Phosphoenolpyruvate transport in the anion transport system of human erythrocyte membranes

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TIBS 1 2 - May 1987

Reviews Phosphoenolpyruvate transport in the anion transport system of human erythrocyte membranes Naotaka Hamasaki and Youichi Kawano Phosphoenolpyruvate, supphed exogenously, ts accumulated m human erythrocytes attd metabohzed by glycolyac enzymes Ttm muque phenomenon ts possible because the morgamc ~ u m transport system is cupable of transferring this metabolae across the erythrocyte membrane Phosphoenolpyruvme ~ the only glycolyac intermediate transported by tim system

Erythrocyte membranes have generally been regarded to be impermeable to phosphorylated glycolyhc intermediates, a condition that would be important for the conservatson of these compounds within intact red cells Similarly, phosphorylated glycolytlc intermediates supphed exogenously are also not usually metabolized by these cells We have shown, however, that exogenously administered phosphoenolpyrurate was metabolized rapidly to ATP, pyruvate and 2,3-blsphosphoglycerate within human erythrocytes 12 A major requirement for this unusual phenomenon was the capabthty of the red cell membrane to transport phosphoenolpyruvate into the red cell ~ 4 This transport ~s specific for phosphoenolpyruvate among the glycolyt~c Intermediates. the red cell membrane was impermeable to 2-phosphoglycerate, whose molecular weight and pKa value are similar to those of phosphoenolpyruvate Here we review the mechamsm underlying phosphoenolpyruvate transport and indicate possible chnical applications of this transport phenomenon Phosphoenolpyruvate transport across the erythrocyte membrane Phosphoenolpyruvate entered erythrocytes incubated in an isotomc sucrose medium containing this substrate t 2 The accumulation rate was decreased when sucrose was partly replaced by KCI or NaCI (Ref 5) The inhlbltors of anion transport, DIDS. DNDS and SITS*.

N ttama~akzmul Y kmt ano are at the Deparnneut of Biochemistry. Fukuoka Unners:t~ S~hool of Mt'thtme Fug.oka 814.01 Japan

completely inhibited phosphoenolpyrurate entry, whereas agents that inhibit the transport of the nonlonlc form of organic acids or of monocarboxylates had no stgmficant effect 34 These findrags indicated that phosphoenolpyruvate accumulation in erythrocytes was consequence of the career-mediated transport of this substance across the red cell membrane Non-mvaslve ~ i p - N M R studies demonstrated direct penetrauon of phosphoenolpyruvate into red cells and also showed that metabolism was not associated with the entry process6 Although phosphoenolpyruvate transport appeared to be active rather than passive, the process was not energyrequmng and the distribution of this compound across the cell membrane was explained by the Gibbs-Donnan equthbnum

The transport phenomenon was specific for phosphoenolpyruvate among all of the phosphorylated glycolyhc intermediates tested Monophosphoglycerales. hexose blsphogphates or nucleitides did not penetrate the erythrocyte membrane, although the m i n i - and blsphosphoglycerates competmvely mlublted phosphoenolpyruvate transport ~ Compounds denved from phosphoenolpyruvate by replacing the methene group with similarly hydrophoblc groups such as hydrogen or methyl (i e phosphoglycolate or 2-phospholactate) were permeant but one with the hydrophlhc hydroxymethyl group (i e 2-phosphoglycerate) was not ~

Kinetic properties of phosphoenolpyruvate transport The rate of phosphoenolpyruvate influx into erythrocytes followed saturation kinetics The phosphoenolpyruvate concentration at half-maximal velocity (Kin) was 8 mM in a sucrose medium and 62 mM in a citrate medium (the high K m in the latter instance was due to the inhibitory effect of citrate on pbosphoenolpyruvate transportS) The transport rate was temperature dependent and the activation energy was about 138 kJ/rail (33 kCal/mol), which was ssmdar to the activation energy for the transport of inorganic anions across the erythrocyte membrane 9 Inorganic phosphate and p~ndoxalphosphate competltlvel~ inhibited pbosphoenolpyruvate transport x~lth K, values of 24 mM and 0 2 ms1, respectively, with pyndoxal, no effect was noted until a concentration of I m~l was exceeded The transport was non-competmvely inhibited by L(+)-Ia,:tate with a K, value of 37 mM (Ref 4) Pyndoxal-phosphate was also transported across the erythrocyte membrane by the inorganic anion transport system "~ Moreover, It could be covalently linked to the reactive lyslne residue of Band 3 protein by reducing the Schtffs base with sodium borohydnde, thereby causing irreversible inhibition of phosphoenolpyruvate transport The transport rate of inorganic phosphate was also inhibited by 0 2 mM pyndoxal-phosphate, the K. value similar to that previously determined for phosphoenolpyruvate4 A reversible inhibitor for the inorganic anion transport system, DNDS, protected both transport activities against pyndoxalphosphate/sodium borohydnde treatmint 4 Accordingly. it may be concluded that phosphoenolpyruvate transport is mediated by the inorganic anion transport system in erythrocyle membranes

pH effects on phosphoenolpyruvate and

inorganic phosphate transport Although phosphoenolpyruvate and inorganic phosphate were transported across er)~hrocyte membranes by the same system, the pH profiles of the transport rates were different for the two anions (Fig I) Phosphoenolpyruvate transport steadily increased as pH ~DIDS 4 4 -dusathloc~anostdbene-22 -dtsulfomc decreased from 7 4 to 4 5 (Ref 3) aid DNDS 4 4 -dmltrosiilbene-22'-dtsulfomc acid SITS 4-acetamtdo-4'-tsothlOC~ano~tflbene- whereas inorgamc phosphate transport exhibited a bell-shaped curve with 2 2'-dlsulk,mc acid C~) 19'4"/E.I~.II.T Pobll~,llhlfls ( Ambnt[g~.

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T I B S 1 2 - M a y 1987

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mammum at pH 6 4 (Ref 11) This suggests that the mechanism underl3nng the transport of these two anions may be different even though both movements are mediated by Band 3 Amen transport appears to be greatly influenced by ionic composmon and pH of the media both reside and outside the cells 12-14, however, it is difficult to control these parameters in both intracellular and extracellular compartments when intact erythrocytes are used for the transport measurements Therefore, the effects of pH on phosphoenolpyruvate and morgame phosphate transport have been reinvestigated in resealed erythrocyte ghosts, in which the control of ]omc composmon on both sides of the membrane is possible Two conditions were employed ( 1 ) A transmembrane gradient was created the external pH (pile) was vaned from 6 0 to 7 5 whde internal pH (pH.) was maintained at 7 2 or 6 2 (2) The external and internal pH were adjusted initially to the same value over a range of 6 0 to 7 5 When the pH. was held at 7 2 and the pHe vaned the pH profile for phosphoenolpyrovate transport was different from that of inorganic phosphate 3 I t as described above for intact erythrocytes In the absence of a transmembrane pH gradient, the pH profile for phosphoenolpyruvate transport was bellshaped with a maximum at pH 6 8, essentially the same pattern was seen with morgamc phosphate transport The morgamc phosphate transport rates below pH 6.8 were higher in the presence (Fig 2, curve 1) rather than in the absence (Fig 2, curve 2) of a gradient In the case of phosphoenolpyruvate transport, essentially the same difference was observed t5 Since the expenmental condttions were essentially the same with the two amons, th~s indicated that the presence of a pH gradient favored transport

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of both morgamc phosphate and phosphoenolpyruvate In the absence of a pH gradient, it is conceivable that the transport rates below pH 6 8 decrease due to protonatton of a residue located at the inner surface of the erythrocyte membrane If the pH, ts held at 7 2, the proposed protonatton does not occur at any value of pHe This view was supported by data from another experiment in which the pH~ was maintained at 6 2, the transport rates were strongly depressed over the entire pile range from 6 0 to 7 5 (Fig 2, curve 3) The residue at the tuner surface of the membrane was protonated even when pHe was higher than 6 8, because pHt was fixed at 6 2 One of the possible candidates for the protonated residue was the lundazole group of htstidme Below pile 6 4, morgarnc phosphate transport of intact erythrocytes was retarded whereas phosphoenolpymvate transport was accelerated The depressed morgantc phosphate entry may be due to protonatton of carboxyl groups at the extracellular surface which participates closely in chloride exchange t2

Functional amino acids and phosphoenolpyruvate transport The rates of phosphoenolpyruvate and morganic phosphate transport were inhibited when erythrocytes were tre tied with phenylglyoxal, suggestmg that an argmme residue(s) parUctpated m the transport of phosphoenolpyruvate as well as of inorganic anions tz, Inhibition of phosphoenolpyruvate transport by the stdbene compounds indicated that a lysme residue(s) may also be involved in this process as in the transport of inorganic anions The pH profiles inorganic phosphate and phosphoenolpyruvate transport in resealed ghosts (Fig 2) implied that htstidme residues were revolved in the transport of these anions

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across the cell membrane Thus, argmlne, lysme and hlsttdine residues all appear to parttapate in phosphoenolpyruvate entry as well as m lnorgamc anion transport However, extracellular carboxyl groups, whose involvement has been suggested in the transport of chloride t2 and morganlc phosphate, did not appear to be concerned with phosphoenolpyruvate transport is.

Pyridoxal.phosphate binding site on Band 3 protein The evidence revmwed above mdtcites that the mechamsm for phosphoenolpyruvate transport across the erythrocyte membrane ts essentially the same as that for inorgamc phosphate transport The Band 3 protein is involved m the transport of inorganic anions such as chloride, phosphate and sulfate¢ t2 t4 t6 as well as of phosphoenolpyruvate The location of the active center within Band 3 has been investigated In this connection, pyndoxalphosphate was a good probe because it itself was transported across the cell ~embrane by the anion transport system tt~ and could be covalently linked to the reactive lysme residue of the protein molecule Two membrane-spanning domains of Band 3 (M r 17000 and 38 000) were labeled by pyndoxal-phosphate and radioactive sodium borohydride Erythrocytes labeled in both domains transported anions such as pyndoxalphosphate, morgamc phosphate and phosphoenolpyruvate at a decreased

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T I B S 1 2 - M a y 1987

rate When erythrocytes were treated with these agents in the presence of the anion transport inhlbltors, DIDS or DNDS, only the 17 kDa domain was labeled If the anion transport inhibitor was subsequently removed, cells labeled m only the 17 kDa domain were obtained which had essentially the same transport activity as control cells Thus, the critical amon transport s~tes were temporanly protected by DNDS in th~s expenment The affinity labehng of erythrocytes with pyndoxal-phosphate allowed us therefore to recognize the 38 kDa domain as contmmng at least a part of the actwe center for the anion transport system ~7 A hydrophob]c peptlde (M~ 5300) vdthln the 38 kDa domain was the affinity labeling site of Band 3 Since the functional amino acids of Band 3 recognize pyrldoxal-phosphate as an anion through ItS phosphate group, the covalent bmdmg s~te m the pepttde (-CHO) may not be the functional site of the transport system However, the chemically reactive group of pyndoxalphosphate ~s only 5 2 A from the phosphate group and so stenc hindrance may account for the transport inhibition Thus, the 5300-Da pept~de vathln the 38 kDa domain should be closely located to the active center of the transport system m sttu This peptlde was punfied from CNBr fragments of Band 3 w~th a combination of gel permeation and reversephase, high-performance hquld chromatography ls and its primary structure ts presently under investigation

Clinical applications of phosphoenolpyruvate transport Phosphoenolpyruvate incorporated into erythrocytes was metabolized to monophosphoglycerates, 3-phosphoglyceroyl phosphate, 2,3-blsphosphoglycerate and ATP by glycolyttc enzymes (Fig 3) The concentratlen of 3-phosphoglyceroyl phosphate in cells treated with phosphoenolpyruvate was more than I00 ames its physiological leveP9 As a consequence, the physiological concentratlon of 2,3-blsphosphoglycerate increased more than threefold 2° This compound ~s physiologically important m oxygen transport through its action as a potent allostenc effector of oxygen binding to hemoglobin 2122 Dunng blood storage voth hquld preservatives, the 2,3-bisphosphoglycerate concentrai o n decreases, severely comprising the oxygen delivery function of the preserved erythrocytes A solution containing phosphoenolpyruvate has been developed as a new preservative for blood storage to maintain 2,3-blsphosphoglycerate and ATP concentrations at

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P-enolpyruvate Fig 3 Metabohsm of phosphoenolpyruvafe incorporated into eryrhrocvtes P-enolpyruvme, phosphoenolpyruvate, 2-PG, 2-phosphoglvcerate 3-PG, 3-phosphoglvcerate, 3-phosphoglycerovI-P. 3-phosphoglycerovl phosphate, GA3P, gtyceraldeh~de 3-pho'.phate, 2,3-DPG. 2,3 blsphosphogl~cerate, PGK, phosphoglycerme kmase, PK, pyruvate kmase, GA3PDH, glyceraldehyde-3-phosphate deh~drogenase, LDH. lactate dehvdrogenme

a high level in preserved red blood cells2 z0 23 The rate of phosphoenolpyruvat~ transport m erythrocytes from patients with hereditary spherocytosls was found to be about half of the normal rate, and the metabolic fate of the mcorporated phosphoenolpyruvate was d~fferent from that of normal red cells24 The lower rate of phosphoenolpyruvate transport may reflect some abnormality of the red cell membrane in hereditary spherocytosls, suggesting that this condition could be of potential diagnostic value A more general apphcation of phosphoenolpyruvate transport may involve the m vtvo increase of ATP concentratlon in erythrocytes of patients with enzymopathles of the EmbdenMeyerhof pathway Acknowledgments We wish to thank Professor A Omachl (Department of Physiology and Biophysics, Umverslty of Illinois at the Medical Center) for reading the manuscript Tlus work was supported by grants from the Mimstry of Education, Science and Culture of Japan and from the Central Research Institute of Fukuoka Umverslty

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Chtgla C (1983)Eur J Btochem 132 531536 5 Hamasakl N Tomoda, A Harasakl, H and Mmakamt S (1977)J BJochem 81 15051509 6 Hamasakl N, Wywacz A M, Lubanks) H J and Omaehl A (1981) Biochem Biophvs Res Commun 1O0 879-887 7 Hamasakl N (1984)Bzochem Btoph~s Res Commun 122 609-612 8 Hamasakl N (1984)Selkagaku56 388-401(m Japanese) Q Cabantehlk Z I Knauf I' F and Rothstem A (1978) BIoclnm BIophvs 4eta 515 239-302 10 Nann H , Hdmasakl N and Mmtkaml S (1983)J Btol Chem 258 5985-5989 II Deuttcke B (1970) Namr.lssenschafien 57 172-179 12 Wteth, J O , Andersen O S Brahm J Bierrum, P J .Borders C L Jr (1982)Phd Tran~ R Soc London Ser B 299 383-399 13 Knauf P A Tarshls T Gnnstem S Furuya W (1980) m Membrane Transport m Ervlhrocytes (Lassen, U V Ussmg H H Wleth J O eds) pp 389.-.408 Munksgaard 14 Jenmngs M L and Adams, M F (1981) Biochemtstr) 20 7118-7123 15 Matsuyama, H Kawano Y and Hamasakl N (1986)J Btochem 99 495-501 16 Jennmgs M L (1985)Annu Re1 Phvsrol 47 519-533 17 Matsuyama, H Ka~ano Y and Hamasakt N (1983)3 Btol Chem 258 15376-15381 18 Kawano, Y Hamasakl N (19~)J Biochem 100, 191-199 19 Inoue, H , Monyasu M and Hama~akl N 3 Biol Chem (m press) ?~ Hamasakl, N and Hirota-Chlglla C (1983) Transfusion 23 I-7 21 Chaantm A and Curmsh R R (1967)Arch B~ochem B~ophvs 121 96-102 22 Benesch R and Benesch R E (1969),Vat.re 221,618-622 23 Yonenaga K Todorokt H Tokunaga K and Hamasakl N (1986)Transftmon26 194198 24 Ideguchl H Hama~akl N aud Ikehara Y (1981) Blood58 426-.430