Immunocytochemical localization of a plasma membrane calcium pump in the insect (Lymantria dispar) Malpighian tubule

The Malpighian tubules of several insects are known to secrete Ca²⁺ against its electrochemical potential, however the molecular identities of the Ca²⁺ transport pathways have not been determined. We have investigated the identity of the active transport step for calcium secretion in the larval gypsy moth Malpighian tubule, using a monoclonal antibody produced against the human erythrocyte plasma membrane calcium pump (PMCA). Western blot analysis of partially-purified cell membranes from Malpighian tubules showed that the antibody binds to a single prominent protein band with a molecular mass of approx. 143 kDa. This molecular mass is identical to that of the predominant immunoreactive protein in partially-purified membranes from the human erythrocyte. In immunocytochemical studies the anti-PMCA antibody bound predominantly to epitopes positioned along the apical domain of the transporting cells of the larval Malpighian tubule. These epitopes lie along the entire length of the tubule. A phylogenetically conserved PMCA is a likely candidate for active calcium transport across the apical membrane. Immunoreactive proteins were undetectable in Western analyses of partially-purified cell membranes from tubules of the adult gypsy moth, and immunostaining of adult tissue sections was weak or nonexistent. A reduction in immunoreactive protein density in the adult tubule may reflect structural modifications which occur late in the last larval instar or pupal stage.