The influence of isolation conditions on the molecular weight of bovine alpha-crystallin.

Oetelaar, Piet J.M. van den; Clauwaert, J.; Laethem, Marc Van; Hoenders, Herman J.

doi:10.1016/s0021-9258(17)38680-5

Cited by 54 publications

(2 citation statements)

References 32 publications

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“…An earlier electron microscopy study has revealed that the recombinant α-crystallin complexes have a polydisperse morphology () and the oligomeric sizes depend on the physicochemical conditions ( , ). The mutant R116C homoaggregates were shown to be highly polydisperse in nature ( , ).…”

Section: Discussionmentioning

confidence: 99%

A Positive Charge Preservation at Position 116 of αA-Crystallin Is Critical for Its Structural and Functional Integrity

et al. 2002

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An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported. Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size. The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys. To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells. The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding. Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes. The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure. Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin.

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Section: Discussionmentioning

confidence: 99%

A Positive Charge Preservation at Position 116 of αA-Crystallin Is Critical for Its Structural and Functional Integrity

et al. 2002

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“…The resultant protein concentration of the resultant supernatant was adjusted to 50 mg of protein/mL. A total of 30 mL of supernatant was loaded onto a 5 cm X 100 cm Sepharose-CL6B (Phamacia-LKB) gel filtration column (Van den Oetelaar et al, 1985), eluted at a flow rate of 1 mL/min, and monitored by absorbance at 280 nm. a-Crystallin eluting as a single symmetrical peak at 1200 mL corresponding to an apparent molecular mass of 800000 Da was collected, extensively dialyzed against water, lyophilized, and stored at -20 °C.…”

Section: Methodsmentioning

confidence: 99%

Photooxidation of specific residues in .alpha.-crystallin polypeptides

McDermott¹,

Chiesa²,

Roberts³

et al. 1991

Biochemistry

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Singlet oxygen is a biologically important, photochemically generated species that preferentially oxidizes His, Trp, and Met residues of protein molecules. Calf alpha-crystallin was photooxidized with use of meso-tetra(p-sulfonatophenyl)porphyrin (TPPS) and uroporphyrin (UP) as singlet oxygen generators. The effects of photooxidation were monitored by analyzing the changes in alpha-crystallin peptide maps obtained by reversed-phase HPLC using a photodiode array absorbance detector. The reaction led to the loss of six specific peptides, five of which contained photooxidizable residues. Peptides containing His-97 and His-154 from the A chain and Met-68 from the B chain are preferentially photooxidized, suggesting that those residues have access to singlet oxygen. Trp residues in the N-terminal region are converted to NFK, whereas Trp-60 in the B chain is not photooxidized strongly suggesting that the former are close to the surface of alpha-crystallin while the latter Trp residue is buried. Only one peptide that is lost from the peptide maps does not contain a photooxidizable group; however, this peptide does contain an apparently undigested Lys residue. It is suggested that it forms a cross-link with a photooxidized His residue.

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Lens

Berman¹

1991

Biochemistry of the Eye

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The influence of isolation conditions on the molecular weight of bovine alpha-crystallin.

Cited by 54 publications

References 32 publications

A Positive Charge Preservation at Position 116 of αA-Crystallin Is Critical for Its Structural and Functional Integrity

A Positive Charge Preservation at Position 116 of αA-Crystallin Is Critical for Its Structural and Functional Integrity

Photooxidation of specific residues in .alpha.-crystallin polypeptides

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