Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.     

Evaluating 50 years of time-series soil radiocarbon data: towards routine calculation of robust C residence times

In 1959, Athol Rafter began a substantial programme of monitoring the flow of ¹⁴C produced by atmospheric thermonuclear tests through New Zealand’s atmosphere, biosphere and soil. By building on the original measurements through ongoing sampling, a database of over 500 soil radiocarbon measurements spanning 50 years has now been compiled. The datasets, including an 11-point time series, allow strong focus on the robust quantification of residence times ranging from years to decades. We describe key aspects of the dataset, including the ability to identify critical assumptions inherent in calculating soil C residence times. The 3 most critical assumptions relate to: (1) the proportion of old C (“fraction passive”), (2) the lag time between photosynthesis and C entering the modeled pool, and (3) changes in the rates of C input (i.e., steady state). We demonstrate the ability to compare residence times in contrasting sites, such Andisols and non-Andisols, and the ability to calculate residence times across a range of soil depths. We use ¹⁴C in a two-box model to quantify soil carbon turnover parameters in deforested dairy pastures under similar climate in the Tokomaru silt loam (non-Andisol) versus the Egmont black loam (Andisol), originally sampled in 1962, 1965 and 1969, and resampled again in 2008. The ¹⁴C-based residence times of the main soil C pool in surface soil (~8 cm) are ~9 years in the Tokomaru soils compared to ~17 years for the Egmont soils. This difference represents nearly a doubling of soil C residence time, and roughly explains the doubling of the soil C stock. Passive soil C comprises 15% of the soil C pool in Tokomaru soils versus 27% in Egmont soils. A similar difference in residence times is found in a second surface soil comparison between the Bruntwood soil (Andisol) and the Te Kowhai soil (non-Andisol) with residence times of 18 and 27 years, respectively. The comparisons support evidence that C dynamics do differ in Andisols versus non-Andisols, as a result of both the mineral allophane and Al complexation. Expanding our calculations beyond surface soil, we show that thickening the calculation depth by combining horizons allows robust residence times to be calculated at a range of depths. Overall, the large and systematically collected dataset demonstrate that soil C residence times of the main soil C pool can be routinely calculated using ¹⁴C wherever samples collected 10 or more years apart in New Zealand grassland soils are available, and presumably under similar circumstances in other soils worldwide.     

Metabolic evidence that serosal sodium does not recycle through the active transepithelial transport pathway of toad bladder

Summary The possibility that sodium from the serosal bathing medium back-diffuses into the active sodium transport pool within the mucosal epithelial cell of the isolated toad bladder was examined by determining the effect on the metabolism of the tissue of removing sodium from the serosal medium. It was expected that if recycling of serosal sodium did occur through the active transepithelial transport pathway of the isolated toad bladder, removal of sodium from the serosal medium would reduce the rate of CO₂ production by the tissue and enhance the stoichiometric ratio of sodium ions transported across the bladder per molecule of sodium transport dependent CO₂ produced simultaneously by the bladder (J _Na/J _{CO 2}). The data revealed no significant change in this ratio (17.19 with serosal sodium and 16.13 after replacing serosal sodium with choline). Further, when transepithelial sodium transport was inhibited (a) by adding amiloride to the mucosal medium, or (b) by removing sodium from the mucosal medium, subsequent removal of sodium from the serosal medium, or (c) addition of ouabain failed to depress the basal rate of CO₂ production by the bladder [(a) rate of basal, nontransport related, CO₂ production (J _CO2 ^b ) equals 1.54±0.52 with serosal sodium and 1.54±0.37 without serosal sodium; (b)J _CO2 ^b equals 2.18±0.21 with serosal sodium and 2.09±0.21 without serosal sodium; (c) 1.14±0.26 without ouabain and 1.13±0.25 with ouabain; unite ofJ _CO2 ^b are nmoles mg d.w.^–1 min^–1]. The results support the hypothesis that little, if any, recycling of serosal sodium occurs in the toad bladder.