PREPARATION OF HIGHLY PHOSPHORYLATING MITOCHONDRIA FROM THE YEAST SCHIZOSACCHAROMYCES-POMBE

Schizosaccharomyces pombe yeast cells grown on either fermentable or r espiratory media were efficiently converted to stable spheroplasts by the alpha-(1 --> 3)-glucanase Novozym 234 in the presence of 1.2 M sor bitol. Lysis of spheroplasts by gentle homogenization in dilute sorbit ol resulted in the preparation of mitochondria with a structure simila r to that observed within the starting yeast cells. The isolated mitoc hondria exhibited high oxidation rates with various respiratory substr ates, NADH being the most efficient. The mitochondria appeared well co upled since the second State 4 rate observed after ADP consumption was identical to the initial one. The State 3 rate in the presence of ADP was completely inhibited by low oligomycin concentrations, similarly to the concomitant ATP synthesis of 900 nmol/min x mg protein. These N ADH oxidation and dependent ATP-synthesis activities are much higher t han those previously described for mitochondria isolated from Schizosa ccharomyces pombe, and similar to the highest values reported for Sacc haromyces cerevisiae.