Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 2. Identification of the transporter

Abstract

Maltosyl isothiocyanate (MITC), a potent irreversible inhibitor of glucose transport in human erythrocytes [Mullins, R. E., & Langdon, R. G. (1980) Biochemistry (preceding paper in this issue)], has been found to react almost exclusively with band 3 of the human erythrocyte membrane. The incorporation of [14C]MITC into band 3 was found to be antagonized by transportable sugars or competitive inhibitors of transport. On the basis of [14C]MITC incorporation into band 3 and MITC inhibition of transport, it is estimated that there are 3 x 10(5) glucose transporters present in the erythrocyte membrane. It was found that [14C]MITC-labeled band 3 could be converted into 14C-labeled band 4.5 during the Triton X-100 extraction procedure described by Kasahara & Hinkle [Kasahara, M., & Hinkel, P. C. (1977) J. Biol. Chem. 252, 7384]. On the basis of the evidence presented here and in the preceding paper, it is suggested that in the native erythrocyte membrane a component of band 3 is the glucose transport protein and that during purification with nonionic detergents the transport protein may be enzymatically degraded with some retention of activity.

The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles

“…Chemicals and radiochemicals used were from the following sources: Na35SO4 (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) (10.7 Ci/nmol) from New England Nuclear. Stractan (arabino galactan) from Sigma.…”

“…The other native transport function of band 3 [27,37,38] or band 4.5 [39][40][41], which are both present in the vesicles, is D-glucose transport. To check whether this transport function in the recombinant vesicles is intact, two approaches have been followed.…”

The glucose‐specific carrier of the Escherichia coli phosphotransferase system

“…Similarly modified carbohydrates have been used previously to study sugar‐recognizing enzymes, e.g. epoxypropyl derivatives of N ‐acetyl‐ d ‐glucosamine to label lysozyme [16], maltosyl isothiocyanate to label the human erythrocyte Glc transporter [17], and N ‐bromoacetylglucosamine to label hexokinase [18]. The glucose analogues have been characterized as pseudosubstrates and as reversible and irreversible inhibitors of EII Glc and EII Man .…”

Partial purification of the D‐glucose transport protein from human erythrocyte membranes by affinity chromatography on wheat germ lectin‐sepharose

“…This is supported by the results of labelhng with maltosylisothiocyanate, an irreversible inhibitor of the glucose-transport protein [6,7]. Possibly the 4.5component [l-3] is a degradation product of the band 3-polypeptide [5,7,8]. However, antibodies against the purified transport component have been reported to react only with polypeptides in the 4.5-region, even when membranes were prepared from fresh blood [9] and in the presence of protease inhibitors [lo].…”

“…Some evidence indicates that the polypeptides of the native transporter migrate as band 3-polypeptides in SDS electrophoresis, with& -90 000, like the anion transporter of human erythrocyte membranes. This is supported by the results of labelhng with maltosylisothiocyanate, an irreversible inhibitor of the glucose-transport protein [6,7]. Possibly the 4.5component [l-3] is a degradation product of the band 3-polypeptide [5,7,8].…”