Puromycin reaction system

It is usually accepted that the 16-membered-ring macrolides inhibit peptidyltransferase activity [79, 88, 89], because they inhibit puromycin reaction although poorly. The reaction is used as an assay system for peptidyltransferase activity, because puromycin characteristically interrupts peptide bond formation by virtue of its structural similarity to the 3 end of aminoacyl-tRNA. Puromycin enters the A site (the so-called aminoacyl site) on the ribosome and is incorporated into either a nascent polypeptide or into A-acylaminoacylate, consequently causing premature release of puromycinyl polypeptide or A-acylaminoacyl-puromycin from the ribosome. 

The reactions of phase 2 relate to the attachment of the bridge-carbohydrate residues to the polypeptide chain. There is evidence showing that this addition occurs while the polypeptide chain is still attached to, or perhaps still being synthesized on, the ribosomes.101-103 Thus, 14C-labeled 2-amino-2-deoxy-D-glucose, injected into the circulatory system of the rat, was incorporated into protein in the ribosomes of the rough endoplasmic-reticulum of the liver. Administration of puromycin caused release of the 14C-labeled glycoprotein, which could be isolated by acid-precipitation methods. Examination of the radioactivity data revealed that the subcellular structures most actively involved in glycoprotein synthesis were the ribosomes bound to the membrane, and not free polysomes. 

Fragment reaction a reaction used to assay the activity of peptidyl transferase. In a cell-free system containing 708 or 80 S ribosomes, the growing peptide chain is transferred to Puromycin (see) and released as peptidyl-puromycin. 

A very simple system for peptide-bond synthesis involves N-formylmet-AC C A A C (the extremity of formylmet-transfer RNA released by Ti-ribonuclease action), puromycin and 50 S ribosomal subunits. The reaction product is N-formyl-met-puromycin, and its formation is inhibited by chloramphenicoL It seems that the antibiotic acts at the level of the peptidyl transferase, a specific constituent of the SO S particle. 

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