Journal of Experimental Botany, Vol. 52, No. 359, pp. 1165-1172,
June 1, 2001
© 2001 Oxford University Press
Original Papers |
Antibodies to assess phosphorylation of spinach leaf nitrate reductase on serine 543 and its binding to 14-3-3 proteins
Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
Received 11 August 2000; Accepted 14 January 2001
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAppendixReferences |
|---|
To monitor site-specific phosphorylation of spinach leaf nitrate reductase (NR) and binding of the enzyme to 14-3-3 proteins, serum antibodies were raised that select for either serine 543 phospho- or dephospho-NR. The dephospho-specific antibodies blocked NR phosphorylation on serine 543. The phospho-specific antibodies prevented NR binding to 14-3-3s, NR inhibition by 14-3-3s, NR dephosphorylation on serine 543, and did not precipitate 14-3-3s together with NR. Together, this confirms that 14-3-3s bind to NR at hinge 1 after it has been phosphorylated on serine 543. The amounts of individual NR forms were determined in leaf extracts by immunoblotting and immunoprecipitation. The phosphorylation state of NR on serine 543 increased 2–3-fold in leaves upon a light/ dark transition. Before the transition, one-third of NR was already phosphorylated on serine 543 but was not bound to 14-3-3s. Phosphorylation of serine 543 seems not to be enough to bind to 14-3-3s in leaves.
Key words: 14-3-3 proteins, nitrate reductase, protein phosphorylation, protein:protein interactions, enzyme inhibition.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
|---|
The metabolic enzyme nitrate reductase (NR; EC 1.6.6.1) is regulated by a phosphorylation-dependent binding to 14-3-3 proteins (MacKintosh et al., 1995
; Kaiser et al., 1999). 14-3-3s are conserved eukaryotic binding proteins that, although their exact function is unknown, are involved in almost every cellular process (Shaw, 2000). Phosphorylation of spinach leaf NR in the hinge 1 region on serine 543 creates a binding site in NR for 14-3-3s and inhibits the enzyme in the presence of 14-3-3s and divalent cations (Moorhead et al., 1996; Bachmann et al., 1996a; Weiner and Kaiser, 2000). This usually occurs in leaves upon a light/dark transition. Reillumination activates the enzyme via dephosphorylation of serine 543 by a protein phosphatase 2A and release of the 14-3-3s. A change in the phosphorylated state of NR on serine 543 can therefore be recognized as a change of NR activity which became widely used to detect NR dephosphorylation or phosphorylation (MacKintosh et al., 1995; Bachmann et al., 1995; Kaiser and Huber, 1997). A number of problems are associated with this approach, however, since it depends on NR binding to 14-3-3s rather than on NR phosphorylation on serine 543 per se. NR activity-based results can give rise to erroneous interpretations with respect to phosphorylation or dephosphorylation of serine 543 because the latter and NR activity can change independently as evidenced by (I) NR phosphorylation seems not to correlate with NR binding to 14-3-3s in leaves (Weiner and Kaiser, 1999); (II) NR is activated if 14-3-3s dissociate from NR, in particular in highly diluted enzyme preparations, as can be inferred from the stability of the 14-3-3 complex of NR (Moorhead et al., 1999; Lillo et al., 1997) and (III) partial purification can remove 14-3-3s from NR and thus activate NR (MacKintosh et al., 1995; Glaab and Kaiser, 1995).
Clearly, binding to 14-3-3s and NR phosphorylation need to be examined independently and directly in order to understand the regulation of NR more fully. The classical and most direct way to assess the phosphorylated state of a particular site in a protein involves metabolic labelling, fragmentation of the protein, and mapping of the phosphopeptides (van der Geer et al., 1998). This procedure is often time-consuming, tedious to perform and requires proper facilities. More recent developments in mass spectrometry allow the detection of site-specific phosphorylation in proteins without radiochemical labelling (Resing and Ahn, 1997). This requires rather expensive equipment, however. A convenient alternative to these procedures is a technique that relies on antibodies that can recognize if a certain residue in a protein is phosphorylated or not (Czernik et al., 1991; Weiner, 1995; Ueno et al., 2000). Here, such a technique was used, NR phosphorylation or dephosphorylation on serine 543 was examined and how this affects NR binding to 14-3-3s was investigated.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
|---|
Materials, extraction, NR activity assay
Growth of spinach, protein extraction from leaves (10 mg ml-1 buffer), desalting over Sephadex G25 (medium grade), and NR activity assays in the presence of 10 mM MgCl2 or 15 mM EDTA (3 min assays without preincubation of the extract) were done as previously (Kaiser and Huber, 1997
), except that the following extraction buffer was used: 50 mM MOPS/NaOH, 5 mM MgCl2, 150 mM NaCl, pH 7.5, 1 mM DTT, 0.5% (v/v) Triton X-100, and 0.5 mM freshly prepared PMSF.
Protein purification and detection, antibody production
Gel electrophoresis (Laemmli, 1970), Western transfer and immunodecoration were as described earlier (Weiner, 1995). To determine the relative amount of NR or 14-3-3s, immunoprecipitated, partially purified or crude proteins (0.05–5 µg) were gel-fractionated in parallel to serial dilutions of purified NR or 14-3-3s. NR or 14-3-3 polypeptides were then measured densitometrically (Image Master, Pharmacia, Uppsala, Sweden) after they were stained with Coomassie Brilliant Blue R 250 or blotted and immunodecorated. The relative amount of NR or 14-3-3s was eventually read from standard curves of the purified proteins.
Serum antibodies were raised in rabbits (New Zealand Whites). Peptide antigens were made by linking synthetic peptides (DRQYHPAPMSGVVRTP, close to the N-terminus of NR) to Keyhole limpet haemocyanin (Harlow and Lane, 1988) or (GPTLKRTASTPFMNTTS, around serine 543 of NR, ‘S-peptide’; GPTLKRTADTPFMNTTS, ‘D-peptide’) to partially purified protein derivative of tuberculin (Weiner, 1995). Antisera to 14-3-3s were raised using purified 14-3-3s as antigen. They were purified from spinach leaves (Bachmann et al., 1996a). Immunoprecipitation of NR was done with immobilized antibodies (Weiner and Kaiser, 1999), using antisera to the N-terminus or to the D- or S-peptide. All NR antibodies cross-reacted only with a single 110 kDa band, since such band was not recognized in the presence of antigenic peptides. 14-3-3-free NR preparations that contained NR-kinase and NR-phosphatase were partially purified with polyethylene glycol and by ion exchange chromatography (Bachmann et al., 1996b).
Tryptic NR fragments, oxidation with performic acid and phosphopeptide mapping on cellulose thin-layer plates with a Hunter HTLE 7000 apparatus were done as previously (van der Geer et al., 1998). A synthetic peptide corresponding to residues 535–552 of spinach NR was phosphorylated and purified (Bachmann et al., 1996b).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
|---|
The peptide antibodies could distinguish between serine 543 phospho- and dephospho-NR
To monitor site-specific phosphorylation and dephosphorylation of NR serum antibodies were raised against two synthetic peptides corresponding to the phosphorylation site of spinach leaf NR around serine 543. One peptide carried a serine residue corresponding to serine 543 as a potential phosphate acceptor. The other peptide had an aspartate instead of the serine residue to provide a stable mimetic of the phosphorylated state. As shown in Fig. 1
, antibodies were obtained that can detect NR phosphorylation over a broad range as evidenced by the changes in NR immunodecoration on Western blots and the incorporation of phosphate into dephosphorylated NR during a time-course of NR phosphorylation using partially purified leaf enzymes. The D-antibodies weakly decorated NR before NR was back-phosphorylated. This decoration disappeared if NR was dephosphorylated before back-phosphorylation for a longer period of time than in the experiment shown in Fig. 1. So the weak initial decoration was probably due to the presence of a minor portion of serine 543 phospho-NR as a result of incomplete dephosphorylation. Phosphate incorporation into NR seemed to occur exclusively and efficiently on serine 543 because (I) the stoichiometry of NR phosphorylation at the end of the time-course reached almost 1 mol phosphate mol-1 NR (Fig. 1), (II) after phosphopeptide mapping of tryptic NR fragments by two-dimensional separation on thin-layer plates, the NR label appeared as a single spot that co-migrated with a synthetic peptide that corresponds to residues 535–552 of spinach NR and that was phosphorylated and treated in the same way as NR (Fig. 2) and (III) NR labelling matched NR decoration with the antibodies that preferred either serine 543 phospho- or dephospho-NR as shown by the inhibition of the immunodecoration of dephospho-NR by the serine peptide and of phospho-NR by the aspartate peptide (Fig. 3). Both antibodies seemed to bind to their corresponding antigenic peptides at least 20-fold more strongly than to the other peptide derivative as can be inferred from the difference in the peptide concentrations that caused 50% inhibition of the immunodecoration. The strong preference of the antibodies was confirmed by the following: (I) the S-antibodies failed to recognize phosphorylated NR that had been immunoprecipitated with the D-antibodies and, vice versa, the D-antibodies did not detect dephosphorylated NR that had been precipitated with the S-antibodies (Fig. 4), (II) unlike the D-antibodies, the S-antibodies did not precipitate the phosphorylated synthetic peptide (Fig. 5) or the phosphorylated NR fragment (not shown) that were mentioned above and (III) this phosphopeptide prevented, unlike the dephosphopeptide, NR immunoprecipitation with the D-antibodies (Fig. 4).
|
32P-ATP (3000 cpm pmol-1) to back-phosphorylate NR. Aliquots were taken from the incubation mix during back-phosphorylation as indicated in order to (I) immunodecorate the 110 kDa NR subunit with S- or D-antibodies following fractionation of proteins on 7% Laemmli gels and their transfer to an Immobilon P membrane, and (II) measure the incorporation of label into NR after addition of 2 mM cold ATP, immunoprecipitation of NR with antibodies to the N-terminus, gel-fractionation of the immunoprecipitate, and Cerenkov counting of the gel piece after Coomassie Blue staining and excision from the dried gel. The stoichiometry of NR phosphorylation was 1.0 and 0.90 mol phosphate mol-1 NR in two independent experiments at the end of the time-course.
|
|
|
|
The antibodies prevented NR binding to 14-3-3s, NR phosphorylation and, unlike 14-3-3s, NR dephosphorylation
Next, it was tested whether the peptide antibodies affect NR phosphorylation and dephosphorylation and NR binding to 14-3-3s using partially purified leaf proteins. As one might expect from the specificity of both antibodies (Figs 1, 3
–5), the S-antibodies inhibited NR phosphorylation (Fig. 6A) and the D-antibodies inhibited NR dephosphorylation (Fig. 6B). Such dephosphorylation was, however, not inhibited by 14-3-3s (Fig. 6B). NR dephosphorylation was catalysed by a protein phosphatase 2A because less than 1% of the phosphate counts that were released from NR after 20 min were released in the presence of 5 nM okadaic acid.
|
The D-antibodies reduced 14-3-3 binding to NR (Fig. 6C
) or 14-3-3-dependent inhibition of NR (Table 1). Conversely, 14-3-3s reduced NR binding to the D-antibodies as evidenced by (I) the reduction of NR immunoprecipitation by the D-antibodies in the presence of 14-3-3s and (II) the inability of the D-antibodies to pull down 14-3-3s together with NR (Fig. 7). Such effects were not observed if antibodies were used to the N-terminus of NR (Fig. 7) or to the C-terminus (not shown) instead of D-antibodies. So the D-antibodies recognized free phospho-NR, but not the 14-3-3 complex of NR.
|
|
In leaves, the phosphorylation state of NR on serine 543 increased only moderately upon a light/dark transition
NR is well known to be rapidly phosphorylated, bound to 14-3-3s and inactivated in spinach leaves upon a light/ dark transition. As recently shown, 14-3-3s are bound to NR in extracts from darkened leaves but not in extracts from illuminated leaves (Weiner and Kaiser, 1999). The amounts of serine 543 phospho- and dephospho-NR before or after a light/dark transition are unknown, however. Based on phosphopeptide mapping (Huber et al., 1992), only a 2–3-fold increase in the phosphorylation state of NR on serine 543 upon a light/dark transition was predicted. This was analysed next with the S- and D-antibodies. Crude protein extracts were prepared from illuminated or darkened leaves and immunodecorated phospho- or dephospho-NR on Western blots after the proteins were gel-fractionated and transferred (Fig. 8; upper panel). In addition, phospho- or dephospho-NR were immunoprecipitated from such extracts (Fig. 8; lower panel). the proportions of NR forms in leaves were then determined from the change in the amount of each NR form upon the light/dark transition (Appendix). As shown in Table 2, the results matched our prediction: about 1/3 of NR was already phosphorylated on serine 543 before the light/dark transition after which NR became almost fully phosphorylated on this site and bound to 14-3-3s. Notably, most of the phospho-NR was bound to 14-3-3s after, but not before the transition.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
|---|
Phospho- and dephospho-specific antibodies were raised that allowed the determination of the amounts of individual NR forms in tissues. In addition, the antibodies were suitable for testing NR regulation in vitro (see below). The antibody techniques in Figs 1 and 6
demonstrate how site-specific dephosphorylation, phosphorylation and binding to 14-3-3s can be monitored directly. Since the techniques are direct, they are more suitable than previous approaches (see Introduction) to assess NR regulation. Such antibody techniques should be particularly useful for characterizing protein phosphatases and kinases that are involved in NR regulation since the physiological substrate is used. As far as is known, this is the first report showing that a peptide that carries an aspartate instead of a phosphoserine residue gives rise to antibodies to the phosphorylated protein. A serine to aspartate exchange is widely used in site-directed mutagenesis to mimic a permanently phosphorylated serine residue in a protein. This has prompted the authors to make antibodies to the aspartate peptide rather than to the phosphoserine peptide. The latter usually leads to the production of antibodies to both the corresponding phospho- and dephospho-protein and requires harsh purification steps to separate phospho- from dephospho-specific antibodies (Czernik et al., 1991). The most useful antibodies can frequently not be recovered after such purification (Harlow and Lane, 1988; Weiner, 1995). Given the size and the similarity of the S- and the D-peptide, one might expect that both peptides contain regions that form identical epitopes. However, for as yet unknown reasons, this was obviously not the case. Consistent with an earlier observation (Weiner, 1995), most if not all antibodies seemed to be directed to phosphorylation-sensitive regions that may (or may not) include the phosphate acceptor residue.
These results confirm that 14-3-3s bind to NR at hinge 1 after it has been phosphorylated on serine 543 (Moorhead et al., 1996; Bachmann et al., 1996a). The 14-3-3 complex of NR was possibly not recognized by the D-antibodies (Fig. 7 and Fig. 8, lower panel) as a result of surface occupancy in the association of NR with 14-3-3s. The 14-3-3s possibly impeded access for the D-antibodies but not for protein phosphatase 2A (Fig. 6B) to the microenvironment around serine 543. It was observed that 14-3-3s promote the ATP-dependent inactivation of NR in the presence of protein kinases and -phosphatases (Bachmann et al., 1996b). These authors therefore concluded that 14-3-3s block NR dephosphorylation on serine 543. The results of the direct approach used here to assess such dephosphorylation (Fig. 6B) does not support such a conclusion, however. Unlike the 14-3-3s, the D- or S-antibodies did not influence NR activity (Table 1). It is therefore unlikely that the inhibition of NR dephosphorylation by the D-antibodies (Fig. 6B) or the reduction of 14-3-3 binding to NR (Fig. 6C) was the result of an indirect conformational effect. Further, the results in Table 1 do not support that hinge 1 region ligands other than 14-3-3s inhibit NR. Given the size and the structural flexibility of the synthetic peptides that were used as antigens, the antibodies did not perhaps bind to the same site in hinge 1 to that which 14-3-3s bind. However, it is also possible that a not yet identified structural feature is involved in NR inhibition by 14-3-3s (Nussaume et al., 1995; Pigaglio et al., 1999). Three findings are remarkable in this context: (I) the 14-3-3 binding stoichiometry of NR is almost two 14-3-3 monomers per NR subunit (Weiner and Kaiser 1999), (II) 14-3-3s are dimers and capable of binding two ligands simultaneously (Shaw, 2000) and (III) 14-3-3s bind to more than one sequence motif in a phosphorylation-dependent or -independent manner (Petosa et al., 1998; Masters et al., 1999). So the next step might be to find out whether or not 14-3-3s bind to NR at a site in addition to hinge 1, even if such a site, unlike the site in hinge 1, cannot simply be predicted from the primary sequence of NR.
The magnesium inhibition of NR (Table 2) increased as expected upon the light/dark transition (Kaiser and Huber, 1997). This reflects that all three forms of NR, in particular the 14-3-3 complex, are inhibited by magnesium (Weiner and Kaiser, 2000). A major portion of NR was phosphorylated on serine 543 in extracts from illuminated leaves but was not bound to 14-3-3s (Table 2). So, although necessary (Moorhead et al., 1996; Bachmann et al., 1996a), phosphorylation of serine 543 seems not to be enough to bind to 14-3-3s in leaves. Why were the 14-3-3s lacking? The reason is unknown. It is perhaps the result of a cellular redistribution of 14-3-3s (Tzivion et al., 2000) that occurs upon a light/ dark transition in leaves and that, given the abundance of 14-3-3s in leaves (Weiner and Kaiser, 1999), makes 14-3-3s less available for certain ligands.
|
Appendix |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
|---|
This appendix develops the theoretical basis for determining the proportions of different NR forms (Table 2
). The calculation of such proportions is based on the assumptions:
 | (1) |
 | (2) |
:
 | (3) |
 | (4) |
 | (5) |
can be exchanged with their corresponding values for x, y and z according to equations 3 and 4 to give
 | (6) |
can be reduced to y+z=0.88 (equation 7). From equations (2–4) and (7) it follows that v+w=0.34, x=0.12, and u=0.66. Since w<0.05z (Weiner and Kaiser, 1999) equation (5) leads to a range of values for v, y or z (0.30–0.34, 0.18–0.20 and 0.68–0.70) for that 0.32, 0.20 and 0.69 were eventually taken.
|
Acknowledgments |
|---|
This work was supported by the European Community (EC Bio4-ct-2231) and by the Deutsche Forschungsgemeinschaft WE 1449/4-1. We thank Heike Weiner for the artwork and Professor Nigel M Stitt for making laboratory facilities available to us.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +49 931 888 6158. E-mail: weiner{at}botanik.uni\|[hyphen]\|wuerzburg.de
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
|---|
Bachmann M, McMichael Jr RW, Huber JL, Kaiser WM, Huber SC. 1995. Partial purification and characterization of a calcium-dependent protein kinase and an inhibitor protein required for inactivation of spinach leaf nitrate reductase. Plant Physiology 108, 1083–1091.[Abstract]
Bachmann M, Huber JL, Athwal GS, Wu K, Ferl RJ, Huber SC. 1996a. 14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-432 by endogenous protein phosphatases. FEBS Letters 398, 26–30.[Web of Science][Medline]
Bachmann M, Shiraishi N, Campbell WH, Yoo B-C, Harmon AC, Huber SC. 1996b. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. The Plant Cell 8, 505–517.[Abstract]
Czernik AJ, Girault J-A, Nairn AC, Chen JC, Snyder G, Kebabian J, Greengard P. 1991. Production of phosphorylation state-specific antibodies. Methods in Enzymology 201, 264–283.[Web of Science][Medline]
Glaab J, Kaiser WM. 1995. Inactivation of nitrate reductase involves NR-protein phosphorylation and subsequent ‘binding’ of an inhibitor protein. Planta 195, 514–518.
Harlow E, Lane D. 1988. In: Antibodies: a laboratory manual. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press.
Huber JL, Huber SC, Campbell WH, Redinbaugh MG. 1992. Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation. Archives of Biocheimistry and Biophysics 296, 58–65.
Kaiser WM, Huber SC. 1997. Correlation between apparent activation state of nitrate reductase (NR), NR hysterisis and degradation of NR protein. Journal of Experimental Botany 48, 1367–1374.
Kaiser WM, Weiner H, Huber SC. 1999. Nitrate reductase in higher plants: a case study for transduction of environmental stimuli into control of catalytic activity. Physiologia Plantarum 105, 385–390.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]
Lillo C, Kazazaic S, Ruoff P, Meyer C. 1997. Characterization of nitrate reductase from light- and dark-exposed leaves. Plant Physiology 114, 1377–1383.[Abstract]
MacKintosh C, Douglas P, Lillo C. 1995. Identification of a protein that inhibits the phosphorylated form of nitrate reductase from spinach (Spinacia oleracea) leaves. Plant Physiology 107, 451–457.[Abstract]
Masters SC, Pederson KJ, Zhang L, Barbieri JT, Fu H. 1999. Interaction of 14-3-3 with a non-phosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa. Biochemistry 38, 5216–5221.[Medline]
Moorhead G, Douglas P, Cotelle V, Harthill J, Morrice N, Meek S, Deiting U, Stitt M, Scarabel M, Aitken A, MacKintosh C. 1999. Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. The Plant Journal 18, 1–12.[Web of Science][Medline]
Moorhead G, Douglas P, Morrice N, Scarabel M, Aitken A, MacKintosh C. 1996. Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin. Current Biology 6, 1104–1113.[Web of Science][Medline]
Nussaume L, Vincentz M, Meyer C, Boutin J-P, Caboche M. 1995. Post-transcriptional regulation of nitrate reductase by light is abolished by an N-terminal deletion. The Plant Cell 7, 611–621.[Abstract]
Petosa C, Masters SC, Bankston LA, Pohl J, Wang B, Fu H, Liddington RC. 1998. 14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove. Journal of Biological Chemistry 273, 16305–16310.
Pigaglio E, Durand N, Meyer C. 1999. A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding. Plant Physiology 119, 219–229.
Resing KA, Ahn NG. 1997. Protein phosphorylation analysis by electrospray ionization-mass spectrometry. Methods in Enzymology 283, 29–44.[Web of Science][Medline]
Shaw A. 2000. The 14-3-3 proteins. Current Biology 10, R400.[Web of Science][Medline]
Tzivion G, Luo ZJ, Avruch J. 2000. Calyculin A induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo. Journal of Biological Chemistry 275, 29772–29778.
Ueno Y, Imanare E, Yoshizawa-Kumagaye K, Nakajima K, Inami K, Shiba T, Sakakibara H, Sugiyama T, Izui K. 2000. Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide. The Plant Journal 21, 17–26.[Web of Science][Medline]
Van der Geer P, Luo K, Sefton BM, Hunter T. 1998. Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates. In: JE Celis, ed. Cell biology, 2nd edn. Vol. 4. San Diego: Academic Press, 310–334.
Weiner H. 1995. Antibodies that distinguish between the serine-158 phospho- and dephospho-form of spinach leaf sucrose-phosphate synthase. Plant Physiology 108, 219–225.[Abstract]
Weiner H, Kaiser WM. 1999. 14-3-3 proteins control proteolysis of nitrate reductase in spinach leaves. FEBS Letters 455, 75–78.[Web of Science][Medline]
Weiner H, Kaiser WM. 2000. Binding to 14-3-3 proteins is not sufficient to inhibit nitrate reductase in spinach leaves. FEBS Letters 480, 217–220.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Allegre, J. Silvestre, P. Morard, J. Kallerhoff, and E. Pinelli Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia J. Exp. Bot., December 1, 2004; 55(408): 2625 - 2634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. M. Kaiser, H. Weiner, A. Kandlbinder, C.-B. Tsai, P. Rockel, M. Sonoda, and E. Planchet Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction J. Exp. Bot., April 15, 2002; 53(370): 875 - 882. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||