Arthur I. Stern

Oxidative Phosphorylation Coupled to Respiration in Highly Purified Intact Euglena Mitochondria

Summary Improved methods for isolation of mitochondria from Euglena gracilis var. bacillaris are presented based on Yeda press breakage of protease-prepared sphaeroplasts of low vitamin B12 grown cells. Active preparations are obtained from dark-grown cells of wild-type or mutants W3BUL or W10BSmL but W1o is preferred since it lacks proplastids that can contaminate the mitochondrial preparations. Crude mitochondria obtained by differential centrifugation are further purified on Percoll gradients yielding five fractions (in order of decreasing density): A) enriched in clean contracted mitochondria; B) enriched in clean orthodox mitochondria; and C) D) and E) which contain broken mitochondria and mitochondrial membrane vesicles. All fractions show succinic dehydrogenase activity but only fractions A and B are intact based on retention of fumarase activity. Fraction A contains the bulk of the protein and consistently shows the highest respiratory activity, the activity of fraction B is somewhat lower and resembles the activity of crude mitochondria; fractions C, D and E are inactive. Oxygen uptake (unstimulated by ADP) by fraction A mitochondria is inhibited 100 % by 0.1 mM KCN, about 30 % by 6.6 μM antimycin A and about 50 % by 5 mM NaN3. True uncoupling is observed for the first time in Euglena mitochondria since dinitrophenol (DNP) (0.16 mM) and carbonylcyanide mtrichlorophenylhydrazone (m-CCCP) (0.2 μM) increase O2 uptake by about 250 %. P to O ratios of 2 - 3 are obtained. Electron microscopy and plating on rich media indicate that these preparations are free of significant contamination with other cell fractions or with microorganisms.

Taurine conjugates in the lipid fraction of Euglena cells and their mitochondria

Summary: Dark-grown resting (non-dividing) cells of Euglena gracilis var. bacillaris and mutants W3BUL (with a proplastid remnant) and W10BSmL (lacking plastids) incubated with 35SO2- 4 form a series of labelled lipids which are low or absent in dividing cells. These lipids all release labelled taurine on mild acid-hydrolysis. Treatment of the labelled lipids with 2,4-dinitrofluorobenzene (DNFB) followed by acid hydrolysis does not yield labelled dinitrophenyltaurine (DNP-taurine), but treatment with DNFB after hydrolysis readily forms labelled DNP-taurine, indicating that taurine is linked to the lipids by at least the amino group. Illumination increases the labelling of these taurolipids in plastid-containing cells (wild-type and W3BUL) but has little effect in cells lacking plastids (W10BSmL); labelling is highest in W10 cells irrespective of illumination. This indicates that the presence of a plastid may exert a negative control on taurolipid formation which is relieved by light. The same series of labelled lipids is found in isolated purified mitochondria from mutant W10, indicating that this organelle is a site for taurolipid deposition. The formation of taurolipids under non-dividing conditions may be a response to nutritional stress and these negatively charged constituents (as well as the thylakoid sulpholipid) may serve to protect membranes by repelling deleterious negatively charged oxygen species.