Preparation of asymmetric,cerebroside sulfate-containing phospholipid vesicles

A procedure is described which inserts asymmetrically cerebroside sulfate (‘sulfatide’) into the outer leaflet of bilayered phospholipid vesicles. Cerebroside sulfate is adsorbed onto a cellulose, filter-paper support and, when incubated with phosphatidylcholine vesicles is transferred to and inserted into the outer leaflet of these vesicles. This transfer occurs at, or above the transition temperature of the phospholipid and follows a similar pattern with small or larger (‘fused’) unilamellar vesicles. The transfer is linear with time for 1–2 h and is maximal after about 6 h, when the sulfatide content reaches about 6 mol% of the total quantity of phospholipid, corresponding to about 10 mol% of the phospholipids present in the outer layer. Initial rates of sulfatide transfer were somewhat increased when the vesicles contained a positively charged lipid (e.g. stearylamine) and decreased when this lipid was negatively charged (e.g. dicetyl phosphate) or hydrophobic (e.g. cholesterol). Divalent ions markedly inhibited sulfatide transfer and monovalent ions did so to a lesser degree. Once incorporated into the outer leaflet of the vesicle, the sulfatide could not be removed by washing with buffer, 1 M NaCl or 1 M urea.     

Effects of ambient PO2 and temperature on oxygen uptake in Nautilus pompilius

This study employs closed-circuit respirometry to evaluate the effect of declining ambient oxygen partial pressure (PO₂) and temperature on mass specific rates of oxygen uptake (V˙O₂) in Nautilus pompilius. At all temperatures investigated (11, 16, and 21 °C), V˙O₂ is relatively constant at high PO₂ (oxyregulation) but declines sharply at low PO₂ (oxyconformation). The critical PO₂ below which oxyconformation begins (P _c) is temperature dependent, higher at 21 °C (49 mmHg) than at 11 °C or 16 °C (21.7 mmHg and 30.8 mmHg respectively). In resting, post-absorptive animals, steady-state resting V˙O₂ increases significantly with temperature resulting in a Q₁₀ value of approximately 2.5. The metabolic strategy of N. pompilius appears well suited to its lifestyle, providing sufficient metabolic scope for its extensive daily vertical migrations, but allowing for metabolic suppression when PO₂ falls too low. The combination of low temperatures and low PO₂ may suppress metabolic rate 16-fold (assuming negligible contributions from anaerobic metabolism and internal O₂ stores), enhancing hypoxia tolerance. Accepted: 20 January 2000     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.