Studies on amino acid activation and protein synthesis

Abstract

This thesis concerns the overall subject of protein synthesis in plants. Several different experimental approaches to the problem are described. The initial studies concern the incorporation of amino acids into tissue sections and homogenates. Tissue sections show a constant rate of incorporation of labeled amino acids into protein for periods of time up to two hours. The rate of incorporation then decreases with time. Conversely, tissue homogenates show ever increasing rates of incorporation of labeled amino acids into protein. This incorporation is indicative of incorporation of labeled amino acids into bacteria within the homogenate rather than incorporation into plant protein. Sterile homogenate preparations do not show the kinetics of incorporation shown by other homogenate preparations. The incorporation of labeled amino acids into acid insoluble material obtained from sterile tissue homogenates is a function of the washing procedure used in the assay. Amino acid activation has been studied by a hydroxylamine trapping reaction and by pyrophosphate exchange. Plant tissues contain amino acid activating enzymes which may be detected by either method of assay. The pyrophosphate exchange assay is however more sensitive. The hydroxamate assay suffers from some added complications common to plant systems. The specific nature of amino acid activation in plant preparations has been studied with enzymes isolated from spinach leaves. The activation reaction requires ATP and amino acids, the products being pyrophosphate and probably a mixed anhydride between the 5' phosphate of AMP and the carboxyl group of the amino acid. The enzymes occur in all plant tissues tested and appear to be a part of the soluble fraction of cells. Purification procedures have been developed which make it possible to remove the free amino acids from the preparations. The purified preparation is capable of activating all of the naturally occurring (in protein) amino acids except serine. The possible activation of serine is-not excluded. Preliminary evidence is presented which indicates that each amino acid is activated by its own specific amino acid activating enzyme. The possibility of linking the process of amino acid activation to protein synthesis is considered. Crude particulate containing preparations of spinach exhibit an ATP and RNA dependent incorporation of leucine. Attempts to obtain an amino acid dependent exchange of AMP into ATP with spinach preparations were unsuccessful. Even so it has been possible to demonstrate an ATP dependent RNAase sensitive incorporation of labeled leucine into acid and alcohol washed material.