COVI, J.A.*; HAND, S.C.: Vacuolar proton-ATPase subunit B mRNA expression in Artemia franciscana gastrulae.

Exposure of A. franciscana gastrulae to anoxia induces a profound metabolic and developmental depression known as quiescence. This fully reversible transition is characterized by an extraordinary intracellular acidification. However, a significant proportion of the protons required for this event are of undetermined origin. We have examined embryonic expression of the V-ATPase as a first step in evaluating the role of this enzyme in generating proton gradients useful during initiation of quiescence. We have cloned and sequenced a 394bp gene fragment for the B-subunit of this enzyme from aerobically incubated embryos. Sequence alignments indicate that the A. franciscana gene shares a great deal of identity with sequences reported for other organisms. Furthermore, Northern blots probed with radiolabeled oligonucleotides synthesized from the B-subunit gene fragment reveal two distinct transcripts, each exhibiting distinct temporal expression during early development. In an attempt to identify V-ATPase protein expression, membrane and cytosolic fractions from early gastrulae to 36 hour larvae were subjected to Western blot analysis using an monoclonal antibody against the homologous B-subunit of the yeast enzyme. An immunoreactive band was visualized with an apparent molecular mass of 75 kDa. This mass exceeds the expected value of 53-60 kDa. Consequently, definitive identification of this protein awaits further analyses. Expression of the V-ATPase within the plasma membrane during preemergence development of A. franciscana offers a potential mechanism for generating an extracellular proton store that could be used in conjunction with other known proton generating mechanisms to acidify the intracellular space at the onset of anoxia-induced quiescence. (Supported by NSF grant IBN-9723746)