Sult1e1

FIGURE 4.4 SULT1A3 rabbit reticulocyte lysate (RRL) protein degradation study. 35S-Methionine radioactively labeled SULT1A3*1 and *2 were incubated in an RRL for 24 hr, and loss of the protein was determined by SDS-PAGE. Each point represents the average of four independent experiments (mean ± SEM). * = p < 0.02, ** = p < 0.05, *** = p < 0.005 when compared with the * 1 allozyme at the same incubation time. (From Thomae et al., 2003, Journal of Neurochemistry, 87, 809-819. With the permission of Blackwell Publishing.)

FIGURE 4.4 SULT1A3 rabbit reticulocyte lysate (RRL) protein degradation study. 35S-Methionine radioactively labeled SULT1A3*1 and *2 were incubated in an RRL for 24 hr, and loss of the protein was determined by SDS-PAGE. Each point represents the average of four independent experiments (mean ± SEM). * = p < 0.02, ** = p < 0.05, *** = p < 0.005 when compared with the * 1 allozyme at the same incubation time. (From Thomae et al., 2003, Journal of Neurochemistry, 87, 809-819. With the permission of Blackwell Publishing.)

Human SULT1E1 Polymorphisms

Asp22Tyr

CAU JW

1Ala32Val I |~j/D~| I Pro253His

4 4 il 4T4T7

tataaa

FIGURE 4.5 Human SULT1E1 genetic polymorphisms. A schematic representation of the human SULT1E1 gene with the locations of polymorphisms indicated by arrows. Black rectangles represent the open reading frame, and white rectangles represent portions of exons that encode an untranslated region (UTR) sequence. Arrows indicate the locations of SNPs and a single indel (I/D). AA represents data obtained with DNA from African-American subjects, and CAU represents data obtained using DNA from Caucasian-American subjects. Changes in encoded amino acids resulting from the presence of nonsynonymous cSNPs are also indicated. (From Adjei et al., 2003, British Journal of Pharmacology, 139, 1373-1382. With the permission of Nature Publishing Group.)

in these samples. Three of the SULT1E1 cSNPs were nonsynonymous and resulted in the following alterations in encoded amino acids: Asp22Tyr, Ala32Val, and Pro253His (Adjei et al., 2003). Those three alleles, two of which were observed in DNA from Caucasian-American subjects and one, that encoding Tyr22, in DNA from an African-American subject, were transiently expressed in COS-1 cells. The Tyr22 allozyme showed a striking decrease in level of enzyme activity and immu-noreactive protein; the Val32 variant displayed a decrease of approximately 50% in both; and the His253 allozyme had no changes in level of either enzyme activity or immunoreactive protein as compared to the wild type (WT) allozyme (Figure 4.6; Adjei et al., 2003). Although apparent Km values for both cosubstrates showed slight differences among these variant allozymes, the most striking alteration — other than

FIGURE 4.6 Human SULT1E1 pharmacogenetics. (A) Recombinant human SULT1E1 allozyme enzyme activity. Average levels of enzyme activity for each of the recombinant SULT1E1 allozymes assayed with E2 as the sulfate acceptor substrate. All values have been corrected for transfection efficiency. Each bar represents the average of six independent transfections (mean ± SEM). * = p < 0.001 when compared to the WT allozyme. (B) Recombinant human SULT1E1 allozyme western-blot analysis. Average levels of immunoreactive SULT1E1 protein for each of the recombinant allozymes, expressed as a percentage of WT protein. Each bar represents the average of three independent transfections (mean ± SEM). * = p < 0.0006 when compared to the WT level of immunoreactive activity. (From Adjei et al., 2003, British Journal of Pharmacology, 139, 1373-1382. With the permission of Nature Publishing Group.)

FIGURE 4.6 Human SULT1E1 pharmacogenetics. (A) Recombinant human SULT1E1 allozyme enzyme activity. Average levels of enzyme activity for each of the recombinant SULT1E1 allozymes assayed with E2 as the sulfate acceptor substrate. All values have been corrected for transfection efficiency. Each bar represents the average of six independent transfections (mean ± SEM). * = p < 0.001 when compared to the WT allozyme. (B) Recombinant human SULT1E1 allozyme western-blot analysis. Average levels of immunoreactive SULT1E1 protein for each of the recombinant allozymes, expressed as a percentage of WT protein. Each bar represents the average of three independent transfections (mean ± SEM). * = p < 0.0006 when compared to the WT level of immunoreactive activity. (From Adjei et al., 2003, British Journal of Pharmacology, 139, 1373-1382. With the permission of Nature Publishing Group.)

the differences in level of activity — was a significant decrease in thermal stability for the Tyr22 variant, the allozyme that displayed striking decreases in levels of both enzyme activity and immunoreactive protein (Figure 4.6). Preliminary, unpublished studies showed that the SULT1E1 Tyr22 variant, like the SULT1A3 Asn234 variant allozyme, is more rapidly degraded in a rabbit reticulocyte lysate than is the WT allozyme (Liewei Wang and Yvette Martin, 2003, unpublished observations).

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