Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes

Archives of Biochemistry and Biophysics
H TamuraM Matsui

Abstract

The St-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS). ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol. To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli. Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities. Others lost all HS-ST activity. Analysis revealed that a central region (region III spanning amino acids 102-164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165-219) are unimportant with regard to substrate preference. It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity. Photoaffinity labeling with [35S]3'-phosphoadenosine 5...Continue Reading

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Jun 1, 1991·Chemical & Pharmaceutical Bulletin·H HommaM Matsui
Nov 30, 1989·Biochemical and Biophysical Research Communications·K OguraR Kato
Oct 1, 1994·The International Journal of Biochemistry·M Matsui, H Homma
Jun 1, 1994·Chemico-biological Interactions·Y YamazoeR Kato
Jun 1, 1994·Chemico-biological Interactions·D M Otterness, R Weinshilboum
Aug 3, 1993·Biochemical Pharmacology·M MatsuiH Homma
Sep 5, 1996·Biochimica Et Biophysica Acta·H HommaM Matsui

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Citations

Jan 12, 1999·The Biochemical Journal·L A BrixM E McManus

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