Abstract
Lubrol solubilized microsomes of liver from JJ and jj rats were salt fractionated and the 60% (NH4)2 SO4 precipitate was redissolved, dialyzed and further fractionated by DEAE cellulose chromatography (DE-52). After initial protein elution with 10 mM PO4 buffer, pH8.0, a linear salt gradient from 0-0.5M KCl was begun. Three separate elution fractions (F1, F2, F3) were collected with BGt activity for bilirubin (B). The DE-52, F3 fractions from both JJ and jj rats activated Che BGt activity of the F1 fraction of JJ rats 5-10 fold (n=20). The A was separated from the BGt in F3 by Sephacryl-300 (S-300) sieving into two molecular weight fractions BGt≅230 kD and A≅60 kD. Further purification of the BGt from F1 and F3 of JJ liver was performed by affinity chromatography. After adsorption on UDP-hexanolamine Sepharose 4-B, the purified BGt was eluted by 5mM UDPGA which showed by polyacrylamide gel electophoresis (PAGE) a subunit couplet of 52 and 54 kD. This purified BGt was activitated 5-8 fold by the A from the S-300 eluate of both JJ and jj microsomes. The subunit size of A by PAGE is similar to its size after S-300 sieving (≅60 kD). It is heat labile, has no BGt activity, is precipitated by TCA, inactivated by alkylation but not by trypsin or iodoacetamide. The A has no effect on the purified transferase (Gt) for p-nitrophenol. Thus in addition to separate Gts in microsomes additional cofactors appear important in determining their substrate specificity. The A increases the formation of both the mono- and diglucuronides of B by BGt.
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Odell, G., Mogilevsky, W. ISOLATION OF THE ACTIVATOR (A) OF BILIRUBIN GLUCURONYL TRANSFERASE (BGt) ROM NORMAL (JJ) AND HOMOZYGOUS (jj) GUNN RATS. Pediatr Res 21 (Suppl 4), 274 (1987). https://doi.org/10.1203/00006450-198704010-00639
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DOI: https://doi.org/10.1203/00006450-198704010-00639