PMID: 7541350May 15, 1995Paper

Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase

European Journal of Biochemistry
V Riveros-MorenoS Moncada

Abstract

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 micrograms/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 ...Continue Reading

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Citations

Nov 2, 2001·The Journal of Biological Chemistry·Eric BlaskoJohn F Parkinson
Nov 21, 2007·The Journal of Comparative Neurology·Carmen Romero-GrimaldiCarmen Estrada
May 18, 1999·The Journal of Biological Chemistry·C R Nishida, P R Ortiz de Montellano
Oct 9, 1998·The Journal of Biological Chemistry·S J Lee, J T Stull
Jan 5, 1999·The Journal of Biological Chemistry·N SennequierD J Stuehr
Oct 14, 2004·Microscopy Research and Technique·María Luisa Del MoralMaría Angeles Peinado
Nov 15, 1997·The American Journal of Physiology·W J PearceS Ashwal
Dec 10, 1999·American Journal of Respiratory and Critical Care Medicine·M KakuyamaP Vallance
Oct 22, 2002·American Journal of Physiology. Heart and Circulatory Physiology·Bernard A CoertFredric B Meyer
Jan 1, 1997·Annual Review of Pharmacology and Toxicology·D J Stuehr
Jun 28, 2011·Journal of Enzyme Inhibition and Medicinal Chemistry·Eden PadayacheeChris G Whiteley
Mar 29, 2003·American Journal of Respiratory and Critical Care Medicine·Perenlei EnkhbaatarDaniel Traber
Dec 5, 2003·Journal of Hypertension·Ana Patricia FernándezEduardo Nava
Sep 28, 1998·Biochimica Et Biophysica Acta·A SchrammelB Mayer
Jun 29, 2001·Journal of Chemical Neuroanatomy·A JiménezM A Peinado
Jun 27, 2003·Nitric Oxide : Biology and Chemistry·Susana Castro-BlancoJosé Rodrigo
Apr 25, 2003·Brain Research. Developmental Brain Research·Ana Patricia FernándezJosé Rodrigo
Jun 26, 2010·BMC Neuroscience·Santos BlancoMaria A Peinado

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