Reevaluation of use of retardation chromatography to demonstrate selective monosaccharide “binding” by erythrocyte membranes

Abstract

Retardation chromatography has been reported to show stereoselective binding of the preferred sugar-transport substrate,D-glucose, by human red cell "ghosts" or by certain protein extracts of such membranes. However, no detectable differential elution of glucose (as compared with the poorly transported analogue,L-sorbose) was observed with columns of diethylaminoethanol-cellulose impregnated with ghost proteins prepared by either of the recommended procedures (using Triton X-100, or NaI followed by extensive dialysis). Celite columns bearing intact ghosts did show marked initial relative delay in glucose emergence, but this persisted only partially through the elution peak, to be followed by a nearly equivalent and much more protracted retardation of the sorbose. Moreover, this differentiation required that the columns be handled sufficiently gently as to leave intact whole-cell membranes or fairly gross vesicles; freeze-thaw treatment or abrasive stirring abolished both the early glucose retardation and the subsequent sorbose retention. Although conflicting seriously with the simple selective-binding interpretation, these phenomena accord with the suggestion that the sugarcomplexing entities on the columns continue to operate as "carriers" mediating sugar access into membrane-enclosed compartments. Effects of experimental manipulation of elution rates and of applied sugar levels lend further support to this interpretation. The capacity of the vesicular spaces so vastly exceeds that of the binding itself that resolution of the latter cannot be achieved through this approach.