97, 400-402 (1979)
of Anionic Sites on the Surfaces of Baby Hamster Cells Using Radiolabeled Polycationic Ferritin FREDERICKGRINNELLAND DONALD C.HAYS
Department of Cell Biology, The University of Texas, Health Science Center at Dallas, Dallas, Texas 75235 Received December 19, 1978 A method is described to determine relative numbers of anionic sites on the surfaces of cells under physiological conditions by binding studies with radiolabeled polycationic ferritin. Labeling of cells by polycationic ferritin occurred very rapidly even at 2°C and was essentially complete within 1 min. At 22°C a rapid initial phase of labeling was followed by a second, slower binding phase. The interaction of rapidly labeled cell surface anionic sites with polycationic ferritin had a binding constant of 3.6 x 106 M-’ (measured at 2’C) and there were about 4 x lo6 of these sites per cell.
All mammalian cells so far studied have a net negative surface charge (1). The magnitude of this charge has been quantitated by electrophoretic analysis (2). By the use of this technique, it has been found that more than 90% of the total cell surface charged groups are anionic. These are believed to consist primarily of the carboxyl groups of neuraminic acid, the y-carboxyl groups of aspartic acid and glutamic acid (1,2), and the carboxyl and sulfate groups of glycosaminoglycans (3). A direct method for studying the ultrastructural topography of cell surface anionic sites has been described (4). This method utilizes polycationic ferritin (PCF)’ as an electron-dense probe for the anionic sites. We have previously used PCF to study the distribution of anionic sites on baby hamster kidney (BHK) cells in suspension and attached to a substratum (5-7). We now describe a method to use radiolabeled polycationic ferritin (*PCF) to directly quantitate the anionic sites on BHK cells to which polycationic ferritin binds. * Abbreviations used: PCF, polycationic ferritin; BHK, baby hamster kidney; *PCF, radiolabeled PCF; PBS, phosphate-buffered saline. 0003-2697/79/120400-03$02.00/O Copyright AII rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
The basis for the cationization of ferritin is the exchange of amino groups for carboxyl groups by the formation of peptide linkages between diamino compounds and ferritin y-carboxyls using carbodiimide as a catalyst (4). This was accomplished as follows, using radioactive putrescine as the diamino compound. A 0.5-ml reaction mixture contained 15 mg of ferritin (Polysciences), 25 &i of 2,3-[N-3H]putrescine-2HC1 (20.64 CYmmole, New England Nuclear), and 40 mg of I-ethyl-3-(3-dimethyl)-carbodiimideHCl (Pierce Chemical) adjusted to ph 6.5 with 0.2 M HCl. After 30 min at 22°C 2 ml of 2 M putrescine-2HCl (Sigma Chemical) and 160 mg of 1-ethyl-3-(3-dimethyl)carbodiimide-HCl were added to the incubation. The pH was maintained at 6.5 by the addition of 0.2 M HCl for an additional 2 h . The incubation was continued at 22°C overnight, and then the solution was exhaustively dialyzed at 4°C against 0.15 M NaCl. In three different preparations, the final radioactivity of the product was 480, 422, and 462 cpm per pg of ferritin (E279= 79.9). *PCF was stored at 4°C. 400
SITES ON HAMSTER
All cell-labeling experiments with *PCF 22--c 0 were carried out in 0.5 ml of modified Dulbecco’s PBS consisting of 150 mM NaCl, 3 mM KCl, 1 mM CaCl,*2&0, 0.5 mM MgC12.6Hz0, 6 mM NaHPO,, and 1 mM KH2POI, pH -7. The incubations contained 2.5 x lo6 BHK cells (subline BHK0 21-13s described in Ref (5)). These condi01 5 10 15 20 tions are similar to those we have preMINUTES viously used in ultrastructural studies FIG. 1. Time dependence of PCF binding of using PCF. BHK cells. The incubations were carried out at 2 or 0
RESULTS AND DISCUSSION
In order to accurately quantitate *PCF binding, it was desirable to be able to rapidly stop the reaction of *PCF with the cells. Initially, we tried to prevent the reaction by the addition of formaldehyde or by dilution of the reaction mixtures; however, these treatments only partially blocked *PCF binding to the cells (Table 1, Expt 1). The addition of excess nonradiolabeled TABLE EFFECT
OF VARIOUS CONDITIONS BINDING TO BHK CELLS”
Control + formaldehyde, 1.5% diluted 20-fold at time 0 + PCF, 1.1 mg
2058 991 1023 616
0 51 SO 70
Control + heparin, + heparin, + heparin, + heparin,
1049 1162 663 233 332
0 0 37 78 68
0.1 units 1.0 units 10 units 100 units
a The incubations were carried out for 1 min at 2°C as described in the text. The reactions were initiated by the addition of 0.025 ml of *PCF (Expt 1: 88 pg, 480 cpm/pg; Expt 2: 43 pg, 422 cpm/pg) and stopped by the addition of 5.0 ml of 0.15 M NaCl. The diluted samples were centrifuged at 800g for 2 mitt, washed with an additional 5.0 ml of 0.15 M NaCl, recentrifuged, and the pellets were resuspended and analyzed for radioactivity in a Nuclear Chicago Mark II scintillation spectrophotometer.
2FC as indicated, and the reactions were stopped after the time periods shown by dilution with 0.15 M NaCl containing 20 units/ml of heparin. The *PCF concentration in the incubations was 43 pg (422 cpm/l.cg). Other details are in Table 1 and the text.
PCF inhibited the reaction more completely, but the high levels of PCF required made this technique very costly. Subsequently, we found that the reaction between *PCF and the cells could be 7080% inhibited by the addition of heparin (20 units/ml) to the reaction mixtures (Table 1, Expt 2), and control experiments demonstrated that heparin did not cause removal of already bound *PCF. Therefore, our standard method for stopping the reactions became: dilution of the reaction mixtures 20-fold with 0.15 M NaCl containing 20 units/ ml of heparin. This technique was quite effective, as shown by our ability to quantitate differences in *PCF binding to BHK cells in short-term incubations (e.g., Fig. 1: 0 time, 10 s, 30 s, 1 min). 3000
50 pG ‘PCF
FIG. 2. Effect of *PCF concentration on *PCF binding to cells. The radioactivity of the PCF used was 462 cpm/pg. Other details are in the text and legend to Fig. 1.
100 (A) MOLES’
FIG. 3. constants analyzed x (l/Z’) +
Double-reciprocal plot to determine kinetic for PCF binding. The data in Fig. 2 were according to the equation, l/B = (l/K .T) l/T. This was derived for the reaction, F+P& B. The symbols are B, bound anionic sites; K, binding constant; P, *PCF concentration; T, total anionic sites; and F, free anionic sites. We assume that *PCF binding to the cells is a measure of Z?, that free anionic sites equals total less bound anionic sites, and that the *PCF concentration is much greater than the amount of *PCF which is bound. The graph is for l/B vs l/P anionic sites. Other details are in the text and legend to Fig. 2.
BHK cells were incubated with *PCF for various time intervals and the incubations were carried out at both 2 and 22°C. The results are shown in Fig. 1. The rate of initial labeling was very fast. At 2°C labeling was more than 50% complete in 10 s and essentially reached its full extent in 1 min. Labeling at 22°C was biphasic. The initial rapid phase of labeling was followed by a second, slower binding phase. The rapidity of binding is in good agreement with our previous ultrastructural studies in which we demonstrated extensive labeling of BHK cells with PCF after a 10 s incubation (5). Cells were incubated with various concentrations of *PCF ranging from 9 to 180 pg. The experiments were carried out for 1 min at 2°C; therefore, only the rapid binding reaction was measured. The results are shown in Figs. 2 and 3. Saturation of *PCF binding sites appeared to occur at high *PCF concentrations (Fig. 2). The data from Fig. 2 were converted to moles, assuming a
molecular weight for *PCF of 467,000, and replotted in a double-reciprocal plot as shown in Fig. 3. A least-squares analysis of the data indicated that the slope of the line was 8.28 and the y intercept was 58.9 x log mol-l. They intercept is the reciprocal of total moles *PCF bound per 2.5 x lo6 cells from which it can be calculated that there are about 4.1 x lo6 binding sites/cell. To calculate the binding constant, the y intercept was corrected to molar concentration (+2000 = 29.5 x lo6 M-‘). K equals 3.6 x lo6 M-r, the y intercept divided by the slope. These results indicate that radiolabeled polycationic ferritin can be used to quantitate the cell surface anionic sites to which this ligand binds. This method can easily be used to compare the relative number of anionic sites on cells under various conditions, e.g., before and after transformation, during development, etc. Also, because *PCF can also be used as an ultrastructural probe, it will be possible in the future to do simultaneous quantitative and topographical analyses of cell surface anionic sites. ACKNOWLEDGMENTS This research was supported by National Institutes of Health Grants CA 14609 and GM 21698.
REFERENCES 1. Weiss, L. (1%9) Znt. Rev. Cyrol. 26, 63-105. 2. Merishi, J. N. (1972) Prog. Biophys. Mol. Biol. 25, l-68. 3. Kraemer, P. M. (1971) Biochemistry 10, 14371445. 4. Danon, D. L., Goldstein, L., Marikovsky, K., and Skutelsky, E. (1972) J. Ultrasrruct. Res. 38, 500-510. 5. Grinnell, F., Tobleman, M. Q., and Hackenbrock, C. R. (1975) J. Cell Biol. 66, 470-479. 6. Grinnell, F., Tobleman, M. Q., and Hackenbrock, C. R. (1976) J. Cell Biol. 70, 707-713. 7. Grinnell, F., Anderson, R. G. W., and Hackenbrock, C. R. (1976) Biochim. Biophys. Acta 426, 772-775.