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Paul David Hamilton, Brittney McGlasson, Usha P Andley; α-Crystallin binding and associations with putative substrates. Invest. Ophthalmol. Vis. Sci. 2016;57(12):724.
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© ARVO (1962-2015); The Authors (2016-present)
Proteomic analysis of the lens shows an increase in the expression of histones in knock-in mice carrying α-crystallin (AC) mutations. To further understand the relationship between AC chaperone function, binding of substrate proteins and cataract, we are performing analysis of AC and substrate proteins interactions to gain insight into their regulation and function.
Lens AC was isolated from pig eyes collected at a local abattoir and was purified by centrifugation and gel permeation chromatography (GPC). Histone was obtained from commercial sources. AC was incubated at 37oC for 1 hour in the presence of histone and ATP at varying concentrations. Interactions of the soluble fractions of the incubations were obtained by centrifugation or use of 0.22 micron centrifuge filters and were studied using GPC chromatography in combination with a refractive index (RI), low and right angle light scattering (LALS and RALS) and viscosity detection systems. Experiments were also performed using denaturing and non-denaturing gel electrophoresis and Isothermal Calorimetry (ITC).
GPC showed that upon adding histone, at very low concentrations of histone (molar ratio of AC subunits : histone = 231), a peak with an increase in molecular weight (MW) of 30% was observed, presumably from binding interactions of the AC with histones. At higher concentration of histones (up to a molar ratio of AC subunits : histone = 2.6), there was a gradual, sustained decrease in MW of the AC as shown by multi-angle light scattering, that was accompanied by a 50% decrease in MW. Concurrently, there was a decrease in the amount of AC present in the soluble fraction, indicating that part of the AC was distributed in the insoluble fraction. Electrophoresis indicated that the AC had no contamination with beta crystallin, and at the higher histone concentrations, histone bands were detectable. ITC analysis confirmed that binding between the AC and histone is occurring, indicating that ITC is a good way to analyze AC binding with its putative substrates.
Binding interactions between the AC and histone suggest that AC may undergo a partial disassembly to oligomers with fewer subunits.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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