ABSTRACT
A fluorometric assay for determination of glutathione-S-epoxide transferase (GSH-T) activity in freshly isolated human hair follicles or cultured cells is described. With this assay, basal levels of the enzyme in hair follicles have been compared between smokers and non-smokers. No significant difference between both groups could be detected. GSH-T was not elevated after treatment of cultured hair follicle keratinocytes with benz[a]anthracene or clotrimazole. The same result was obtained with cultured skin fibroblasts from Ah-responsive and Ah-non-responsive mice. The possible significance of basal GSH-T levels for assessment of the risk of individuals for the carcinogenic action of polycyclic aromatic hydrocarbons is discussed.
Subject(s)
Glutathione Transferase/metabolism , Hair/enzymology , Adult , Benz(a)Anthracenes/pharmacology , Cells, Cultured , Clotrimazole/pharmacology , Female , Humans , Male , SmokingABSTRACT
Citraconic anhydride reacts readily with alpha-crystallin's lysine residues at pH 7.4. Upon addition of 2 equivalents of citraconic anhydride per equivalent lysine, 24% of the lysine residues were modified without disrupting the native quaternary structure. Further citraconylation led to dissociation into 10 S aggregates. Complete dissociation into subunits (1.4 S) occurred after adding 100 equivalents of citraconic anhydride, resulting in 98% modification. Decitraconylation did not lead to reaggregates identical with the native ones. The unmodified and the once and twice citraconylated alpha-crystallin subunits were discerned by isoelectric focusing according to their theoretical isoelectric points. In the native alpha-crystallin aggregates, nearly all B chains and approx. 60% of the A chains were found to possess at least one surface-exposed lysine residue. No differences between the susceptibilities to citraconylation of the in vivo deamidated (A1 and B1) and the de novo synthesized (A2 and B2) subunits were found. These results support the three-layer spherical assembly model for the alpha-crystallin quaternary structure.
