Jaya Bhattacharyya

A peptide sequence-YSGVCHTDLHAWHGDWPLPVK [40-60]-in yeast alcohol dehydrogenase prevents the aggregation of denatured substrate proteins.

The structural and functional characteristics of a yeast alcohol dehydrogenase (ADH) peptide (YSGVCHTDLHAWHGDWPLPVK, residues 40-60) have been studied in detail. The peptide is hydrophobic in nature, binds the hydrophobic probe bis-ANS, and is mostly present in a random coil conformation. It shows chaperone-like activity by preventing dithiothreitol (DTT)-induced aggregation of insulin at 27 degrees C, oxidation-induced aggregation of gamma-crystallin at 37 degrees C, and aggregation of thermally denatured ADH and beta(L)-crystallins at 52 degrees C. However, the ADH peptide does not solubilize protein aggregates as do surfactants. Substitution of Pro for His in the ADH peptide leads to diminished anti-aggregation activity. Further, analysis of ADH incubated at 47 degrees C suggests that a significant portion of the enzyme remains as soluble inactive protein with negligible conformational change. Therefore, we propose that the residues 40-60 in native protein may be an intramolecular chaperone site of yeast ADH.

Evaluation of Hydrophobicity Versus Chaperonelike Activity of Bovine αA- and αB-Crystallin

Calf lens αA-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than αB-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to αA-crystallin than to αB-crystallin at room temperature. Bis-ANS binding to both αA- and αB-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of αA-crystallin is lower than that of αB-crystallin whereas at higher temperatures, αA-crystallin shows significantly higher protection against aggregation of substrate proteins compared to αB-crystallin. Therefore, calf lens αA-crystallin is more hydrophobic than αB-crystallin and chaperone-like activity of α-crystallin subunits is not quantitatively related to their hydrophobicity.

Tryptophan Metabolites from Young Human Lenses and the Photooxidation of Ascorbic Acid by UVA Light

PURPOSE To determine whether there are UVA light-responsive sensitizers in young human lenses capable of initiating the oxidation of ascorbic acid in the absence of oxygen. METHODS Lens homogenates were fractionated, and low-molecular-weight (LMW) components were separated from the proteins by filtration through a 3000-MWt cutoff filter. Aliquots of each fraction were assayed for sensitizer activity by UVA irradiation (337-nm cutoff filter) with 0.1 mM ascorbic acid, measuring ascorbate oxidation by loss of absorbance at 265 nm. Two major peaks were isolated from a human lens water-soluble (WS)-LMW fraction on a reversed-phase column and were identified by mass spectrometry. RESULTS All human lens fractions oxidized ascorbate when irradiated by UVA light. Most of the sensitizer activity in young human lenses was in the LMW fractions. An action spectrum for the WS-LMW fraction from human lens showed activity throughout the UVA region. Assays with and without oxygen showed little or no difference in ascorbate oxidized, arguing for a direct transfer of an electron in a so-called type 1 reaction. A human lens WS-LMW fraction contained two major peaks of activity. The greater peak was identified as 3-hydroxykynurenine glucoside (3OHKG) by mass spectrometry and its absorption spectrum, whereas the lesser peak was identified as 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG). The activities were 1.1 and 2.8 nmol of ascorbate oxidized in 30 minutes/nmol 3OHKG and AHBG, respectively. CONCLUSIONS The filter compounds present in human lenses can absorb UVA light and cause the oxidation of ascorbic acid in the presence and absence of oxygen, possibly initiating the glycation of lens proteins.

Interactions of chlorpromazine with α-, β- and γ-crystallins

The binding parameters (binding affinity constant, K and number of binding sites, p) has been determined spectrofluorometrically for chlorpromazine (CPZ) binding to the lens proteins - αL-crystallin, βL-crystallin and γ-crystallin. The binding affinity constants for CPZ binding to αL- and γ-crystallins are higher than the binding affinity constants for βL-crystallin, although the number of CPZ binding sites for βL-crystallin is comparatively higher than the number for the other two lens proteins. CPZ causes local conformational changes around the tryptophan moieties of the protein molecules but does not cause any gross conformational change within the protein moieties. Binding of CPZ to αL-crystallin does not significantly alter the anti-aggregation properties of the molecular chaperone, α-crystallin against oxidation-induced aggregation of γ-crystallin at 37°C and thermal aggregation of alcohol dehydrogenase (ADH) at 48°C. Therefore, CPZ induced alteration in chaperone activity of αL-crystallin is probab...