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Notes Effect of Glass on the Polymerization of G-Actin to F-Actin Priya S. Niranjan,† Jeffrey G. Forbes,† and Sandra C. Greer*,†,‡ Department of Chemistry and Biochemistry and Department of Chemical Engineering, The University of Maryland at College Park, College Park, Maryland 20742-2111 Received March 6, 2000 Revised Manuscript Received April 17, 2000
1. Introduction Under appropriate conditions of temperature, concentration of actin, and concentration of salts, the globular protein G-actin aggregates reversibly to form the filamentary polymer F-actin.1 Since actin is an important protein, this process has been studied extensively, but the details of this reaction mechanism are still not well understood.2 The polymerization of actin, in vivo and in vitro, is induced by the presence of salts. We report here new experiments which show that in the absence of such added salts, the polymerization of rabbit muscle actin is induced by borosilicate glass containers but is not induced by vitreous silica containers. This result is of considerable importance in studies of actin polymerization, since the container is normally assumed to be inert. We have studied the fluorescence of pyrene-labeled rabbit muscle G-actin as a function of temperature.3 At low temperatures, the actin is below its polymerization temperature and does not polymerize.4,5 As the temperature increases and exceeds the polymerization or “floor” temperature, an increase in fluorescence indicates that polymerization is taking place.6 When KCl is added to freshly prepared actin in buffer solution in a vitreous silica vessel, we see clear evidence of polymerization of the G-actin; this has been observed by neutron scattering4 as well as by fluorescence labeling.3 For freshly prepared G-actin in a vitreous silica cell and in the absence of any added salts, we observe no evidence of significant polymerization at temperatures above the polymerization temperature. However, when the actin is contained in a glass cell in the absence of added salt, we see clear evidence of polymerization at the higher temperatures. 2. Materials and Methods 2.1. General. The fluorescence of pyrene-labeled actin in buffer was measured as the temperature was increased. The samples were contained in cells made from vitreous silica, from glass, and from silanized glass. * Corresponding author. Telephone: (301) 405-1895. Fax: (301) 4050523. Email:
[email protected]. † Department of Chemistry and Biochemistry. ‡ Department of Chemical Engineering.
2.2. Actin Preparation. As in our previous work,4 rabbit muscle acetone powder was prepared as described by Pardee and Spudich.7 The actin was extracted from the acetone powder into buffer A (4 mM Tris, 0.2 mM Na2ATP, 0.5 mM 2-mercaptoethanol, 0.2 mM CaCl2, 0.005% azide, in H2O). The resulting G-actin solution was polymerized, by adding KCl to a final concentration of 100 mM and by increasing the ATP concentration to 1 mM and the MgCl2 concentration to 2 mM, and then stored at 4 °C as F-actin stock solution at about 3 mg/mL actin. The stock solution was diluted to about 0.5 mg/mL; more KCl, ATP, and MgCl2 were added to ensure full polymerization, and the solution was ultracentrifuged at 150 000g to make a pellet of F-actin. The pellet was resuspended in buffer A and then depolymerized by dialysis in a collodion bag (10 000 Da cut off) against buffer at 4 °C with rapid stirring, for approximately 12 h. The resulting G-actin solution was then centrifuged at 120 000g for 1.5 h at 4 °C to pellet any remaining F-actin. The supernatant solution of G-actin was further purified by size exclusion chromatography with Sephadex G-150, using the same buffer A. The purified G-actin was studied within 48 h. All the vessels used in the actin purification were of plastic, except for the glass Sephadex column. G-Actin concentrations were obtained from the UV absorbance at 290 nm, using an extinction coefficient of 290 ) 0.63 cm2/mg.8,9 Actin purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The actin purity was analyzed before and after the experiments described below; both analyses showed very pure (>95%) actin. The fluorescence of pyrene-labeled actin is the most sensitive and accurate assay for actin polymerization.6 The F-actin was labeled for the fluorescence studies using the method used by Kouyama and Mihashi.10 F-Actin stock solution was diluted to 0.5 mg/mL, completely polymerized by again adding KCl, ATP, and MgCl2, and then dialyzed against buffer F (which is the same as buffer A but contains no 2-mercaptoethanol). N-(1-Pyrenyl)iodoacetamide (Molecular Probes, Eugene, OR) was added to the dialyzed G-actin solution in a 4:1 molar ratio of dye to actin and allowed to react overnight on ice. Dithiothreitol was added to a final concentration of 1 mM to remove unreacted dye. The sample was then ultracentrifuged at 120 000g for 1.5 h at 4 °C. A yellow pellet was obtained which was homogenized and depolymerized by dialysis against buffer A as described above. The dialyzed, labeled G-actin was purified on a Sephadex column as above. The labeled G-actin concentration was calculated by measuring the UV absorbance at 344 nm and using an extinction coefficient of 2.2 × 104 M-1 cm-1.10 The labeled and purified G-actin was
10.1021/bm005553g CCC: $19.00 © 2000 American Chemical Society Published on Web 06/10/2000
Notes
mixed with unlabeled purified G-actin to produce a mixture of 3% labeled actin and 97% unlabeled actin. The total G-actin concentration in the samples studied here was 3.1 mg/mL, and 0.25 mL of sample was injected into each cell. We chose this rather high concentration of actin in order to be able to compare these experiments to our earlier neutron scattering experiments, for which we needed high concentrations to have large scattering signals.4 2.3. Cells. The spectrometer cells (Starna Cells, Inc, Atascadero, CA) were made from (1) “special optical glass”, (2) “Spectrosil vitreous silica”, and (3) silanized “special optical glass” (see below). “Special optical glass” is a borosilicate glass (Schott Glass, Mainz). The optical windows of the Spectrosil cells are of Spectrosil and the other walls are of Vitreosil. “Spectrosil vitreous silica” is a synthetic quartz made from silicon dioxide formed by vapor phase hydrolysis of silicon. All cells had interior dimensions of 4 mm × 4 mm × 45 mm and nominal volumes of 0.56 mL. The cells were constructed by fusing the walls; thus no adhesives were present. The glass and vitreous silica cells were rinsed several times with 10% HCl and then cleaned by sonication in deionized water. All cells were oven-dried at 120 °C before use. Both glass and vitreous silica cells filled with Nanopure water showed no fluorescence at 407 nm. The glass cell to be silanized was first cleaned thoroughly and oven-dried. It was then placed for 20 min in a 2% solution of octadecyltrichlorosilane (Fluka) in hexane, after which it was rinsed in hexane and dried at 120 °C for 30 min. 2.4. Measurement of the Extent of Polymerization. The fluorescence was measured by an Aminco Bowman Series 2 luminescence spectrometer with the excitation wavelength, λex, set at 365 nm, resulting in emission wavelengths at λem ) 387 and 407 nm. The temperature at the start of each experiment was 0.5 °C; the temperature was increased in steps of 2 °C to a maximum of 30 °C, with a 25 min equilibration time after each temperature change. The increase in polymer concentration was determined by measuring the increase in the fluorescence signal at 407 nm. After the temperature reached about 30 °C, the sample was completely polymerized by bringing the concentration of MgCl2 to 15 mM. We then calculated the extent of polymerization of actin from the ratio of the fluorescence at a given temperature to the fluorescence of the fully polymerized sample.
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Figure 1. Extent of polymerization as a function of temperature for samples of 3.1 mg/mL rabbit muscle G-actin in buffer (see text), as measured by labeled fluorescence spectroscopy. The actin samples were held in cells made of (1) Spectrosil vitreous silica, (2) borosilicate glass, and (3) silanized borosilicate glass. Also shown are (4) measurements of the extent of polymerization of 2.9 mg/mL G-actin in buffer containing 9 mM KCl as initiating salt.3 The nonzero extent of polymerization at low temperatures reflects the fluorescence of G-actin, which has not been subtracted.
3. Results
cell. The nonzero extent of polymerization for that sample (and the other samples) reflects the fluorescence of G-actin, which has not been subtracted. Second, it is clear that considerable polymerization of the actin occurred in the borosilicate glass cell (2) as the temperature was increased, even though no initiating salts had been added to the actin. Compare the polymerization measured in sample (4) of 2.9 mg/mL G-actin in buffer A with 9 mM (0.04%) KCl added as an initiator for the polymerization. Third, the sample in the silanized borosilicate glass (3) also polymerized, but the polymerization began at a higher temperature than for the unsilanized borosilicate glass. Samples 1-3 were later analyzed by atomic absorption spectroscopy (Galbraith Laboratories) for Ca2+, Na+, and K+; analysis showed no presence of these species in any of the samples, at detection limits of 0.01%, except that Na+ was detected at 0.01% for the unsilanized borosilicate glass sample. The behavior in Figure 1 was reproduced with a second preparative batch of G-actin.
Figure 1 shows the extent of polymerization of G-actin containing no initiating salt as a function of temperature for samples in cells made of (1) Spectrosil vitreous silica, (2) borosilicate glass, and (3) silanized borosilicate glass. The measurements shown were all made on aliquots of G-actin taken from the same preparative batch. Also shown in Figure 1 for comparison are (4) our measurements of the extent of polymerization of G-actin from another preparative batch of 2.9 mg/mL actin with 9 mM KCl as initiating salt.3 We first note that negligible polymerization took place in the G-actin sample (1) that was studied in the vitreous silica
Figure 2 shows the time development of samples 1, 2, and 4 in Figure 1, after an increase in temperature of 2 °C from 20 °C. Sample 1 in a vitreous silica cell without salt remained unpolymerized.Sample 4 containing KCl in a vitreous cell showed an increase in extent of polymerization and reached a new equilibrium after about 30 min. By contrast, sample 2 in the glass cell with no salt showed an increase in extent of polymerization which had not reached equilibrium even after 70 min. Thus the data in Figure 1 for the glass cell (2) do not represent an equilibrated system.
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cleaning with acid serves to remove alkali metals.12 Spectrosil vitreous silica (or fused silica) is nearly pure SiO2, with less than 0.1 ppm Na, 0.3 ppm Ca, 0.1 ppm K, and 0.5 ppm Mg.13,14 Vitreosil is similar to Spectrosil, but does not have as good optical properties; Vitreosil has less than 2 ppm Na, 1.5 ppm Ca, 1.8 ppm K, and no significant Mg.14 Vitreous silica “is relatively unaffected by any acidic medium” except HF; HCl can remove traces of alkaline materials from vitreous silica.13 We suggest that studies of the polymerization of actin are better done in vitreous silica or plastic containers and that the inertness of any cell should be verified. Acknowledgment. This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, a part of the National Institutes of Health. We thank N. Blough for the use of his luminescence spectrometer and A. K. Hulme of Optiglass Ltd. (Essex, U.K.) for information on the cell materials. Figure 2. Extent of polymerization as a function of time for samples of 3 mg/mL rabbit muscle G-actin in buffer, as in Figure 1, after an increase in temperature of 2 °C from 20 °C.
4. Discussion and Conclusions Borosilicate glass vessels can cause the polymerization of rabbit muscle actin. We do not know the exact mechanism of this process. Silanization of the glass produces a hydrophobic coating which prevents the actin from contacting the glass surface. The actin in the silanized cell still polymerized, but that polymerization began at a temperature about 12 °C higher than in the case of the unsilanized cell. This result suggests that the polymerization in the borosilicate glass cell is due to the leaching of ions from the glass into the solution, rather than the polymerization of the actin by a process on the surface of the cell. This view is supported by the detection of a small amount of Na+ in the sample from the unsilanized cell and by the failure of that sample to reach an equilibrated level of polymerization even after 70 min. The known properties of borosilicate glass and of vitreous silica are consistent with our observations. Borosilicate glass generally contains Na2O, K2O, B2O3, and Al2O3, in addition to SiO2.11,12 It is known that water leaches ions from glass, that the alkali metals are preferentially removed, and that
References and Notes (1) Oosawa, F.; Asakura, S. Thermodynamics of the Polymerization of Protein; Academic Press: New York, 1975. (2) Sheterline, P.; Clayton, J.; Sparrow, J. C. Actins; 3rd ed.; Academic Press: San Diego, CA, 1996. (3) Niranjan, P. S.; Forbes, J. G.; Greer, S. C. To be published. (4) Ivkov, R.; Forbes, J. G.; Greer, S. C. J. Chem. Phys. 1998, 108, 5599-5607. (5) Greer, S. C. J. Phys. Chem. 1998, 102, 5413-5422. (6) Cooper, J. A.; Pollard, T. D. In Methods in Enzymology: Structural and Contractile Proteins, Part B, The Contractile Apparatus and the Cytoskeleton; Frederiksen, D. W., Cunningham, L. W., Eds.; Academic Press: New York, 1982; Vol. 85, pp 182-210. (7) Pardee, J. D.; Spudich, J. A.; In Methods in Enzymology: Structural and Contractile Proteins, Part B, The Contractile Apparatus and the Cytoskeleton; Frederiksen, D. W., Cunningham, L. W., Eds.; Academic Press: New York, 1982; Vol. 85, pp 164-181. (8) Maclean-Fletcher, S.; Pollard, T. D. Biochem. Biophys. Res. Commun. 1980, 96, 18-27. (9) Houk, T. W.; Ue, K. Anal. Biochem. 1974, 62, 66-74. (10) Kouyama, T.; Mihashi, K. Eur. J. Biochem. 1981, 114, 33-38. (11) Moore, J. H.; Davis, C. C.; Coplan, M. A. Building Scientific Apparatus: A Practical Guide to Design and Construction; 2nd ed.; Addison-Wesley: New York, 1989. (12) Adams, P. B. Ultrapurity Methods and Techniques; Zief, M., Speights, R., Eds.; Marcel Dekker: New York, 1972; pp 293-351. (13) Hetherington, G.; Bell, L. W. In Ultrapurity Methods and Techniques; Zief, M., Speights, R., Eds.; Marcel Dekker: New York, 1972; pp 353-400. (14) Hulme, A. K. Personal communication.
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