Primary structure of rat spermidine synthase: An example of refining the cDNA-derived amino acid sequence

The primary structure of rat spermidine synthase having the N-terminal acet ylated methionine and 98.7% homology with that of the mouse enzyme is prese nted using a limited amount of the homogeneous enzyme. The study strategy w as principally to compare the molecular masses of liberated peptides determ ined by three specific cleavage methods with those expected from known cDNA -derived amino acid sequences of mouse and human enzymes using matrix-assis ted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF -MS). The cleavage methods involved two enzymatic methods using lysylendope ptidase and arginylendopeptidase, and a chemical method for cleaving at the cysteine residue using 2-nitro-5-thiocyanobenzoic acid. Their usefulness w as clearly demonstrated. Column-switching semimicro reversed-phase HPLC, wh ich permits application of the entire reaction mixture, was useful for coll ecting a small amount of peptides containing the N-terminal amino acid, to confirm acetylation of the N-terminal methionine by MALDI TOF-MS. It was ne cessary in this approach to examine the amino acid sequence of certain pept ides. The Edman method was used for the sequence analysis, and this will be replaced by an improved MALDI TOF-MS now available in a few laboratories.