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Functional heterogeneity and pH-dependent dissociation properties of human transferrin
766
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 7 6 6 - - 7 7 1 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s
BBA 27882
F U N C T I O N A L H E T E R O G E N E I T Y AND pH-DEPENDENT DISSOCIATION PROPERTIES OF HUMAN T R A N S F E R R I N
J O S E P H V. P R I N C I O T T O a n d E D W A R D J. Z A P O L S K I
Department of Physiology and Biophysics, Georgetown University, Schools of Medicine and Dentistry, 3900 Reservoir Rd., Washington, D.C. 20007 (U.S.A.) (Received October 20th, 1975)
Summary Human diferric transferrin was partially labeled with SgFe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59Fe uptake experiments with chemically labeled preparations indicated that iron bound at near neutral pH was more readily incorporated by reticulocytes than iron b o u n d at low pH. The pH
Introduction Human transferrin binds two Fe 3÷ at apparently equivalent but slightly differing iron-binding sites [1,2], which surrender these Fe3+to rabbit r~ticulocytes in a non-equivalent manner. One binding site is a better iron donor than the other [3,4]. In a recent study of the pH-dependent dissociation characteristics of human diferric transferrin we observed a distinct difference in the binding properties of each site, one site binds ferric iron at a lower pH than the Abbreviation: HEPES, N-2-hydroxyethylpiperazine-N L2-ethanesulfonic acid.
767 other site [ 5]. The present experiments were undertaken to determine whether rabbit reticulocyte utilization of human transferrin-bound iron is related to the different acid-base properties of each site. Materials and Methods
Preparation of partially labeled trans[errin solutions (chemically labeled). Human apotransferrin, treated as described previously [5] was utilized to prepare two chemically labeled diferric preparations by a slight modification of procedures used in an earlier study [5]. Apotransferrin was partially labeled by reaction with S9Fe-labeled ferric dinitrilotriacetate at pH 5.5 (1.0 Fe/mol transferrin), resin treated (BioRad AG 1-X4, C1-form), adjusted to pH 7.4 with dilute NaHCO3, saturated by adding a slight excess (5% of total iron-binding capacity) of unlabeled ferric dinitrilotriacetate and resin treated. In this preparation (Solution A), 31% of the total diferric iron was S9Fe labeled, a n d w a s b o u n d at the acidic iron-binding site. Solution B was similarly prepared with unlabeled ferric dinitrilotriacetate (1.5 Fe/mol transferrin) b u t was adjusted from pH 5.5 to 6.0 with bicarbonate before resin treatment and final saturation with [S7Fe]ferric dinitrotriacetate at pH 7.4. This adjustment provided for complete occupancy of the acidic binding site by unlabeled iron so that 42% of the total diferric iron was SgFe labeled, occupying the more neutral binding site. A control, uniformly S9Fe-labeled diferric transferrin was also prepared (Solution C). Preparation of Biologically labeled transferrin solutions. Labeled or unlabeled diferric transferrin was incubated with rabbit reticulocytes and iron which was donated to the cells was then replaced by the alternate isotope, using the same procedures described previously [4] except that ferric dinitrilotriacetate rather than ferrous ammonium sulfate/ascorbic acid was employed as the iron source. In Solution D, S7Fe Was removed by reticulocytes (50%) and this iron was replaced by unlabeled iron. Solution E was labeled by the reverse sequence. Experiments. Rabbit reticulocyte collection, handling and incubation procedures employed in the SgFe uptake experiments have been described previously [4]. (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)buffered, rather than phosphate-buffered, saline was employed in all experiments). Two volumes of a 50% cell suspension were incubated with one volume of each transferrin solution (0.005 mM/1) and the percent STFe present in washed cells were determined. The techniques used for the pH-dependent dissociation studies were as described previously [5]. Buffer solutions (0.10 and 0.01 M) were prepared from stock Tris/maleic acid solution (0.20 M each) by dilution and adjustment of pH with 0.20 or 0.02 M NaOH. 1 ml of each transferrin solution (A--E, 0.002 mM/1) was mixed with 0.5 ml of 0.01 M buffer. An aqueous slurry of washed AG l-X4 resin was distributed into small disposable Pasteur pipets (loosely plugged with fiberglass), rinsed with 0.5 ml of 0.10 M buffer, three 0.5-ml washes of 0.01 M buffer and the final eluate pH was checked. The equilibrated columns were transferred to clean tubes and 1.0 ml of the transferrin/buffer mixture was applied. (Approx. 45 min was required to set up
768
duplicate determinations for the transferrin solutions tested). Each column was then washed with three 0.5-ml aliquots of 0.01 M buffer. Both eluant and suspended resin were counted and the percent SgFe recovered in eluate was calculated. Protein recovery was ascertained in separate experiments by taking the eluates (Solution C), adjusting the pH to 7.4 with a HEPES/NaHCO3, adding S9Fe-labeled ferric dinitrilotriacetate and 30 min later removing u n b o u n d iron with resin. The activity in these solutions was within +2% of appropriately diluted control Solution C, indicating that no protein was lost to the resin column and the iron binding of the transferrin was unaltered. Results
Chemically labeled transferrin solutions Percent S7Fe recovered in washed cells (15% reticulocytes) after incubation for 1 h with transferrin Solutions A, B and C are shown in Fig. 1. In Solution B, the acidic transferrin iron-binding site was occupied by unlabeled iron and +gFe was bound at the more neutral binding site. This S9Fe3+ is incorporated
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.
.
~
.
.
+
k
~
C
%59Fe
40
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I
20
i
40
i
60
TIME,MINUTES
Fig. 1. Rabbit r e t i c u l o c y t e percent S9Fe u p t a k e from chemically labeled h u m a n diferric transferrin. A, 59Fe bound at pH 5.5, saturated with unlabeled Fe at pH 7.4; B, labeled by reverse sequence; C, uniformly labeled SgFe at pH 7.4.
769 more rapidly by the reticulocytes (75% in washed cells by 20 min, Solution B) than is the SgFe 3+b o u n d at low pH (Solution A, 45% by 20 min). The pH-dependent dissociation curves obtained from these solutions are not illustrated. They were similar to previous findings [5]. At pH 5.6--5.7, control SgFe-labeled diferric transferric (C) was half dissociated whereas 90% of the isotope in Solution A (SgFe labeled at pH 5.5) was still b o u n d to the transferrin. Only 42% of the SgFe in Solution B remained b o u n d to transferrin.
Biologically labeled transferrin solutions Reticulocytes percent uptake of SgFe transferrin-bound iron from Solutions D and E as well as control (C) was similar to our previous reported findings [4]. Cells incorporated SgFe more rapidly from Solution E (where unlabeled iron that was initially removed by reticulocytes was replaced with SgFe) than from Solution D (labeled by the reverse procedure). After 10 min incubation, cells t o o k up 72% SgFe from uniformly SgFe-labeled diferric transferrin (C), 58% SgFe from Solution D and 82% from Solution E. The percentages of SgFe recovered in resin elutate from samples C, D and E, as a function of pH (5.2--6.8), are illustrated in Fig. 2. SgFe in Solution D (labeled reticulocyte supernatant which was resaturated with unlabeled iron) did not dissociate until the pH was below 5.6--5.7, while nearly 60% of the SgFe in Solution E (SgFe-labeled supernatant whose donated iron was replaced
I00 -
80
c 59
/
Fe
ELUATE
60
40 |
5.2
6.0
|
|
6.8
pH Fig. 2. Dissociation of 5 9 F e from biologically labeled h u m a n diferric transferrin as a function of pH. Percent 5 9 F e in resin eluate. C, uniformly labeled 59Fe; D, S 9 F e paxtially removed by reticulocytes w a s replaced with unlabeled Fe; E, unlabeled Fe, partially r e m o v e d by reticulocytes was replaced with 59Fe.
770 with unlabeled Fe 3+) dissociated above pH 5.6--5.7. At this pH control, uniformly SgFe-labeled diferric transferrin (C) was half dissociated. Thus, the dissociation findings with Solution E were similar to those observed for Solution B and each of these preparations yielded SgFe iron to reticulocytes more readily than did preparations A or D. Discussion In our initial study of the pH-dependent iron dissociation properties we suggested that one iron-binding site of human transferrin retained its binding ability at low pH and that this difference in iron-binding ability might account for observed selective rabbit reticulocyte utilization of Fe 3÷ from one preferred site. The results obtained in the present study support this suggestion. Comparison of the rabbit reticulocyte SgFe uptake and pH-dependent dissociation findings indicate that the human transferrin-binding site which binds iron at lo~v pH is n o t the site which initially surrenders Fe3*to rabbit reticulocytes. The other, more neutral binding site is involved in this reaction. Diferric transferrin, where STFe was bound at low pH, did not dissociate 57Fe until the pH was lowered to 5.6--5.7 and was the least efficient reticulocyte SgFe iron donator (Fig. 1, Solution A). Moreover, after reticulocytes removed half the iron from SgFe-labeled diferric transferrin and this donated iron was replaced by unlabeled iron, similar reticulocyte uptake and dissociation data were observed {Fig. 2, Solution D). The data obtained from preparations B and E, which were selectively labeled in the reverse sequence by these chemical and biological procedures also concur. These findings identify that the transferrin iron-binding site which begins to dissociate as the pH is lowered from 7.4 to near 5.6 is the transferrin iron-binding site from which reticulocytes more readily incorporate Fe 3+. The maleate utilized in the buffer solutions is a potential iron chelator and may possibly effect dissociation by competitive chelation. At low pH, the concentration of bicarbonate, necessary for the formation of the Fe3+-carbonate transferrin complex, is reduced and may also contribute to increase dissociation of the transferrin complex. However, both these effects would be expected to randomly influence dissociation from either site, rather than be directed toward one unique site. Conformational changes in the protein, induced by changes in the pH, might occur, possibly leading to a masking of one site from the aqueous environment. Such an effect could also account for the two different dissociation patterns observed for the partially labeled preparations (A and B) and the acid-base properties of the two sites may be nearly identical. In order for this effect to occur and account for our observations, at neutral pH, one binding site should be exposed and the other masked, facilitating removal of Fe 3+ from this site as the pH is lowered. Then, a conformational change could reverse the masking, " b u r y i n g " this site and exposing the second site. Thus at low pH, this second site would reversibly bind Fe 3÷ and as the pH is increased it becomes masked and the other site is opened. However, such an effect would n o t be in keeping with observations that Fe 3+are randomly bound by each site at neutral pH [4]. Hence, both sites must be unmasked at neutral pH. In either case, whether the effect is due to protein conformational changes
771
or if there are different acid-base properties of each site, one site does preferentially donate its iron to the rabbit reticulocyte and can be identified by its ability to dissociate its Fe 3+at a higher pH than does the other site. The precise mechanism by which human transferrin-bound iron is ultimately transferred into the rabbit reticulocyte cell for heme synthesis is n o t yet fully known. An anion-detaching e n z y m e which cleaves carbonate (or bicarbonate) from the ternary transferrin complex and permits facile release of the Fe 3+has been suggested to account for the removal of transferrin-bound iron [6--8]. The more neutral iron-binding site may be kinetically more favorable as this enzyme's substrate than the low pH binding site because of the acid base ironbinding properties of each site or possible protein conformational changes and thus account for the functional heterogeneity of the binding sites. It is also possible that two observations are unrelated. Functional heterogeneity, which may be unique only to this particular system is observed at physiological pH. Dissociation p h e n o m e n o n at non-physiologic pH may or may not have any bearing on events that occur at the cell surface.
Acknowledgements Supported in part by a grant from the National Institutes of Health, Research Grant RO1 AM 15553 from the National Institute of Arthritis, Metabolism and Digestive Diseases and a grant from the Washington Heart Association. References 1 2 3 4 5 6 7 S
Aasa, R., Malmstrom, B.G.. Saltman, P. and Vangaard, T. (1963) Biochim. Biophys. Acta 75, 203--222 Aasa, R. (1972) Biochem. Biophys. Res. C o m m u n . 49,206--212 Fletcher,J. and Huehns, E.R. (1967) Nature 215, 584--586 Zapolski, E.J., Ganz, R. and Princiotto,J.V. (1974) A m . J. Physiol. 226,334--339 Princiotto,J.V. and Zapolski, E.J. (1975) Nature 255, 87--88 Aisen, P. and Leibman, A. (1973) Biochim. Biophys. Acta 304, 797--804 Egyed, A. (1973) Biochim. Biophys. Acta 304, 805--813 Martinez-Medellin, J. and Schulman, J. (1973) Biochem. Biophys. Res. C o m m u n . 53.32--38
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 7 6 6 - - 7 7 1 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s
BBA 27882
F U N C T I O N A L H E T E R O G E N E I T Y AND pH-DEPENDENT DISSOCIATION PROPERTIES OF HUMAN T R A N S F E R R I N
J O S E P H V. P R I N C I O T T O a n d E D W A R D J. Z A P O L S K I
Department of Physiology and Biophysics, Georgetown University, Schools of Medicine and Dentistry, 3900 Reservoir Rd., Washington, D.C. 20007 (U.S.A.) (Received October 20th, 1975)
Summary Human diferric transferrin was partially labeled with SgFe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59Fe uptake experiments with chemically labeled preparations indicated that iron bound at near neutral pH was more readily incorporated by reticulocytes than iron b o u n d at low pH. The pH
Introduction Human transferrin binds two Fe 3÷ at apparently equivalent but slightly differing iron-binding sites [1,2], which surrender these Fe3+to rabbit r~ticulocytes in a non-equivalent manner. One binding site is a better iron donor than the other [3,4]. In a recent study of the pH-dependent dissociation characteristics of human diferric transferrin we observed a distinct difference in the binding properties of each site, one site binds ferric iron at a lower pH than the Abbreviation: HEPES, N-2-hydroxyethylpiperazine-N L2-ethanesulfonic acid.
767 other site [ 5]. The present experiments were undertaken to determine whether rabbit reticulocyte utilization of human transferrin-bound iron is related to the different acid-base properties of each site. Materials and Methods
Preparation of partially labeled trans[errin solutions (chemically labeled). Human apotransferrin, treated as described previously [5] was utilized to prepare two chemically labeled diferric preparations by a slight modification of procedures used in an earlier study [5]. Apotransferrin was partially labeled by reaction with S9Fe-labeled ferric dinitrilotriacetate at pH 5.5 (1.0 Fe/mol transferrin), resin treated (BioRad AG 1-X4, C1-form), adjusted to pH 7.4 with dilute NaHCO3, saturated by adding a slight excess (5% of total iron-binding capacity) of unlabeled ferric dinitrilotriacetate and resin treated. In this preparation (Solution A), 31% of the total diferric iron was S9Fe labeled, a n d w a s b o u n d at the acidic iron-binding site. Solution B was similarly prepared with unlabeled ferric dinitrilotriacetate (1.5 Fe/mol transferrin) b u t was adjusted from pH 5.5 to 6.0 with bicarbonate before resin treatment and final saturation with [S7Fe]ferric dinitrotriacetate at pH 7.4. This adjustment provided for complete occupancy of the acidic binding site by unlabeled iron so that 42% of the total diferric iron was SgFe labeled, occupying the more neutral binding site. A control, uniformly S9Fe-labeled diferric transferrin was also prepared (Solution C). Preparation of Biologically labeled transferrin solutions. Labeled or unlabeled diferric transferrin was incubated with rabbit reticulocytes and iron which was donated to the cells was then replaced by the alternate isotope, using the same procedures described previously [4] except that ferric dinitrilotriacetate rather than ferrous ammonium sulfate/ascorbic acid was employed as the iron source. In Solution D, S7Fe Was removed by reticulocytes (50%) and this iron was replaced by unlabeled iron. Solution E was labeled by the reverse sequence. Experiments. Rabbit reticulocyte collection, handling and incubation procedures employed in the SgFe uptake experiments have been described previously [4]. (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)buffered, rather than phosphate-buffered, saline was employed in all experiments). Two volumes of a 50% cell suspension were incubated with one volume of each transferrin solution (0.005 mM/1) and the percent STFe present in washed cells were determined. The techniques used for the pH-dependent dissociation studies were as described previously [5]. Buffer solutions (0.10 and 0.01 M) were prepared from stock Tris/maleic acid solution (0.20 M each) by dilution and adjustment of pH with 0.20 or 0.02 M NaOH. 1 ml of each transferrin solution (A--E, 0.002 mM/1) was mixed with 0.5 ml of 0.01 M buffer. An aqueous slurry of washed AG l-X4 resin was distributed into small disposable Pasteur pipets (loosely plugged with fiberglass), rinsed with 0.5 ml of 0.10 M buffer, three 0.5-ml washes of 0.01 M buffer and the final eluate pH was checked. The equilibrated columns were transferred to clean tubes and 1.0 ml of the transferrin/buffer mixture was applied. (Approx. 45 min was required to set up
768
duplicate determinations for the transferrin solutions tested). Each column was then washed with three 0.5-ml aliquots of 0.01 M buffer. Both eluant and suspended resin were counted and the percent SgFe recovered in eluate was calculated. Protein recovery was ascertained in separate experiments by taking the eluates (Solution C), adjusting the pH to 7.4 with a HEPES/NaHCO3, adding S9Fe-labeled ferric dinitrilotriacetate and 30 min later removing u n b o u n d iron with resin. The activity in these solutions was within +2% of appropriately diluted control Solution C, indicating that no protein was lost to the resin column and the iron binding of the transferrin was unaltered. Results
Chemically labeled transferrin solutions Percent S7Fe recovered in washed cells (15% reticulocytes) after incubation for 1 h with transferrin Solutions A, B and C are shown in Fig. 1. In Solution B, the acidic transferrin iron-binding site was occupied by unlabeled iron and +gFe was bound at the more neutral binding site. This S9Fe3+ is incorporated
100 - -
$0
60
-
+
,
//17
.
.
~
.
.
+
k
~
C
%59Fe
40
2O
I
20
i
40
i
60
TIME,MINUTES
Fig. 1. Rabbit r e t i c u l o c y t e percent S9Fe u p t a k e from chemically labeled h u m a n diferric transferrin. A, 59Fe bound at pH 5.5, saturated with unlabeled Fe at pH 7.4; B, labeled by reverse sequence; C, uniformly labeled SgFe at pH 7.4.
769 more rapidly by the reticulocytes (75% in washed cells by 20 min, Solution B) than is the SgFe 3+b o u n d at low pH (Solution A, 45% by 20 min). The pH-dependent dissociation curves obtained from these solutions are not illustrated. They were similar to previous findings [5]. At pH 5.6--5.7, control SgFe-labeled diferric transferric (C) was half dissociated whereas 90% of the isotope in Solution A (SgFe labeled at pH 5.5) was still b o u n d to the transferrin. Only 42% of the SgFe in Solution B remained b o u n d to transferrin.
Biologically labeled transferrin solutions Reticulocytes percent uptake of SgFe transferrin-bound iron from Solutions D and E as well as control (C) was similar to our previous reported findings [4]. Cells incorporated SgFe more rapidly from Solution E (where unlabeled iron that was initially removed by reticulocytes was replaced with SgFe) than from Solution D (labeled by the reverse procedure). After 10 min incubation, cells t o o k up 72% SgFe from uniformly SgFe-labeled diferric transferrin (C), 58% SgFe from Solution D and 82% from Solution E. The percentages of SgFe recovered in resin elutate from samples C, D and E, as a function of pH (5.2--6.8), are illustrated in Fig. 2. SgFe in Solution D (labeled reticulocyte supernatant which was resaturated with unlabeled iron) did not dissociate until the pH was below 5.6--5.7, while nearly 60% of the SgFe in Solution E (SgFe-labeled supernatant whose donated iron was replaced
I00 -
80
c 59
/
Fe
ELUATE
60
40 |
5.2
6.0
|
|
6.8
pH Fig. 2. Dissociation of 5 9 F e from biologically labeled h u m a n diferric transferrin as a function of pH. Percent 5 9 F e in resin eluate. C, uniformly labeled 59Fe; D, S 9 F e paxtially removed by reticulocytes w a s replaced with unlabeled Fe; E, unlabeled Fe, partially r e m o v e d by reticulocytes was replaced with 59Fe.
770 with unlabeled Fe 3+) dissociated above pH 5.6--5.7. At this pH control, uniformly SgFe-labeled diferric transferrin (C) was half dissociated. Thus, the dissociation findings with Solution E were similar to those observed for Solution B and each of these preparations yielded SgFe iron to reticulocytes more readily than did preparations A or D. Discussion In our initial study of the pH-dependent iron dissociation properties we suggested that one iron-binding site of human transferrin retained its binding ability at low pH and that this difference in iron-binding ability might account for observed selective rabbit reticulocyte utilization of Fe 3÷ from one preferred site. The results obtained in the present study support this suggestion. Comparison of the rabbit reticulocyte SgFe uptake and pH-dependent dissociation findings indicate that the human transferrin-binding site which binds iron at lo~v pH is n o t the site which initially surrenders Fe3*to rabbit reticulocytes. The other, more neutral binding site is involved in this reaction. Diferric transferrin, where STFe was bound at low pH, did not dissociate 57Fe until the pH was lowered to 5.6--5.7 and was the least efficient reticulocyte SgFe iron donator (Fig. 1, Solution A). Moreover, after reticulocytes removed half the iron from SgFe-labeled diferric transferrin and this donated iron was replaced by unlabeled iron, similar reticulocyte uptake and dissociation data were observed {Fig. 2, Solution D). The data obtained from preparations B and E, which were selectively labeled in the reverse sequence by these chemical and biological procedures also concur. These findings identify that the transferrin iron-binding site which begins to dissociate as the pH is lowered from 7.4 to near 5.6 is the transferrin iron-binding site from which reticulocytes more readily incorporate Fe 3+. The maleate utilized in the buffer solutions is a potential iron chelator and may possibly effect dissociation by competitive chelation. At low pH, the concentration of bicarbonate, necessary for the formation of the Fe3+-carbonate transferrin complex, is reduced and may also contribute to increase dissociation of the transferrin complex. However, both these effects would be expected to randomly influence dissociation from either site, rather than be directed toward one unique site. Conformational changes in the protein, induced by changes in the pH, might occur, possibly leading to a masking of one site from the aqueous environment. Such an effect could also account for the two different dissociation patterns observed for the partially labeled preparations (A and B) and the acid-base properties of the two sites may be nearly identical. In order for this effect to occur and account for our observations, at neutral pH, one binding site should be exposed and the other masked, facilitating removal of Fe 3+ from this site as the pH is lowered. Then, a conformational change could reverse the masking, " b u r y i n g " this site and exposing the second site. Thus at low pH, this second site would reversibly bind Fe 3÷ and as the pH is increased it becomes masked and the other site is opened. However, such an effect would n o t be in keeping with observations that Fe 3+are randomly bound by each site at neutral pH [4]. Hence, both sites must be unmasked at neutral pH. In either case, whether the effect is due to protein conformational changes
771
or if there are different acid-base properties of each site, one site does preferentially donate its iron to the rabbit reticulocyte and can be identified by its ability to dissociate its Fe 3+at a higher pH than does the other site. The precise mechanism by which human transferrin-bound iron is ultimately transferred into the rabbit reticulocyte cell for heme synthesis is n o t yet fully known. An anion-detaching e n z y m e which cleaves carbonate (or bicarbonate) from the ternary transferrin complex and permits facile release of the Fe 3+has been suggested to account for the removal of transferrin-bound iron [6--8]. The more neutral iron-binding site may be kinetically more favorable as this enzyme's substrate than the low pH binding site because of the acid base ironbinding properties of each site or possible protein conformational changes and thus account for the functional heterogeneity of the binding sites. It is also possible that two observations are unrelated. Functional heterogeneity, which may be unique only to this particular system is observed at physiological pH. Dissociation p h e n o m e n o n at non-physiologic pH may or may not have any bearing on events that occur at the cell surface.
Acknowledgements Supported in part by a grant from the National Institutes of Health, Research Grant RO1 AM 15553 from the National Institute of Arthritis, Metabolism and Digestive Diseases and a grant from the Washington Heart Association. References 1 2 3 4 5 6 7 S
Aasa, R., Malmstrom, B.G.. Saltman, P. and Vangaard, T. (1963) Biochim. Biophys. Acta 75, 203--222 Aasa, R. (1972) Biochem. Biophys. Res. C o m m u n . 49,206--212 Fletcher,J. and Huehns, E.R. (1967) Nature 215, 584--586 Zapolski, E.J., Ganz, R. and Princiotto,J.V. (1974) A m . J. Physiol. 226,334--339 Princiotto,J.V. and Zapolski, E.J. (1975) Nature 255, 87--88 Aisen, P. and Leibman, A. (1973) Biochim. Biophys. Acta 304, 797--804 Egyed, A. (1973) Biochim. Biophys. Acta 304, 805--813 Martinez-Medellin, J. and Schulman, J. (1973) Biochem. Biophys. Res. C o m m u n . 53.32--38