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Kevin L. Schey, Mark Little, John G. Fowler, Rosalie K. Crouch; Characterization of Human Lens Major Intrinsic Protein Structure. Invest. Ophthalmol. Vis. Sci. 2000;41(1):175-182.
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© ARVO (1962-2015); The Authors (2016-present)
purpose. To determine the primary covalent structure of human lens major
intrinsic protein (MIP) in lenses of varying age.
methods. MIP was isolated from single human lenses of various ages (7–86 years)
by homogenization of the lenses, followed by centrifugation and urea
washes of the membranes. Proteins present in the membrane preparation
were reduced, alkylated, and cleaved by CNBr. Peptide fragments were
fractionated by reverse-phase high-performance liquid chromatography,
and the primary structures of the peptides were determined by tandem
mass spectrometry and Edman sequencing.
results. Complete coverage of the human MIP sequence was observed in the form of
CNBr fragments. In addition, peptide structures resulting from in vivo
heterogeneous N- and C-terminal cleavage were characterized. The amount
of intact MIP decreased with lens age; however, the pattern of
truncation did not change from 7 to 86 years. The major site of
phosphorylation was identified as serine 235. Asparagine residues 246
and 259 were completely deamidated by age 7 years.
conclusions. The major structural modifications of human lens MIP have been
determined. Human MIP is heterogeneously modified in lenses ranging in
age from 7 to 86 years of age by N- and C-terminal truncation,
phosphorylation, and deamidation, resulting in decreased levels of
native intact MIP with age.
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