Materials.

Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). Oligo(dT)-cellulose was purchased from Boehringer Mannheim (La Jolla, CA). Nitroplus 2000 nitrocellulose hybridization paper was purchased from μm Separations Inc. (Westwood, MA). The Erase-a-Base DNA sequence kit was obtained from Promega (Madison, WI), and DNA sequencing kits were purchased from United States Biochemical (Cleveland, OH). DNA nick translation kits, [α-³²P]dCTP (3000 Ci/mmol), and [³⁵S]dATPαS (400 Ci/mmol) were purchased from Amersham Corp. (Arlington Heights, IL). Uridine diphosphate glucuronic acid [glucuronyl-¹⁴C(U)] (225 mCi/mmol, ammonium salt) and Trans³⁵S-label ([³⁵S]l-methionine, [³⁵S]l-cysteine) were obtained from ICN Radiochemicals (Costa Mesa, CA). The eukaryotic expression vector pSVL was purchased from Pharmacia (Piscataway, NJ). Aglycone substrates, uridine 5′-diphosphoglucuronic acid (triammonium salt), and formalin-fixed Staphylococcus aureus cells were obtained from Sigma (St. Louis, MO). Glass-backed linear-K thin-layer chromatography (TLC) plates with preabsorbent strips were purchased from Whatman Labsales, Inc. (Hillsboro, OR). The SuperScript Preamplification System for First Strand cDNA Synthesis was purchased from Life Technologies (Gaithersburg, MD). Cloned Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). Bio-X-ACT DNA polymerase was obtained from ISC BioExpress (Kaysville, UT). All oligonucleotides were manufactured with an Applied Biosystems model 380B DNA synthesizer from the UCSD Cancer Center Molecular Biology Core laboratory.

Southern Blot Analysis.

Genomic DNA was isolated from rabbit liver or human peripheral blood samples by the method of Blin and Stafford (1976), and approximately 10 μg of DNA was digested with restriction endonucleases. The digested DNA fragments were electrophoresed in a 1.0% agarose gel containing 50 mM Tris-HCl (pH 8.4), 20 mM sodium acetate, 18 mM sodium chloride, and 2 mM EDTA. After denaturing and neutralizing, DNA was transferred from the gel to MSI NitroPlus 2000 nitrocellulose filters. Filters were prehybridized and then hybridized at 42°C for 18 h with 10⁷cpm/ml ³²P-labeled probe DNA in 6× SSC (1× SSC: 15 mM sodium citrate, 150 mM sodium chloride, pH 7.0), 5× Denhardt's solution (1× Denhardt's solution: 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin fraction V), 100 mg/ml denatured salmon sperm DNA, 0.1% sodium dodecyl sulfate (SDS), and 50% deionized formamide. The filter was washed twice at room temperature in 2× SSC and 0.1% SDS for 5 min, then twice at 55°C in 0.1× SSC and 0.1% SDS for 30 min. The filter was exposed to X-ray film with intensifying screens at −70°C.

Long-Range Polymerase Chain Reaction (LR-PCR).

LR-PCR analysis was undertaken to determine whether the rabbitUGT1A6-like exons were linked. Two oligonucleotides, 5′-gagcgggcctcagacggaatacaggaataa-3′ (1A6-SL), which corresponded to bases 396–425, and 5′-gagcgagcctcagacggaatacaggaaact-3′ (1A6-AL), which corresponded to bases 563–534 of the rabbit UGT1A6 cDNA (Lamb et al., 1994), were synthesized and used as primers to amplify rabbit genomic DNA. LR-PCR was performed with a kit from ISC BioExpress (Kaysville, UT). The 50-μl reaction mixture included 10 ng of rabbit genomic DNA, 1× OptiPerform (ISC) buffer, 2 mM magnesium chloride, 250 mM dNTPs, and 200 nM 1A6-SL and 1A6-AL oligonucleotides. The reaction profile included 2 min at 94°C, 25 cycles at 94°C for 10 s, then 65°C for 30 s, followed by 68°C for 15 min. The amplified products were electrophoresed in a 0.6% agarose gel.

Genomic Cloning of Rabbit UGT1A6-like Genes.

To amplify rabbit UGT1A6-like genes from genomic DNA, oligonucleotides 5′-actggatccaggatggcctgcctg-3′ and 5′-agacctcgagtccggcttcttgcagttga-3′, which matched the 5′ and 3′ regions of the coding region represented by rabbit UGT1A6exon 1, were synthesized and used as primers for PCR. There are no restriction BamHI or XhoI endonuclease-specific sites, so recognition sites for these enzymes were introduced into the 5′ ends of these two oligonucleotides. In a 100-μl reaction buffer containing 20 mM Tris-HCl (pH 7.8), 2 mM MgSO₄, 10 mM KCl, 10 mM (NH₄)₂SO₄, 200 nM primers, 200 mM dNTPs, 500 ng of rabbit genomic DNA, and 5 units cloned Pfu DNA polymerase was denatured at 94°C for 1 min, annealed at 58°C for 1 min, and polymerized at 72°C for 1 min, and the reaction was repeated for 30 cycles. The PCR products were purified and digested with BamHI and XhoI and cloned into these same sites in pBluescript-KS+. More than 20 recombinants were sequenced using primers that recognized the T3 and T7 promoter regions.

Screening Rabbit Liver cDNA Library.

The New Zealand White adult rabbit λ-ZAP cDNA library, previously constructed in this laboratory (Tukey et al., 1993), was screened with a³²P-labeled 700-bp 3′ (BsaI-1294/XmnI-1943) conserved portion of the rabbit UGT1A6 cDNA. The cDNA probe was labeled by nick translation to an approximate specific activity of 1 × 10⁸ cpm/μg of DNA. The cDNA library was plated on 100-mm LB plates containing ampicillin at a density of 2–3 × 10³ pfu/plate. Each filter was annealed with 5 × 10⁶ cpm/ml of labeled DNA at 42°C for 16 h in 50% deionized formamide, 5× SSC, 100 mg/ml sonicated salmon sperm DNA, and 20 mM potassium phosphate buffer (pH 7.4). The filters were first washed at room temperature in 2× SSC containing 0.1% SDS for 15 min, followed by washing in 0.1× SSC and 0.1% SDS for 2 h, with several changes of this wash solution. Each positive cDNA recombinant was isolated and placed in 0.5 ml of water and rescreened as outlined. This procedure was repeated until each λ-ZAP cDNA was demonstrated to be 100% pure, as judged by positive hybridization. Those positive clones with inserted DNA larger than 1600 bp were chosen for further studies. As outlined by Stratagene, each recombinant λ-ZAP cDNA clone was recovered as a double-stranded plasmid. Using both T3 and T7 primers, we sequenced the plasmids at their 5′ and 3′ regions, as outlined in the protocols from the United States Biochemical DNA sequencing kits. The cDNA clones of interest were selected for further characterization.

DNA Sequence Analysis.

The cDNA fragments were individually sequenced using primers that recognized the T3 and T7 promoter regions. In addition, a series of clones with progressively overlapping deletions from the 5′ end were constructed by removing portions of the cDNA by exonuclease III and S₁-nuclease digestion, as outlined with the Erase-A-Base kit supplied by Promega. Colonies were prepared using conventional mini plasmid preparations, and each clone was sequenced using T7 or T3 primers.

Construction of Plasmids for Expression in COS-1 Cells.

The entire coding region of rabbit UGT1A3 cDNA was removed by digesting the plasmid with XbaI andEcoRV, and then cloning this fragment into theXbaI and SmaI sites in the eukaryotic expression vector pSVL. This recombinant plasmid was identified as rabbit p1A3.SVL. Furthermore, the coding region of rabbit UGT1A6αcDNA was removed from pBluescript plasmid by digestion withXhoI and BamHI, and then subcloned into the same sites in pSVL, and the plasmid was referred to as rabbit p1A6α.SVL. The expression vectors p1A4.SVL and p1A6.SVL have previously been constructed (Lamb et al., 1994; Philipp et al., 1994).

The Expression of Rabbit UGTs in COS-1 Cells.

The rabbit expression vectors, p1A3.SVL, p1A4.SVL, p1A6.SVL, and p1A6α.SVL, were transfected into COS-1 cells as outlined (Zuber et al., 1986). COS-1 cells were grown to 50% confluency in Dulbecco's modified Eagle's medium in 150-mm diameter plates, supplemented with 5% fetal calf serum and 5% Nu-serum. After being washed twice with phosphate-buffered saline (PBS), the cells were transfected by incubation with Dulbecco's modified Eagle's medium, 5 mg/ml plasmid DNA, 0.25 mg/ml DEAE-dextran (MW 500,000) and 20 mMN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (pH 7.5). The transfection medium was aspirated and cells were incubated for 5 h in medium containing 100 mM chloroquine. The cells were washed twice with PBS and further incubated for 48 h in culture medium.

³⁵S-Labeling and Immunoprecipitation.

Forty-eight hours after transfection, COS-1 cells from a single 35-mm tissue culture plate were washed three times with Hanks' balanced salt solution and incubated for 4 h in methionine-free modified Eagle's medium supplemented with 100 mCi/ml Trans³⁵S-label. The cells were washed twice in PBS, and the cells were lysed on ice in 600 μl of RIPA solution containing 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, 150 mM sodium chloride, 1 mM phenylmethanesulfonyl fluoride, and 50 mM Tris-HCl (pH 7.5). Cellular DNA was broken by shearing through a 25-gauge needle and was removed with other cellular debris by centrifugation at 16,000g. The cleared supernatant was incubated for 4 h at 4°C with 22 mg of sheep anti-rabbit UGT1A6 Ig (Lamb et al., 1994), followed by 80 μl of a 10% suspension of formalin-fixed S. aureus cells. After the cells were washed four times in RIPA and once in TSA buffer [50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride], the S. aureus. cells were suspended in 100 μl of loading buffer containing 63 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 5% β-mercaptoethanol, and 0.02% bromophenol blue, and the radiolabeled antigens were released by boiling the sample for 5 min, followed by removal of the cells by centrifugation. The immunoprecipitated proteins were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was soaked with 1 M sodium salicylate for 30 min before drying, and the radiolabeled bands were detected by fluorography at −70°C overnight.

UDP-Glucuronosyltransferase Activity Assays.

UGT activities were determined according to the method of Bansal and Gessner (1980), as modified (Tukey et al., 1993). Transfected COS-1 cells were homogenized in 5 volumes of ice-cold 50 mM Tris-HCl (pH 7.5) containing 10 mM MgCl₂. In a total volume of 100 μl, 100 μg of cell extract, 100 μM UDP-glucuronosyltransferase activity (UDPGA), and 0.04 mCi of UDP-[¹⁴C]glucuronic acid, 10 mg of phosphatidylcholine, and 100 mM substrate in 50 mM Tris-HCl (pH 7.5) containing 10 mM MgCl₂ were incubated at 37°C for 1 h and then extracted with 200 μl of 100% ethanol. The protein was removed by centrifugation at 10,000 rpm for 10 min in an Eppendorf centrifuge, and the supernatant was dried and resuspended in 30 μl of methanol for application to a Whatman glass-backed linear K preabsorbent TLC plate. Chromatography was performed in a mixture of 1-butanol/acetone/acetic acid/water (35:35:10:20). The TLC plate was dried, sprayed with a thin layer of EN³HANCE, and exposed to X-ray film at −70°C. The appropriate portions of the TLC cellulose that represented the glucuronides were scraped into glass scintillation vials and dissolved in 0.5 ml of water, and then 12 ml of EcoLite scintillation liquid was added. The vials were shaken and counted in a Beckman scintillation counter. For each substrate, control experiments were conducted with mock-transfected COS-1 cells, and the area of the TLC plate corresponding to the Rf value of the glucuronide from transfected COS-1 cells was scraped and quantitated. Those values were subtracted from the values obtained from transfected cells. We observed no detectable glucuronidation for any of the substrates in nontransfected cells.

Genetic Analysis of the Rabbit UGT1A Locus.

The human UGT1A locus has evolved several conserved clusters of exon 1 sequences that are encoded by UGT1A1, UGT1A3-UGT1A5, UGT1A6, and UGT1A7-UGT10. The exon 1 sequences within each cluster are more conserved in DNA sequence than between the different clusters. Recombinant cDNAs that encode rabbit UGT1A4, UGT1A6, and UGT1A7 have been identified from rabbit liver cDNA libraries (Lamb et al., 1994; Philipp et al., 1994; Bruck et al., 1997). We have recently demonstrated that rabbit UGT1A7 is most homologous to human UGT1A7 (Bruck et al., 1997). In continuing experiments to examine the genetic composition of the rabbitUGT1A locus, genomic DNA was digested with a panel of restriction endonucleases and subjected to Southern blot analysis using³²P-labeled cDNA fragments from the first exon sequences of rabbit UGT1A4 and UGT1A6. Most of the restriction enzymes that were selected do not contain cleavage sites within the exon sequences encoded by the cDNA probes. Thus the appearance of multiple genomic fragments indicates the identity of highly related genes. Each rabbit genomic Southern blot was directly compared to Southern blots, using human genomic DNA that was probed with comparable exon 1 sequences from human UGT1A4 andUGT1A6 cDNAs (Figs. 1 and2). As predicted, rabbitUGT1A4 identifies several related gene fragments, most likely those that encode UGT1A3, UGT1A4, andUGT1A5. However, the banding patterns associated with UGT1A4 in rabbits indicate a greater multiplicity of homologous genes than observed in human. This result indicates that the rabbitUGT1A4 gene has undergone additional gene duplication events.

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Figure 1

The genetic diversity of rabbit and human UGT1A4, as detected by Southern blot analysis. Rabbit and human liver DNA was isolated and digested with a panel of restriction enzymes. After transfer of the DNA to nitrocellulose, the rabbit DNA was hybridized with an EcoRI fragment isolated from the rabbitUGT1A4 cDNA that corresponds to bases 1–668. The filter containing the human DNA was hybridized with a humanNsiI UGT1A4 cDNA fragment corresponding to bases 1–781. Neither of the restriction enzymes that were used to digest rabbit or human DNA was recognized within the cDNA fragments used for hybridization. The patterns of hybridization demonstrated in this figure are reproducible with DNA isolated from other rabbit and human tissue. Thus the incidence of the multiplicity as observed most likely is not the result of incomplete restriction enzyme digestion.

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Figure 2

The genetic diversity of rabbit and human UGT1A6 as detected by Southern blot analysis. The Southern blot containing the rabbit DNA was hybridized with an SacI fragment isolated from the rabbit UGT1A6 cDNA that corresponds to bases 1–796. The human DNA was hybridized with a PvuII cDNA fragment isolated from the human UGT1A6 cDNA that corresponds to bases 1–778. The human UGT1A4 cDNA used in Fig. 1 and the UGT1A6 cDNA employed in this figure have been isolated previously in this laboratory from human liver mRNA by reverse transcription, followed by polymerase chain reaction using specific UGT1A4 and UGT1A6oligonucleotides.

In contrast, the exon sequence of human UGT1A6 recognizes only single gene fragments, consistent with the finding that otherUGT1A exon sequences are not closely related to the more divergent UGT1A6 exon (Fig. 2). This finding is in direct contrast to those results from rabbit UGT1A6 cDNA (Fig. 2), where anywhere from five to nine individual gene fragments can be identified relative to the different restriction endonucleases. This result indicates that the location of the UGT1A6 exon on theUGT1A locus has undergone repetitive duplication events.

Identification of UGT1A6 Exon 1 Gene Sequences.

To examine the potential linkage of the UGT1A6 exons, extended PCR reactions were developed with a pair of sense (1A6-SL) and antisense oligonucleotide primers (1A6-AL). The positions of these oligonucleotides are in divergent regions of the UGT1A6 exon 1. We predicted that if the UGT1A6 exons are arranged in tandem, sense and antisense primers generated to similar regions of the exon would prime and extend the sense position of one exon to the antisense position of the flanking exon. With the pair of primers (sense and antisense) made to exon 1 of UGT1A6, PCR analysis using rabbit genomic DNA generated three DNA transcripts ranging from approximately 9 to 15 kb (Fig. 3). Similar size transcripts were generated when primers made to the 5′ and 3′ regions of the UGT1A6 exon 1 cDNA were used. When we eluted and purified these larger bands from the agarose gels and used the DNA as a substrate, PCR experiments using 5′ and 3′ oligonucleotides to UGT1A6 resulted in the generation of UGT1A6 exon fragments, as confirmed by DNA sequence analysis. These results provide indirect evidence that the UGT1A6-like exons are located on both sides of these large DNA fragments and support the Southern blot analysis, indicating the existence of multipleUGT1A6-like exon sequences.

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Figure 3

Long-range polymerase chain reaction (LR-PCR) of the rabbit UGT1A6 gene. Two oligonucleotides specific to rabbit UGT1A6, the sense 1A6-SL and antisense 1A6-AL (described in Experimental Procedures), were used in a PCR reaction using rabbit liver DNA. The reaction profile for LR-PCR included 2 min at 94°C followed by 25 cycles that included 10 s at 94°C, 30 s at 68°C, and 15 min at 68°C. The amplified products were subjected to electrophoresis in a 0.6% agarose gel. Shown in the figure are photographs taken during short and extended electrophoresis.

The exon sequences were further characterized, generating a library ofUGT1A6 exon 1 fragments by PCR, using rabbit genomic DNA and primers to the 5′ and 3′ positions of the UGT1A6 exon 1 sequence. DNA sequence analysis from over 20 genomic clones identified five unique exons, one of which encoded the original rabbitUGT1A6 cDNA (Lamb et al., 1994). Their predicted amino acid sequences are shown in Fig. 4. Each of the predicted UGT1A6 sequences is more than 98% homologous. Because we obtained five exon sequences generated by PCR analysis and three different genomic fragments, it can be predicted that at least four of the UGT1A6 exons are located in tandem and separated by 9–15 kb of DNA, as shown in Fig. 3.

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Figure 4

Alignment of the predicted amino acid sequences of the rabbit UGT1A6 exon sequences. Using rabbit DNA and oligonucleotides (see Experimental Procedures) specific for the UGT1A6 exon 1 sequence, we cloned PCR products into Stratagene pBluescript II KS+ plasmid. From a library of recombinants, 20 randomly selected plasmids were purified, and the DNA inserts were sequenced. These recombinants were grouped into one of five categories based upon their sequence. Shown in the figure is the predicted amino acid sequence of these five recombinant subgroups. The original UGT1A6 cloned in this laboratory is shown on top, and those amino acids that differ from this sequence are shown.

Identification of Additional UGT1A cDNAs.

To determine whether additional UGT1A6 RNA transcripts are transcribed from the UGT1A6 exons in rabbit liver, a New Zealand White adult rabbit λ-ZAP cDNA library constructed from the sample of tissue used for the Southern blot analysis was screened with a radioactive probe encoding common exons 2–5. This probe would identify all of the UGT1A RNAs represented in the cDNA library. The positive clones with inserts larger than 1600 bp were sequenced with T3 and T7 primers to identify 5′ and 3′ DNA sequences. Recombinant cDNAs encoding the previously identified UGT1A4 (Philipp et al., 1994), UGT1A6 (Lamb et al., 1994), and UGT1A7 (Bruck et al., 1997) were identified. In addition, a cDNA that encoded UGT1A3 and a UGT1A6 cDNA that was identical in sequence to exon UGT1A6α (Fig.4) were also identified. The UGT1A3 cDNA is 2017 bases in length and contains an open reading frame that encodes a protein of 533 amino acids. The deduced amino acid sequence of UGT1A3 is 80.1, 78.9, and 77.7% similar to those of human UGT1A3, UGT1A4, and UGT1A5, respectively, and less than 70% homologous to the other UGT1A isoforms. The predicted amino acid sequence of UGT1A6α is 98.2% similar to that of UGT1A6. This latter result demonstrates that multiple UGT1A6-like gene products are transcribed from theUGT1A6 locus in rabbit liver.

To examine the functional properties of UGT1A6 and UGT1A6α, the cDNAs were recloned into eukaryotic expression plasmid pSVL, and the cDNAs were transfected transiently into COS-1 cells. For comparison, enzyme analysis was also conducted in COS-1 cells that had been transfected with the rabbit UGT1A3 and UGT1A4 cDNAs. To examine the expression levels, transfected cells were labeled with [³⁵S]methionine/cysteine, followed by immunoprecipitation with a polyclonal anti-UGT antibody (Lamb et al., 1994), to confirm that each of the cells transfected with the cDNAs was manufacturing UGTs of the appropriate length (Fig.5). Total cell extracts were used to examine phenolic, steroid, and quaternary amine glucuronidation patterns (Table 1). UGT1A3 was active toward bulky phenolic compounds such as 4-hydroxybiphenyl, octylgallate, and propylgallate, as well as several steroids like β-estradiol, estriol, and androstane. Like previous reports on the activity of human UGT1A3 (Mojarrabi et al., 1996), rabbit UGT1A3 was also active toward estrone. UGT1A4 catalyzed the glucuronidation of androstane, estriol, and androsterone, but was not active toward estrone. We have previously demonstrated that rabbit UGT1A4 catalyzes the glucuronidation of tricyclic antidepressants such as imipramine (Bruck et al., 1997), but it is also active in the presence of other tertiary amines such as amitriptyline. The catalytic profiles of rabbit UGT1A3 and UGT1A4 are similar to those observed for their human orthologs.

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Figure 5

Immunoprecipitations of radiolabeled COS-1 cell extracts. pSVL expression plasmids containing rabbit UGT1A3, UGT1A4, UGT1A6, and UGT1A6α were transfected in COS-1 cells. Control cells were transfected with only the pSVL plasmid. Approximately 48 h after transfection, the cells were labeled for 4 h with 100 μCi/ml [³⁵S]methionine/cysteine, as previously outlined (Lamb et al., 1994). The cells were lysed, and the extracts were incubated with antibody. The antigen/antibody complexes were then incubated withS. aureus cells, and the complex was precipitated and extensively washed. The cell pellet was boiled, and a portion was subjected to SDS-PAGE on an 8% acrylamide gel. The gel was soaked in 1 M sodium salicylate as a fluorographic enhancer, dried, and exposed to X-ray film overnight at −70°C.

UGT1A6 is most active toward small phenolic substrates such as 1,5-isoquinoline, 2-hydroxybiphenyl, 4-isopropylphenol, 4-methylumbelliferone, and 4-tert-butylphenol (Table 1). Although UGT1A6 and UGT1A6α are more than 98% homologous in amino acid sequence, UGT1A6α metabolized these phenolics poorly in comparison to the catalytic activities attributed to UGT1A6. Interestingly, bulky phenols such as octylgallate and propylgallate as well as flavones such as 7-hydroxyflavone are excellent substrates for glucuronidation by UGT1A6α, although being poor substrates, in comparison, for UGT1A6. Figure6demonstrates this dramatic difference, as observed for glucuronide formation by TLC analysis. Plasmids were transfected into COS-1 cells, and UGT analysis was conducted in the presence of substrate and 100 μM UDPGA. When these experiments are carried out with 1 mM UDPGA, dilution of the labeled UDPGA in the reaction leads to a dramatic reduction in detectable 2-hydroxybiphenyl or 4-isopropylphenol catalyzed by UGT1A6, yet significant 4-methylumbelliferone glucuronide is still observed. Likewise, when 1 mM UDPGA is used to assay UGT1A6α catalysis, only octylgallate and 7-hydroxyflavone glucuronide formation is detectable (data not shown). This result, along with the data presented in Table 1 and Fig. 6, clearly demonstrates that the six amino acid differences between UGT1A6 and UGT1A6α dramatically affect the efficiency of glucuronidation and substrate specificity of these two highly homologous rabbit UGTs.

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Figure 6

Differential expression of UGT1A6 and UGT1A6α. The expression plasmids encoding UGT1A6 and UGT1A6α were transiently transfected into COS-1 cells, and total cell extracts were used to analyze patterns of glucuronidation, using 4-methylumbelliferone (4 MU), 2-hydroxybiphenyl (2OH-biphenyl), 4-isopropylphenol (4-IP-phenol), octylgallate, and 7-hydroxyflavone (7OH-flavone). Assays were conducted as outlined in Experimental Procedures, using similar concentrations (100 μg) of cell extract. The specific activities (pmol/mg protein/min) are shown beneath the respective lanes of the TLC. These experiments were run independently from those reported in Table 1.