Polytomella spp. growth on ethananol - Extracellular pH affects the accumulation of mitochondrial cytochrome c(550)

A defined medium with ethanol as sole carbon source was devised for growth of the colorless, unicellular alga Polytomella spp. Cell density on this ca rbon source was related to extracellular pH. An acidic pH was required for ethanol utilization; best yields were obtained at pH 3.7. Spectroscopic ana lysis of the cells showed that the concentration of cytochrome c per cell w as 40% higher than at pH 6.0; the concentrations of cytochrome a(606) (cyto chrome c oxidase) and b(566) (cytochrome bc(1) complex) were the same. A so luble cytochrome c(550) was purified from cells grown at pH 3.7 and charact erized by peptide sequencing as the 12-kDa cytochrome c(550) of the mitocho ndrial respiratory chain. Immunoblots of total cell proteins showed higher accumulation of cytochrome c(550) at pH 3.7 than at pH 6.0. RNA blot analys is gave clear evidence of the abundance of c(550) transcript in cells grown at pH 3.7. The amount of mitochondrial proteins obtained from cells grown at pH 3.7 was twofold higher than that of cells grown at pH 6.0. Mitochondr ia isolated from both cell types readily oxidized succinate, malate or etha nol. The rates of oxygen uptake were 20-25% higher in mitochondria from cel ls grown at pH 3.7. Cyanide and antimycin A inhibited respiration with succ inate up to 95% in both types of mitochondria. The participation of cytochr ome c(550) in mitochondrial electron transport from succinate to oxygen was shown by spectral measurements.