Present address: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4.
Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays
Article first published online: 3 MAR 2003
European Journal of Biochemistry
Volume 270, Issue 6, pages 1356–1362, March 2003
How to Cite
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: 1356–1362. doi: 10.1046/j.1432-1033.2003.03509.x
- Issue published online: 3 MAR 2003
- Article first published online: 3 MAR 2003
- (Received 15 November 2002, revised 8 January 2003, accepted 10 February 2003)
- nucleotide pyrophosphatase (nucleotide pyrophosphohydrolase);
- nitrate reductase;
- glutamine synthetase;
- nudix hydrolase
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.