Subcellular Fractionation by Differential and Zonal Centrifugation of the Trypanosomatid Crithidia fasciculata

Abstract

Homogenates of Crithidia fasciculata were fractionated by differential centrifugation. Mitochondria were sedimented quantitatively at 10⁴ g-min and accounted for approximately 10% of the total recovered protein. Catalase was found exclusively in the supernatant fraction whilst NADH: cytochrome c oxidoreductase and p-nitrophenylphosphatase were found in all the fractions. Zonal centrifugation confirmed that catalase was non-sedimentable. Clean separation of mitochondria was obtained in both high-speed and rate zonal experiments, but no NADH: cytochrome c oxidoreductase activity could be detected in these organelles. Separation of large lysosomal vacuoles which contained p-nitrophenylphosphatase activity was obtained and these were clearly resolved from mitochondria by both highspeed and rate zonal centrifugation.