A fluorescence-quenching assay for measuring permeability of reconstituted lens MIP26

Abstract

A sensitive fluorometric assay for measuring permeability of liposomes containing bovine lens MIP26 has been developed and characterized. Lens membrane proteins were isolated and incorporated into artificial membranes composed of bovine brain phospholipids, sphingomyelin and the fluorescent probe, N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidylethanolamine (NBD-PE). Quenching of the probe with cobalt chloride allowed the measurement of liposome permeability in the presence and absence of lens membrane proteins. Liposomes containing the putative gap junctional polypeptide known as Major Intrinsic Protein 26 (MIP26) were shown to be more permeable than those not containing the MIP26 protein. Permeability was shown to be positively correlated with the amount of MIP26 in the liposomes and with increasing purity of the protein. This method offers a sensitive assay for channel function of the putative junction protein, and provides further evidence that MIP26 from lens membranes is a gap junctional polypeptide.