Localization of pyrophosphatase in membranes of cauliflower inflorescence cells

Using a polyclonal antiserum specific for the tonoplastic H+-pyrophosphatas e (tPPase), significant amounts of antigenic polypeptides of the correct mo lecular mass were detected in Western blots of plasma membrane isolated fro m cauliflower (Brassica oleracea L.) inflorescence by phase-partitioning an d subsequent sucrose density centrifugation. Potassium iodide-stripped plas ma membranes continued to give a strong positive signal, indicating that th e PPase antigen detected was not a result of contamination through soluble PPase released during homogenisation. The same preparation contained neglig ible vacuolar (v)H+-ATPase activity and the A subunit of the vATPase could not be detected by immunoblotting. Plasma membrane fractions exhibited a pr oton-pumping activity with ATP as substrate, but such an activity was not m easurable with pyrophosphate, although the hydrolysis of this substrate was recorded. By contrast, pyrophosphate supported proton pumping in tonoplast -containing fractions. Immunogold electron microscopy confirmed the presenc e of PPase at the plasma membrane as well as at the tonoplast, trans Golgi network, and multivesicular bodies. The density of immunogold label was hig her at the plasma membrane than at the tonoplast, except for membrane fragm ents occurring in the lumen of the vacuoles which stained very conspicuousl y.