Characterisation of Phospho<i>enol</i>pyruvate Transport across the Erythrocyte Membrane

Matsuyama, Hiroyuki; Hirota-Chigita, C

doi:10.1111/j.1432-1033.1983.tb07394.x

Cited by 26 publications

(5 citation statements)

References 27 publications

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“…PEP transport in erythrocytes is also competitively inhibited by Pi and pyridoxal-P. Incubation with pyridoxal-P and [3H]NaBH4 specifically and irreversibly labels band 3 in the erythrocyte membrane and inhibits PEP transport. Preincubation with DIDS prevents this inhibition (16).…”

Section: Resultsmentioning

confidence: 99%

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O₂ Evolution in Isolated Chloroplasts

Rumpho

Edwards²

1985

Plant Physiol.

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ABSTRACr 3-Phosphoglycerate (PGA)-dependent 02 evolution by mesophyll chloroplasts of the C4 plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of 02 evolution. The extent of inhibition of 02 evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophylL concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA.Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent 02 evolution. Protection inst this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, ifthe chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorgnic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent 02 evolution by isolated chloroplasts of the C3 plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C4 chloroplasts.Oxalacetate-dependent 02 evolution and its stimulation by the uncoupler, NHCICL were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C4 crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA.Phosphenolpyruvate is a key regulatory metabolite of the C4 pathway of photosynthesis. It is formed in the mesophyll chlo-

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Section: Resultsmentioning

confidence: 99%

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O₂ Evolution in Isolated Chloroplasts

Rumpho

Edwards²

1985

Plant Physiol.

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“…As discussed above, variations in binding affinity among different substrates are smaller than the variations in translocation rates. Anions as large as phosphoenolpyruvate ( 240 ), pyridoxal phosphate ( 241 ), and N -(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-3-diazole (NBD)-taurine ( 242 ) are band 3 substrates. Modeling studies will be necessary to gain insight into how anions of various sizes and geometries fit into the binding pocket.…”

Section: Band 3 Structurementioning

confidence: 99%

Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1

Jennings

2021

American Journal of Physiology-Cell Physiology

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The major transmembrane protein of the red blood cell, known as band 3, AE1, and SLC4A1, has two main functions: 1) catalysis of Cl-/HCO3- exchange, one of the steps in CO2 excretion; 2) anchoring the membrane skeleton. This review summarizes the 150 year history of research on red cell anion transport and band 3 as an experimental system for studying membrane protein structure and ion transport mechanisms. Important early findings were that red cell Cl- transport is a tightly coupled 1:1 exchange and band 3 is labeled by stilbenesulfonate derivatives that inhibit anion transport. Biochemical studies showed that the protein is dimeric or tetrameric (paired dimers) and that there is one stilbenedisulfonate binding site per subunit of the dimer. Transport kinetics and inhibitor characteristics supported the idea that the transporter acts by an alternating access mechanism with intrinsic asymmetry. The sequence of band 3 cDNA provided a framework for detailed study of protein topology and amino acid residues important for transport. The identification of genetic variants produced insights into the roles of band 3 in red cell abnormalities and distal renal tubular acidosis. The publication of the membrane domain crystal structure made it possible to propose concrete molecular models of transport. Future research directions include improving our understanding of the transport mechanism at the molecular level and of the integrative relationships among band 3, hemoglobin, carbonic anhydrase, and gradients (both transmembrane and subcellular) of HCO3-, Cl-, O2, CO2, pH, and NO metabolites during pulmonary and systemic capillary gas exchange.

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“…How-80%. In addition, pyridoxal-P can permeate membranes (13) and n comparison could inhibit 3-PGA dependent 02 evolution through its effect rocedures. To on the stromal reducing enzymes.…”

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confidence: 99%

Specific Labeling of the Phosphate Translocator in C₃ and C₄ Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2′ -Disulfo-4,4′ -Diisothiocyano] Diphenylethane)

Rumpho¹,

Edwards²,

Yousif³

et al. 1988

Plant Physiol.

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The phosphate translocator protein of C3 and C4 mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-dititio1,2-(2,2'sdisuufo4,4'-diisothiocyano) diphenylethane (pH]2-DIDS). The regulation of metabolite traffic across the chloroplast envelope through carrier mediated transport is a common feature of both the C4 and C3 pathways of photosynthesis and there is some evidence for differences in transport between chloroplast types (6,14). Our limited understanding of chloroplast metabolite transport has been summarized recently by Heldt and Flugge (16) and Flugge and Heldt (11).The phosphate translocator of spinach (C3) chloroplasts, which catalyzes the one-to-one exchange of triose-P,2 Pi, and 3-PGA, (10) have also obtained 70 to 80% pure 29-kD protein, incorporated this fraction into liposomes, and measured transport activity. However, further purification resulted in a total loss of activity. While it is generally accepted that the 29-kD polypeptide is the phosphate translocator in spinach chloroplasts, the protein has not been purified to homogeneity and reconstitution has not been achieved with the purified, active protein.With respect to organic phosphate transport in C4 mesophyll chloroplasts, studies using the silicone oil technique have shown that triose-P, 3-PGA, and Pi are all exchanged (5) and, in contrast to C3 chloroplasts, PEP is also rapidly transported across the C4 mesophyll chloroplast envelope (5,18,33). The majority of evidence suggests that PEP, 3-PGA, triose-P, and Pi all compete for the same substrate binding site on a common phosphate translocator in C4 mesophyll chloroplasts (5,28,29). It is probable that recognition of PEP by C4 chloroplasts evolved from a modification of the substrate binding site of the C3 phosphate translocator. However, since it has been demonstrated with isolated C4 mesophyll chloroplasts that PEP can also exchange directly with Pi (18) and ADP or ATP (33), a more direct method to identify the transport protein is needed.The definitive identification and isolation of transport proteins in chloroplasts of either C3 or C4 plants has been hampered, at least in part, by the lack of a suitable marker, or inhibitor of the protein (11). We recently reported, however, on a very potent inhibitor, DIDS, which will specifically, irreversibly, and totally inhibit 3-PGA dependent oxygen evolution at micromolar concentrations in both C3 and C4 mesophyll chloroplasts (29). Of particular importance was the observation that preincubation with PEP protected against DIDS inhibition in the C4 mesophyll chloroplasts, but not in the C3 chloroplasts. In the experiments reported in this paper we isolated C3 and C4 mesophyll chloroplasts, incubated them with [3H]2-DIDS, and compared the labeling pattern of solubilized envelopes with that of pea chloroplasts incubated with pyridoxal-P/NaB3H4.

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Characterisation of Phosphoenolpyruvate Transport across the Erythrocyte Membrane

Cited by 26 publications

References 27 publications

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O₂ Evolution in Isolated Chloroplasts

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O₂ Evolution in Isolated Chloroplasts

Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1

Specific Labeling of the Phosphate Translocator in C₃ and C₄ Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2′ -Disulfo-4,4′ -Diisothiocyano] Diphenylethane)

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Characterisation of Phosphoenolpyruvate Transport across the Erythrocyte Membrane

Cited by 26 publications

References 27 publications

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O2 Evolution in Isolated Chloroplasts

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O2 Evolution in Isolated Chloroplasts

Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1

Specific Labeling of the Phosphate Translocator in C3 and C4 Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2′ -Disulfo-4,4′ -Diisothiocyano] Diphenylethane)

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Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O₂ Evolution in Isolated Chloroplasts

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O₂ Evolution in Isolated Chloroplasts

Specific Labeling of the Phosphate Translocator in C₃ and C₄ Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2′ -Disulfo-4,4′ -Diisothiocyano] Diphenylethane)