Cells dissociated with 0.25% crude trypsin from the muscle tissue of 9-day-old chick embryos were employed to investigate the effect of puromycin on cellular metabolism. Parallel studies were also made, using the gyratory shaker, to confirm the effectiveness of puromycin in inhibiting cell aggregation and protein synthesis.
Puromycin when introduced at a concentration of 10µg/ml into a suspension of cells in Eagle's MEM did not completely inhibit cell aggregation. Small aggregates were formed in the first 4 h of the experiment. Protein synthesis of the rotated cells, as measured by the incorporation of L-[α-14C]leucine into proteins, was arrested by 91.7% within 15 min of introducing puromycin into a cell suspension. The antibiotic retained its inhibitory effect on protein synthesis for the 24-h period of rotation.
Puromycin inhibited the cellular oxygen uptake and carbon dioxide evolution of the rotated cells by 40% within 4 h of its introduction. However, treated cells were still respiring, though at a much reduced rate, at the end of the 24-h experimental period. The release of radioactive carbon dioxide by puromycin-treated cells was also inhibited by 40% at the 4-h stage but after 8 h no further 14CO2 was evolved. The presence of the antibiotic markedly inhibited the uptake of glucose by trypsin-dissociated cells.
The level of glycogen and lactate in cells suspended in Eagle's MEM was reduced very considerably over a 24-h period. The presence of puromycin accelerated glycogen utilization over the first 6 h of rotation but at 24 h there was a difference of only 0.6% between the glycogen content of treated cells and controls. At 24 h 11.3% less lactate remained in the puromycin-treated cells than in the controls.
The ATP/ADP ratio of trypsin-dissociated cells decreased from an initial value of 2.59 to 1.45 after rotation for 24 h. In the presence of puromycin the ATP/ADP ratio was 0.62 at 4 h and had further declined to 0.48 by 24 h.
The effects of puromycin on the aggregation, protein synthesis and cellular metabolism of trypsin-dissociated cells are discussed in relation to cellular adhesive mechanisms.