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Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary Kl cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP-and GTP-binding proteins and is located near the C terminus of all steroid and phenol (aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8-to 28-fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate (>30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site.The biotransformation of compounds by sulfonation occurs widely, effecting a marked change in the physicochemical properties of the sulfonated products. Sulfonation of drugs and xenobiotics functions primarily to inactivate and clear these generally hydrophobic compounds, although there are examples where the active form of a drug is sulfonated (1). Likewise, the sulfonation of endogenous substances is a fundamental metabolic mechanism that covers an extended range of molecules of diverse size, structure, chemistry, and function. Membrane and secretory proteins are post-translationally modified by sulfonation. Macromolecules such as glycosaminoglycans and proteoglycans, components of cell surface and connective tissue structures, are subjected to modification by the addition of sulfonate groups. Importantly, a multitude of low molecular weight compounds, including neurotransmitters and hormones-e.g., catecholamines, iodothyronines, and steroids, are modified by sulfoconjugation.Inherently, the enzymes that carry out the transfer of a sulfonate group (SO3), termed sulfotransferases, must interact with two substrates, namely, the sulfonate donor and acceptor molecules. In mammals, the universal activated sulfonate donorThe publication costs of this article were defrayed in part by page charge payment. This article must therefor...

Contralateral uninephrectomy attenuates unilateral ischemic injury. The present work was performed to elucidate whether the beneficial effect of uninephrectomy was associated with the modification of ischemia-induced changes in plasma or renal renin activity. A 60-min left renal artery occlusion was conducted in right nephrectomized (Nx) and sham-nephrectomized (Sham-Nx) rats. The decline in inulin clearance 48 h after ischemia was significantly less in Nx rats than in Sham-Nx animals (0.50 ± 0.10 vs. 0.052 ± 0.029 ml/min/kidney, p < 0.05). Following ischemia, plasma renin activity (PRA) significantly increased in Sham-Nx (from 5.4 ± 0.9 to 15.5 ± 1.4 ng Al/ml/min, p < 0.01) but not in Nx (from 3.5 ± 0.5 to 5.0 ± 1.0 ng AI/ml/ min) animals. PRA and renal cortical renin content (2,200 ± 225 vs. 1,257 ± 187 ng Al/h/mg protein, p < 0.05) were significantly less in Nx rats than in Sham-Nx animals 48 h after renal ischemia. The decrease in body weight was greater in Nx rats than in Sham-Nx animals. Plasma atrial natriuretic peptide (ANP) (195 ± 30 vs. 302 ± 40 pg/ml, p < 0.05) and renal dopamine (DA) content(3.2 ± 0.5 vs. 13.7 ± 1.3 ng/g tissue, p < 0.01) were rather lower in the Nx group when compared with the Sham-Nx group. No significant difference was found in the intrarenal content of norepinephrine (NE) between two ischemic groups. These findings suggested that uninephrectomy prevents the ischemia-induced increase in renin activity. The prevention of the increase in renin activity in Nx rats is not be mediated through the modulation of ischemia-induced changes in sodium balance, plasma ANP level and/or intrarenal contents of NE and DA.

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