Stability of the glycerol facilitator in detergent solutions.

Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence. Protein tetramers were prepared in neutral dodecyl maltoside (DDM) and in zwitterionic lysomyristoylphosphatidylcholine (LMPC) detergent solutions that are stable during SDS-PAGE. Thermal unfolding experiments show that the protein is more stable in LMPC than in DDM. Tertiary structure unfolds before quaternary and some secondary structure in LMPC, whereas unfolding is more cooperative in DDM. The high stability of the protein in DDM is evident from the unfolding half-life of 8 days in 8 M urea, suggesting that hydrophobic interactions contribute to the stability. The protein unfolds readily in LMPC below pH 6, whereas the tetramer remains intact at pH 4 in DDM. At pH 4 in DDM, the protein is more sensitive than at neutral pH to unfolding by SDS and the effect is reversible. At pH 3 in DDM, the tetramer unfolds, losing its tertiary structure but retaining native helical structure which melts at significantly lower temperatures than in the native tetramer. The glycerol facilitator prepared in SDS is mainly monomeric and has ~10% less alpha-helix than the native protein. CD suggests that it forms a condensed structure with non-native tertiary contacts highly similar to the state observed in LMPC at low pH. The implications of the results for in vitro and in vivo folding of the protein are discussed.

Detection of moderate regional ischemia in pig hearts in vivo by near-infrared and thermal imaging: effects of dipyridamole.

Effects of coronary vasodilator, dipyridamole, on epicardial oxygenation and flow were investigated under conditions of moderate coronary occlusion using near-infrared spectroscopic (NIRS) and thermal imaging. In anesthetized open chest pigs an inflatable occluder and flow probe were placed around the left anterior descending artery (LAD). In the ischemic group (n = 11) LAD occlusion (50% flow, 80 min) was followed by complete occlusion (10 min, n = 4), and reflow. Dipyridamole was infused (0.14 mg/min/kg/4 min) intravenously during 50% occlusion. In the control group (n = 6) LAD flow was temporarily increased (hyperemic response) by two 2-min periods of complete LAD occlusion applied 120 min apart, with a 4-min period of dipyridamole infusion between the two occlusions. NIRS and thermal images were acquired throughout the protocol. Maps of subepicardial oxygen saturation parameter (OSP), and epicardial temperature (T) were obtained. Partial occlusion reduced OSP and the temperature by 0.23 +/- 0.08 and 0.88 +/- 0.39 degrees C versus remote region, respectively. Dipyridamole decreased systolic blood pressure by 36%, which caused further decline in the LAD flow to 18% and OSP and T by 0.37 +/- 0.01 and 2.46 +/- 0.32 degrees C, respectively. Reflow restored OSP and T to their baseline levels. In control group dipyridamole and hyperemia increased LAD flow 2-4-fold associated with moderate increase in OSP and T. OSP and T showed linear dependence on the flow below 100%, which is leveled-off at flows above normal. Dipyridamole increases differences in the epicardial oxygenation and T between normal and moderately ischemic areas due to enhancement of disparity in perfusion of these areas.

Visualization and grading of regional ischemia in pigs in vivo using near-infrared and thermal imaging.

Reductions in regional coronary flow result in tissue deoxygenation and decrease in surface temperature, changes detectable by near-infrared spectroscopic (NIRS) and thermal imaging, respectively. In anesthetized open-chest pigs, an inflatable occluder and flow probe were placed around the left anterior descending artery. Gated NIRS and nongated thermal images were acquired at baseline, partial (17% and 50%), and complete occlusion and reflow. At each step, dobutamine was infused (10 microg.min(-1).kg(-1)) for 7-9 min to increase blood pressure and flow. Changes in the oxygen saturation parameter, rate of indocyanine green flow tracer passage, and the surface temperature were correlated with the measured left anterior descending artery flow. Location and sizes of the areas of reduced oxygenation, indocyanine green uptake, and temperature were similar. Decrease in the coronary flow to 50% and 17% of baseline resulted in progressive decrease in the above parameters, whereas increase in flow from 75% to approximately 250% achieved by dobutamine and reactive hyperemia did not significantly change them. Dobutamine increased total and epicardial flow in ischemic areas and increased subepicardial oxygenation. NIRS and thermal imaging provide epicardial maps of oxygen saturation and perfusion that reveal ischemic areas. Combination of these techniques may be useful in the coronary artery bypass graft (CABG) surgery setting.