No abstract

Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of v-primary or v-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-p interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant K m (BAL) increases and V max /K m (BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the V max /K m (BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other v-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.

No abstract

The renal-specific Na(+)-K(+)-2Cl(-) cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na(+)-Cl(-) cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by (86)Rb(+) influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced approximately 20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl(-) affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161-F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na(+) than for Cl(-), with apparent Michaelis-Menten constant (K(m)) values of 58.2 +/- 7.1 and 22.1 +/- 4.2 mM, respectively. These K(m) values are significantly higher than those observed in rTSC. The Na(+) and Cl(-) affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10(-9) to 10(-7) M) resulted in activation of the cotransporter, whereas higher concentrations (10(-6) to 10(-4) M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na(+) or the Cl(-) concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg(2+), with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.