Biochemistry 1985, 24, 6095-6107 Martinez, O., Vazquez, D., & Modolell, J. (1978) FEBS Lett. 87, 21-25. Melangon, P., Boileau, G., & Brakier-Gingras, L. (1984) Biochemistry 23, 6697-6703. Nierhaus, K. H. (1982) Curr. Top. Microbiol. Immunol. 97, 81-155. Ofengand, J . (1980) in Ribosomes: Structure, Function and Genetics (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L., & Nomura, M., Eds.) pp 497-529, University Park Press, Baltimore, MD. Olson, H . M., Grant, P. G., Cooperman, B. S . , & Glitz, D. G. (1982) J . Biol. Chem. 257, 2649-2656. Ozaki, M., Mizushima, S., & Nomura, M. (1969) Nature (London) 222, 333-339. Schreiner, G., & Nierhaus, K. H. (1973) J . Mol. Biol. 81, 71-82. Schwartz, I., Vincent, M., Strycharz, W. A., & Kahan, L. (1983) Biochemistry 22, 1483-1489. Skold, S . E. (1981) Biochimie 63, 53-60. Stoffler, G . , & Wittmann, H. G. (1977) in Molecular Mechanisms of Protein Biosynthesis (Weissbach, H., & Pestka, S., Eds.) pp 117-202, Academic Press, New York. Stoffler-Meilicke, M., Epe, B., Steinhauser, K. G . , Woolley,
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P., & Stoffler, G. (1983a) FEBS Lett. 163, 94-98. Stoffler-Meilicke, M., Noah, M., & Stoffler, G. (1983b) Proc. Natl. Acad. Sci. U.S.A. 80, 6780-6784. Swank, R. T., & Munkres, K. D. (1971) Anal. Biochem. 39, 462-477. Takahashi, K. (1968) J . Biol. Chem. 243, 6171-6179. Tolan, D. R., Lambert, J. M., Boileau, G., Fanning, T. G., Kenny, J. W., Vassos, A., & Traut, R. R. (1980) Anal. Biochem. 103, 101-109. Traut, R. R., Lambert, J. M., Boileau, G., & Kenny, J. W. (1980) in Ribosomes: Structure, Function and Genetics (Chambliss, G.,Craven, G. R., Davies, J., Davis, K., Kahan, L., & Nomura, M., Eds.) pp 89-1 10, University Park Press, Baltimore, MD. Tritton, T. R. (1978) Biochemistry 17, 3959-3964. Vazquez, D. (1979) Mol. Biol., Biochem. Biophys. 30, 89-95. Wallace, B. J., Tai, P. C., & Davis, B. D. (1979) in Antibiotics I/-Mechanism of Action of Antibacterial Agents (Hahn, F. E., Ed.) pp 272-303, Springer-Verlag, Berlin. Wittmann, H. G., & Wittmann-Liebold, B. (1974) in Ribosomes (Nomura, M., Tissisres, A., & Lengyel, P., Eds.) pp 115-140, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
H2 Histaminic Receptors in Rat Cerebral Cortex. 1. Binding of [ 3H]Histaminet Glenn H. Steinberg, J. Gary Eppel, Marianne Kandel, Stephen I. Kandel, and James W . Wells* Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 1A1 Received December 18, 1984; Revised Manuscript Received April 18, 1985
ABSTRACT:Saturable binding of [3H]histamine in equilibrium with homogenates of rat cerebral cortex reveals
Hill coefficients between 0.4 and 1.O, depending upon the conditions. Data from individual experiments are well described assuming one or two classes of sites. Only the sites of higher affinity (Kpl = 3.9 f 0.5 n M ) are observed when binding is measured by isotopic dilution a t a low concentration of the radioligand ( 0.15) protein; for G M P - P N P added with the radioligand (.-D-), koff = when the value of nH1 is determined by regression The mean 0.0080 f 0.0013 s-' and B,,,, = 21.6 f 0.3 pmol/g of protein. Zero Hill coefficient is 0 . 9 6 f 0.03 (Table II), and the data thus percent on the ordinate is taken as the level of binding in control are well described assuming a single population of sites. The experiments containing 1.0 m M unlabeled histamine added at the same time as the radioligand; absolute values are 18.9 (O), 17.8 ( O ) , and mean value of KpI obtained from eq 6 (n = 1) is 3.8 f 0.8 nM One hundred percent on the ordinate 16.5 pmol/g of protein (0). (log KpI = -8.42 f 0.09; Table 11). 'Ihe affinity of histamine is taken as B,,, for the experiment without GMP-PNP (54.3 pmol/g determined by isotopic dilution thus is virtually identical with of protein) and for the experiment in which G M P - P N P was added that determined by varying the corice~itrationof the radiowith unlabeled histamine (35.4 pmol/g of protein). For the experiment ligand. A combination of results from the 11 experiments in which G M P - P N P was added with the radioligand, 100% is taken as the level of binding after 45 min (45.3 pmol/g of protein) in a control summarized in Table I1 yields a mean value for Kp, of 3.90 sample from which the nucleotide was omitted; B,,, in the presence f 0.53 nM (log Kpl = -8.409 f 0.059) Equation 6 also can of the nucleotide was 35.2 pmol/g of protein. be used to estimate the capacity from data obtained by isotopic dilution, and values range from 40 to 207 pmol/g of protein et al. ( 1 9 8 0 ) thus arises from a noncompetitive effect on the rate of dissociation. GMP-PNP reduces but does not eliminate for the seven experiments summarized in Table 11. Owing to binding at equilibrium (Barbin et al., 1980); it thus is possible the low concentrations of [ 3 ~ 1histamine ] relative to Kpl, to add the nucleotide with the radioligand rather than with however, [R,], and K,, are highly correlated in individual unlabeled histamine and thereby to measure the rate of disexperiments. This dependence has been avoided by estimating sociation for binding that coexists with the nucleotide. The [R,], according to eq 5 (n = 1 ) with K p , fixed at the mean rate constant determined in this manner is not significantly value of 3.9 nM. Capacities estimated in this way tend to show
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LOG CHISTQMINE? Inhibition of [3H]histamineby unlabeled histamine at four concentrations of the radioligand. Total binding was measured following incubation of the suspension with 1.22 ( O ) , 3.49 (0), 5.84 (O),or 10.1 nM (A)[3H]histaminetogether with unlabeled histamine at the concentrations shown on the abscissa. The lines represent the best fit of eq 4 (n = 2) to the four sets of data taken together, with a common value of K2 and separate values of K 1 ,F2,B,, and Bmin. Fz was set to zero for the data at 1.22nM [3H]histamine. Parametric values obtained by regression are as follows: log K2 for all curves, -6.63 i 0.03; (0) log K , = -8.27 f 0.02; (0) log K1 = -8.61 i 0.02 and Fz = 0.18 f 0.01; (0)log K l = -8.80 f 0.03 and Fz = 0.24 f 0.01; (A)log Kl = -9.05 i 0.05 and Fz= 0.61 i 0.01. Values plotted on the ordinate are normalized at 100%and 0% to the values of B,,, and B,,, respectively, obtained from eq 4; successive curves are offset by 25% for clarity. Points at the lower limit on the abscissa represent binding in the absence of unlabeled histamine. log K 1 and log K2 are indicated by the arrows and F2 by the dashed lines. FIGURE4:
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rc1TRL C 3 H 3 H I S T R M I N E (nM1 FIGURE 3 . Binding of [3H]hi~tamine at different concentrations of
the radioligand with and without GMP-PNP. Total binding (A) was measured following equilibration of the suspension (0.90 mg of protein/mL) with [3H]histamine alone (0),in the presence of 0.1 mM GMP-PNP ( O ) , or in the presence of 1.0 mM unlabeled histamine (0). The lines represent the best fit of eq 6 (n = 1) to the experimental data taken together, with a common value of KpI and separate values of [R,], for data in the absence and presence of GMP-PNP parametric values obtained by regression are as follows: Kpl = 6.12 f 0.06 nM, [R,], (without GMP-PNP) = 214 f 11 pmol/g of protein, [RJ, (with GMP-PNP) = 137 f 8 pmol/g of protein, and C = 16.4 f 0.2 pmol g-l nM-'. The data also were fitted assuming separate values for both Kp, and [R,], (fit not shown), with little change in the variance of residuals (P = 0 58); parametric values obtained by regression are as follows: without GMP-PNP, Kpl = 6.3 i 0.7 nM and [R,], = 217 f 13 pmol/g of protein; with GMP-PNP, Kpl = 5.7 f 0.9 nM and [R,]! = 132 f 12 pmol/g of protein; C = 16.4 f 0.2 pmol g-I nM-'. Specific binding (B) represents total binding less the fitted estimate of nonspecific binding and is corrected for the small increase in the latter that occurs in the presence of excess, unlabeled histamine (Seeman et al., 1984) less variability; for the data summarized in Table 11, the range is from 66 to 188 pmol/g of protein, and the mean value is 104 f 16 pmol/g of protein. The mean capacity from the 11 experiments listed in Table I1 is 107 f 15 pmol/g of protein a t the sites corresponding to KpI. The consistent results obtained from two approaches to the estimation of KPl,together with the good agreement between fitted curves and the experimental data, suggest that binding is to a uniform population of mutually independent sites. This consistency is lost, however, when isotopic dilution is carried out a t concentrations of the radioligand exceeding 1.5 nM. The data illustrated in Figure 4 are representative of the results from eight experiments performed a t four concentrations of [3H]histamine. The Hill coefficient decreases from 0.95 to about 0.46 (Table III), and the binding patterns become distinctly biphasic, as the concentration of radioligand increases from below 1.5 to 10 nM. At 3.3 n M [3H]hi~tamine and
above, good but empirical descriptions of the data are obtained with eq 4 assuming two classes of sites differing in affinity for the unlabeled drug. There is excellent agreement between duplicate experiments a t each concentration of the radioligand: parametric values are virtually identical (Table 111), and the data are superimposable when normalized on the ordinate to the fitted values of B,,, and Bmin(not shown). Moreover, the increase in the variance of residuals a t each concentration of [3H]hi~tamineis negligible (P4> 0.5) when eq 4 is fitted to the two sets of data taken together rather than separately and assuming common rather than separate values of K , , K2, and F2.
The fitted curves in Figure 4 are obtained from simultaneous analysis of the four sets of data assuming a single value of K2 common to the three curves wherein it appears but separate values of K , , F,, B-, and B-; the global variance of residuals is not increased significantly (P = 0.30) over that obtained assuming separate values for all parameters, indicating that K2 is independent of the concentration of [3H]hi~taminefor the data shown in the figure. A similar result is obtained for all eight experiments summarized in Table 111. Simultaneous analysis assuming a single value of K2 and four values of K , and F2,one for the pair of curves a t each concentration of [3H]histamine, yields a variance of residuals not significantly greater (P= 0.57) than that obtained assuming three values of K2. If the inhibition corresponding to F2 is competitive, the absence of any change in K2 implies that the concentration of radioligand is well below its equilibrium dissociation constant (Kps) for the sites under consideration. For the two experiments a t about 10.5 n M [3H]histamine, the highest concentration used, a mean value of 226 n M can be calculated for Kp2 (log Kp2 = -6.65 f 0.01) from the expression Kp2 = K 2 - [free [3H]histamine], which assumes that the labeled and unlabeled forms of the drug bind with the same affinity. A similar calculation yields a mean value of 247 n M (log Kp2
V O L . 2 4 , N O . 22, 1 9 8 5
CHARACTERIZATION O F H2 RECEPTORS
6101
Table 11: Mean Parametric Values for the Specific Binding of Histamine with and without Magnesium and GMP-PNP" vary [,H]histamineb vary unlabeled histamine' eq 6d eq sef eq 6e [GMP-PNP] [Rilr eq 3d eq 4e B11r [R2lr (mM) N -log K p I bmoI/g) N nHI F, -log Kpl -Ion K p , (umol/n) ( p m o l / d 0 4 8.40 f 0.07 65-217 7h 0.96 f 0.03 g 8.42 f 0.09 g 66-188 g 0.1 2 8.33 f 0.09 30-132 5' 0.66 f 0.05 0.25 f 0.06 8.52 f 0.10 6.57 f 0.10 17-68 432-1340 0 0 2 8.35 f 0.17 54-75 2' 0.64 f 0.04 0.48 f 0.02 8.38 f 0.04 6.83 f 0.15 26-48 1090-1900 "Values listed in the table reflect best fits of each equation to the data from independent experiments; the number (N)of independent experiments is indicated. The reaction mixture in all experiments contained 100 mM Tris and 1.0 mM EDTA. bTotal binding was measured at various concentrations of the radioligand with and without 1.0 mM unlabeled histamine (e.g., Figure 3). 'Total binding was measured at a single concentration of the radioligand (1.0-1.5 nM) and various concentrations of unlabeled histamine (e.g., Figure 6). dThe expression was fitted assuming a single class of sites ( n = 1). eThe expression was fitted assuming two classes of sites ( n = 2) except where indicated otherwise. 'Values of log KpI and log Kp2 were fixed at -8.409 and -6.655, respectively, as described in the text. gF2 was fixed at zero. *Values of PI:>0.2 (6), 0.023; P2: >0.15 (6), 0.016. 'Values of PI:>0.1 (2),
