The preparation of large-scale synchronous cultures of the trypanosomatid, Crithidia fasciculata, by cell-size selection: changes in respiration and adenylate charge through the cell-cycle

SUMMARY: Large-scale synchronous cultures of Crithidia fasciculata were prepared by a sedimentation—velocity-size-selection method using a non-osmotic gradient in a zonal rotor. Synchrony indices of up to 0.68 were obtained (average length of cell-cycle, 5.1 h). Dry weight, protein and RNA increased continuously so as to double in one cell-cycle. Rates of oxygen uptake/ml culture increased overall so as to double over a cell-cycle, but rose to maxima at five periods in the cell-cycle. KCN gave a similar degree of inhibition throughout, and so did not alter the periodicity or amplitude of the oscillations. Total adenylates doubled over a cycle, and complex changes in pool sizes of ATP, ADP and AMP were temporally interrelated and were correlated with changes of oxygen uptake rates rather than with changes in biosynthetic requirements. Adenylate charge varied between 0.47 and 0.66. Discontinuous respiratory activity of mitochondria through the cell-cycle and possible mechanisms for its control are discussed with reference to previous data