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C.S. Ricard, A. Mukherjee; Triton X–114 Phase Separation of Three Cell–Associated Human Versus Canine Myocilin Species . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1264.
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Human myocilin possesses two translation start sites, resulting in three bands resolved by SDS PAGE. A 55/57 kDa doublet plus a 67 kDa species that corresponds to N–glycosylated forms. The 57 kDa would be expected to be more hydrophobic than the 55 kDa due to the additional N–terminal amino acids, and the 67kDa form(s) would be expected to be the most hydrophilic owing to the presence of carbohydrates. In contrast, canine myocilin possesses only one translational start codon, corresponding to the 55 kDa form that is further post–translationally modified.
Triton X–114 is a nonionic detergent that has been used to separate proteins on the basis of hydrophobicity imparted by modifications such as glycosylphosphatidylinositol anchors or palmitoylation. Triton X–114 differs from other nonionic detergents in that it remains in one phase until warmed to room temperature or higher. Then it separates into two phases with proteins partioned according to hydrophobicity. Radiolabeled cell cultures were treated and fractionated with this detergent, myocilin was immunoprecipitated from each phase and analyzed by SDSPAGE.
Separate species of myocilin were obtained by this method. As expected the lower phase contained the more hydrophobic 57 kDa species of human myocilin, while the 55 kDa species remained in the upper detergent phase and the glycosylated forms were in the non–detergent fraction. Canine myocilin partitioned similarly except for the 57 kDa form, which was not detectable.
Human myocilin is constitutively produced in cultured astrocytes and three sizes are produced in apparently equal amounts. The degree of hydrophobicity of each species as well as post–translational processing result in different roles for each species depending upon the cellular fraction with which the protein species associates. This represents the first nondenaturing method to separate native myocilin species. This purification method will permit extremely pure, native myocilin for structural as well as functional analysis, and to analyze the interesting modifications of myocilin proteins.
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