Biochemical energy consumption by wheat germ extract during cell-free protein synthesis

Abstract

The biochemical energy level in a cell-free translation system regulates the rate of protein synthesis. ATP and GTP hydrolysis rates in wheat germ extract (WGE) were measured in the presence or absence of protein synthesis. ATP and GTP were hydrolyzed in WGE at high rates independently of protein synthesis. The ATP hydrolysis rate was expressed using 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 energy 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 al. was maintenance of the high energy charge by the supply of fresh ATP and GTP, and continuous removal of their hydrolyzates. The mechanism underlying the marked increase in the protein synthesis rate with increasing feed rate in the CFCF system was also explained by the increasing energy charge level with increasing feed rate. ATP inhibited protein synthesis at concentrations greater than 2 mM. The optimal feed conditions 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 prepared by the authors.