Functional properties of mitochondria isolated from murine L5178 lymphoblasts grown in cell culture

Abstract

Mitochondria were isolated from lymphoblasts grown for 5 days in cell culture. Measurement of mitochondrial respiratory activity revealed poor response to adenosine 5'-diphosphate with reduced nicotinamide adenine dinucleotide-linked substrates but well-coupled active respiration with succinate as substrate. These mitochondria also exhibited rapid initial rates of respiration-supported calcium uptake as measured by dual-beam spectrophotometry. H+/2e- and Ca2+/2e- ratios were in normal limits for the lymphoblast mitochondria during calcium uptake in the presence of phosphate. In the absence of phosphate no calcium uptake, H+ ejection, or stimulation of oxygen consumption was observed. However, the lack of discharge of the accumulated calcium from the lymphoblast mitochondria upon inhibition of respiration suggests possible different mechanisms of cation transport compared to mitochondria from normal, mammalian cell types. Electron microscopy of freshly prepared mitochondrial suspensions revealed preparations with intact outer membranes and abundant cristae and that were relatively free of other cellular structures. These studies demonstrate the feasibility of obtaining intact respiring mitochondria from cultured lymphoid cells and indicate that active ion transport in these mitochondria may be significantly different from "normal" cell mitochondria.