The Isolation of Plasma Membrane from the Diatom PhaeoductyIum tricornutum Using an Aqueous Two-Polymer Phase System

Plasma membrane of the marine diatom Phaeodactylum tricornutum was purified by the application of the microsomal fraction to an aqueous two-polymer phase system containing 5.7 % (w/w) each of polyethylene glycol ( M, 3340) and dextran T500, plus 50 mM-NaC1 in a Tris/maleate buffer (pH 7.3) with 500 mM-sorbitol. 5’-Nucleotidase, used as a marker for the plasma membrane, partitioned differentially into the upper phase, and chlorophyll into the lower phase. In sectioned intact cells only the plasma membrane and tonoplast stained with periodic acid-chromic acid-phosphotungstic acid as viewed by electron microscopy. All of the membranous material which partitioned into the upper phase of the two phase system stained with this procedure, whilst much of the material in the lower phase did not. This result indicated the presence of plasma membrane and/or tonoplast only in the upper phase; the increased specific activity of 5’-nucleotidase indicated a 25-fold purification of plasma membrane in this fraction compared to broken cells. In order to differentiate between plasma membrane and tonoplast, vanadate-sensitive, nitrate-insensitive, azide-insensitive, molybdate-insensitive K+, Mg2+-ATPase was used as an additional marker for plasma membrane, and acid phosphatase and nitrate-sensitive ATPase were used as markers for tonoplast. Use of these markers indicated the presence of some tonoplast membrane in the upper phase. It is concluded that the procedure used separates plasma membrane almost completely from organelle membranes and partially from tonoplast