Structural insights into sodium transport by the oxaloacetate decarboxylase sodium pump

The oxaloacetate decarboxylase sodium pump (OAD) is a unique primary-active transporter that utilizes the free energy derived from oxaloacetate decarboxylation for sodium transport across the cell membrane. It is composed of 3 subunits: the α subunit catalyzes carboxyl-transfer from oxaloacetate to biotin, the membrane integrated β subunit catalyzes the subsequent carboxyl-biotin decarboxylation and the coupled sodium transport, the γ subunit interacts with the α and β subunits and stabilizes the OAD complex. We present here structure of the Salmonella typhimurium OAD βγ sub-complex. The structure revealed that the β and γ subunits form a β3γ3 hetero-hexamer with extensive interactions between the subunits and shed light on the OAD holo-enzyme assembly. Structure-guided functional studies provided insights into the sodium binding sites in the β subunit and the coupling between carboxyl-biotin decarboxylation and sodium transport by the OAD β subunit.

Uptake and metabolism of putrescine in confluent LLC-PK1 cells

Putrescine is taken up by confluent pig kidney (LLC-PK1) cells at roughly equal rates over both Na(+)-dependent and Na(+)-independent pathways. The former is sensitive to 1 mM amiloride, but the latter is not. Uptake rates are similar at both the apical and basolateral surfaces. The principal fate of the putrescine is oxidative deamination, yielding a product that appears to be either gamma-aminobutyraldehyde or delta 1-pyrroline. Most of the remainder is converted to products tentatively identified as spermidine, spermine, or another unidentified product; these products as well as putrescine itself are lost from the cell at either surface. Changing the extracellular pH in the range of 6.8-8.0 has no affect on putrescine uptake. Cells acidified to intracellular pH 6.8 show a reduced capacity to incorporate radioactivity, an effect that may be due to inhibition of diamine oxidase. Depletion of ATP stores by treating cells with 2-deoxy-D-glucose and NaN3 does not reduce putrescine uptake, suggesting that the mechanism is not a primary active transporter. The Na(+)-dependent component of uptake is inhibited by 5-50 microM Hg2+ in a dose-dependent manner. p-Chloromercuribenzene sulfonic acid (p-CMBS) at high concentrations (500-1,000 microM) does not affect Na(+)-independent uptake but in the presence of Na+ depresses total uptake more than Na+ depletion alone, suggesting that Na+ enhances the binding of p-CMBS to both transporters. Spermidine and spermine compete with putrescine for uptake, but a variety of other organic bases and amino acids do not, indicating that polyamines are transported by mechanisms distinct from the transporters for those other compounds.