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

Riveros-Moreno, V; Heffernan, B; Torres, B; Chubb, A; Charles, IG; Moncada, S

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

Riveros-Moreno, V Heffernan, B Torres, B Chubb, A Charles, IG Moncada, S

Permalink

Publisher:: Wiley-Blackwell Publishing Ltd.
Publication Type:: Journal Article
Citation:: European Journal Of Biochemistry, 1995, 230 (1), pp. 52 - 57
Issue Date:: 1995-01

Closed Access

	Filename	Description	Size
	2009004970OK.pdf		3.38 MB	Adobe PDF	View/Open

Copyright Clearance Process

Recently Added
In Progress
Closed Access

This item is closed access and not available.

Full metadata record

Field	Value	Language
dc.contributor.author	Riveros-Moreno, V	en_US
dc.contributor.author	Heffernan, B	en_US
dc.contributor.author	Torres, B	en_US
dc.contributor.author	Chubb, A	en_US
dc.contributor.author	Charles, IG	en_US
dc.contributor.author	Moncada, S	en_US
dc.date.issued	1995-01	en_US
dc.identifier.citation	European Journal Of Biochemistry, 1995, 230 (1), pp. 52 - 57	en_US
dc.identifier.issn	0014-2956	en_US
dc.identifier.uri	http://hdl.handle.net/10453/13302
dc.description.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 ?g/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol · min<sup>-1</sup> · mg<sup>-1</sup>, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol · min<sup>-1</sup> · mg<sup>-1</sup>) 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; H<sub>4</sub>biopterin, 0.32, with a specific activity of 1500 nmol · min<sup>-1</sup> · mg<sup>-1</sup>. 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	en_US
dc.publisher	Wiley-Blackwell Publishing Ltd.	en_US
dc.relation.ispartof	European Journal Of Biochemistry	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.title	Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase	en_US
dc.type	Journal Article
utslib.citation.volume	1	en_US
utslib.citation.volume	230	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	1101 Medical Biochemistry and Metabolomics	en_US
utslib.for	0304 Medicinal and Biomolecular Chemistry	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	closed_access
pubs.consider-herdc	false	en_US
pubs.issue	1	en_US
pubs.volume	230	en_US

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 ?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

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10453/13302