Acetate Metabolism in Cell Suspension Cultures

Abstract

Cell suspension cultures of Paul's Scarlet rose were grown over a 14-day period, during which a 50-fold increase in fresh weight occurred. Three phases could be recognized from weight, DNA determinations, and microscopic examination. From days 0 to 7, cell division was accompanied by cell expansion; from days 7 to 10, only cell expansion occurred; and from days 10 to 14, there was no further growth.When acetate-1l'4C was supplied continuously to 4-day and 12-day cells, 14C was readily incorporated into lipid, C02, organic acids, amino acids, and protein. In the older cells, relatively greater amounts of 14C were recovered in organic and amino acids, and accumulation of 14C in these components continued after a steady rate of incorporation of 14C into protein and 14CO2 had been established.Glutamate-14C was the most heavily labeled compound recovered after a pulse of acetate-1-'4C in both cell types and was depleted when acetate-1-14C was removed, particularly rapidly in 4-day cells. 14C was rapidly lost from the malate-14C labeled during the pulse of acetate-1-'4C in 4-day cells, whereas malate-'4C continued to increase in 12-day cells after the pulse. Glutamate-14C was shown to be the source of the 14C accumulating in malate in 12-day cells.Several other soluble amino acids were labeled during a pulse of acetate-'4C in both cell types. After the pulse most of the 14C lost from these was recovered in the corresponding amino acid in protein, showing that only protein precursor pools had been labeled. The behavior of asparagine was exceptional since its 14C content increased after the pulse and no turnover was apparent. The kinetics of labeling of aspartate, malate, and CO2 showed that oxaloacetate generated in the tricarboxylic acid cycle following the pulse was not in equilibrium with aspartate.Several authors (2, 15, 18) have elaborated on the advantages of cell suspension cultures for metabolic studies. Street and his colleagues (5, 16) have shown that in such cultures of Acer pseudoplatanus the growth is marked by distinct phases, and from Dougall's work (3) with Paul's Scarlet rose a similar conclusion may be drawn.In the present study with rose cells, growth and DNA measurements during a 14-day period under standard conditions established that cell division and enlargement were occurring in 4-day-old cultures whereas both of these processes had ceased in 12-day-old cultures. Labeling Experiments. In continuous feeding experiments a series of 15-ml Millipore filter assemblies were connected to an air line to provide aeration. Each filter held 5 ml of growth medium and 2 gc of labeled substrate. At 0 time, 1 g of cells was placed in each filter and a CO2 trap was connected to the gas output from each filter. The trap consisted of 10 ml of hyamine held in a tube fitted with a Vigreaux column. Individual samples of cells were removed at 30-min intervals by drawing off the medium through the bottom of the filter and washing the cells several times with water. The medium and washings were sampled and the ...

Contribution of Nonautotrophic Carbon Dioxide Fixation to Protein Synthesis in Suspension Cultures of Paul's Scarlet Rose

“…Suspension cultures of Paul's Scarlet rose were grown in the dark in 250-ml flasks containing 80 growth conditions and composition of the medium were the same as used previously (5,14), with two exceptions; the concentration of molybdic acid in the medium was 0.01 mg/l, and potassium iodide was 0.5 mg/l. One-gram samples of cells were harvested at selected times and incubated in 25-ml Erlenmeyer flasks containing 5 ml of the medium (pH 7.5).…”

“…The alcohol-soluble compounds were filtered from each extract and fractionated into lipids, amino acids, and organic acids (5). The protein present in the insoluble residue was hydrolyzed as described by Fletcher and Beevers (5).…”

Influence of Cycloheximide on the Synthesis and Utilization of Amino Acids in Suspension Cultures

“…Suspension cultures of Paul's Scarlet rose were grown as previously described (9). The investigation was performed on cells harvested after 4 days of growth, at a time when the cells were undergoing rapid cell division (9).…”

“…The investigation was performed on cells harvested after 4 days of growth, at a time when the cells were undergoing rapid cell division (9). The sterility of the cells and the medium used in the labeling experiments was established by plating each separately on Difco Bacto Nutrient Agar and incubating at 25 C for 96 hr.…”

“…The sterility of the cells and the medium used in the labeling experiments was established by plating each separately on Difco Bacto Nutrient Agar and incubating at 25 C for 96 hr. The alcohol extracts recovered from the samples were fractionated and the distribution of '4C in the individual soluble amino acids and those in the protein were determined as previously described (9). The amount of "4C incorporated into the amino acids of the cycloheximide-treated cells was compared with that of the control cells.…”