This study describes the successful subcellular fractionation of Crithidia fasciculata, a trypanosomatid parasite, using differential and zonal centrifugation techniques. The researchers developed a gentle cell disruption method using digitonin treatment followed by a Chaikoff press, which preserved subcellular organelles better than previously reported harsh methods. Through differential centrifugation, mitochondria were isolated as a pellet accounting for approximately 10% of total protein. Subsequent high-speed and rate zonal centrifugation on sucrose gradients achieved clean separation of multiple organelles. Catalase was found to be entirely soluble and non-sedimentable, with no association to microbodies. Mitochondria were successfully isolated and characterized by marker enzyme activity, sedimenting at density 1.20-1.21 g/ml. Large lysosomal vacuoles containing p-nitrophenylphosphatase activity were separated from mitochondria, sedimenting at approximately 1.22 g/ml. The study demonstrates that gentle disruption methods preserve organellar integrity better than previously used harsh techniques, enabling future detailed biochemical characterization of trypanosome organelles and their components.

Key findings

• Gentle digitonin-based cell disruption preserves subcellular organelles of Crithidia fasciculata better than harsh methods like French press or sonication
• Catalase in C. fasciculata is soluble and not associated with microbodies or peroxisomes
• Mitochondria account for approximately 10% of total protein and sediment at density 1.20-1.21 g/ml, with succinate dehydrogenase as a reliable marker enzyme
• Large lysosomal vacuoles containing p-nitrophenylphosphatase activity sediment at density 1.22 g/ml and are cleanly separated from mitochondria by zonal centrifugation
• NADH:cytochrome c oxidoreductase shows unexpected distribution across fractions and is not reliably associated with mitochondria in this organism