PURIFICATION TO HOMOGENEITY AND CHARACTERIZATION OF RAT-BRAIN RECOMBINANT NITRIC-OXIDE SYNTHASE

We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. and Riveros-Moreno, V. (1993) Biochem. Bi ophys, Res. Commun. 196, 1481-1489], where a small proportion of the e xpressed enzyme was soluble and active, but the majority was insoluble (approximately 1546 of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addi tion to calmodulin and NADPH. Here, we report that a substantial propo rtion of the total NO synthase produced becomes soluble following addi tion of hemin (2.5 mu g/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nm ol.min(-1).mg(-1), after ADP-Sepharose affinity purification. Full act ivity (approximately 800 nmol.min(-1).mg(-1)) could, however be obtain ed by including precursors for the cofactors, nicotinic acid, riboflav in, and sepiapterin in the culture medium. We demonstrate that the enz yme activity is exclusively associated with the dimeric form of the en zyme, which had the following molar ratios for the cofactors: heme, 0. 92; FAD, 0.57; FMN, 0.34; H(4)biopterin, 0.32, with a specific activit y of 1500 nmol.min(-1).mg(-1). The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studie s on the relationship between enzyme structure and its mechanism of ca talysis.