Abstract
The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7‐day‐old oat ( Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg 2+ . NR sensitivity to Mg 2+ remained unchanged when 10 m M nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg 2+ inhibition of NR, which was strongly diminished in the presence of 10 m M NO 3 − + 100 µ M NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg 2+ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0‐40% saturation fraction (F1). After incubation of F1 with ATP, the sensitivity of the enzyme to Mg 2+ inhibition remained low, but it strongly increased after mixing F1 with a 45‐60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho‐NR, would be required to keep the phosphorylated enzyme inactive in a +Mg 2+ medium. Addition of 10 m M NO 3 − + 100 µ M NADH together with desalted F2 did not prevent Mg 2+ inhibition suggesting that NO 3 − did not interfere with IP binding to phospho‐NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg 2+ , After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 m M NO 3 − at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho‐NR induced by the substrates. The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase‐ or the kinase‐proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.
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Claims 13
Claim · model-written summary
ATP strongly phosphorylated nitrate reductase in oat leaf crude extracts, indicated by Mg2+ inhibition of activity.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg 2+ .”
e7“7‐day‐old oat”
Claim · model-written summary
Nitrate added after ATP did not change NR sensitivity to Mg2+.
- Taxon
- oat
Avena sativae7 - Treatment
- 10 m M nitratee0
- Outcome
- NR sensitivity to Mg 2+e0
- Direction
- remained unchangede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“NR sensitivity to Mg 2+ remained unchanged when 10 m M nitrate was added to crude extracts after ATP.”
e7“7‐day‐old oat”
Claim · model-written summary
Nitrate before or with ATP slightly decreased Mg2+ inhibition of NR.
- Taxon
- oatAvena sativae7
- Treatment
- nitrate before or simultaneously with ATPe0
- Outcome
- Mg 2+ inhibition of NRe0
- Direction
- slightly decreasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Addition of nitrate before or simultaneously with ATP slightly decreased Mg 2+ inhibition of NR”
e7“7‐day‐old oat”
Claim · model-written summary
Nitrate and NADH together strongly diminished Mg2+ inhibition of NR.
- Taxon
- oatAvena sativae7
- Treatment
- 10 m M NO 3 − + 100 µ M NADHe0
- Outcome
- Mg 2+ inhibition of NRe1
- Direction
- strongly diminishede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“which was strongly diminished in the presence of 10 m M NO 3 − + 100 µ M NADH.”
e1“Mg 2+ inhibition of NR”
e7“7‐day‐old oat”
Claim · model-written summary
NADH alone did not affect Mg2+ inhibition susceptibility.
- Taxon
- oatAvena sativae7
- Treatment
- NADH alonee0
- Outcome
- susceptibility to Mg 2+ inhibitione0
- Direction
- did not affecte0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Incubation with NADH alone did not affect the enzyme's susceptibility to Mg 2+ inhibition.”
e7“7‐day‐old oat”
Claim · model-written summary
Adding the F2 fraction to ATP treated F1 strongly increased NR sensitivity to Mg2+ inhibition.
- Taxon
- oatAvena sativae7
- Treatment
- mixing F1 with a 45‐60% saturation fraction (F2)e0
- Outcome
- sensitivity of the enzyme to Mg 2+ inhibitione0
- Direction
- strongly increasede0
- Comparator
- F1 with ATPe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“After incubation of F1 with ATP, the sensitivity of the enzyme to Mg 2+ inhibition remained low, but it strongly increased after mixing F1 with a 45‐60% saturation fraction (F2)”
e7“7‐day‐old oat”
Claim · model-written summary
Nitrate and NADH added with F2 did not prevent Mg2+ inhibition.
- Taxon
- oatAvena sativae7
- Treatment
- 10 m M NO 3 − + 100 µ M NADH together with desalted F2e0
- Outcome
- Mg 2+ inhibitione0
- Direction
- did not prevente0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Addition of 10 m M NO 3 − + 100 µ M NADH together with desalted F2 did not prevent Mg 2+ inhibition”
e7“7‐day‐old oat”
Claim · model-written summary
Both substrates during F1 phosphorylation kept NR active after F2 was added despite Mg2+.
- Taxon
- oatAvena sativae7
- Treatment
- both substrates during in vitro phosphorylatione0
- Outcome
- enzyme activee0
- Direction
- kept the enzyme activee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg 2+”
e7“7‐day‐old oat”
Claim · model-written summary
After phosphorylation NR was hardly reactivated alone or with nitrate at 30°C.
- Taxon
- oatAvena sativae7
- Outcome
- NR in crude extracte0
- Direction
- hardly reactivatede0
- Qualifier
- at 30°Ce0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 m M NO 3 − at 30°C.”
e7“7‐day‐old oat”
Claim · model-written summary
Nitrate and NADH together caused strong, rapid NR reactivation.
- Taxon
- oatAvena sativae7
- Treatment
- both nitrate and NADHe0
- Outcome
- reactivatione0
- Direction
- strong and very rapide0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH”
e7“7‐day‐old oat”
Claim · model-written summary
Microcystine inhibited substrate induced reactivation of phospho NR.
- Taxon
- oatAvena sativae7
- Treatment
- Microcystinee0
- Outcome
- reactivation of phospho‐NRe0
- Direction
- inhibitede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho‐NR induced by the substrates.”
e7“7‐day‐old oat”
Claim · model-written summary
Substrate binding appears to prevent phosphorylation and promote dephosphorylation by changing the NR protein.
- Taxon
- oatAvena sativae7
- Gene
- NR proteine0
- Outcome
- NR phosphorylatione0
- Direction
- prevente0
- Qualifier
- only after their binding to the NR proteine0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase‐ or the kinase‐proteins.”
e7“7‐day‐old oat”
Claim · model-written summary
The authors propose that nitrate induced conformational change blocks kinase access and stimulates NR dephosphorylation.
- Taxon
- oatAvena sativae7
- Gene
- NRe0
- Outcome
- NR phosphorylatione0
- Direction
- prevente0
- Qualifier
- this conformational changee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.”
e7“7‐day‐old oat”
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