BIOCHEMICAL ENERGY-CONSUMPTION BY WHEAT-GERM EXTRACT DURING CELL-FREEPROTEIN-SYNTHESIS

The biochemical energy level in a cell-free translation system regulat es the rate of protein synthesis. ATP and GTP hydrolysis rates in whea t germ extract (WGE) were measured in the presence or absence of prote in synthesis. ATP and GTP were hydrolyzed in WGE at high rates indepen dently of protein synthesis. The ATP hydrolysis rate was expressed usi ng a first order equation of ATP concentration with a rate constant k of 0.06 min(-1). Protein synthesis stopped when the biochemical energy charge defined by Atkinson declined to 0.85, and restarted when the e nergy charge was raised to almost 1.0 by replacement of the liquid in the translation system with fresh buffer containing ATP and GTP. These results suggest that the principal factor in the success of the CFCF (continuous flow cell-free translation) system reported by Spirin et a l. was maintenance of the high energy charge by the supply of fresh AT P and GTP, and continuous removal of their hydrolyzates. The mechanism underlying the marked increase in the protein synthesis rate with inc reasing feed rate in the CFCF system was also explained by the increas ing energy charge level with increasing feed rate. ATP inhibited prote in synthesis at concentrations greater than 2 mM. The optimal feed con ditions for the CFCF system were an hourly feed rate of three reactor volume, and an ATP concentration of 2 mM for the case using WGE prepar ed by the authors.