Immunoaffinity purification and biochemical characterization of plasma membrane arabino-galactan-rich glycoproteins of Nicotiana glutinosa

Abstract

Monoclonal antibody PN 16.4B4 reacts with an epitope present on the external face of the plasma membrane as shown by immunofluorescent staining of Nicotiana glutinosa L. protoplasts (Norman et al. 1986, Planta 167, 452–459). We show here that this epitope is present in a glycan moiety and defines a family of surface glycoproteins with molecular masses in the range 135–180 kilodalton (kDa). These glycoproteins are exclusively associated with the plasma membrane as demonstrated by immunostaining of highly purified plasma membrane vesicles obtained by aqueous two-phase partitioning of microsomal fractions. The bulk of these glycoproteins were not released by high-salt washing, sonication or hypotonie shock treatment of plasma membrane vesicles, demonstrating a tight association with the membrane. Triton X-114 partitioning of plasma membrane vesicles indicates that these antigens are hydrophilic, peripheral membrane glycoproteins. The glycoproteins were purified by immunoaffinity chromatography following solubilization in sodium dodecyl sulfate and shown to contain glycan moieties abundant in arabinose and galactose linked to a 50-kDa polypeptide rich in alanine, glycine, serine and threonine.