COMMUNICA TION
J. Biochem. 108, 505-506 (1990)
Human Plasma Geisolin Binds Adenosine Triphosphate Hideo Yamamoto,* Hiroaki I to,* Hideji Nakamura,* Eijiro Hayashi,* Susumu Kishimoto,* Tadao Hashimoto,** and Kunio Tagawa** * Third Department of Internal Medicine, Osaka University School of Medicine, Fukushima-ku, Osaka, Osaka 553; and "Department of Physiological Chemistry, Osaka University School of Medicine, Kita-ku, Osaka, Osaka 530 Received for publication, June 7, 1990
Binding studies of human plasma geisolin with ATP were done by equilibrium dialysis. Analysis of the binding data showed that plasma geisolin had one class of ATP binding site with Ka = 2.8 X 10~7 M, which saturated at an ATP/gelsolin ratio of 0.6. The bioluminescent assay for ATP with luciferin and firefly luciferase confirmed that the protein contained a nucleotide as ATP.
Geisolin is a multifunctional actin-modulating protein of mammalian cytoplasm (1). First, it severs actin filaments by breaking the non-covalent bond between actin-actin monomers within an actin filament. Second, it caps the fast-growing end of actin filament, blocking monomer exchange from that end. Third, it binds to actin monomers and oligomers to nucleate actin assembly. A variant of geisolin containing a 25-amino acid extension at the NH2-terminus is present in human plasma, and is functionally similar to the cytoplasmic counterpart {2-4). The role of plasma geisolin in blood remains unknown, although the possibility has been proposed that it may play a role in the clearance of actin from circulation during tissue damage or normal cell turnover. To understand the structural basis for geisolin function and its regulation, the actin binding and functional properties of proteolytically derived fragments of human geisolin have been extensively studied. Investigations thus far have revealed that geisolin function is activated by micromolar Ca2+, and inhibited by phosphatidyl inositol 4,5 bisphosphate and phosphatidyl inositol monophosphate (5-10). Geisolin has three actin binding sites, two of which bind to actin monomers and a third which binds to actin molecules within a filament (11, 12). It has been proposed that the third may be the actin binding domain that allows geisolin to bind to the side of the filament before severing. Despite all of these findings, the overall mechanism by which geisolin regulates actin filament length has yet to be elucidated, and it may be conceivable that there remain unknown functional domains on the geisolin molecule that have yet to be brought to light for the final elucidation of the mechanism. Recently we have demonstrated that human plasma geisolin was specifically eluted from a Cibacron Blue F3GA affinity column with 1 mM adenosine, guanosine, cytidine, and uridine di- and triphosphates except for cytidine 5'-diphosphate, but neither mononucleoside 5'monophosphate nor dinucleotide (NAD and NADP) did so, indicating that the interaction of plasma geisolin and the dye-ligand is biospecific (23, 14). If this interaction is indeed biospecific, it is reasonable to expect that the protein will exhibit at least one binding site for mononucleoside polyphosphate. Since ATP is one of the major factors that regulate actin polymerization, the possible interaction of geisolin and ATP may be related to the geisolin funcVol. 108, No. 4, 1990
tionality. Therefore, the speculation that geisolin may exhibit binding sites for ATP provides a matter of considerable interest. The purpose of this communication is to describe direct evidence for the binding of ATP by human plasma geisolin. Human plasma geisolin was prepared for ATP binding studies as previously described (13), except that 25 mM Tris-HCl, 1 mM EGTA, 0.2 mM dithiothreitol, 5% glycerol, pH 8.0 was used as the buffer solution. Briefly, geisolin was purified from approximately 300 ml of citrated plasma by 30-45% ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Affi-Gel Blue chromatography coupled with affinity elution with 1 mM ATP. To prepare ATP-free gelsolin, active fractions from an Affi-Gel Blue column were combined and brought to 70% saturation by the addition of solid ammonium sulfate. The precipitate was redissolved to 1-2 mg/ml in approximately 10 ml of 25 mM Tris-HCl, 1 mM EGTA, 0.2 mM dithiothreitol, 5% glycerol, 135 mM NaCl, pH 7.5, and extensively dialyzed against 500 ml of the same buffer with 7 changes over a 4-day period to ensure complete removal of the ATP. The dialyzed geisolin preparation was centrifuged to remove any precipitates. Then, ATP in the geisolin preparation was assayed bioluminescently with luciferin and firefly luciferase [EC 1.13.12.7] (15, 16), and was found to be undetected. The geisolin preparation thus obtained was virtually homogeneous as judged by Coomassie Blue staining of SDS-polyacrylamide gels (14). The geisolin concentration was estimated using an extinction coefficient, E^° of 117,580 M"1 • cm"1, calculated from the number of tyrosine and tryptophan residues present in the human plasma geisolin (11). The binding of ATP to geisolin was examined by equilibrium dialysis. [2,8-'H] ATP, mixed with unlabeled ATP in the dialysis buffer, was used for the binding studies, and the ATP concentration in the reaction mixtures ranged from 0.1 to 10/*M. The purified geisolin was subjected to equilibrium dialysis at 4'C over a 3-day period, and the dialysis cell was rotated throughout the dialysis period. At the end of dialysis, aliquots of 0.1 ml of the dialyzates and the dialysis buffers were taken and counted for radioactivity in a liquid scintillation counter. The concentrations of free and protein-bound ATP were calculated, and binding constants were determined by the Scatchard method (17). 505
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Fig. 1. A: Binding of ATP to human plasma gelsolin. Gelsolin was mixed with [2,8-JH]ATP to make up the mixtures of IB5.7 //M of gelsolin and the indicated concentrations of ATP in 0.7 ml of 25 mM TrisHC1, 1 mM EGTA, 0.2 mM dithiothreitol, 1.55% glycerol, 135 mM NaCl, pH 7.5, and LJ each was subjected to equilibrium dialysis LJ against 2.8 ml of the same concentration of L- 10ATP solutions, respectively. At the end of Q dialysis, aliquots of 0.1 ml of the dialyzed Z preparations and the dialysis buffers were D O 0.5taken, counted for radioactivity, and then m the concentrations of free and proteinbound ATP were calculated. At the same time, gelsolin concentrations were mea04 0.6 0 02 sured to ensure that loss of the protein did BOUND ( A T P / G E L S O U N ) FREE ATP not occur by adsorption to the dialysis cell or the membrane during the dialysis period. Moles of ATP bound per mol of gelsolin were plotted as a function of free ATP concentration in micromolars. B: Scatchard plot of ATP binding to human plasma gelsolin. Bound ATP was expressed as mol of ATP per mol of gelsolin. Free ATP concentration was expressed in micromolars. The line was fitted by linear regression analyses and the coefficient of correlation was -0.99. 06-
A
I
Figure 1A shows that saturable binding of ATP to gelsolin occurred over the range of ATP concentrations indicated. The maximum ATP bound was 0.6 mol per mol of gelsolin based on linear regression, and the dissociation constant was calculated to be approximately 0.28 //M (Fig. IB). At concentrations ranging from 20 to 100 //M ATP, additional binding experiments yielded constant binding ratios of 0.64 ±0.04 (mean±SD) mol ATP per mol of gelsolin. This confirms that the saturation of ATP binding to gelsolin occurred around 10 //M as shown in Fig. 1A. The observed binding stoichiometry was consistently lower than the expected value of 1.0. Since the equilibrium analyses were performed over the range of 0.1 to 100 // M ATP, the effects of ligand self-association are unlikely at such low concentrations. It is, however, conceivable that the observed low binding ratios are likely to result from a substantial proportion of the inactive form in the gelsolin preparation which might have occurred during the preparation of ATP-free gelsolin. Alternatively, the gelsolin concentration may be overestimated by the above-mentioned theoretical determination. Anyhow, it should be mentioned that we confirmed the binding of ATP to gelsolin by the centrifuge column procedure described by Penefsky (28). Gelsolin was incubated overnight with various concentrations of radiolabeled ATP, and aliquots of the reaction mixture of gelsolin and ATP were applied to centrifuge columns to separate free and protein-bound ATP. Appropriate aliquots of column effluent were taken for measurement of protein and ATP, and the ratios of ATP bound to gelsolin were calculated. The results showed that the binding ratios were slightly underestimated by the centrifuge column procedure as compared to those obtained by equilibrium analyses (data not shown). The above-mentioned bioluminescent assay for ATP showed that the gelsolin contained a nucleotide as ATP. Additionally, plasma gelsolin did not show ATPase activity when it was measured for ATPase activity by coupling the reaction to the pyruvate kinase [EC 2.7.1.40] and lactic dehydrogenase [EC 1.1.1.27] systems at measuring the oxidation of NADH spectrophotometrically at 340 nm. At present, it is unknown whether the nucleotide interaction with gelsolin regulates the functional activity of the protein. However, it seems quite unlikely that a nucleotide
binding site of such high affinity would exist on gelsolin without being involved in eliciting some of the multiple functions attributed to this protein. Such a speculation comes from previous observations in which the ATP bound to the gelsolin-actin complex was nonexchangeable with free ATP while the ATP bound to the actin monomer was readily exchangeable (19-21). The ATP binding site on gelsolin proposed here may play a role in the nonexchangeability of ATP bound to the complex. Further studies are needed to clarify the affinity of gelsolin to various nucleotides and to locate the binding site on the gelsolin molecule. These points are currently under investigation. We are grateful to Mr. Seiichi Kohda of Oriential Yeast Co., Ltd. for assisting us in the bioluminescent assay for ATP. REFERENCES 1. Yin, H.L. (1987) BioEetays 7, 176-179 2. Norberg, R., Thorstensson, R., Utter, G., & Fagraeus, A. (1979) Eur. J. Biochan. 100, 575-583 3. Thorstensson, R., Utter, G., & Norberg, R. (1982) Eur. J. Biochem. 126, 11-16 4. Yin, H.L., Kwiatkowski, D.L., Mole, J.E., & Cole, F.S. (1984) J. BioL Chan. 269, 5271-5276 5. Yin, H.L. & Stossel, T.P. (1979) Nature 281, 583-586 6. Yin, H.L. & Stossel, T.P. (1980) J. Biol. Chan. 265, 9490-9493 7. Janmey, P.A. & Stossel, T.P. (1987) Nature 326, 362-364 8. Janmey, P.A., Iida, K., Yin, H.L., & Stossel, T.P. (1987) J. BioL Chan. 262, 12228-12236 9. Janmey, P.A. & Stossel, T.P. (1989) J. BioL Chan. 264, 48254831 10. Bryan, J. & Hwo, S. (1986) J. Cell BioL 102, 1439-1446 11. Yin, H.L., Iida, K., & Janmey, P.A. (1988) J. Cell Biol. 106, 805-812 12. Bryan, J. (1988) J. Cell BioL 106, 1553-1562 13. Yamamoto, H., Terabayashi, M., Egawa, T., Hayashi, E., Nakamura, H., &Kishimoto, S. (1989) J. Biochem. 105,799-802 Ito, H., Yamamoto, H., Kimura, Y., Kambe, H., Okochi, T., & Kishimoto, S. (1990) J. Chromatogr. 526, 397-406 Kricka, L.J. (1988) AnaL Biochem. 175, 14-21 Jabs, CM., Ferrell, W.J., & Robb, H.J. (1977) Clin. Chan. 23, 2254-2257 Scatchard, G. (1949) Ann N. Y. Acad. Sci. 51, 660-672 Penefsky, H.S. (1977) J. BioL Chan. 262, 2891-2899 Harris, H.E. (1985) FEBS Lett. 190, 81-83 Tellam, R.L. (1986) Biochemistry 25, 5799-5804
J. Biochan.
J. Biochem. 108, 505-506 (1990)
Human Plasma Geisolin Binds Adenosine Triphosphate Hideo Yamamoto,* Hiroaki I to,* Hideji Nakamura,* Eijiro Hayashi,* Susumu Kishimoto,* Tadao Hashimoto,** and Kunio Tagawa** * Third Department of Internal Medicine, Osaka University School of Medicine, Fukushima-ku, Osaka, Osaka 553; and "Department of Physiological Chemistry, Osaka University School of Medicine, Kita-ku, Osaka, Osaka 530 Received for publication, June 7, 1990
Binding studies of human plasma geisolin with ATP were done by equilibrium dialysis. Analysis of the binding data showed that plasma geisolin had one class of ATP binding site with Ka = 2.8 X 10~7 M, which saturated at an ATP/gelsolin ratio of 0.6. The bioluminescent assay for ATP with luciferin and firefly luciferase confirmed that the protein contained a nucleotide as ATP.
Geisolin is a multifunctional actin-modulating protein of mammalian cytoplasm (1). First, it severs actin filaments by breaking the non-covalent bond between actin-actin monomers within an actin filament. Second, it caps the fast-growing end of actin filament, blocking monomer exchange from that end. Third, it binds to actin monomers and oligomers to nucleate actin assembly. A variant of geisolin containing a 25-amino acid extension at the NH2-terminus is present in human plasma, and is functionally similar to the cytoplasmic counterpart {2-4). The role of plasma geisolin in blood remains unknown, although the possibility has been proposed that it may play a role in the clearance of actin from circulation during tissue damage or normal cell turnover. To understand the structural basis for geisolin function and its regulation, the actin binding and functional properties of proteolytically derived fragments of human geisolin have been extensively studied. Investigations thus far have revealed that geisolin function is activated by micromolar Ca2+, and inhibited by phosphatidyl inositol 4,5 bisphosphate and phosphatidyl inositol monophosphate (5-10). Geisolin has three actin binding sites, two of which bind to actin monomers and a third which binds to actin molecules within a filament (11, 12). It has been proposed that the third may be the actin binding domain that allows geisolin to bind to the side of the filament before severing. Despite all of these findings, the overall mechanism by which geisolin regulates actin filament length has yet to be elucidated, and it may be conceivable that there remain unknown functional domains on the geisolin molecule that have yet to be brought to light for the final elucidation of the mechanism. Recently we have demonstrated that human plasma geisolin was specifically eluted from a Cibacron Blue F3GA affinity column with 1 mM adenosine, guanosine, cytidine, and uridine di- and triphosphates except for cytidine 5'-diphosphate, but neither mononucleoside 5'monophosphate nor dinucleotide (NAD and NADP) did so, indicating that the interaction of plasma geisolin and the dye-ligand is biospecific (23, 14). If this interaction is indeed biospecific, it is reasonable to expect that the protein will exhibit at least one binding site for mononucleoside polyphosphate. Since ATP is one of the major factors that regulate actin polymerization, the possible interaction of geisolin and ATP may be related to the geisolin funcVol. 108, No. 4, 1990
tionality. Therefore, the speculation that geisolin may exhibit binding sites for ATP provides a matter of considerable interest. The purpose of this communication is to describe direct evidence for the binding of ATP by human plasma geisolin. Human plasma geisolin was prepared for ATP binding studies as previously described (13), except that 25 mM Tris-HCl, 1 mM EGTA, 0.2 mM dithiothreitol, 5% glycerol, pH 8.0 was used as the buffer solution. Briefly, geisolin was purified from approximately 300 ml of citrated plasma by 30-45% ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Affi-Gel Blue chromatography coupled with affinity elution with 1 mM ATP. To prepare ATP-free gelsolin, active fractions from an Affi-Gel Blue column were combined and brought to 70% saturation by the addition of solid ammonium sulfate. The precipitate was redissolved to 1-2 mg/ml in approximately 10 ml of 25 mM Tris-HCl, 1 mM EGTA, 0.2 mM dithiothreitol, 5% glycerol, 135 mM NaCl, pH 7.5, and extensively dialyzed against 500 ml of the same buffer with 7 changes over a 4-day period to ensure complete removal of the ATP. The dialyzed geisolin preparation was centrifuged to remove any precipitates. Then, ATP in the geisolin preparation was assayed bioluminescently with luciferin and firefly luciferase [EC 1.13.12.7] (15, 16), and was found to be undetected. The geisolin preparation thus obtained was virtually homogeneous as judged by Coomassie Blue staining of SDS-polyacrylamide gels (14). The geisolin concentration was estimated using an extinction coefficient, E^° of 117,580 M"1 • cm"1, calculated from the number of tyrosine and tryptophan residues present in the human plasma geisolin (11). The binding of ATP to geisolin was examined by equilibrium dialysis. [2,8-'H] ATP, mixed with unlabeled ATP in the dialysis buffer, was used for the binding studies, and the ATP concentration in the reaction mixtures ranged from 0.1 to 10/*M. The purified geisolin was subjected to equilibrium dialysis at 4'C over a 3-day period, and the dialysis cell was rotated throughout the dialysis period. At the end of dialysis, aliquots of 0.1 ml of the dialyzates and the dialysis buffers were taken and counted for radioactivity in a liquid scintillation counter. The concentrations of free and protein-bound ATP were calculated, and binding constants were determined by the Scatchard method (17). 505
H. Yamamoto et al.
506 15-
Fig. 1. A: Binding of ATP to human plasma gelsolin. Gelsolin was mixed with [2,8-JH]ATP to make up the mixtures of IB5.7 //M of gelsolin and the indicated concentrations of ATP in 0.7 ml of 25 mM TrisHC1, 1 mM EGTA, 0.2 mM dithiothreitol, 1.55% glycerol, 135 mM NaCl, pH 7.5, and LJ each was subjected to equilibrium dialysis LJ against 2.8 ml of the same concentration of L- 10ATP solutions, respectively. At the end of Q dialysis, aliquots of 0.1 ml of the dialyzed Z preparations and the dialysis buffers were D O 0.5taken, counted for radioactivity, and then m the concentrations of free and proteinbound ATP were calculated. At the same time, gelsolin concentrations were mea04 0.6 0 02 sured to ensure that loss of the protein did BOUND ( A T P / G E L S O U N ) FREE ATP not occur by adsorption to the dialysis cell or the membrane during the dialysis period. Moles of ATP bound per mol of gelsolin were plotted as a function of free ATP concentration in micromolars. B: Scatchard plot of ATP binding to human plasma gelsolin. Bound ATP was expressed as mol of ATP per mol of gelsolin. Free ATP concentration was expressed in micromolars. The line was fitted by linear regression analyses and the coefficient of correlation was -0.99. 06-
A
I
Figure 1A shows that saturable binding of ATP to gelsolin occurred over the range of ATP concentrations indicated. The maximum ATP bound was 0.6 mol per mol of gelsolin based on linear regression, and the dissociation constant was calculated to be approximately 0.28 //M (Fig. IB). At concentrations ranging from 20 to 100 //M ATP, additional binding experiments yielded constant binding ratios of 0.64 ±0.04 (mean±SD) mol ATP per mol of gelsolin. This confirms that the saturation of ATP binding to gelsolin occurred around 10 //M as shown in Fig. 1A. The observed binding stoichiometry was consistently lower than the expected value of 1.0. Since the equilibrium analyses were performed over the range of 0.1 to 100 // M ATP, the effects of ligand self-association are unlikely at such low concentrations. It is, however, conceivable that the observed low binding ratios are likely to result from a substantial proportion of the inactive form in the gelsolin preparation which might have occurred during the preparation of ATP-free gelsolin. Alternatively, the gelsolin concentration may be overestimated by the above-mentioned theoretical determination. Anyhow, it should be mentioned that we confirmed the binding of ATP to gelsolin by the centrifuge column procedure described by Penefsky (28). Gelsolin was incubated overnight with various concentrations of radiolabeled ATP, and aliquots of the reaction mixture of gelsolin and ATP were applied to centrifuge columns to separate free and protein-bound ATP. Appropriate aliquots of column effluent were taken for measurement of protein and ATP, and the ratios of ATP bound to gelsolin were calculated. The results showed that the binding ratios were slightly underestimated by the centrifuge column procedure as compared to those obtained by equilibrium analyses (data not shown). The above-mentioned bioluminescent assay for ATP showed that the gelsolin contained a nucleotide as ATP. Additionally, plasma gelsolin did not show ATPase activity when it was measured for ATPase activity by coupling the reaction to the pyruvate kinase [EC 2.7.1.40] and lactic dehydrogenase [EC 1.1.1.27] systems at measuring the oxidation of NADH spectrophotometrically at 340 nm. At present, it is unknown whether the nucleotide interaction with gelsolin regulates the functional activity of the protein. However, it seems quite unlikely that a nucleotide
binding site of such high affinity would exist on gelsolin without being involved in eliciting some of the multiple functions attributed to this protein. Such a speculation comes from previous observations in which the ATP bound to the gelsolin-actin complex was nonexchangeable with free ATP while the ATP bound to the actin monomer was readily exchangeable (19-21). The ATP binding site on gelsolin proposed here may play a role in the nonexchangeability of ATP bound to the complex. Further studies are needed to clarify the affinity of gelsolin to various nucleotides and to locate the binding site on the gelsolin molecule. These points are currently under investigation. We are grateful to Mr. Seiichi Kohda of Oriential Yeast Co., Ltd. for assisting us in the bioluminescent assay for ATP. REFERENCES 1. Yin, H.L. (1987) BioEetays 7, 176-179 2. Norberg, R., Thorstensson, R., Utter, G., & Fagraeus, A. (1979) Eur. J. Biochan. 100, 575-583 3. Thorstensson, R., Utter, G., & Norberg, R. (1982) Eur. J. Biochem. 126, 11-16 4. Yin, H.L., Kwiatkowski, D.L., Mole, J.E., & Cole, F.S. (1984) J. BioL Chan. 269, 5271-5276 5. Yin, H.L. & Stossel, T.P. (1979) Nature 281, 583-586 6. Yin, H.L. & Stossel, T.P. (1980) J. Biol. Chan. 265, 9490-9493 7. Janmey, P.A. & Stossel, T.P. (1987) Nature 326, 362-364 8. Janmey, P.A., Iida, K., Yin, H.L., & Stossel, T.P. (1987) J. BioL Chan. 262, 12228-12236 9. Janmey, P.A. & Stossel, T.P. (1989) J. BioL Chan. 264, 48254831 10. Bryan, J. & Hwo, S. (1986) J. Cell BioL 102, 1439-1446 11. Yin, H.L., Iida, K., & Janmey, P.A. (1988) J. Cell Biol. 106, 805-812 12. Bryan, J. (1988) J. Cell BioL 106, 1553-1562 13. Yamamoto, H., Terabayashi, M., Egawa, T., Hayashi, E., Nakamura, H., &Kishimoto, S. (1989) J. Biochem. 105,799-802 Ito, H., Yamamoto, H., Kimura, Y., Kambe, H., Okochi, T., & Kishimoto, S. (1990) J. Chromatogr. 526, 397-406 Kricka, L.J. (1988) AnaL Biochem. 175, 14-21 Jabs, CM., Ferrell, W.J., & Robb, H.J. (1977) Clin. Chan. 23, 2254-2257 Scatchard, G. (1949) Ann N. Y. Acad. Sci. 51, 660-672 Penefsky, H.S. (1977) J. BioL Chan. 262, 2891-2899 Harris, H.E. (1985) FEBS Lett. 190, 81-83 Tellam, R.L. (1986) Biochemistry 25, 5799-5804
J. Biochan.
