16] Actin amino-terminal acetylation and processing in a rabbit reticulocyte lysate

Abstract

Publisher Summary This chapter describes method for inhibiting the acetylation of proteins in the reticulocyte lysate. It also illustrates procedure for assaying the acetylation of the actin NH2-terminal methionine residue and the subsequent removal of acetylmethionine from the completed actin polypeptide chain. A nonreactive analog of acetyl-CoA, S-acetonyl-CoA, is synthesized to serve as a potential competitive inhibitor of the protein acetyltransferase. If the reticulocyte lysate is first treated according to Palmiter and then S-acetonyl-CoA is added, then 85-95% inhibition of acetylation could be achieved. It has been demonstrated that NH2-terminal acetylation can occur as a cotranslational process after the first 30-40 amino acids of the nascent polypeptide chain have been polymerized. The chapter considers using acetylation inhibition system to make actin as a completed 43,000-dalton polypeptide with a free NH2- terminal methionine. Following this, the acetylation of the NH2-terminal methionine can occur in a fully posttranslational acetyl- CoA-dependent reaction in the reticulocyte lysate.