Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. II. Formation of membrane leaks in ghost membranes after limited proteolysis of skeletal proteins by trypsin.
Limited proteolysis of human erythrocyte ghost membranes by low levels of trypsin (10-240 ng/ml) added bilaterally at 0 degrees C together with the proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF) before resealing at 37 degrees C leads to a graded digestion of spectrin and ankyrin and the disappearance of band 4.1 protein, while band 3 is cleaved only to a very low extent. These alterations are accompanied by an increase of membrane permeability of the resealed ghosts to hydrophilic nonelectrolytes (erythritol to sucrose), taken to reflect impaired resealing. Moreover, the membrane begins to vesiculate. Shedding of vesicles during the efflux measurements can not be responsible for the increased release of test solutes, since the ghosts do not loose hemoglobin and discriminate the nonelectrolytes according to their size. Moreover, the vesiculation site itself does not seem to act as the leak site, since ghosts prepared from erythrocytes pretreated with a carbodiimide which induces membrane rigidification still exhibit a pronounced protein degradation and vesiculation while the permeability enhancement induced by trypsination is markedly suppressed. The trypsin-induced leak has the properties of an aqueous pore as indicated, besides size selectivity, by its inhibition by phloretin and the very low activation energy. In analogy with concepts developed in the preceding paper (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)) the impaired resealing after limited proteolysis is assumed to be related to a perturbation of interactions of membrane skeletal elements with themselves and/or with the bilayer domain constituting the permeability barrier.
Klonk
S
S
Institut für Physiologie, Medizinische Fakultät, Rheinisch-Westfälische Technische Hochschule Aachen, Germany.
Deuticke
B
B
D002234:Carbodiimides / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*
D002234:Carbodiimides; D003598:Cytoskeletal Proteins; C030840:ethyldiaminopropylcarbodiimide; D010664:Phenylmethylsulfonyl Fluoride; D014357:Trypsin