Novel support for membrane enzyme immobilization: gel beads containing polymerized phospholipid vesicles

The present study has demonstrated a novel immobilization support for membrane enzymes; the support is composed of agarose gel beads and polymerized phospholipid vesicles contained within the beads. A phosphatidylcholine analogue, bis-[12-(methacryl-oyloxy)dodecanoyl]-L-alpha-phosphatidylcholine (BMPC) [Regen, Singh, Oehme and Singh (1982) J. Am. Chem. Sec. 104, 791-795], was contained in Sepharose CL-6B beads through the formation and simultaneous entrapment of vesicles by a combination of phospholipid solubilization in organic solvent with the beads, complete removal of the solvent and sonication in a buffer. The vesicle membranes were then polymerized by UV irradiation, which stabilized the hybrid-type support. gamma-Glutamyl transpeptidase, a membrane enzyme from bovine kidney, was immobilized in the beads by reconstitution in the polymerized BMPC vesicles contained within the beads, The enzyme catalytic activity, as indicated by apparent Michaelis-Menten kinetics, was almost identical with that of the enzyme reconstituted in unpolymerized BMPC vesicles, Polymerized BMPC vesicles significantly stabilized the enzyme to heat treatment when compared with unpolymerized BMPC vesicles and egg yolk phospholipid liposomes.