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+ ag ainst its electrochemical potential, however the molecular identities of the Ca2+ transport pathways have not been determined. We have inves tigated the identity of the active transport step for calcium secretio n in the larval gypsy moth Malpighian tubule, using a monoclonal antib ody produced against the human erythrocyte plasma membrane calcium pum p (PMCA). Western blot analysis of partially-purified cell membranes f rom Malpighian tubules showed that the antibody binds to a single prom inent protein band with a molecular mass of approx. 143 kDa. This mole cular mass is identical to that of the predominant immunoreactive prot ein in partially-purified membranes from the human erythrocyte. In imm unocytochemical 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 l ength of the tubule. A phylogenetically conserved PMCA is a likely can didate for active calcium transport across the apical membrane. Immuno reactive proteins were undetectable in Western analyses of partially-p urified cell membranes from tubules of the adult gypsy moth, and immun ostaining of adult tissue sections was weak or nonexistent, A reductio n in immunoreactive protein density in the adult tubule may reflect st ructural modifications which occur late in the last larval instar or p upal stage.