated mitochondria have been maintained active and coupled for 72 hours at 25 C. Survival (retention of respiratory control) is a function of incubation temperature and dependent upon aeration and substrate. ATP does not entirely substitute for substrate, indicating a need for products of active metabolism other than energy. An improvement in respiratory control is often observed during the first several hours of incubation. Sedimentation and resuspension at 24-hour intervals prolonged survival. As revealed by electron microscopy, mito-chondria maintained their basic structure during a 72-hour period at 25 C. Survival is a dynamic, energy-requiring process and must be distinguished from so-called "aging" of organelles at ice temperatures. As a manifestation of partial autonomy, survival may prove useful in assessing aspects of mitochondrial function and the mitochondrial-cellular interrelationship. The prospects of prolonged incubation of isolated organelles were heightened by the reports of Ridley and Leech (7) and Giles and Sarafis (2) demonstrating the survival of isolated chloroplasts for several days. In earlier experiments (8) we have shown that mitochondria will retain respiratory control for 20 hr at 25 C when supplied with substrate and appropriate cofactors. The present paper describes the influence of physical and energy parameters that further extend the retention of respiratory control, referred to hereafter as survival, to at least 72 hr at 25 C. A preliminary report of this work has appeared (9). MATERIALS AND METHODS Avocados (Persea americana "Fuerte" Mill) were obtained from orchards in southern California or from local wholesale markets and used as a source of mitochondria. Isolation Techniques. The isolation procedure used throughout these experiments was essentially that described by Lance et al. (3) with minor modifications. Peeled and pitted pieces of avocado were grated into isolation medium (1:3, w/v) composed of 0.25 M sucrose, 0.05 M potassium phosphate buffer (pH 7.2), 5 mm EDTA, 0.2% polyvinylpyrollidone (40,000 mol wt), 0.1 % bovine serum albumin, and 5 mM /3-mercapto-ethanol. Grating was done below the liquid surface (10). Cellular debris was removed by centrifugation at 1,200g for 10 min. After additional centrifugation at 14,000g for 15 min the mitochondrial pellet was resuspended in "wash" medium (0.25 M sucrose, 0.05 M phosphate buffer (pH 7.2), 0.1% bovine serum albumin, and S mm /3-mercaptoethanol). The suspension was centrifuged at 1,000g for 5 min and the resultant supernatant fraction at 8,000g for 10 min. The final pellet was resuspended in a few tenths of a milliliter of wash medium and held at 0 C, for a period seldom exceeding 1 hr, until the mitochondria were assayed or incubated at 25 C. Incubation and Assay. As a standard incubation procedure based on earlier experiments (8), the mitochondria were suspended in 3 ml of reagent mixture (see legend, Fig. 1) contained in a 20-ml beaker. At the start of the incubation 0.4 ,umole of ADP was also added, and the beaker was covered with parafilm and then placed in a shaking water bath at 25 C. To assay for oxygen consumption, the incubated mixture was transferred to a closed vessel equipped with a polarographic oxygen electrode. As the objective of this work was to test additional incubation parameters, the latter varied as described in each experiment. However, in all instances the incubation conditions were normalized (temperature adjusted, substrate added, etc.) just before the polarographic assay that was always conducted under the conditions described in the legend of Figure 1. Respiratory control was expressed in the manner described by Chance and Williams (1). Specific activities were based on Lowry protein assays according to a modification by Miller (4). In preparation for embedding and electron microscopy mitochondria suspended in incubation medium were transferred into a test tube containing 11o volume of 25% glutaral-dehyde and placed at 0 C for 2 to 5 hr. After centrifugation at 1 0,000g for 10 min the pellet was rinsed and then resuspended in 2 ml of 2.5% glutaraldehyde in 0.1 M phosphate, pH 7.2. The fixed mitochondria were stained with 1% OSO4 (in 0.1 M phosphate, pH 7.2) for 1.5 hr at 0 C and centrifuged, and the pellet was dehydrated with increasing concentrations of alcohol. RESULTS Survival Capacity of Various Intracellular Fractions. As an expedient in studying the effect of incubation conditions on survival, commonly used isolation procedures (3, 10) were adopted. One exception was the selection of centrifugation forces to obtain the final mitochondrial pellet. A comparison of organelle fractions obtained at increasing centrifugal forces indicated (Table I) that the organelle fraction sedimented at 10OOg or at forces above 8000g had a low respiratory activity and little or no RC.' On the basis of these and other corrobora-tive data the fraction sedimenting between 1000 and 8000g 1 Abbreviations: RC: respiratory control; RCR: respiratory control ratio. 702