May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Coiled–coil interactions of native myocilin
Author Affiliations & Notes
  • W. Stamer
    Departments of Ophthalmology and Pharmacology,
    University of Arizona, Tucson, AZ
  • D.J. Rak
    Department of Ophthalmology,
    University of Arizona, Tucson, AZ
  • B.S. McKay
    Departments of Ophthalmology and Cell Biology,
    University of Arizona, Tucson, AZ
  • Footnotes
    Commercial Relationships  W. Stamer, None; D.J. Rak, None; B.S. McKay, None.
  • Footnotes
    Support  EY12797, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3658. doi:
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      W. Stamer, D.J. Rak, B.S. McKay; Coiled–coil interactions of native myocilin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3658.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: The function of myocilin (MYOC), the protein product of the first gene to link to primary open–angle glaucoma, is unknown. The purpose of the present study was to test the hypothesis that native MYOC participates in high affinity protein–protein interactions (likely via its two coiled–coil domains) by determining its fluid phase structural properties. Methods: Retinas from human donor eyes were homogenized in buffer containing 0.1% SDS, N–Ethyl maleimide and protease inhibitors. Soluble proteins were fractionated by either glycerol gradient sedimentation (linear 10–40%) or gel exclusion chromatography (Sephadex G400). Fractionated proteins were subjected to nonreducing SDS–PAGE followed by either western blotting to identify MYOC or silver staining to illustrate protein fractionation and standard protein distribution. Results: Soluble MYOC exists in two cellular pools separable by both glycerol gradient sedimentation or gel exclusion chromatography. One pool exists as a 6–6.3 particle with an apparent MW of 106 kD. This particle has an S (max)/S ratio of 1.38, suggesting a globular shape. The second pool of MYOC exists in a large particle with a 15.3s value and an apparent MW of 405–440 kD. The relative partitioning of MYOC between the two pools was roughly equivalent between tissue preparations (n=3). In all fractions, MYOC was identified by characteristic migration as 55–57 kD doublet using western blot analysis. Conclusions: Native MYOC participates in two different protein complexes stable in SDS, suggesting high affinity coiled–coil interactions. The small complex likely represents a myocilin dimer, while the identity of the large complex remains unknown. Disulfide bonds do not contribute to MYOC interactions because reduction was unnecessary to identify monomeric MYOC by SDS PAGE.

Keywords: proteins encoded by disease genes • protein purification and characterization • retina 
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