Ultrastructural localisation and further biochemical characterisation of prolyl 4-hydroxylase from Phaseolus vulgaris: comparative analysis.
Prolyl 4-hydroxylase (EC 1.14.11.2), the enzyme responsible for the post-translational hydroxylation of peptide proline, has been well described in animals but has been little studied in plants. The best characterised example is the enzyme from French bean (Phaseolus vulgaris). In this study, the biochemical properties of this plant enzyme were examined in more detail and, using specific polyclonal antibodies, the localisation of the enzyme was determined. Both alpha- and beta-subunits did not show multiple forms, suggesting a relatively broad specificity of the enzyme complex with respect to the target hydroxylated amino acid sequences. Antibodies to the mammalian and Chlamydomonas enzymes cross-react with the higher plant subunits, indicating that some epitopes are highly conserved. The P. vulgaris enzyme was inhibited by analogues of oxoglutarate, but was not susceptible to doxorubicin. Inhibition of the bean enzyme by an oxaloglycine derivative resulted in the retention of the target (hydroxy)proline-rich protein in the endomembrane system. Immunolocalisation of the enzyme showed close association with the endoplasmic reticulum and Golgi apparatus in root tip cells of P. vulgaris or Tropaeolum majus. This localisation was particularly pronounced in Golgi-associated vesicles of young root tip cells of T. majus, cell types where rapid synthesis and deposition of wall material was observed. These data are consistent with the hypothesis, proposed by Bolwell [G.P. Bolwell, Dynamic aspects of the plant extracellular matrix, Int. Rev. Cytol. 146 (1993) 261-324], that protein hydroxylation must be completed before the glycosylation of the target (hydroxy)proline-rich glycoproteins in the Golgi stack.
Wojtaszek
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P
Division of Biochemistry, School of Biological Sciences, Royal Holloway and Bedford New College, University of London, Egham, Surrey, UK.
Smith
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CG
Bolwell
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GP
D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D006023:Glycoproteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006909:Hydroxyproline / Q000378:metabolism; D007150:Immunohistochemistry; D010835:Phytohemagglutinins / Q000378:metabolism* / Q000648:ultrastructure; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure*
D006023:Glycoproteins; D010835:Phytohemagglutinins; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline