Purification to Homogeneity and Characterisation of Rat Brain Recombinant Nitric Oxide Synthase
| Autor: | Salvador Moncacdta, Valentina Riveros-Moreno, Ann Chubb, Brian Heffernan, Belkis Torres, I. G. Charles |
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| Rok vydání: | 1995 |
| Předmět: |
Biochemistry & Molecular Biology
DNA Complementary Biochemistry Cofactor chemistry.chemical_compound Affinity chromatography medicine Animals Heme chemistry.chemical_classification biology Brain Tetrahydrobiopterin Hydrogen-Ion Concentration Molecular biology Recombinant Proteins Enzyme structure Enzyme assay Rats Nitric oxide synthase Enzyme Solubility chemistry biology.protein Hemin Amino Acid Oxidoreductases Nitric Oxide Synthase Baculoviridae medicine.drug |
| Zdroj: | European Journal of Biochemistry. 230:52-57 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1995.0052i.x |
| Popis: | 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. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% 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 addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 ?g/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol · min-1 · mg-1, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol · min-1 · mg-1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol · min-1 · mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis |
| Databáze: | OpenAIRE |
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