Effect of temperature on sulfate movements across chemically or enzymatically modified membranes of human red blood cells

Abstract

The effect of temperature on sulfate equilibrium exchange was measured in cells which were modified by papain, pronase, salicylate, or i-fluoro-2,4-dinitrobenzene (FDNB). Following modification at 37°C with the enzymes of FDNB the excess modifiers were removed and sulfate exchange was measured at 4°C and 34°C. The reversibly bound salicylate was also present during the exchange. With increasing concentrations of salicylate of papain, SO 4 2−-permeability shows a saturation type decrease at both temperatures. The apparent activation enthalpy ( E A) does not change significantly. After treatment with FDNB or pronase, saturation type inhibition is observed at 37°C. If measured at 4 °C, sufficiently high concentrations of both modifiers accelerate SO 4 2− transfer. At pH 7.2, E A decreases from 33 to 10 kcal per mole per °K. If measured at 37 °C, in FDNB or pronase treated cells the rate of anion movement decreases with increasing pH. If measured at 4°C, the pH dependence is reversed. E A is correspondingly lowered with increasing pH down to 6.4 kcal per mole per °K at pH 8.3. In contrast, the pH dependence of untreated cells is generally similar at both temperatures. At 37 °C a maximum is observed at pH 6.2. This is shifted to pH 5.8 at 4 °C. Variations of E A with pH are relatively small over the range pH 5.8 to 8.0. A study of the effects of purified pronase subfractions showed that neither aminopeptidase nor carboxypeptidase activity is responsible for the change of E A. Among the endopeptidases those with activity towards casein and the ester p-nitrophenyl acetate were found to be particularly potent.