Techniques Used In Sult Research

To further our understanding of the role of sulfation during human development, researchers have studied the expression and relative activities of the major SULT isoforms in human adult and fetal tissues using a variety of biochemical and molecular techniques, which are outlined below.

A vital feature of the human SULT enzyme family is the degree of substrate specificity demonstrated by the individual isoforms, and this feature has been exploited in enzyme activity assays. SULT activity can be determined in tissue cytosol using specific substrates at concentrations selective for each isoform. SULT1A1 can be measured by virtue of its high affinity (low Km) for 4-nitrophenol compared to other SULTS. At low micromolar concentrations, SULT1A1 selectively sulfates 4-nitrophenol, whereas at similar concentrations SULT 1 A3, which has a major role in the sulfation of catecholamines, preferentially sulfates dopamine. SULT1E family members preferentially sulfate endogenous (e.g., estrone and 17^-estradiol) and xenobiotic (e.g., 17a-ethinylestradiol) estrogens, generally with very high affinity (Aksoy et al., 1994; Coughtrie et al., 1994, 1998; Falany 1997; Falany et al., 1994). Although SULT1A1 and SULT2A members also sulfate estrogens, the affinity for endogenous estrogens is typically several orders of magnitude lower than the affinity of SULT1E1 for those substrates. The SULT2 family (SULTs 2A and 2B) can catalyze sulfation of steroids and sterols, such as dehydroepiandrosterone (DHEA), pregnenolone, and cholesterol. Sulfation of 4-nitrophenol and dopamine can be determined using PAPS35S as originally described by Foldes and Meek (1973). Sulfation of estrone, 17^-estradiol, 17a-ethinylestradiol, and DHEA can be determined using 3H-labelled substrates as previously described (Borthwick et al., 1993; Sharp et al., 1993). Sulfation of iodothyronines T2, T3, rT3, and T4 can be measured using 125I-labelled substrates (as detailed in Kaptein et al., 1997). At the time of writing, substrates selective for SULTs 1B1, 1C2, and 1C4 have not yet been identified.

To determine SULT enzyme protein expression and, more importantly, to assess the expression of SULTs 1B1 and 1C2 (for which no specific substrates are available), western-immunoblot analysis can be used. Cytosolic fractions can be resolved by SDS-PAGE and transferred to nitrocellulose as first described by Laemmli (1970) and Towbin et al. (1979), respectively. Antibodies against purified, recombinant human SULT isoforms can be raised in sheep by immunization, using an adaptation of the method of Vaitukaitis (1981). Immunological detection of SULT proteins can be achieved using the enhanced chemiluminescence method, which is commercially available and has been described by several manufacturers. Our research group has successfully produced anti-SULT1A3, which has been described previously (Dajani et al., 1998; Richard et al., 2001; Stanley et al., 2001). This preparation cross-reacts with SULTs 1A1 and 1A2 on immunoblot and ELISA analysis (these isoforms share >93% amino acid sequence identity), thus it is possible to use this preparation to assess the expression of all three isoforms. We have also made anti-SULT1B1 (Kester et al., 1999a), 1C2 (Stanley, 2001), and 1E1 (Rubin et al., 1999) preparations.

To localize the sites of SULT expression, immunohistochemical analysis can be performed using the SULT antibody preparations detailed above. Tissue sections can be cut from formalin-fixed, paraffin-embedded material, and a standard perox-idase-antiperoxidase technique can be used incorporating 3,3'-diaminobenzidine as a developing agent (Sternberger et al., 1990). For more complex tissues, such as placenta, immunofluorescence can be performed using frozen sections (Stanley et al., 2001). The use of frozen sections generally does not provide as high resolution staining as formalin-fixed, paraffin-embedded material. Control sections with replacement of the primary antibody with nonimmune IgG should be performed in tandem for both immunohistochemical techniques.

To rapidly screen a large panel of human tissues for SULT expression, mRNA dot-blot analysis can be conducted. Dot-blot analysis is performed using poly(A)+RNA isolated from a number of human tissues, and expression is detected using SULT cDNA as a probe. The probe can be labeled with, for example, [a-32P]-dCTP by random priming using a commercially available kit. This technique is described in further detail in Her et al. (1997).

To detect expression of SULT genes, a PCR-based approach, also known as expression profiling, can be adopted. Total RNA can be extracted from tissue using a commercially available kit and cDNA generated by RT-PCR. Alternatively, cDNA can be purchased directly from a commercial supplier. PCR is performed on cDNA using isoform-specific primer pairs, designed specifically to avoid amplification of other SULT isoforms (as detailed in Dooley et al., 2000). Newer applications, such as real-time PCR analysis with the TaqMan® 5'-nuclease system (PE Applied Biosystems) and with the Light Cycler™ system (Roche), can be utilized for a more quantitative estimation of SULT expression levels.

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