Localization of Erythrocyte Membrane Sulfhydryl Groups Essential for Glucose Transport

Abstract

The reactions of three organic mercurial compounds, chlormerodrin, parachloromercuribenzoate (PCMB), and parachloromercuribenzenesulfonate (PCMBS) with intact red blood cells, hemolyzed red cells, hemoglobin solutions, and hemoglobin-free ghosts have been characterized. Both PCMB and PCMBS react with only 2 to 3 sulfhydryl groups per mole of hemoglobin in solution, whereas chlormerodrin reacts with 6 to 7. In hemoglobin-free ghosts, however, all three reagents react with a similar number of sulfhydryl groups, approximately 4 X 10-17 moles per cell, or about 25 per cent of the total stromal sulfhydryl groups, which react with inorganic mercuric chloride. In the intact cell the membrane imposes a diffusion barrier; chlormerodrin and PCMB penetrate slowly, whereas PCMBS does not. Kinetic studies of chlormerodrin binding to intact cells reveal that the majority of stromal sulfhydryl groups is located inside the diffusion barrier, with only 1 to 1.5 per cent (or 1 to 1,400,000 sites per cell) located outside of this barrier. Reaction of PCMBS with intact cells is limited to this small fraction on the outer membrane surface. All three reagents are capable of inhibiting glucose transport in the red cell. With chlormerodrin and PCMBS it was demonstrated that the inhibition results from interactions with the sulfhydryl groups located on the outer surface of the membrane.

Mechanisms of hypochlorite injury of target cells.

“…Lost intracellular NAD could be quantitatively recovered in the extracellular fluid, indicating that it was not degraded, but lost due to permeability changes in the plasma membrane. same changes were observed in the presence ofpCMBS, which acts primarily with plasma membrane -SHs (44,45,52). The additive effect of HOCI and pCMBS on 06Rb efflux, aminoisobutyric acid uptake and increase in cell volume suggests that the two reagents act upon functionally similar cellular targets, presumably extracellularly accessible -SH groups.…”

“…If cells were lysed with 0.01% digitonin before the addition of HOCI, 90% inactivation of GAPDH was seen with 10 gM HOCl, indicating that the cytoplasmic location protects GAPDH from inactivation in intact cells to a considerable degree, but not completely. pCMBS similarly inactivated GAPDH (59% inactivation at 10 gM) in spite of its poor membrane permeability (44). (Table III), but may have been due to inhibition of glucose uptake rather than GAPDH inactivation.…”

“…Since HOCI is a potent -SH oxidizing reagent and since reduced thiols are essential for the function of various cellular transport systems (44)(45)(46), the effect of HOCI on some of these transporter activities was assessed. Effect ofHOC!…”

Thermal damage to red cells

“…Such an effect may be explicable in terms of differing heat sensitivities ofthevarious enzymes, depending upon their direction towards the inside or outside of the membrane. Vansteveninck, Weed, and Rothstein (1965) have shown that only about 5% of the sulphydryl groups of the red cells are directed towards the outside of the membrane, Fig. 8 Bottom 9% of whole normal blood heated in vitro for 10 minutes at 480C.…”

Cytochemical Studies on the Intramembranous Particles of Erythrocyte Membranes

“…Physiological experiments have proved that SH groups in human erythrocyte membrane are involved in many membrane functions (see review (29)); permeability of cations (31), transport of sugars (35) and water (22) and activity of membrane enzymes (such as Na-K-ATPase) (38). These functions are disturbed by chemical reagents with a degree of specificity for SH groups.…”