Immunological detection of a vacuolar-type H(+)-ATPase in vanadocytes of the ascidian Ascidia sydneiensis samea.

Ascidians belonging to the family Ascidiidae are known to accumulate vanadium from seawater in their blood cells, concentrating vanadium by a factor of 10(7). Among several different types of blood cell, the signet ring cells have both high levels of vanadium and a low pH. These observations suggest the possibility that proton ions concentrated by a H(+)-ATPase are energetically linked to the accumulation of vanadium. In the present experiments, therefore, we made an immunological search for a H(+)-ATPase in the vacuolar membranes of the signet ring cells, as a first step in our attempts to clarify the energetics of the accumulation of vanadium by these cells. Antibodies raised against the 72-kDa and 57-kDa subunits of a vacuolar-type H(+)-ATPase from bovine chromaffin granules reacted with the vacuolar membranes of signet ring cells. Immunoblotting analysis confirmed that specific antigens in ascidian blood cells actually reacted with the antibodies. Furthermore, addition of bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibited the uptake of protons by the vacuoles of signet ring cells. Thus, the addition of bafilomycin A1 inhibited the pumping function of the vacuoles of signet ring cells, with resultant neutralization of the contents of the vacuoles.