The volumes of human erythrocytes suspended in solutions of varying concentrations of sodium chloride and sucrose were measured by a Coulter Channelyzer Model H4 with appropriate corrections. The cells showed greatly restricted volume changes at osmolarities between 200-700 mOsm. At osmolarities outside this limit, on the other hand, the cells showed nonrestricted volume changes following essentially the predictions of an ideal osmometer. This unexpected volume response was not spuriously due to changes in shape or to a changing orientation of the cells as they traversed the aperture. The restricted volume change observed was abolished when the cells had previously been treated with diamide or had been heated for 60 minutes at 50 degrees C, conditions that are known to disturb the spectrin-actin network. The possibility must be considered that the osmotic behavior of human erythrocytes may be nonideal and that this nonideal behavior is primarily due to mechanical restriction provided by the spectrin-actin network of the membrane cytoskeleton.
In normal whole human blood in vitro, the source of the enzyme controlling the hydrolysis of aspirin (ASA) was found to be the erythrocyte (RBC). Experiments were carried out to determine whether this enzyme was membrane-bound or free in the lysate. The mean rates of ASA hydrolysis in comparable concentrations of intact RBC (1.61 f 0.20 pmole/liter/minute, n = 12 and 1.23 f 0.17 pmole/liter/minute, n = 5) were much faster than that measured in isolated RBC membranes (0.23 f 0.08 pmole/liter/minute, n = 6, P = <0.001). Detailed study showed that the RBC-related ASA esterase is located intracellularly and is not related to membrane acetylcholinesterase. The ASA esterase from crude lysate was purified 900-fold by means of DEAE Sephacel chromatography of active enzyme recovered from a 50% saturated ammonium sulfate fraction. Non-SDS polyacrylamide gel electrophoresis (pH 8.3 and 9.0) resulted in one major band and one or more small minor protein bands. When esterase activity was assayed in a non-stained gel, ASA depeletion and salicylate production corresponded exactly to the major stained band. This band was eluted from another unstained gel, concentrated, and applied to an SDS gel. This yielded a single band with a molecular weight of
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