Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Rat Small Intestine

Glucose-6-phosphate dehydrogenase (G6PD) was purified from rat small intestine with 19.2% yield and had a specific activity of 53.8 units per miligram protein. The pH optimum was determined to be 8.1. The purified rat small intestinal G6PD gave one activity, one protein band on native PAGE. The observation of one band on SDS/PAGE with an Mr of 48 kDa and a specific activity lower than expected may suggest the proteolytically affected enzyme or different form of G6PD in the rat small intestine. The activation energy, activation enthalpy, Q10, and optimum temperature from Arrhenius plot for the rat small intestinal G6PD were found to be 8.52 kcal/mol, 7.90 kcal/mol, 1.59, and 38 degrees C, respectively. The Km values for G6P and NADP+ were 70.1 +/- 20.8 and 23.2 +/- 7.6 microM, respectively. Double-reciprocal plots of 1/Vm versus 1/G6P (at constant [NADP+]) and of 1/Vm versus 1/NADP+ at constant [G6P]) intersected at the same point on the 1/Vm axis to give Vm = 53.8 U/mg protein.     

Natriuretic peptide receptor A activation stabilizes a membrane-distal dimer interface

We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp(74) in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp(74) from both subunits drastically differs, depending on whether the V-shaped (84 A) or the A-shaped (8 A) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRA(W74C) mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRA(W74C). Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRA(W74C). The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.     

Endemic goiter, thiocyanate overload, and selenium status in school-age children

Iodine deficiency (ID) and related disorders are still major, yet unresolved health concerns. Recently, in a systematic survey of schoolage children (SAC), we reported severe to moderate ID, in Ankara and three cities from Black Sea region of Turkey. The current study attempted to evaluate selenium (Se) status, thiocyanate (SCN⁻) overload, and their possible contribution to the goiter endemics and thyroid hormone profile observed in these cities. Thyroid ultrasonography was performed and serum Se, SCN⁻, thyroid hormones, sensitive TSH (sTSH) levels, and urinary iodine concentrations (UICs) were determined from 251 SAC (9–11 yr old). Thyroid volumes (TVs) exceeding recommended upper normal limits and median UIC indicated goitre endemics and moderate to severe ID in the areas studied. Mean serum SCN⁻ concentrations were found to be greater than the controls from the literature. The UIC/SCN⁻ ratio was found to be lowest in Bayburt and Trabzon denoting that SCN⁻ overload may contribute to the goiter endemics. Serum Se concentrations represent a marginal deficiency in the four areas studied. No significant correlations between serum Se concentrations and the other parameters studied (i.e., TV, SCN⁻, thyroid hormones, sTSH, UIC) was detected. In conclusion, this study showed that selenium is also marginally deficient in the iodine-deficient endemic areas studied, but this has little or no impact on the thyroid hormone profile and the goiter endemics. SCN⁻ overload may contribute to the endemics, especially for the areas where iodine is severely deficient. An effective iodine supplementation program will not only resolve the goiter endemics but also the consequence of SCN⁻ overload as well in the endemic goiter areas studied.