George L. Dale

Density fractionation of erythrocytes by Percoll/hypaque results in only a slight enrichment for aged cells

Density fractionation has served for many years as a standard procedure for the isolation of aged erythrocyte populations; however, a quantitative evaluation of the technique has not been available. This report describes such an analysis. Rabbits were infused intravenously with N-hydroxysuccinimido biotin to biotinylate greater than 90% of all circulating erythrocytes; the enumeration of these biotinylated cells was possible by monitoring their ability to bind avidin-coated microspheres. The biotinylated erythrocytes were shown to have a normal in vivo survival. As a result of the normal senescence process, only aged red cells would have membrane-bound biotin 50 days after biotinylation. At this time, the rabbit red cells were fractionated over Percoll/hypaque to determine the ability of density fractionation to enrich for the aged, biotinylated cells. Density fractionation resulted in an average enrichment for aged cells of only 2-3-fold above that present in a random population.

Studies on glutathione transport utilizing inside-out vesicle prepared from human erythrocytes

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Lineweaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25 degrees C, this transport activity was easily lost during the storage of vesicles at 4 degrees C. The Km for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation fo transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37 degrees C, this transport activity was stable during storage of vesicles at 4 degrees C for several days. The Km for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the Km, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.

Phosphatidylinositol 4-phosphate kinase is associated with the membrane skeleton in human erythrocytes

Phosphatidylinositol 4-phosphate kinase was eluted from human erythrocyte stroma by three separate and distinct techniques which are known to disrupt the membrane skeleton. In addition, this kinase was found to be associated with the intact skeletons prepared by Triton X-100 extraction of stroma. Phosphatidylinositol 4-phosphate kinase which has been extracted from the membrane is a freely soluble protein with poor enzymatic activity toward added phosphatidylinositol-4-phosphate; however, the enzyme was shown to reassociate with skeleton-depleted stroma and then regain full enzymatic activity toward stromal bound substrate.

Simple and rapid purification of inside-out vesicles from human erythrocytes

The preparation of inside-out vesicles from human erythrocytes requires their separation from contaminating right-side-out vesicles. We have taken advantage of the fact that there are no glycoproteins on the internal side of the erythrocyte membrane; therefore, inside-out vesicles do not interact with the lectin, concanavalin A, while right-side-out vesicles do interact. A concanavalin A-cellulose affinity matrix has been utilized to separate easily inside-out vesicles with a purity comparable to those prepared by prolonged centrifugation.

The effect of phlebotomy as a treatment of Fabry disease

Senescent erythrocytes are considered a major source of the ceramide trihexoside which accumulates in Fabry disease patients. We have evaluated weekly phlebotomy as a method to reduce the catabolic load imposed by the senescence of erythrocytes and thereby ceramide trihexoside in one Fabry patient; no change was observed in either plasma or urinary level of ceramide trihexoside. The implications of this observation are discussed with regard to glycolipid metabolism in man.

Antithrombin III does not have bound glucocerebroside

Purified antithrombin III has been reported to have bound glucocerebroside, the major glycolipid of plasma. We have separated whole plasma by ultracentrifugation into lipoprotein-rich and lipid-deficient fractions and demonstrated that glucocerebroside and antithrombin III clearly separate into different fractions. Antithrombin III does not have glucocerebroside associated with it.