Heterogeneity of the vacuolar pyrophosphatase protein from Chenopodium rubrum

Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of variou s metabolites, organic and inorganic ions in the plant vacuole. We used ani on exchange chromatography, liquid-phase isoelectric focusing (IEF), and co ntinuous-elution native polyacrylamide gel electrophoresis (preparative PAG E) to enrich the V-PPase from solubilized tonoplast proteins from suspensio n cultured cells of Chenopodium rubrum L. The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological r eactivity with an antibody raised against mung bean V-PPase. Alt these diff erent methods used for the separation of solubilized tonoplast proteins rev ealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the acti ve V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the obs ervation of high recoveries of enzymatic activity after different preparati ons we suggest that the V-PPase proteins separated may represent physiologi cally occurring forms of the enzyme which cannot be distinguished by SDS-PA GE and Western blot.