C Ehnes | PubMed Health

PubMed2002

Functional studies on a split type II Na/P(i)-cotransporter.

Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/P(i)-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane distribution of this split NaP(i)-IIa transporter we followed two strategies. In one strategy we expressed the transporter as two complementary parts (p40 and p45) in Xenopus laevis oocytes and as another strategy we cleaved the WT protein with trypsin. Both strategies resulted in a split NaP(i)-IIa protein located in the plasma membrane. The two domains were tied together by a disulfide bridge, most likely involving the cysteines 306 and 334. Surface expression of the NaP(i)-IIa fragments was dependent on the presence of both domains. If both domains were coexpressed, the transporter was functional and transport characteristics were identical to those of the WT-NaP(i)-IIa protein. Corresponding to this, the transporter cleaved by trypsin also retains its transport capacity. These data indicate that cleavage of the type IIa Na/P(i)-cotransporter at ECL-2 is compatible with its cotransport function.

Amino Acid Sequence↗

PubMed2000

Binding of xenobiotics to hepatic estrogen receptor and plasma sex steroid binding protein in the teleost fish, the common carp (Cyprinus carpio).

Competitive receptor binding assays have been suggested as an in vitro screening tool for assessing the activity of alleged estrogenic substances. In this study, we determined the ability of steroidal and nonsteroidal substances to inhibit the binding of [(3)H]17 beta-estradiol (E2) to the hepatic estrogen receptor (ER) and the plasma sex steroid binding protein (SBP) of the teleost fish, the common carp (Cyprinus carpio). The objectives of the study were (1) to characterize ER binding in the liver cytosol of male and female carp, (2) to establish complete [(3)H]E2 displacement curves from carp ER for a range of natural and xenobiotic substances and to compare the ligand data of carp ER with published data from other vertebrate species to reveal possible species differences, and (3) to determine the interaction of natural and xenobiotic substances with the steroid binding site of SBP in carp plasma. The results indicate the presence of a single class of estrogen binding sites with high affinity and limited capacity in liver cytosol of carp. The various test agents showed partly quantitative differences in their binding affinities, with the xenobiotics generally showing limited ability to displace [(3)H]E2 from the hepatic ER or from plasma SBP of carp. However, we found no evidence that a compound is an ER ligand exclusively in one species. The findings of this study indicate that interspecies extrapolation of steroid receptor binding data is possible on a yes/no basis but not on a quantitative basis.

Animals↗