Tissue specificity of E subunit isoforms of plant vacuolar H+-ATPase and existence of isotype enzymes

Immunoblot analyses and partial amino acid sequencings revealed that both t he 40- (E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms. Similarly, both the 35- (D1) and 29-kDa (D2) subunits we re D subunit isoforms, although the similarity of the amino acid sequences is still unknown. In immunoblot analyses, two or three E subunit isoforms w ith molecular masses ranging from 29 to 40 kDa were detected in other plant s. Two isotypes of V-ATPase from the pea epicotyl were separated by ion exc hange chromatography and had subunit compositions differing only in the rat io of E1 and E2. There was a difference in the V-max and K-m of ATP hydroly sis between the two isotypes. E1 was scarcely detected in crude membrane fr actions from the leaf and cotyledon, while E2 was detected in fractions fro m all of the tissues examined. The compositions of D subunit isoforms in th e leaf and epicotyl were different, and the vacuolar membrane in the leaf d id not contain D2. The efficiency of H+ pumping activity in the vacuolar me mbrane of the leaf was higher than that of the epicotyl. The results sugges t that the presence of the isoforms of D and E subunits is characteristic t o plants and that the isoforms are closely related to the enzymatic propert ies.