Localization and partial characterization of the extracellular proteins centrifuged from pea internodes

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Abstract
The proteins removed from the extracellular space of dark‐grown pea (Pisum sativum L. cv. Alaska) internode sections by centrifugation were studied. A large number of proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These proteins ranged in size from 10 to 150 kdalton and their removal from the cell wall was greatly facilitated by the presence of salts of divalent and trivalent cations in the infiltration medium. Pulse‐labelling experiments with [35S)‐methionine showed that many of the proteins extracted from the cell wall incorporated radioactivity and that treatment with indoleacetic acid (IAA) altered the pattern of radiolabel incorporation. One of the proteins centrifuged from pea internode sections possessed per‐oxidase (EC 1.11.1.7) activity. The activity of this peroxidase increased less in auxin‐treated internode segments than in untreated controls. Antibodies were raised to the total protein fraction extracted by centrifugation and used to localize antigens on protein blots. Most of the proteins centrifuged from pea internode sections were stained by a dye coupled to the cell wall antiserum. Light microscopic immunohistochemical studies showed that the proteins centrifuged from dark‐grown pea internodes were localized almost exclusively in the cell wall and intercellular spaces of pea internode tissue. Light microscopic immunohistochemistry also showed that antibodies to extracted proteins penetrate into the apoplast of abraded pea internode segments and split pea stems. These antibodies did not influence growth of IAA‐treated or control tissue.