LOCALIZATION OF MEMBRANE PYROPHOSPHATASE ACTIVITY IN RICINUS-COMMUNISSEEDLINGS

The membrane localization of the Mg2+K+-pyrophosphatase (PPase) activi ty in Ricinus communis was investigated, following density gradient ce ntrifugation and phase partitioning of membrane fractions, by a combin ation of marker enzyme analysis and immunological characterization and by using immunogold techniques. In membrane fractions isolated from R icinus cotyledons, PPase activity comigrated with the plasma membrane marker, vanadate-sensitive ATPase, following sucrose and Dextran gradi ent 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 o r could not be detected in the cotyledon fractions. The higher activit y of the plasma membrane H+-ATPase and PPase in the upper phase follow ing phase partitioning was correlated with a stronger staining reactio n in this fraction using polyclonal antibodies to the Arabidopsis plas ma 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 va cuolar H+-ATPase, and the mung bean vacuolar channel protein, VM23. Im munogold studies using the mung bean vacuolar H+-PPase showed a strong staining at the cell surface of phloem sieve elements in cotyledons a nd 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.