Synthesis of Na+/K+ ATPase by the preimplantation rabbit blastocyst

Abstract

The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (β- and γ-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished ~79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (<0.06 mM) and stage-related differences in blastocoele volume. Analysis of β- and γ-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P<0.001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst.