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Stimulation of 32Pi transport into human erythrocyte ghosts and reconstituted vesicles by Mg2+ and hemoglobin
I Ducis1, A Kandrach, E Racker
1Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Abstract:
Human erythrocyte ghosts catalyze a low rate of 32Pi uptake. A severalfold stimulation of 32Pi uptake was observed after exposure of the membranes to an erythrocyte lysate or to hemoglobin in the presence of Mg2+. Ghosts prepared from erythrocytes that had been exposed to 10 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked reduction in 32Pi uptake. Reconstitution of membranes with added phospholipids by freezing and thawing, by octylglucoside dilution or by cholate dialysis, yielded vesicles that catalyzed 32Pi uptake. When membranes were incubated with hemoglobin and Mg2+ prior to reconstitution, the rate of uptake was increased severalfold. The inhibition of hemoglobin and Mg2+-dependent uptake of 32Pi by chloride suggests that the transport in the reconstituted vesicles is catalyzed by the classical inorganic anion transporter.
Insights
Human erythrocyte ghosts show low phosphate uptake, but this increases significantly with hemoglobin and Mg2+. Inhibition by chloride suggests the inorganic anion transporter is involved in this process.
Area of Science:
- Biochemistry
- Cell Biology
- Membrane Transport
Background:
- Human erythrocyte ghosts exhibit basal inorganic phosphate (32Pi) uptake.
- Erythrocyte membranes play a crucial role in cellular ion transport.
- Understanding phosphate transport mechanisms is vital for cellular function.
Purpose of the Study:
- To investigate factors stimulating 32Pi uptake in human erythrocyte ghosts.
- To characterize the inorganic anion transporter in erythrocyte membranes.
- To explore the role of hemoglobin and Mg2+ in modulating phosphate transport.
Main Methods:
- Preparation of human erythrocyte ghosts.
- Incubation with erythrocyte lysate or hemoglobin in the presence of Mg2+.
- Treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS).
- Reconstitution of membranes using freezing/thawing, octylglucoside dilution, or cholate dialysis.
- Measurement of 32Pi uptake rates.
- Inhibition studies using chloride.
Main Results:
- Erythrocyte ghosts showed a low basal rate of 32Pi uptake.
- Hemoglobin and Mg2+ significantly stimulated 32Pi uptake in ghosts.
- DIDS treatment markedly reduced 32Pi uptake, indicating transporter involvement.
- Reconstituted vesicles catalyzed 32Pi uptake, with rates enhanced by prior incubation with hemoglobin and Mg2+.
- Chloride inhibited the hemoglobin and Mg2+-dependent uptake of 32Pi.
Conclusions:
- Hemoglobin and Mg2+ enhance inorganic phosphate uptake in human erythrocyte membranes.
- The inorganic anion transporter is implicated in this stimulated phosphate transport.
- Reconstituted erythrocyte membrane vesicles serve as a functional model for studying phosphate transport.
