Skip to main content
Log in

α-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.) I. In situ localization by indirect immunofluorescence

  • Published:
Planta Aims and scope Submit manuscript

Abstract

Antisera were raised against two forms of α-1,4-glucan phosphorylase (EC 2.4.1.1) which had been purified from leaves of Spinacia oleracea L. Immunoglobulin G preparations were isolated from the antisera, and their specificity was ensured by immunoplobulin G preparations were used for in situ localization of the two phosphorylase forms in spinach leaf thin sections by indirect immuno-fluorescence. Both enzyme forms were present in the palisade and spongy parenchyma and in the guard cells, but their intracellular distribution was complementary. One phosphorylase form (designated as the chloroplastic form) was restricted to the stromal space of chloroplasts whereas the other (the non-chloroplastic form) was present only in the cytoplasm of chloroplast-containing cells. Thus, the phosphorylases represent two distinct compartment-specific enzyme forms which reside within the same photosynthetically active mesophyll cell.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

Abbreviations

DBM:

diazobenzyloxymethyl

FITC:

fluorescein isothiocyanate

IgG:

immunoglobulin G

PMSF:

phenylmethylsulfonyl fluoride

References

  • Alwine, J.C., Kemp, D.J., Stark, G.R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354

    Google Scholar 

  • Edwards, G.E., Robinson, S.P., Tyler, N.J.C., Walker, D.A. (1978) Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat. Plant Physiol. 62, 313–319

    Google Scholar 

  • Gershoni, J.M., Palade, G.E. (1983) Protein blotting: principles and applications. Anal. Biochem. 131, 1–15

    Google Scholar 

  • Johnson, G.D., de Nogueira Araujo, G.M. (1981) A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods 43, 349–350

    Google Scholar 

  • Okita, T.W., Greenberg, E., Kuhn, D.N., Preiss, J. (1979) Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves. Plant Physiol. 64, 187–192

    Google Scholar 

  • Renart, J., Reiser, J., Stark, G.R. (1979) Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA 76, 3116–3120

    Google Scholar 

  • Rick, W., Stegbauer, H.P. (1974) ga-Amylase. Messung der reduzierenden Gruppen. In: Methoden der enzymatischen Analyse, 3rd edn, vol. 1, pp. 918–923, Bergmeyer, H.U., ed., Verlag Chemie, Weinheim

    Google Scholar 

  • Rittenhouse, J., Marcus, F. (1984) Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartylprolyl peptide bonds in sodium dodecyl sulfate-containing buffers. Anal. Biochem. 138, 442–448

    Google Scholar 

  • Sajtos, B. (1985) Untersuchungen zum Polysaccharid-Stoffwechsel in Höheren Pflanzen. Diploma thesis, Münster

  • Schächtele, C., Steup, M. (1983) Glucan phosphorylase forms: immunological studies. Sixth International Congress on Photosynthesis Brussels, Abstract vol. I, 112–130

  • Schneider, E.M., Becker, J.-U., Volkmann, D. (1981) Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods. Planta 151, 124–134

    Google Scholar 

  • Steup, M., Latzko, E. (1979) Intracellular localization of phosphorylases in spinach and pea leaves. Planta 145, 69–75

    Google Scholar 

  • Steup, M., Robenek, H., Melkonian, M. (1983) In-vitro degradation of starch granules isolated from spinach chloroplasts. Planta 58, 428–436

    Google Scholar 

  • Steup, M., Schächtele, C. (1981) Mode of glucan degradation by purified phosphorylase forms from spinach leaves. Planta 153, 351–361

    Google Scholar 

  • Steup, M., Schächtele, C. (1986) α-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.). II. Peptide patterns and immunological properties. A comparison with other phosphorylase forms. Planta 168 (in press)

  • Steup, M., Schächtele, C., Latzko, E. (1980a) Separation and partial characterization of chloroplast and non-chloroplast α-glucan phosphorylases from spinach leaves. Z Pflanzenphysiol. 96, 365–374

    Google Scholar 

  • Steup, M., Schächtele, C., Latzko, E. (1980b) Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves. Planta 148, 168–173

    Google Scholar 

  • Thomas, W., de Fekete, M.A.R., Vieweg, G.H., Kramer, D. (1979) Cytochemische Lokalisierung der Stärkephosphorylase in isolierten Bündelscheidenzellen des Maisblattes. Eur. J. Cell Biol. 19, 73–77

    Google Scholar 

  • van Berkel, J. (1984) Immunchemische Lokalisierung von Proteinen in pflanzlichen Geweben. Diploma thesis, Münster

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schächtele, C., Steup, M. α-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.) I. In situ localization by indirect immunofluorescence. Planta 167, 444–451 (1986). https://doi.org/10.1007/BF00391219

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00391219

Key words

Navigation