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Membrane proteins related to anion permeability of human red blood cells

Effects of proteolytic enzymes on disulfonic stilbene sites of surface proteins

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Summary

The proteolytic enzymes, pronase, chymotrypsin and trypsin, release a small fraction of covalently bonded 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonate or (3H)DIDS, a specific inhibitor of anion permeability, from intact human red cells. The rate of release is parallel to the digestion of the sialoglycoprotein, indicating that the released (3H)DIDS was bound to that component. Most of the label is associated with a protein that behaves on SDS acrylamide gel electrophoresis as though its molecular weight was 95,000 Daltons (95K). Trypsin has no effect on this protein, but after pronase or chymotrypsin treatment of the cells, the label is found in three peaks of 95, 65 and 35K in proportions of 5, 85 and 10%. In parallel, the enzyme treatment results in a shift of most of the 95K protein to 65 and 35K. The digestion of the glycoprotein and splitting of the 95K protein can occur without any appreciable effects of the enzymes on anion permeability or on the inhibitory effects of DIDS treatment either before or after proteolytic attack. It is suggested that the sialoglycoprotein and the 35K segment of the 95K protein are not involved directly in anion permeation. The most likely location of the inhibitory site is in a portion of the 65K segment, exposed to the outside surface. Some additional observations are presented concerning the shielding effects of the negatively charged sialoglycoprotein and the arrangement of the 95K protein in the membrane.

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Abbreviations

(3H)DIDS:

4,4′-Diisothiocyano-2,2′-ditritiostilbene disulfonic acid

SITS:

4-Acetamido-4′-isothiocyano-2,2′-stilbene disulfonic acid

TBS:

Tris-buffer-saline

PBS:

Phosphate buffer saline

PAS:

Periodic Acid Schiff staining (for carbohydrates)

CB:

Coomassie Blue staining (for proteins)

TNBS:

2,4,6-Trinitrobenzene sulfonic acid

FDNB:

1-Fluoro-2,4-dinitrobenzene

EDTA:

Ethylene diamine tetra acetic acid

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Authors and Affiliations

  1. Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada

    Z. I. Cabantchik (was the recipient of an International Atomic Energy Agency Fellowship) & A. Rothstein

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  1. Z. I. Cabantchik
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  2. A. Rothstein
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Cabantchik, Z.I., Rothstein, A. Membrane proteins related to anion permeability of human red blood cells. J. Membrain Biol. 15, 227–248 (1974). https://doi.org/10.1007/BF01870089

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  • DOI: https://doi.org/10.1007/BF01870089

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