Purification, cloning, and expression of nitric-oxide synthase

This chapter describes the purification, cloning, and expression of nitric-oxide synthase (NOS). Calmodulin is added to fractionated tissue extracts to purify NOS. To purify NOS from samples, tissues are homogenized in 10 times their weight in buffer A, and the samples are kept at 4° throughout the following procedure. A two-step polymerase chain reaction (PCR) strategy is used to clone rat n-NOS). NOS is purified from rat cerebella, digested with trypsin, and the peptide fragments purified and sequenced. The development of a swift, sensitive, and specific assay for NOS catalytic activity has facilitated the purification of NOS isoforms and the isolation of NOS cDNA sequences. The identification of NO as an active biological agent and the purification of NOS lead to an understanding of the physiology and pathophysiology of NOS.