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A model for l -lactate binding to Cancer magister hemocyanin
Comp. Biochem. Physiol. Vol. 81B, No. 4, pp. 885-887, 1985 Printed in Great Britain
0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
A MODEL F O R L-LACTATE B I N D I N G TO CANCER MAGISTER H E M O C Y A N I N ROBERT A. GRAHAM* Oregon Institute of Marine Biology, University of Oregon, Charleston, OR 97420, USA (Received 13 November 1984)
Abstract--1. L-Lactate raises the oxygen affinity of Cancer magister hemocyanin. The L-lactate analogs, D-lactate, glycolate and 2-methyl-lactate cause smaller increases in an oxygen binding affinity. Other analogs have no detectable effect. 2. These data suggest that L-lactate binds to the hemocyanin at all four positions around the chiral carbon. 3. The carboxyl and hydroxyl groups are required for activity. The protein can only partially distinguish between the methyl group and hydrogen atom.
INTRODUCTION L-Lactate, which accumulates in the blood of crustaceans during hypoxia, has been shown to raise the oxygen affinity of hemocyanins from a number of species of crustaceans (Truchot, 1980; Johnson and Becker, 1981; Booth et al., 1982; Graham et al., 1982, 1983; Mangum, 1983a; Johnson et aL, 1984). In the hemocyanins of the crabs, Cancer magister (Graham et aL, 1982, 1983) and Callinectes sapidus (Johnson et aL, 1982, 1984) L-lactate was shown to be a more effective modulator of oxygen affinity than glycolate or D-lactate. Graham et al. (1983) also found that a variety of other L-lactate analogs and organic acids showed no effect on the binding of oxygen by C. magister hemocyanin. However, 2-methyl-lactate (2-hydroxy-2-methylpropanoate) modulated hemocyanin-oxygen binding to a degree similar to glycolate and o-lactate. From these experiments Graham et al. (1983) concluded that a hydroxyl group is required for effector activity. It was assumed that the carboxyl group is required. The protein would also have to recognize either the hydrogen atom or methyl group to distinguish between L- and D-lactate. Similar conclusions were reached by Johnson et al., (1982, 1984) from studies of the hemocyanin of another brachyuran crab, Callineetes sapidus. The specificity of the action of L-lactate (Graham et al., 1982, 1983; Johnson and Becker, 1981; Johnson et al., 1982, 1984) and the demonstration that L-lactate binds to the hemocyanin at a specific site other than the oxygen binding site shows that the modulation of the oxygen binding behavior of the protein by L-lactate is allosteric (Johnson et al., 1984). Allosteric modulation of oxygen binding by the vertebrate hemoglobins has been extensively studied (of. Perutz, 1970). Detailed models of these interactions were first proposed from studies of allosteric effectors with different affinities for hemoglobin as well as different effectiveness as modulators of oxygen affinity (Benesch and Benescb, 1969). In this report I
*Present address: Biological Science, Florida State University, Tallahassee, FL 32306, USA.
propose a model for the binding of L-lactate to the 25S C. magister hemocyanin which accounts for the effects of L-lactate and some of its structural analogs on raising the respiratory protein's affinity for oxygen. In this model all four positions around the chiral carbon of L-lactate are involved in binding. This model differs from previous models (Graham et al., 1983; Johnson et al., 1984) in which it was proposed that three of the four carbons around the chiral carbon are involved in binding. Dr. Charlotte Mangum briefly discussed the model presented here at the 1982 meetings in Leeds (Mangum, 1983b).
RESULTS AND DISCUSSION Projections of the structures of the organic molecules which raise the oxygen binding affinity of C. magister hemocyanin and some of the related analogs which do not are shown in Fig. 1. The projections are drawn as they would appear to an observer in the active site of the hemocyanin: the - C O O - and - O H groups are drawn in equivalent positions. A model of how L-lactate might interact with the hemocyanin is presented in Fig. 2. Both Graham et al. (1983) and Johnson et al. (1982, 1984) concluded that the hydroxyl group is required for activity. In Fig. 1 it can be seen that substitution of the hydroxyl group with an H atom (propionate, acetate) results in a loss of effector activity. In the model it is proposed that the hydroxyl group interacts with the hemocyanin via a hydrogen bond. Previously, it has been assumed that the carboxyl group is required for activity (Graham et al., 1983; Johnson et al., 1982, 1984). Direct evidence that this group is required is provided by the observed lack of effector activity of DL-3-hydroxybutyrate (data in Graham, 1983). Lengthening of the distance between the carboxyl and hydroxyl group by one carbon completely eliminates effector activity. Apparently the carboxyl group and hydroxyl group are too far apart in 3-hydroxybutyrate for both to fit within the active site. The interaction between the carboxyl group and the protein is presumed to be electrostatic.
885
886
ROBERT A. GRAHAM
STRONG EFFECT H I
HO--C--CH3
~;O0" L-lactate
WEAK EFFECT
NO EFFECT
HO-COO" D-lactate
H--C--H 40O-
HO-C--HI CO0"
H-C-CH3 CO0"
glycolate
ICH
acetate
propionate
H
there are selection pressures for separate weak effector sites, since these molecules probably do not appear in the blood of C. magister (Graham et al., 1983) or any other crustacean. Another possible model is one in which only three positions around the chiral carbon are involved in L-lactate binding. Such models have been proposed (Graham et al., 1983; Johnson et al., 1984). However, in these models it is difficult to rationalize the similar activities of all three of the weak effectors. Both glycolate and 2-methyl-lactate have three positions in common with L-lactate (Fig. 1). If only three of the four positions in L-lactate were important in binding to the protein, then either glycolate or 2-methyl-lactate might be expected to have effector activity similar to that of L-lactate. Many, but not all crustacean hemocyanins are modulated by L-lactate. The L-lactate responsive hemocyanins include both two-hexamer and onehexamer types. As yet no D-lactate responsive chelicerate or molluscan hemocyanins have been found (Mangum, 1983a). The physiological basis of the L-lactate response seems to be well understood (Booth et al., 1982; Graham et aL, 1983; Mangum, 1983a,b) and the magnitude of the L-lactate effect on most of the L-lactate responsive hemocyanins is remarkably similar (cf. Truchot, 1980; Booth et al., 1982; Graham et al., 1983; Johnson et al., 1984; Mangum, 1983a). Therefore the model presented here is very likely to be applicable to all the hemocyanins which respond to L-lactate. This is the only binding site for an organic effector that is known for an oxygen transporting protein other than the tetrameric vertebrate hemoglobins, or
o--CHcoo. HO-I C -C 2-methyllactate
2-hydroxybutyrate
Y
HO--?--CH3
CH2 CO0" 3-hydroxybutyrate
Fig. 1. Projections of the structures of L-lactate and its structural analogues. The projections are drawn as they would appear to an observer in the active site of the hemocyanin. The hydroxyl and carboxyl groups are drawn in equivalent positions. It is the positions of H atom and methyl group which appear to differ, for example, in D- and L-lactate. The Fischer conventions regarding the three dimensional relationships of the bonds around the chiral carbon are retained. Only the L-forms of the hydroxybutyrates are shown though the D- and L- forms were tested. Based on data from Graham et aL (1983) and the more recent binding that DL-3-hydroxybutyratehas no effect (Graham, 1983).
A specific arrangement of both the H atom and methyl group appears to be required for full activity. The hemocyanin apparently can only partially distinguish between the H atom and the methyl group. Compounds with the H atom and methyl group substituted for one another (D-lactate, glycolate, 2-methyl-lactate) all have similar effector activity which is less than the effector activity of L-lactate. A substitution of either group with an ethyl group (DL-2-hydroxybutyrate) results in the complete loss of activity. The H atom and methyl group of L-lactate are proposed to interact with hydrophobic regions on the protein. Though either H atoms or methyl groups can occupy the hydrophobic sites on the hemocyanin, ethyl groups appear to be too large to fit into these sites. When the glycolate interacts a hydrogen atom is in the methyl site; when 2-methyl-lactate interacts with the protein a methyl group is present in the H atom site; and with D-lactate bound there is a hydrogen in the methyl site and a methyl group in the hydrogen atom site. It is proposed that the activity of the weak effectors reflects the lower affinity of the methyl site for hydrogen atoms and the hydrogen site for methyl groups. Other more complex models for lactate binding will also accomodate the data. For example, since it has not been shown that the effectors in Fig. 1 compete with one another for the same site, it is possible that there are separate binding sites for the weak effectors. On the other hand, it is unlikely that
"-,
. . . , , p" ............... '..
Fig. 2. Proposed binding of L-lactate to C. magister hemocyanin. The open circles represent atoms of L-lactate, identified at the left; the protein is shown stippled or hatched. The effector binds to the protein at all four positions around the chiral carbon. The CO0- interacts by means of electrostatic forces, and the OH by an H bond. The H atom and CH 3 group fit in hydrophobic sites (hatched) which incompletely distinguish between them, thus permitting low activity of the weak effectors.
L-Lactate-hemocyanin binding model for any extracellular oxygen transporting protein. The organophosphate effectors of vertebrate hemoglobins act to lower oxygen binding affinity by binding to a single site on the low affinity, deoxy, form of the hemoglobin (Benesch et al., 1968a,b). L-Lactate presumably binds to the oxy-form of hemocyanin to increase oxygen affinity (Johnson and Becker, 1981; Johnson et al., 1984). Therefore, the modulation of the oxygen binding affinity of hemocyanin by lactate is a fundamentally different process from the modulation by the organophosphate effectors of hemoglobin. There appear to be about four L-lactate binding sites on the hemoeyanin from Callinectes sapidus. There are, therefore, fewer active sites than subunits on the two-hexamer protein (Johnson et al., 1984). Like other crustacean hemocyanins the hemocyanins of Callinectes sapidus (Hamlin and Fish, 1977) and Cancer magister (Larson et al., 1981) are composed of different kinds of subunits. The binding site could be a property of a single subunit type or the binding site could be a property of the oligomer (hexamer or two-hexamer) as it is the organophosphate binding site on the tetrameric vertebrate hemoglobins (Benesch et al., 1968a). The fact that the lactate binding site is not uniformly distributed among the crustacean hemocyanins raises the interesting possibility that the lactate binding site is associated with particular kinds of subunits which appear in some species but not others. There is mounting evidence that subunit heterogeneity is important in determining the oxygen binding behavior of crustacean hemocyanins (Jeffrey and Treacy, 1980; Graham, 1983). The study of the allosteric interactions of L-lactate and crustacean hemocyanins, now in its infancy, should provide an interesting contrast to the extremely well studied allosteric interactions of the vertebrate hemoglobins.
Acknowledgements--This work is taken from my Ph.D. dissertation presented to the University of Oregon in the summer of 1983. Figure 2 is similar to a figure that I drew for Dr. Mangum and which appeared in Mangum (1983b). The figure here is used with permission of the publisher. I am very grateful to Drs R. C. Terwilliger and C. P. Mangum for many helpful discussions during the course of the research and for reviewing the manuscript. This work was supported in part by NSF grant No. PCM 80-12554 to Dr Terwilliger, Oregon Institute of Marine Biology and NIGMASNAS Award No. 5T332 GM 07527.
887 REFERENCES
Benesch R. and Benesch R. E. (1969) Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature 221, 618-622. Benesch R., Benesch R. E. and Enoki Y. (1968a) The interaction of hemoglobin and its subunits with 2,3-diphosphoglycerate. Proc. natl. Acad. Sci. U.S.A. 61, 1102-I 106. Benesch R., Benesch R. E. and Yu C. I. (1968b) Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin. Proc. natl. Acad. Sci. U.S.A. 59, 526-532. Booth C. E., McMahon B. R. and Pinder A. W. (1982) Oxygen uptake and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab, Callinectes sapidus. J. comp. Physiol. 148, 111-121. Graham R. A. (1983) Structure and function of Cancer magister hemocyanin. Dissertation. University of Oregon, Eugene. Graham R. A., Mangum C. P., Terwilliger R. C. and Terwilliger N. B. (1982) Evidence for L-lactate as an allosteric modulator of O2-binding in Cancer magister hemocyanin. Am. Zool. 22, 93. Graham R. A., Mangum C. P., Terwilliger R. C. and Terwilliger, N. B. (1983) The effects of organic acids on oxygen binding of hemocyanin from the crab Cancer magister. Comp. Biochem. Physiol. 74A, 45-50. Hamlin L. M. and Fish W. W. (1977) The subunit characterization of Callinectes sapidus hemocyanin. Biochim. biophys. Acta 667, 44-58. Jeffry P. D. and Treacy G. B. (1982) Hemocyanin from the Australian freshwater crayfish Cherax destructor. Oxygen binding studies of the major components. Biochemistry 19, 5428-5433. Johnson B. P. and Becker D. J. (1981) Lactic acid is an allosteric modifier of the oxygen affinity of blue crab hemocyanin. Am. Zool. 21, 951. Johnson B. A., Bonaventura C. and Bonaventura J. (1982) L-Lactate is an allosteric modifier of the oxygen affinity of blue crab hemocyanin. Fedn Proc. Fedn Am. Socs exp. Biol. 41, 890. Johnson B. A., Bonaventura C. and Bonaventura J. (1984) Allosteric modulation of Callinectes sapidus hemocyanin by binding of L-lactate. Biochemistry 23, 872-878. Larson B. A., Terwilliger N. B. and Terwilliger R. C. (1981) Subunit heterogeneity of Cancer magister hemocyanin. Biochim. biophys. Acta 667, 294-302. Mangum C, P. (1983a) On the distribution of lactate sensitivity among the hemoeyanins. Mar. Biol. Lett. 4, 139-149. Mangum C. P. (1983b) Adaptability and inadaptability among HcO2 transport systems: an apparent paradox. Life Chem. Repts 1, 335-352. Perutz M. F. (1970) Stereochemistry of co-operative effects of haemoglobin. Nature 288, 726-739. Truchot J. P. (1980) Lactate increases the oxygen affinity of crab hemocyanin. J. exp. Zool. 294, 205-208.
0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
A MODEL F O R L-LACTATE B I N D I N G TO CANCER MAGISTER H E M O C Y A N I N ROBERT A. GRAHAM* Oregon Institute of Marine Biology, University of Oregon, Charleston, OR 97420, USA (Received 13 November 1984)
Abstract--1. L-Lactate raises the oxygen affinity of Cancer magister hemocyanin. The L-lactate analogs, D-lactate, glycolate and 2-methyl-lactate cause smaller increases in an oxygen binding affinity. Other analogs have no detectable effect. 2. These data suggest that L-lactate binds to the hemocyanin at all four positions around the chiral carbon. 3. The carboxyl and hydroxyl groups are required for activity. The protein can only partially distinguish between the methyl group and hydrogen atom.
INTRODUCTION L-Lactate, which accumulates in the blood of crustaceans during hypoxia, has been shown to raise the oxygen affinity of hemocyanins from a number of species of crustaceans (Truchot, 1980; Johnson and Becker, 1981; Booth et al., 1982; Graham et al., 1982, 1983; Mangum, 1983a; Johnson et aL, 1984). In the hemocyanins of the crabs, Cancer magister (Graham et aL, 1982, 1983) and Callinectes sapidus (Johnson et aL, 1982, 1984) L-lactate was shown to be a more effective modulator of oxygen affinity than glycolate or D-lactate. Graham et al. (1983) also found that a variety of other L-lactate analogs and organic acids showed no effect on the binding of oxygen by C. magister hemocyanin. However, 2-methyl-lactate (2-hydroxy-2-methylpropanoate) modulated hemocyanin-oxygen binding to a degree similar to glycolate and o-lactate. From these experiments Graham et al. (1983) concluded that a hydroxyl group is required for effector activity. It was assumed that the carboxyl group is required. The protein would also have to recognize either the hydrogen atom or methyl group to distinguish between L- and D-lactate. Similar conclusions were reached by Johnson et al., (1982, 1984) from studies of the hemocyanin of another brachyuran crab, Callineetes sapidus. The specificity of the action of L-lactate (Graham et al., 1982, 1983; Johnson and Becker, 1981; Johnson et al., 1982, 1984) and the demonstration that L-lactate binds to the hemocyanin at a specific site other than the oxygen binding site shows that the modulation of the oxygen binding behavior of the protein by L-lactate is allosteric (Johnson et al., 1984). Allosteric modulation of oxygen binding by the vertebrate hemoglobins has been extensively studied (of. Perutz, 1970). Detailed models of these interactions were first proposed from studies of allosteric effectors with different affinities for hemoglobin as well as different effectiveness as modulators of oxygen affinity (Benesch and Benescb, 1969). In this report I
*Present address: Biological Science, Florida State University, Tallahassee, FL 32306, USA.
propose a model for the binding of L-lactate to the 25S C. magister hemocyanin which accounts for the effects of L-lactate and some of its structural analogs on raising the respiratory protein's affinity for oxygen. In this model all four positions around the chiral carbon of L-lactate are involved in binding. This model differs from previous models (Graham et al., 1983; Johnson et al., 1984) in which it was proposed that three of the four carbons around the chiral carbon are involved in binding. Dr. Charlotte Mangum briefly discussed the model presented here at the 1982 meetings in Leeds (Mangum, 1983b).
RESULTS AND DISCUSSION Projections of the structures of the organic molecules which raise the oxygen binding affinity of C. magister hemocyanin and some of the related analogs which do not are shown in Fig. 1. The projections are drawn as they would appear to an observer in the active site of the hemocyanin: the - C O O - and - O H groups are drawn in equivalent positions. A model of how L-lactate might interact with the hemocyanin is presented in Fig. 2. Both Graham et al. (1983) and Johnson et al. (1982, 1984) concluded that the hydroxyl group is required for activity. In Fig. 1 it can be seen that substitution of the hydroxyl group with an H atom (propionate, acetate) results in a loss of effector activity. In the model it is proposed that the hydroxyl group interacts with the hemocyanin via a hydrogen bond. Previously, it has been assumed that the carboxyl group is required for activity (Graham et al., 1983; Johnson et al., 1982, 1984). Direct evidence that this group is required is provided by the observed lack of effector activity of DL-3-hydroxybutyrate (data in Graham, 1983). Lengthening of the distance between the carboxyl and hydroxyl group by one carbon completely eliminates effector activity. Apparently the carboxyl group and hydroxyl group are too far apart in 3-hydroxybutyrate for both to fit within the active site. The interaction between the carboxyl group and the protein is presumed to be electrostatic.
885
886
ROBERT A. GRAHAM
STRONG EFFECT H I
HO--C--CH3
~;O0" L-lactate
WEAK EFFECT
NO EFFECT
HO-COO" D-lactate
H--C--H 40O-
HO-C--HI CO0"
H-C-CH3 CO0"
glycolate
ICH
acetate
propionate
H
there are selection pressures for separate weak effector sites, since these molecules probably do not appear in the blood of C. magister (Graham et al., 1983) or any other crustacean. Another possible model is one in which only three positions around the chiral carbon are involved in L-lactate binding. Such models have been proposed (Graham et al., 1983; Johnson et al., 1984). However, in these models it is difficult to rationalize the similar activities of all three of the weak effectors. Both glycolate and 2-methyl-lactate have three positions in common with L-lactate (Fig. 1). If only three of the four positions in L-lactate were important in binding to the protein, then either glycolate or 2-methyl-lactate might be expected to have effector activity similar to that of L-lactate. Many, but not all crustacean hemocyanins are modulated by L-lactate. The L-lactate responsive hemocyanins include both two-hexamer and onehexamer types. As yet no D-lactate responsive chelicerate or molluscan hemocyanins have been found (Mangum, 1983a). The physiological basis of the L-lactate response seems to be well understood (Booth et al., 1982; Graham et aL, 1983; Mangum, 1983a,b) and the magnitude of the L-lactate effect on most of the L-lactate responsive hemocyanins is remarkably similar (cf. Truchot, 1980; Booth et al., 1982; Graham et al., 1983; Johnson et al., 1984; Mangum, 1983a). Therefore the model presented here is very likely to be applicable to all the hemocyanins which respond to L-lactate. This is the only binding site for an organic effector that is known for an oxygen transporting protein other than the tetrameric vertebrate hemoglobins, or
o--CHcoo. HO-I C -C 2-methyllactate
2-hydroxybutyrate
Y
HO--?--CH3
CH2 CO0" 3-hydroxybutyrate
Fig. 1. Projections of the structures of L-lactate and its structural analogues. The projections are drawn as they would appear to an observer in the active site of the hemocyanin. The hydroxyl and carboxyl groups are drawn in equivalent positions. It is the positions of H atom and methyl group which appear to differ, for example, in D- and L-lactate. The Fischer conventions regarding the three dimensional relationships of the bonds around the chiral carbon are retained. Only the L-forms of the hydroxybutyrates are shown though the D- and L- forms were tested. Based on data from Graham et aL (1983) and the more recent binding that DL-3-hydroxybutyratehas no effect (Graham, 1983).
A specific arrangement of both the H atom and methyl group appears to be required for full activity. The hemocyanin apparently can only partially distinguish between the H atom and the methyl group. Compounds with the H atom and methyl group substituted for one another (D-lactate, glycolate, 2-methyl-lactate) all have similar effector activity which is less than the effector activity of L-lactate. A substitution of either group with an ethyl group (DL-2-hydroxybutyrate) results in the complete loss of activity. The H atom and methyl group of L-lactate are proposed to interact with hydrophobic regions on the protein. Though either H atoms or methyl groups can occupy the hydrophobic sites on the hemocyanin, ethyl groups appear to be too large to fit into these sites. When the glycolate interacts a hydrogen atom is in the methyl site; when 2-methyl-lactate interacts with the protein a methyl group is present in the H atom site; and with D-lactate bound there is a hydrogen in the methyl site and a methyl group in the hydrogen atom site. It is proposed that the activity of the weak effectors reflects the lower affinity of the methyl site for hydrogen atoms and the hydrogen site for methyl groups. Other more complex models for lactate binding will also accomodate the data. For example, since it has not been shown that the effectors in Fig. 1 compete with one another for the same site, it is possible that there are separate binding sites for the weak effectors. On the other hand, it is unlikely that
"-,
. . . , , p" ............... '..
Fig. 2. Proposed binding of L-lactate to C. magister hemocyanin. The open circles represent atoms of L-lactate, identified at the left; the protein is shown stippled or hatched. The effector binds to the protein at all four positions around the chiral carbon. The CO0- interacts by means of electrostatic forces, and the OH by an H bond. The H atom and CH 3 group fit in hydrophobic sites (hatched) which incompletely distinguish between them, thus permitting low activity of the weak effectors.
L-Lactate-hemocyanin binding model for any extracellular oxygen transporting protein. The organophosphate effectors of vertebrate hemoglobins act to lower oxygen binding affinity by binding to a single site on the low affinity, deoxy, form of the hemoglobin (Benesch et al., 1968a,b). L-Lactate presumably binds to the oxy-form of hemocyanin to increase oxygen affinity (Johnson and Becker, 1981; Johnson et al., 1984). Therefore, the modulation of the oxygen binding affinity of hemocyanin by lactate is a fundamentally different process from the modulation by the organophosphate effectors of hemoglobin. There appear to be about four L-lactate binding sites on the hemoeyanin from Callinectes sapidus. There are, therefore, fewer active sites than subunits on the two-hexamer protein (Johnson et al., 1984). Like other crustacean hemocyanins the hemocyanins of Callinectes sapidus (Hamlin and Fish, 1977) and Cancer magister (Larson et al., 1981) are composed of different kinds of subunits. The binding site could be a property of a single subunit type or the binding site could be a property of the oligomer (hexamer or two-hexamer) as it is the organophosphate binding site on the tetrameric vertebrate hemoglobins (Benesch et al., 1968a). The fact that the lactate binding site is not uniformly distributed among the crustacean hemocyanins raises the interesting possibility that the lactate binding site is associated with particular kinds of subunits which appear in some species but not others. There is mounting evidence that subunit heterogeneity is important in determining the oxygen binding behavior of crustacean hemocyanins (Jeffrey and Treacy, 1980; Graham, 1983). The study of the allosteric interactions of L-lactate and crustacean hemocyanins, now in its infancy, should provide an interesting contrast to the extremely well studied allosteric interactions of the vertebrate hemoglobins.
Acknowledgements--This work is taken from my Ph.D. dissertation presented to the University of Oregon in the summer of 1983. Figure 2 is similar to a figure that I drew for Dr. Mangum and which appeared in Mangum (1983b). The figure here is used with permission of the publisher. I am very grateful to Drs R. C. Terwilliger and C. P. Mangum for many helpful discussions during the course of the research and for reviewing the manuscript. This work was supported in part by NSF grant No. PCM 80-12554 to Dr Terwilliger, Oregon Institute of Marine Biology and NIGMASNAS Award No. 5T332 GM 07527.
887 REFERENCES
Benesch R. and Benesch R. E. (1969) Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature 221, 618-622. Benesch R., Benesch R. E. and Enoki Y. (1968a) The interaction of hemoglobin and its subunits with 2,3-diphosphoglycerate. Proc. natl. Acad. Sci. U.S.A. 61, 1102-I 106. Benesch R., Benesch R. E. and Yu C. I. (1968b) Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin. Proc. natl. Acad. Sci. U.S.A. 59, 526-532. Booth C. E., McMahon B. R. and Pinder A. W. (1982) Oxygen uptake and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab, Callinectes sapidus. J. comp. Physiol. 148, 111-121. Graham R. A. (1983) Structure and function of Cancer magister hemocyanin. Dissertation. University of Oregon, Eugene. Graham R. A., Mangum C. P., Terwilliger R. C. and Terwilliger N. B. (1982) Evidence for L-lactate as an allosteric modulator of O2-binding in Cancer magister hemocyanin. Am. Zool. 22, 93. Graham R. A., Mangum C. P., Terwilliger R. C. and Terwilliger, N. B. (1983) The effects of organic acids on oxygen binding of hemocyanin from the crab Cancer magister. Comp. Biochem. Physiol. 74A, 45-50. Hamlin L. M. and Fish W. W. (1977) The subunit characterization of Callinectes sapidus hemocyanin. Biochim. biophys. Acta 667, 44-58. Jeffry P. D. and Treacy G. B. (1982) Hemocyanin from the Australian freshwater crayfish Cherax destructor. Oxygen binding studies of the major components. Biochemistry 19, 5428-5433. Johnson B. P. and Becker D. J. (1981) Lactic acid is an allosteric modifier of the oxygen affinity of blue crab hemocyanin. Am. Zool. 21, 951. Johnson B. A., Bonaventura C. and Bonaventura J. (1982) L-Lactate is an allosteric modifier of the oxygen affinity of blue crab hemocyanin. Fedn Proc. Fedn Am. Socs exp. Biol. 41, 890. Johnson B. A., Bonaventura C. and Bonaventura J. (1984) Allosteric modulation of Callinectes sapidus hemocyanin by binding of L-lactate. Biochemistry 23, 872-878. Larson B. A., Terwilliger N. B. and Terwilliger R. C. (1981) Subunit heterogeneity of Cancer magister hemocyanin. Biochim. biophys. Acta 667, 294-302. Mangum C, P. (1983a) On the distribution of lactate sensitivity among the hemoeyanins. Mar. Biol. Lett. 4, 139-149. Mangum C. P. (1983b) Adaptability and inadaptability among HcO2 transport systems: an apparent paradox. Life Chem. Repts 1, 335-352. Perutz M. F. (1970) Stereochemistry of co-operative effects of haemoglobin. Nature 288, 726-739. Truchot J. P. (1980) Lactate increases the oxygen affinity of crab hemocyanin. J. exp. Zool. 294, 205-208.