Demonstration of a common antigenic site on endomembrane proteins of Phaseolus vulgaris by a rat monoclonal antibody

Five hybrid myeloma cell lines that secrete antibodies to plant endomembrane-bound proteins have been prepared using rat myelomas and spleen cells from rats immunized against intact endoplasmic-reticulum and Golgi-membrane preparations from Phaseolus vulgaris. Four of these lines produced antibodies which all showed identical binding patterns in Western blots, recognising polypeptides of Mr 35 000, 58 000, 70 000, 91 000 and possibly 117 000 common to both membrane types, while the antibody produced by the fifth line bound to a polypeptide of Mr 57 000. This binding pattern persisted for the antibody produced by all positive clones derived by extensive subcloning of hybridomas 2B3 and 2C3 even with subsequent growth, so these polypeptides, therefore, probably have a common antigenic site. The antibody tested from the hybridoma 2C3 and two subsequent subclones inhibited the arabinosyl transferase involved in the synthesis of arabinan, a component of the primary cell-wall matrix, so that one of these polypeptides probably represents the enzyme. Comparison of the patterns of the changes in enzyme activity with the levels of each individual polypeptide in cells induced to divide and undergo primary growth tentatively identifies the 70 000-Mr polypeptide as the arabinan synthase. Interpretation of this and previous data indicates that the induction of this enzyme activity by plant growth regulators involves de novo synthesis of the protein.Abbreviations PBS phosphate-buffered saline - Ig immuno-globulin - SDS sodium dodecyl sulfate