The beta amino acid taurine is important to the growth and development of the fetus and newborn and is considered to be an essential amino acid in the pre- and immediate postnatal period. Therefore, the objectives of this grant proposal are (1) to define the transport mechanisms for taurine in the intestine and placenta, tissues supplying exogenous taurine during perinatal development and, (2) by using the ontogeny of carrier function as an experimental strategy, to identify, is date and clone the brush border membrane (BBM) carrier protein in order to further examine the regulatory mechanisms controlling developmental expression. Highly purified membrane vesicles prepared from basdateral and brush border membrane domains will be used to characterize membrane transport and identify essential amino acid residues on the carrier at different developmental ages. In addition, BBM is dated from tissue at various ages will be analyzed on SDS-polyacrylamide gels and compared with partially purified protein obtained by affinity chromatography. Identification and isolation of an appropriate ontogenically expressed protein will allow production of specific monoclonal and polyclonal antibodies that can be used to further examine the function and structure of the carrier protein and, by immunochemcial techniques, allow determination of quantitative developmental differences in the carrier protein. Molecular cloning and cDNA sequencing of the BBM carrier protein will be achieved using synthetic oligonucleotide probes prepared from partial sequencing of the purified protein or monospecific antibodies generated to the carrier in order to screen appropriate cDNA libraries. Alternatively, expression of size-selection mRNA encoding the Na+/taurine cotransporter in Xenopus laevis oocytes will provide a method for cloning and cDNA sequencing of the carrier without first purifying the protein. Ultimately, sequencing of the cDNA encoding the BBM carrier will allow further definition of the structural organization of the membrane protein. In addition, hybridization studies cDNA probes will provide a method for examining the mechanism (transcriptional or translational) for ontogenic expression of the transport protein.
| Shneider, B L; Moyer, M S (1993) Characterization of endogenous carrier-mediated taurocholate efflux from Xenopus laevis oocytes. J Biol Chem 268:6985-8 |
| Dumaswala, R; Setchell, K D; Moyer, M S et al. (1993) An anion exchanger mediates bile acid transport across the placental microvillous membrane. Am J Physiol 264:G1016-23 |
| Moyer, M S; Insler, N; Dumaswala, R (1992) The role of chloride in taurine transport across the human placental brush-border membrane. Biochim Biophys Acta 1109:74-80 |
