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

Abstract

S U M M A R YLarge-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 cellcycle. 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 cellcycle and possible mechanisms for its control are discussed with reference t,o previous data.

Oscillations of Respiration and Adenine Nucleotides in Synchronous Cultures of Acanthamoeba castellanii: Mitochondrial Respiratory Control in vivo

“…These respiratory oscillations are clearly different from those previously observed in synchronous cultures of the fission yeast Schizosaccharomyces pombe (growing in the presence of glucose), where a Pasteur effect was invoked to explain the modulation of mitochondria1 activity (Poole et al, 1973), in the trypanosomatid protozoon Crithidia fasciculata (Edwards et al, 1975), or in Tetrahymena pyriformis . The observation of a dramatic fluctuation of adenylate energy charge in the trypanosome (Edwards et al, 1975), in T. pyriformis , and in A .…”

“…The observation of a dramatic fluctuation of adenylate energy charge in the trypanosome (Edwards et al, 1975), in T. pyriformis , and in A . castellanii (this report) is of great interest as it has been suggested that thermodynamic and kinetic considerations of the regulatory mechanisms involved in energy metabolism would predict considerable constraints on variations of this parameter, and a tendency to stabilization of adenylate charge between values of 0.87 and 0.95 (Chapman & Atkinson, 1977).…”

“…Cyanide was added as an aqueous solution (pH 7.4) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was added as an ethanolic solution (the concentration of solvent alone had no effect on respiration). Adenine nucleotide pool levels were measured by the luciferase method (Chapman et al, 1971), as described previously (Edwards et al, 1975), after rapid quenching and extraction in boiling chloroform. Adenylate charge (Atkinson & Walton, 1967) was calculated as:…”

ATP and Energy Charge as Criteria of Growth and Metabolic Activity of Mollicutes: Application to Spiroplasma citri

“…Spiroplasma citri, like many other organisms (Bomsel & Pradet, 1968;Chapman et al, 1971 ;Niven et al, 1977), maintained its energy charge at high values close to 0.9 throughout active growth, though some eukaryotic organisms have been reported to have low energy charge values even when actively metabolizing (Edwards et al, 1975;Edwards & Lloyd, 1977;Lloyd et al, 1978). The importance of energy charge as a metabolic regulator in vivo is still under debate (Atkinson, 1977;Fromm, 1977).…”

Changes in Oxygen Uptake Rates, Enzyme Activities, Cytochrome Amounts and Adenine Nucleotide Pool Levels during Growth of Acanthamoeba castellanii in Batch Culture

“…Intensification factors of 3.7, 6.1 and 13.3 for c, b and a-type cytochromes respectively (Edwards et al, 1977) were used to quantify low temperature spectra. Measurements of adenine nucleotide levels were as described by Edwards, Statham & Lloyd (1975) using luciferase assays on extracts from I ml of culture prepared by chloroform extraction. ATP, ADP and AMP were estimated by the method of Chapman, Fall & Atkinson (1971) using ATP as a calibration standard.…”