Membrane-bound cooperative enzymes. Stokes' radii, Hill plots and membrane fluidity in the regulation of adenosinetriphosphatase from Escherichia coli.
The soluble Ca2+-ATPase from Escherichia coli had a distinctive behavior with respect to inhibition by Na+ measured at 36 degrees C and 19 degrees C. At the first temperature the Hill plots are linear and show a slope of 1.1 while at 19 degrees C the plots are biphasic, with slopes of 1.8 and 0.8 before and after the break, respectively. The break occurs at about 50 nM NaCl. Gel chromatography was performed in jacketed Sepharose 4B columns kept at 2 temperatures in the presence of different concentrations of NaCl. It was found that the Stokes' radius of the enzyme was dependent on the temperature and on the salt concentration. Equilibrium sucrose gradients run at 19 degrees C showed that the sedimentation constant of the enzyme remained constant irrespective of the NaCl concentration used. It is concluded that a "folding" of the enzyme takes place in the presence of NaCl, the process being complete at about 50 mM NaCl at 19 degrees C and at about 20 mM at 36 degrees C. The results are in excellent agreement with the kinetic data: the "folded" or "compact" configuration would show no cooperative response towards Na+ while the "expanded" conformer would present strong cooperativity. This is also in agreement with the results obtained with the enzyme embedded in the membrane: when the membrane is fluid a high n value (Hill coefficient) is found; when the membrane is more rigid the value of n falls. A model explaining all our results is proposed and discussed.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology; D008560:Membrane Fluidity; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013329:Structure-Activity Relationship; D013696:Temperature
D008565:Membrane Proteins; D012965:Sodium Chloride; D000251:Adenosine Triphosphatases