Preparation and properties of mitochondria from mammalian cells cultured in vitro.

Abstract

Summary 1. Intact mitochondria were prepared from various cell-lines cultured in vitro by treatment with Bacillus subtilis proteinase (Nagase). For protection of the mitochondria, mannitol was found to be better than sucrose. 2. As with normal tissue mitochondria, the mitochondria of cultured cells oxidized most intermediates of the Krebs cycle and succinate was especially rapidly oxidized. NADH was not oxidized, but citrate was. Oxidation of β-hydroxybutyrate and glutamate were very slow. 3. The phosphorylation efficiencies (ADP:O ratio) of about 1.9 for succinate, 0.9 for ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and 2.5 to 2.9 for NAD-linked substrates were obtained. High respiratory control ratios were also observed with various substrates; 5.4 for succinate, 5.0 for α-ketoglutarate, 4.0 for malate and fumarate, 3 to 2 for other NAD-linked substrates and 1.4 for ascorbate-TMPD. 4. The oxidative phosphorylation of the present mitochondria were inhibited or uncoupled by various inhibitors or uncouplers in essentially the same way as that of normal tissue mitochondria. 5. HeLa cell mitochondria prepared from cells treated with the phosphate buffered saline, showed sluggish respiration in the initial stage after addition of ADP with succinate, many NAD-linked substrates and an ascorbate-TMPD as respiratory substrates. Addition of serum albumin had no effect on the sluggish respiration. 6. This sluggishness of respiration were progressively lost as the oxidative phosphorylation reaction proceeded. The P:O and respiratory control ratios were improved in parallel to the recovery of respiratory activity. 7. The same phenomena were also observed with mitochondria from FL and Chang cells. However, mitochondria from rat liver slices did not show this phenomenon after the same treatment.