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M. H. Gangadhariah, R. H. Nagaraj; Effect of Methylglyoxal-Modification of Human A-Crystallin on the Ability to Assist in Refolding of Denatured Client Proteins. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4509.
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To determine if the enhancement in chaperone function by chemical modification by methylglyoxal (MGO) affects the ability of human αA-crystallin to assist in the refolding of denatured proteins.
Human αA-crystallin was modified by incubating with 20 to 500 micromolar MGO for 2 days. The chaperone activity of the modified and unmodified protein was determined using citrate synthase (CS) and insulin as client proteins. The αA-crystallin’s ability to assist in refolding of denatured proteins was assessed using malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and CS. The proteins (MDH and LDH) were denatured by incubating with 6M guanidine hydrochloride. Dilution of denatured proteins with buffers in the presence or absence of αA-crystallin and measurement of enzyme activity established the renaturation of proteins. Inhibition of thermal inactivation (incubation at 430C) of CS by αA-crystallin was assessed by measuring its activity using a colorimetric assay. Surface hydrophobicity of proteins was measured by TNS (6-p-toluidinyl naphthalene-2-sulfonate) binding.
As demonstrated by us previously (Nagaraj et al, Biochemistry 42: 10746-55, 2003), MGO-modification enhanced the chaperone function of αA-crystallin against two client proteins and this effect was MGO concentration dependent. The ability of αA-crystallin to assist in refolding of MDH and LDH was unaltered by MGO modification. Similarly, the ability to prevent thermal inactivation of CS was unaffected by MGO modification. The complex of catalytically inactivated MDH and αA-crystallin was isolated by gel filtration and was used in the MDH renaturation assay. The prior binding of MDH completely abrogated the ability of αA-crystallin to assist in renaturation of MDH. TNS binding of αA-crystallin showed that hydrophobic sites exposed as a result of modification by MGO do not take part in the refolding of denatured proteins.
The sites responsible for chaperone function and protein renaturing ability overlap in the native αA-crystallin. Mild modification by MGO enhances the chaperone function of human αA-crystallin against CS and insulin without impacting on its ability to assist in refolding of denatured MDH and LDH. This appears to be due to failure of participation of hydrophobic sites (that are exposed as result of MGO modification) in refolding of denatured proteins.
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