Comparative studies of human UDP-glucuronosyltransferase 1A8 and 1A9 proximal promoters using single base substitutions.

  The nucleotide sequences of the proximal promoters of UDP-glucuronosyltransferase (UGT) 1A8 and 1A9 genes are very similar. However, UGT1A8 and 1A9 are mainly expressed in extra-hepatic and hepatic cells, respectively. Using mutants of UGT1A8 and 1A9 proximal promoters, we revealed their critical differences in terms of promoter activity and the role of the T-repeat region (T-region) conserved in both promoters. In extra-hepatic cells, Caco2, the activity of UGT1A9 proximal promoter increased to 73.4 ± 8.5% of that of the UGT1A8 proximal promoter with only 4 base changes: -160C, -152A, -62T, and -59G. The derivatives of the T-region showed that this region is not necessary for promoter activity, but the length of T repeats influences the activity somewhat. Therefore, the cause of the low activity of the UGT1A9 proximal promoter may be not only 4 base changes, but also the truncation of T repeats. From these results, the UGT1A9 proximal promoter was assumed to change into the non-active form from the original sequence, and this might be one of the reasons for the tissue-specific expression of UGT1A9.

Application of "homogeneous assay for fluorescence concentrated on membrane" to the analysis of the substrate specificity of protease.

The utility of the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) in the analysis of the substrate specificity of protease was investigated using tobacco etch virus (TEV) protease. The V(max) of TEV protease against variants of a substrate was obtained by a simple procedure. It was considered that HAFCOM was more accurate than other endpoint measurements of protease assay.

Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk.

Green fluorescent protein (GFP) is very stable for various proteases. Using this property, three protease assay methods designated the disk separation assay for remaining GFP (DSAR), the disk separation assay for liberated GFP (DSAL), and the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) were developed. These methods employ a nylon membrane designated "Cleave-Checker" on which GFP-SpA(B) (domain B in staphylococcal protein A) is immobilized. The SpA(B) region was used as a substrate for the protease, and the isolation of GFP from the membrane generated by the digestion of the SpA(B) region was detected. In DSAR, it was possible to detect solution of at least 25 ng/ml trypsin or proteinase K by visual observation. The most important feature of DSAR is that the detection of the protease is possible only under UV light. In contrast, DSAL is suitable for a highly sensitive assay. The assay ranges of DSAL were 1.6 to 100 ng/ml in trypsin and 1.6 to 400 ng/ml in proteinase K. HAFCOM does not require bound/free (B/F) separation; thus, the procedure is simpler than that with DSAL and the reproducibility is high. The assay ranges of HAFCOM were 25 to 400 ng/ml in trypsin and 12.5 to 200 ng/ml in proteinase K. The Cleave-Checker used for these methods was stable in a dry state, and long-term preservation for at least several months was possible.