Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays

Moorhead, G. B.G., Meek, S. E.M., Douglas, P., Bridges, D., Smith, C. S., Morrice, N. and MacKintosh, C. (2003) Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays. European Journal of Biochemistry, 270(6), pp. 1356-1362. (doi: 10.1046/j.1432-1033.2003.03509.x)

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Publisher's URL: http://dx.doi.org/10.1046/j.1432-1033.2003.03509.x

Abstract

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Meek, Dr Sarah and MacKintosh, Prof Carol
Authors: Moorhead, G. B.G., Meek, S. E.M., Douglas, P., Bridges, D., Smith, C. S., Morrice, N., and MacKintosh, C.
College/School:College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Journal Name:European Journal of Biochemistry
Publisher:Wiley
ISSN:0014-2956
ISSN (Online):1432-1033

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