Skip to main content
SpringerLink
Log in

Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5′-triphosphate

  • Published:
Planta Aims and scope Submit manuscript

Abstract

Photoaffinity labeling with [32P] 8-azidoadenosine 5′-triphosphate (8-N3ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of 32P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [32P] 8-N3ATP. 32P-labeling was dependent upon ultraviolet irradiation and occurred with [32P] 8-N3ATP labeled in the α-position, indicating covalent incorporation of the photoprobe. Both [32P] 8-N3ATP and [32P] 8-N3GTP were incorporated to a similar extent into the 53-kilodalton (kDa) “large” subunit (LSu), but incorporation of [32P] 8-N3GTP into the 14-kDa “small” subunit (SSu) of RuBPCase was <5% of that measured with [32P] 8-N3ATP. Distinct binding sites for 8-N3ATP on the two subunits were indicated by different apparent K D values, 3 and 18 μM for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg2+, anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N3ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [32P] 8-N3ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N3ATP binds to a site on the SSu that has physiological significance.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

kDa:

kilodalton

LSu:

large subunit

8-N3ATP:

8-azidoadenosine 5′-triphosphate

RuBP:

ribulose-1,5-bisphosphate

RuBPCase:

ribulose-1,5-bisphosphate carboxylase/oxygenase

SSu:

small subunit

References

  • Andersson, I., Knight, S., Schneider, G., Lindqvist, Y., Lindqvist, T., Branden, C.-I., Lorimer, G.H. (1989) Crystal structure of the active site of ribulose-bisphosphate carboxylase. Nature 337, 229–234

    Google Scholar 

  • Andrews, T.J., Ballment, B. (1984) Active-site carbamate formation and reaction-intermediate-analog binding by ribulosebisphosphate carboxylase/oxygenase in the absence of its small subunits. Proc. Natl. Acad. Sci. USA 81, 3660–3664

    Google Scholar 

  • Andrews, T.J., Lorimer, G.H. (1985) Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase. J. Biol. Chem. 260, 4632–4636

    Google Scholar 

  • Andrews, T.J., Lorimer, G.H., Pierce, J. (1986) Three partial reactions of ribulose-bisphosphate carboxylase require both large and small subunits. J. Biol. Chem. 261, 12184–12188

    Google Scholar 

  • Badger, M.R., Lorimer, G.H. (1981) Interaction of sugar phosphates with the catalytic site of ribulose 1,5-bisphosphate carboxylase. Biochemistry 20, 2219–2225

    Google Scholar 

  • Bahr, J.T., Jensen, R.G. (1978) Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light. Arch. Biochem. Biophys. 185, 39–48

    Google Scholar 

  • Bennett, J. (1977) Phosphorylation of chloroplast membrane polypeptides. Nature 269, 344–346

    Google Scholar 

  • Bloom, M.V., Milos, P., Roy, H. (1983) Light-dependent assembly of ribulose-1,5-bisphosphate carboxylase. Proc. Natl. Acad. Sci. USA 80, 1013–1017

    Google Scholar 

  • Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–274

    Article  CAS  PubMed  Google Scholar 

  • Cline, K., Werner-Washburne, M., Lubber, T.H., Keegstra, K. (1985) Prcursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J. Biol. Chem. 260, 3691–3696

    Google Scholar 

  • Crafts-Brandner, S.J., Salvucci, M.E., Egli, D.B. (1990) Changes in ribulosebisphosphate carboxylase/oxygenase and ribulose 5phosphate kinase abundances and photosynthetic capacity during leaf senescence. Photosynth. Res. 23, 223–230

    Google Scholar 

  • Czarnecki, J.J. (1984) Dynamics of the tautomeric equilibria of the 2-azido-adenosine photoaffinity analogs. Biochem. Biophys. Acta 800, 41–51

    Google Scholar 

  • Czarnecki, J.J., Geahlen, R.T., Haley, B.E. (1979) Synthesis and use of azido photoaffinity analogs of adenine and guanine nucleotides. Methods Enzymol. 56, 642–653

    Google Scholar 

  • Czarnecki, J.J., Abbott, M.S., Selman, B.R. (1982) Photoaffinity labeling with 2-azidoadenosine diphosphate of a tight nucleotide binding site on chloroplast coupling factor 1. Proc. Natl. Acad. Sci. USA 79, 7744–7748

    Google Scholar 

  • Evans, R.K., Haley, B.E., Roth, D.A. (1985) Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties. J. Biol. Chem. 260, 7800–7804

    Google Scholar 

  • Foyer, C. (1985) Stromal protein phosphorylation in spinach (Spinacia oleracia) chloroplasts. Biochem. J. 231, 97–103

    Google Scholar 

  • Glynn, I.M., Chappell, J.B. (1964) A simple method for the preparation of 32P-labeled adenosine triphosphate of high specific activity. Biochem. J. 90, 147–149

    Google Scholar 

  • Haley, B.E., Hoffman, J.F. (1974) Interactions of a photoaffinity ATP analog with cation-stimulated ATPase activities of human red cell ghosts. Proc. Natl. Acad. Sci. USA 71, 3367–3371

    Google Scholar 

  • Hatch, A.L., Jensen, R.G. (1980) Regulation of ribulose-1,5-bisphosphate carboxylase from tobacco: changes in pH response and affinity for CO2 and Mg2+ induced by chloroplast intermediates. Arch. Biochem. Biophys. 205, 587–594

    Google Scholar 

  • Incharoensakdi, A., Takabe, T., Akazawa, T. (1986) Role of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in the activation process. Arch. Biochem. Biophys. 248, 62–70

    Google Scholar 

  • Jordan, D.B., Chollet, R. (1983) Inhibition of ribulose bisphosphate carboxylase by substrate ribulose 1,5-bisphosphate. J. Biol. Chem. 258, 13752–13728

    Google Scholar 

  • Jordan, D.B., Ogren, W.L., Cholet, R. (1983) Binding of phosphorylated effectors by active and inactive forms of ribulose 1,5-bisphosphate carboxylase. Biochemistry 22, 3410–3418

    Google Scholar 

  • Julian, D.A., Lehman, I.R. (1987) Photoaffinity labeling of the recBCD enzyme of Escherichia coli with 8-azidoadenosine 5′-triphosphate. J. Biol. Chem. 262, 9044–9051

    Google Scholar 

  • Knight, K.L., McEntee, K. (1985) Covalent modification of the recA protein from Escherichia coli with the photoaffinity label 8-azidoadenosine 5′-triphosphate. J. Biol. Chem. 260, 867–872

    Google Scholar 

  • Laing, W.A., Christeller, J.T. (1984) Chloroplast phosphoproteins: distribution of phosphoproteins within spinach chloroplasts. Plant Sci. Lett. 36, 99–104

    Google Scholar 

  • Lin, Z.-F., Lucero, H.A., Racker, E. (1982) Protein kinases from spinach chloroplasts. I. Purification and identification of two distinct protein kinases. J. Biol. Chem. 257, 12153–12156

    Google Scholar 

  • Lorimer, G.H. (1981) Ribulosebisphosphate carboxylase: amino acid sequence of a peptide bearing the activator carbon dioxide. Biochemistry 20, 1236–1240

    Google Scholar 

  • Lorimer, G.H., Miziorko, H.M. (1980) Carbamate formation on the ξ-amino group of a lysyl residue as the basis for the activation of ribulosebisphosphate carboxylase by CO2 and Mg2+. Biochemistry 19, 5321–5328

    Google Scholar 

  • McCurry, S.D., Pierce, J., Tolbert, N.E., Orme-Johnson, W.H. (1981) On the mechanism of effector-mediated activation of ribulose bisphosphate carboxylase/oxygenase. J. Biol. Chem. 256, 6623–6628

    Google Scholar 

  • Mills, W.R., Joy, K.W. (1980) A rapid method for isolation of purified, physiologically active chloroplasts, used to study the intercellular distribution of amino acids in pea leaves. Planta 148, 75–83

    Google Scholar 

  • Miziorko, H.M., Sealy, R.C. (1980) Characterization of the ribulosebisphosphate carboxylase-carbon dioxide-divalent cation-carboxypentitol bisphosphate complex. Biochemistry 19, 1167–1171

    Google Scholar 

  • Mornet, R., Theiler, J.B., Leonard, N.J., Schmitz, R.Y., Moore, F.H., Skoog, F. (1979) Active cytokinins. Photoaffinity labeling agents to detect binding. Plant Physiol. 64, 600–610

    Google Scholar 

  • Penefsky, H.S. (1977) Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J. Biol. Chem. 252, 2891–2899

    Google Scholar 

  • Pierce, J., Tolbert, N.E., Barker, R. (1980) Interaction of ribulosebisphosphate carboxylase/oxygenase with transition state analogues. Biochemistry 19, 934–942

    Google Scholar 

  • Potter, R.L., Haley, B.E. (1983) Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: techniques and applications. Methods Enzymol. 91, 613–633

    Google Scholar 

  • Robinson, S.P., Portis, A.R. Jr. (1989) Adenosine triphosphate hydrolysis by purified Rubisco activase. Arch. Biochem. Biophys. 268, 93–99

    Google Scholar 

  • Robinson, S.P., Wiskich, J.T. (1977) Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides. Plant Physiol. 59, 422–427

    Google Scholar 

  • Salvucci, M.E., Portis, A.R. Jr., Ogren, W.L. (1985) A soluble chloroplast protein catalyzes ribulosebisphosphate carboxylase/ oxygenase activation in vivo. Photosynth. Res. 7, 201–203

    Google Scholar 

  • Salvucci, M.E., Haley, B.E. (1990) Photoaffinity labeling of the Rubisco active-site and small subunit with 8-azidoadenosine 5′-triphosphate. In: Proc. VIIIth Int. Congr. on Photosynthesis, Stockholm, Sweden. Nijhoff, Dordrecht (in press)

  • Soll, J., Buchanan, B.B. (1983) Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ. J. Biol. Chem. 258, 6686–6689

    Google Scholar 

  • Streusand, V.J., Portis, A.R. Jr. (1987) Rubisco activase mediates ATP-dependent activation of ribulosebisphosphate carboxylase. Plant Physiol. 85, 152–154

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. U.S. Department of Agriculture, Agricultural Research Service, 40546, Lexington, KY, USA

    Michael E. Salvucci

  2. Department of Biochemistry, University of Kentucky, 40546, Lexington, KY, USA

    Boyd E. Haley

Authors
  1. Michael E. Salvucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Boyd E. Haley
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Kentucky Agricultural Experiment Station Journal Article No. 89-3-150

The authors acknowledge the technical assistance of J.C. Anderson. This work was supported in part by National Institute of Health grant GM 35766 to B.E.H.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Salvucci, M.E., Haley, B.E. Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5′-triphosphate. Planta 181, 287–295 (1990). https://doi.org/10.1007/BF00195878

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00195878

Key words

Search

Navigation