LOCALIZATION OF MEMBRANE PYROPHOSPHATASE ACTIVITY IN RICINUS-COMMUNIS SEEDLINGS

The membrane localization of the Mg2+K+-pyrophosphatase (PPase) activity in Ricinus communis was investigated, following density gradient centrifugation and phase partitioning of membrane fractions, by a combination of marker enzyme analysis and immunological characterization and by using immunogold techniques. In membrane fractions isolated from Ricinus cotyledons, PPase activity comigrated with the plasma membrane marker, vanadate-sensitive ATPase, following sucrose and Dextran gradient centrifugation and aqueous two-phase partitioning. However, levels of the tonoplast markers azide-insensitive, nitrate-sensitive ATPase activity or bafilomycin-sensitive ATPase activity were extremely low or could not be detected in the cotyledon fractions. The higher activity of the plasma membrane H+-ATPase and PPase in the upper phase following phase partitioning was correlated with a stronger staining reaction in this fraction using polyclonal antibodies to the Arabidopsis plasma membrane proton pump and the mung bean vacuolar H+-PPase. Although this suggested that a PPase may be associated with the plasma membrane, a stronger reaction in the upper phase than the lower phase was also observed following immunoblotting with antibodies to the Kalanchoe vacuolar H+-ATPase, and the mung bean vacuolar channel protein, VM23. Immunogold studies using the mung bean vacuolar H+-PPase showed a strong staining at the cell surface of phloem sieve elements in cotyledons and roots whereas in the mesophyll and cortical cells of these tissues the staining was associated mainly with the vacuoles. The possibility of a phloem-specific plasma membrane pyrophosphatase is discussed.