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Lipid-protein interactions.I. Role of divalent ions in binding of glycylglycine to phosphatidylserine
Biochimica et Biophysics Acta, 326
BBA Report BBA 51169
Lipid-protein interactions. I. Role of divalent ions in binding of glycylglycine to phosphatidylserine
BERNARD J. BULKIN and ROBERT HAUSER Hunter College of the City Universityof New York, 695 Park Avenue, New York, N. Y. 10021 (U.S.A.) (Received October 5th, 1973)
SUMMARY
The reaction between glycylglycine, divalent metals, and phosphatidylserine has been investigated. Calcium is unique among eight ions in forming a mixed lipid-dipeptideCa2+complex exclusively. The pH dependence of complex formation and results for all eight ions are reported.
The nature of the interactions between phospholipids and proteins has been the study of numerous investigations”‘. The binding of both neutral and anionic lipids utilizing such forces as hydrogen bonding, van der Waals interactions, ionic (electrostatic) attractions, and various hydrophobic interactions have been considered. Undoubtedly, all of these are important in particular protein-lipid complexes. One possible lipid-protein interaction which has been suggested occasionally but which has never, to our knowledge, been studied experimentally, is the role of divalent cations in “bridging” anionic lipid and negatively charged protein sites. Such “tricomplexes” have been proposed by Reynolds and Trayer 3, for example, in discussions of the role of ions in membranes. Closely related to these proposals is the work of several investigators4-’ on the formation of diphospholipid complexes with divalent metals (particularly phosphatidylserine with Ca2+)in monolayers and bilayers. Such complexation, presumably occurring primarily through the anionic carboxyl and secondarily via the phosphate group of the phosphatidylserine, has been well established. In this paper, we present for the first time evidence that, at neutral pH, in the presence of the dipeptide glycylglycine, phosphatidylserine and Ca2+do not associate to the dilipid complex but rather form a tricomplex of stoichiometry Ca2+-phosphatidyl-
se:ine--Gly-GBy-Cl-.
The pH dependence
exation tendencies en a suspension glycylglycice precipitate
stability of &is coqiex,
of phos
as well “s
ylserine in water is mixed with a solution of
in the presence of diss hed CaC!l,, a precipitate
fomm imme
which formed was centrifuged,’
kept at constant precipitate
of the
of other divaient cations (Mg2Ti ST2*5B@+, Ba2+, c.,P,
ried and analyzed. The reac’ciofi mhtrare was ionic strength by use of a 1OS-fo’oB
stoichiometry
was carried out in three
e precipitate
were determined
itative, however, as fro solution could be obtained. amounts detectable constant.
of Ga’+, g~ycy~g~y6~~e amount of gfyc
does Ga’+. At neutral p d the calcium complex.
Initial concentrations
of all reagents were IO+ M. Ionic st~e~~~ ~a~t~~~
from
spectra
by use of 4 M KC1.
BBA REPORT
291
suspended. The precipitate in the Mg2+case was thus a mixture of the tricomplex.and the dilipid complex. The reaction was also attempted with a number of other divalent ions (see Table I). Only in the case of Mn2* and Sr2+could even small amounts of the glycylglycine be precipitated. In these two cases mixtures of the dilipid complex with the tricomplex were formed in the precipitate. For the other metals shown in the table, only the dilipid complex could be prepared. Of the cations examined, it is only in the case of calcium that one can prepare a pure tricomplex at neutral pH. The pH dependence of the stability of the Ca2+and Mg2”complexes were investigated in some detail. If the pH of the supernatant liquid above the Ca2+complex is raised (7.2-7.9) by dropwise addition of 0.1 M NaOH, the tricomplex begins to dissociate. This is evidenced by positive ninhydrin tests and the reappearance of suspended phosphatidylserine. Above pH 8 dissociation is complete. The pH was not raised above pH 8.5 to prevent complications due to Ca(OH), formation and peptide hydrolysis. When the above mixture containing dissociated complex is neutralized by dropwise addition of 0.1 M HCl, the tricomplex again precipitates. Again, no trace of the dilipid complex is found. Further acidification of the complex to pH as low as 5.5 has no effect. As noted above, at pH 7 Mg2+does not completely bind either the phosphatidylserine or glycylglycine in an insoluble complex. When the pH of this solution was raised to pH 8, however, all of the phosphatidylserine precipitated yielding a mixture of tricomplex and dilipid complex. At this point, however, 42% of the glycylglycine originally added still remained in solution. Thus tricomplexation is not favored at higher pH for the Mg2+ complex, although dilipid complex formation is. The pH of the solutions was not raised above pH 8 because of Mg(OH)2 formation, If the pH of the complexed mixture using Mg2+is lowered to pH 6 complete dissociation of all complexes occurs. In this case, however, careful reassociation to neutral pH with NaOH increases the amount of glycylglycine in the supernatant liquid to 88% of that originally present, i.e. the precipitate now contains mostly dilipid complex. Allowing the mixture to stand for 10 days, heating, or further pH variation did not alter the ratio of tricomplex to dilipid complex in the precipitate. In the above discussion, the need for an anionic phospholipid with the COOgroup available for complexation has been assumed. To test this assumption, an analogous series of experiments to those described above was attempted using lecithin in place of phosphatidylserine. Only a small amount of precipitate formed for experiments using Ca2+ and Mg2+as metals. These precipitates contained no glycylglycine, and were the dilipid complex, presumably more weakly associated through the phosphate group. The results suggest that the binding of the original tricomplexes using phosphatidylserine is through the COO- group. This fact could not be ascertained directly from the infrared spectra of the tricomplexes, as the carbonyl stretching vibrations of the solid complexes were too broad for any specific conclusions to be drawn.
I Freeman, M.K., Lindgren, F.T. and Nichols, A.Y. (1963) Prig. Chem. Eilfs L$k2s 6,215 2 Sawer .T. and Levy, M. (1971) Biochim. Biophys. Acta 241,97
4 4 5 6 7
Reynol J.A. and Trayer, W. (1971) J. Biol. Chern.%46,X337 ~ap~~dj~~~u~o~~, D. (1968) him. Biophys. Acta H&3,240 em. Btophyys. Rex Commun. 51, 13% Qhnishi, S. and Ito, T. (1933) Seimiya, T. and Ohki, S. (197 Seimiya, T. and 0
BBA Report BBA 51169
Lipid-protein interactions. I. Role of divalent ions in binding of glycylglycine to phosphatidylserine
BERNARD J. BULKIN and ROBERT HAUSER Hunter College of the City Universityof New York, 695 Park Avenue, New York, N. Y. 10021 (U.S.A.) (Received October 5th, 1973)
SUMMARY
The reaction between glycylglycine, divalent metals, and phosphatidylserine has been investigated. Calcium is unique among eight ions in forming a mixed lipid-dipeptideCa2+complex exclusively. The pH dependence of complex formation and results for all eight ions are reported.
The nature of the interactions between phospholipids and proteins has been the study of numerous investigations”‘. The binding of both neutral and anionic lipids utilizing such forces as hydrogen bonding, van der Waals interactions, ionic (electrostatic) attractions, and various hydrophobic interactions have been considered. Undoubtedly, all of these are important in particular protein-lipid complexes. One possible lipid-protein interaction which has been suggested occasionally but which has never, to our knowledge, been studied experimentally, is the role of divalent cations in “bridging” anionic lipid and negatively charged protein sites. Such “tricomplexes” have been proposed by Reynolds and Trayer 3, for example, in discussions of the role of ions in membranes. Closely related to these proposals is the work of several investigators4-’ on the formation of diphospholipid complexes with divalent metals (particularly phosphatidylserine with Ca2+)in monolayers and bilayers. Such complexation, presumably occurring primarily through the anionic carboxyl and secondarily via the phosphate group of the phosphatidylserine, has been well established. In this paper, we present for the first time evidence that, at neutral pH, in the presence of the dipeptide glycylglycine, phosphatidylserine and Ca2+do not associate to the dilipid complex but rather form a tricomplex of stoichiometry Ca2+-phosphatidyl-
se:ine--Gly-GBy-Cl-.
The pH dependence
exation tendencies en a suspension glycylglycice precipitate
stability of &is coqiex,
of phos
as well “s
ylserine in water is mixed with a solution of
in the presence of diss hed CaC!l,, a precipitate
fomm imme
which formed was centrifuged,’
kept at constant precipitate
of the
of other divaient cations (Mg2Ti ST2*5B@+, Ba2+, c.,P,
ried and analyzed. The reac’ciofi mhtrare was ionic strength by use of a 1OS-fo’oB
stoichiometry
was carried out in three
e precipitate
were determined
itative, however, as fro solution could be obtained. amounts detectable constant.
of Ga’+, g~ycy~g~y6~~e amount of gfyc
does Ga’+. At neutral p d the calcium complex.
Initial concentrations
of all reagents were IO+ M. Ionic st~e~~~ ~a~t~~~
from
spectra
by use of 4 M KC1.
BBA REPORT
291
suspended. The precipitate in the Mg2+case was thus a mixture of the tricomplex.and the dilipid complex. The reaction was also attempted with a number of other divalent ions (see Table I). Only in the case of Mn2* and Sr2+could even small amounts of the glycylglycine be precipitated. In these two cases mixtures of the dilipid complex with the tricomplex were formed in the precipitate. For the other metals shown in the table, only the dilipid complex could be prepared. Of the cations examined, it is only in the case of calcium that one can prepare a pure tricomplex at neutral pH. The pH dependence of the stability of the Ca2+and Mg2”complexes were investigated in some detail. If the pH of the supernatant liquid above the Ca2+complex is raised (7.2-7.9) by dropwise addition of 0.1 M NaOH, the tricomplex begins to dissociate. This is evidenced by positive ninhydrin tests and the reappearance of suspended phosphatidylserine. Above pH 8 dissociation is complete. The pH was not raised above pH 8.5 to prevent complications due to Ca(OH), formation and peptide hydrolysis. When the above mixture containing dissociated complex is neutralized by dropwise addition of 0.1 M HCl, the tricomplex again precipitates. Again, no trace of the dilipid complex is found. Further acidification of the complex to pH as low as 5.5 has no effect. As noted above, at pH 7 Mg2+does not completely bind either the phosphatidylserine or glycylglycine in an insoluble complex. When the pH of this solution was raised to pH 8, however, all of the phosphatidylserine precipitated yielding a mixture of tricomplex and dilipid complex. At this point, however, 42% of the glycylglycine originally added still remained in solution. Thus tricomplexation is not favored at higher pH for the Mg2+ complex, although dilipid complex formation is. The pH of the solutions was not raised above pH 8 because of Mg(OH)2 formation, If the pH of the complexed mixture using Mg2+is lowered to pH 6 complete dissociation of all complexes occurs. In this case, however, careful reassociation to neutral pH with NaOH increases the amount of glycylglycine in the supernatant liquid to 88% of that originally present, i.e. the precipitate now contains mostly dilipid complex. Allowing the mixture to stand for 10 days, heating, or further pH variation did not alter the ratio of tricomplex to dilipid complex in the precipitate. In the above discussion, the need for an anionic phospholipid with the COOgroup available for complexation has been assumed. To test this assumption, an analogous series of experiments to those described above was attempted using lecithin in place of phosphatidylserine. Only a small amount of precipitate formed for experiments using Ca2+ and Mg2+as metals. These precipitates contained no glycylglycine, and were the dilipid complex, presumably more weakly associated through the phosphate group. The results suggest that the binding of the original tricomplexes using phosphatidylserine is through the COO- group. This fact could not be ascertained directly from the infrared spectra of the tricomplexes, as the carbonyl stretching vibrations of the solid complexes were too broad for any specific conclusions to be drawn.
I Freeman, M.K., Lindgren, F.T. and Nichols, A.Y. (1963) Prig. Chem. Eilfs L$k2s 6,215 2 Sawer .T. and Levy, M. (1971) Biochim. Biophys. Acta 241,97
4 4 5 6 7
Reynol J.A. and Trayer, W. (1971) J. Biol. Chern.%46,X337 ~ap~~dj~~~u~o~~, D. (1968) him. Biophys. Acta H&3,240 em. Btophyys. Rex Commun. 51, 13% Qhnishi, S. and Ito, T. (1933) Seimiya, T. and Ohki, S. (197 Seimiya, T. and 0