Pyridoxal phosphate. An anionic probe for protein amino groups exposed on the outer and inner surfaces of intact human red blood cells.

I Z Cabantchik,M Balshin,W Breuer,A Rothstein

doi:10.1016/s0021-9258(19)41287-8

Abstract

Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins in the membrane of the intact red blood cell. It reacts with amino groups with a high degree of specificity, forming a Schiff's base that can be fixed as an irreversible bond upon reduction with NaBH4; its binding site can be identified by use of [3-H]pyridoxal phosphate or NaB3-H4; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake. Pyridoxal phosphate uptake involves a rapid and a slow component. The former represents the binding to the outer surface of the membrane; it is not substantially affected by pH and temperature, but is reduced considerably by pretreatment of cells by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid, a specific inhibitor of anion transport. The slow component represents penetration into the cell; it is blocked by high pH, low temperature, or pretreatment with the disulfonic stilbene. Pyridoxal phosphate itself is also an effective and specific inhibitor of the permeation of other anions. Under conditions of minimal uptake, the only labeled proteins are three glycoproteins and a protein of apparent molecular weight 95,000. Under conditions of substantial uptake into the cell, the other major protein bands seen by staining on acrylamide gels after electrophoresis are labeled. It is concluded that virtually all of the major membrane proteins interact with pyridoxal phosphate from one side of the membrane or the other. The differences in labeling under conditions of minimal or maximal uptake can, therefore, be attributed to the sidedness in the distribution of the membrane proteins rather than to differences in their reactivity.

Highlights

AN ANIONIC PROBE FOR PROTEIN AMINO GROUPS EXPOSED ON THE OUTER AND INNER SURFACES OF INTACT HUMAN RED BLOOD CELLS*
It reacts with amino groups with a high degree of specificity, forming a Schiff’s base that can be fixed as an irreversible bond upon reduction with NaBH,; its binding site can be identified by use of [3H]pyridoxal phosphate or NaB3H,; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake
Studies have been made using preparations of membranes. These include: “leaky ghosts,” in which many new proteinaceous sites are exposed which did not react with the probe in the intact cell [8,9,10,11,12,13,14,15]; “resealed ghosts,” in which the probe can be incorporated in the interior compartment in a nonreactive form and activated [14,15,16,17]; and inside-out vesicles in which the inner surface of the membrane can be directly exposed to the probe [18]

Summary

PROCEDURE

4,4’-Diisothiocyano-2,2’-stilbene disulfonate was synthesized as previously described [7, 9]. The [SH]pyridoxal phosphate uptake was measured under specified conditions of temperature and pH. The reaction was carried out for 10 min at 0”, and was terminated by washing the cell suspension with 10 volumes of Hepes-buffered saline NaBH,, isolation of the membranes, and measurement of tritium or of fluorescence. The latter measurements were carried out on membranes in 20 mosm of phosphate-buffered saline, pH 7.4, containing 0.1%. Amino acid analysis of membranes isolated from pyridoxal phosphate, NaBH,-treated cells was performed as described elsewhere [33], following 3 hours of hydrolysis in ucuxo in 5.7 N HCl at 105”. The effect of pyridoxal phosphate was studied either with the probe present during the efflux measurements, or after reaction with NaBH, and washing of the cells prior to the measurements

RESULTS

C PDP IO mM

DISCUSSION