• Journal of the Korean Magnetic Resonance Society
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• v.21 no.1
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• pp.20-25
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• 2017

The cytoplasmic domains A and B of the mannitol transporter enzyme $II^{Mtl}$ are covalently linked in Escherichia coli, but separately expressed in Thermoanaerobacter Tengcongensis. The phosphorylation of domain B ($TtIIB^{Mtl}$) substantially increases the binding affinity to the domain A ($TtIIA^{Mtl}$) in T. Tengcongensis. To understand the structural basis of the enhanced domain-domain interaction by protein phosphorylation, we obtained NMR backbone assignments of the phospho-$TtIIB^{Mtl}$ using a standard suite of triple resonance experiments. Our results will be useful to monitor chemical shift changes at the active site of phosphorylation and the binding interfaces.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.1
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• pp.42-48
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• 2015

The mannitol transporter Enzyme $II^{Mtl}$ of the bacterial phosphotransferase system has two cytoplasmic phosphoryl transfer domains $IIA^{Mtl}$ and $IIB^{Mtl}$. The two domains are linked by a flexible peptide linker in mesophilic bacterial strains, whereas they are expressed as separated domains in thermophilic strains. Here, we carried out backbone assignment of $IIA^{Mtl}$ from thermophilic Thermoanaerobacter Tencongensis using a suite of heteronuclear triple resonance NMR spectroscopy. We have completed 94% of the backbone assignment, and obtained secondary structural information based on torsion angles derived from the chemical shifts. $IIA^{Mtl}$ of Thermoanaerobacter Tencongensis is predicted to have six ${\beta}$ strands and six ${\alpha}$ helices, which is analogous to $IIA^{Mtl}$ of Escherichia coli.

• Archives of Pharmacal Research
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• v.25 no.6
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• pp.984-988
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• 2002

The issue of whether or not the presence NOx (NO and oxidized metabolites) in the hepatocytes at pathological levels affects the functional activity of transport systems within the sinusoidal membrane was investigated. For this purpose, the effect of the pretreatment of isolated hepatocytes with sodium nitroprusside (SNP), a spontaneous NO donor, on the sinusoidal uptake of tributylmethylammonium (TBuMA) and triethylmethyl ammonium (TEMA), representative substrates of the organic cation transporter (OCT), and taurocholate, a representative substrate of the $Na^+$/taurocholate cotransporting polypeptide (NTCP), was measured. The uptake of TBuMA and TEMA was not affected by the pretreatment, as demonstrated by the nearly identical kinetic parameters for the uptake ($i.e., V_{max}, K_{m} and CL_{linear}$). The uptake of mannitol into hepatocytes was not affected, demonstrating that the membrane integrity remained constant, irregardless of the SNP prutreatment. On the contrary, the uptake of taurocholate was significantly inhibited by the pretreatment, resulting in a significant decrease in V_{max}$, thus providing a clear demonstration that NOx preferentially affects the function of NTCP rather than OCT on the sinusoidal membrane. A direct interaction between NOx and NTCP or a decrease in $Na^+/K^+$ ATPase activity as the result of SNP pretreatment might be responsible for this selective effect of NOx.