Gerald E. Siefring, Jr., Alma B. Apostol, Pauline T. Velasco, and Laszlo Lorand* abstract: The accumulation of Ca2+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heteroge-neous size distribution and is rich in Y-glutamyl-e-lysine cross-links. Isolation of thisisodipeptide, in amounts as high as 6 mol/105 g of protein, confirms the idea [Lorand, L., Weissmann, L. B., Epel, D. L., and Bruner-Lorand, J.(1976), Proc. Natl. Acad. Sci. USA 73, 4479] that the Ca2+-induced

1 he accumulation of Ca2+ ions in intact human erythrocytes leads to the formation of new high-molecular-weight protein polymers in the cell membrane which are produced by covalent bonds other than disulfides (Lorand et al., 1976a, b). Since t From the Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201. Received January 23, 1978. This work was supported by United States Public Health Service Career Award 5K06 HL 03512 and by National Heart, Lung, and Blood Institute Grant HL 02212. This work was presented at the Meetingof the Federation of American Societies for Experimental Biology, April 1977, Chicago, Illinois (Siefring and Lorand, 1977). membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is in-hibited by the additionof small, water-soluble primary amines. Inasmuch as these amines are known to prevent the Ca2+-dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening. formation of the polymer is accompanied by the disappearance of band 4.1 and also by a reduction in spectrin and band 3 materials (for nomenclature, see Fairbanks et al., 1971), at least threeinternal membrane proteins seem to be involved. The polymerization reaction could be induced by Ca2+ con-centrations which are also capable of activating the intrinsic, but otherwise latent, transglutaminase of these cells, suggesting that, similarly to blood clotting (see Lorand, 1972), intermolecular 7-glutamyl-e-lysine bridging might occur. This idea was further strengthened by the finding that polymer formation could be inhibited if, prior to and during loading with Ca2+