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

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.