Proteoliposome interaction with human erythrocyte membranes. Functional implantation of .gamma.-glutamyl transpeptidase

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Abstract
The transfer of detergent solubilized and purified .gamma.-glutamyl transpeptidase (.gamma.-GTase), of hog kidney cortex, from proteoliposomes into human erythrocyte ghost membranes was studied. The transfer of .gamma.-GTase was observed upon incubation of .gamma.-GTase incorporated palmitoylphosphatidylcholine [DPPC] vesicles with erythrocyte ghost membranes at 37.degree. C for 12 h. The extent of transfer was dependent upon the fluidity of donor proteoliposomes, being more when DPPC proteoliposomes were used compared to dimyristoylphosphatidylcholine (DMPC), and intermediate values were observed when binary mixtures and DMPC and DPPC were used. The transfer of .gamma.-GTase was facilitated when rigid basic phospholipid proteoliposomes were used as donor. The transfer of .gamma.-GTase was associated with the removal of intrinsic membrane proteins and lipids from erythrocytes, mainly acetylcholinesterase, sphingomyelin and cholesterol. An enhancement in the fluorescence due to resonance energy transfer was observed when ghost membranes containing fluorescent donor probe were incubated with proteoliposomes containing fluorescent acceptor probe, indicating that fusion but not adsorption of vesicles occurs during the transfer process. The inability of entrapped [14C]sucrose delivery from proteoliposomes into ghost membrane vesicles suggests that fusion per se is not primarily involved in the transfer process. The transfer of .gamma.-GTase apparently occurs by a collisional transfer process resulting in intermembrane protein transfer. The .gamma.-GTase implanted ghost membranes exhibited the uptake of L-glutamate which was inhibited by serine-borate, an inhibitor of transpeptidase activity. The uptake of L-glutamate was inhibited by the dipeptide .gamma.-glutamyl-L-glutamate, thus supporting the proposed role of .gamma.-GTase in the uptake of amino acids in biological membranes.