Intracellular phosphorylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis.

A procedure was developed for the preparation of rough and smooth microsomes from small quantities of liver obtained from estrogen-stimulated Xenopus laevis females. The separation involves th centrifugation of a postmitochondrial supernatant over a CsCl-containing, discontinuous sucrose gradient. Morphological, biochemical, and enzymatic characterization of these fractions indicates that excellent separation of rough microsomes from smooth microsomes is achieved. In addition, pulse-chase experiments in which (5-min) pulses with [3H]leucine are used demonstrate that rough and smooth microsomes each exhibit the predictable patterns of incorporation characteristic of secretory protein synthesis and intracellular translocation. This procedure was combined with suitable incubation conditions for pulse-chase experiments which demonstrate the subcellular sites of vitellogenin phosphorylation. The data presented indicate that approximately 70% of the phosphate residues are covalently attached to vitellogenin during its intracellular translocation through the smooth microsomes, while the rough microsomes can account for the remainder of the total incorporated phosphate. This is further supported by the analysis of newly synthesized and assembled [3H,32P]vitellogenin on sodium dodecyl sulfate-polyacrylamide gels and measurements of protein kinase activity in microsomal subfractions.