Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays.
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.
Moorhead
Greg B G
GB
MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, UK.
Meek
Sarah E M
SE
Douglas
Pauline
P
Bridges
Dave
D
Smith
Catherine S
CS
Morrice
Nick
N
MacKintosh
Carol
C
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001681:Biological Assay; D001937:Brassica / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment
D004791:Enzyme Inhibitors; D010940:Plant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase; D005974:Glutamate-Ammonia Ligase