Comp. Biochem. Physiol. Vol. 76B. No. 3, pp. 529-533, 1983
0305-0491/83 $3.00+0.00 ~ 1983 Pergamon Press Ltd
Printed in Great Britain
R A N A CA T E S B E I A N A TAIL FIN LACTOGENIC H O R M O N E RECEPTOR: H Y D R O D Y N A M I C C H A R A C T E R I Z A T I O N RANDAL C. JAFFE Department of Physiology and Biophysics, University of Illinois at Chicago, Health Sciences Center, 901 South Wolcott Street, Chicago, IL 60612 USA (Tel: 312 996-7620)
(Received 13 April 1983) The hydrodynamic properties of the 125I-hGH-receptor-detergent complex of the tadpole tail fin were determined. 2. Based on the distribution coefficient from chromatography on Sepharose 6B the Stokes' radius of the complex was 5.03 nm. 3. Using centrifugation through sucrose/H20 and sucrosePH20 gradients the complex was determined to have a sedimentation coefficient of 4.05 S and a partial specific volume of 0.810cm3/g. 4. The ~25I-hGH-receptor-detergent complex was calculated to have a mol wt of 121,000 and a frictional ratio of 1.38. 5. Based on a calculated detergent content of 0.430g/g complex the mol weight of the 125I-hGHreceptor was 69,100 and the mol wt of the receptor alone was 47,100.
INTRODUCTION Mammalian prolactin causes increases in tadpole tail length and body weight (Bern et al., 1967; Etkin and Gona, 1967) and stimulates an increase in protein synthesis (Frye et al., 1972; Yoshizato and Yasumasu, 1972). Prolactin also inhibits thyroid h o r m o n e induced changes in metamorphosis including tail regression (Bern et al., 1967; Etkin and G o n a , 1967) and increases the activity of several tail lysosomal enzymes (Blatt et al., 1969; Jaffe and Geschwind, 1974). Neutralization of endogenous prolactin in the tadpole by administration of antisera to ovine (Eddy and Lipner, 1975) or frog (Clemons and Nicoll, 1977) prolactin accelerates metamorphosis. The action of prolactin and other peptide hormones in mammals is generally believed to be initiated by the binding of the h o r m o n e to specific binding proteins, termed receptors, in the plasma membrane. Specific prolactin binding sites in plasma membranes have previously been identified in the tadpole tail fin (White and Nicoll, 1979; Carr and Jaffe, 1980) which have similar characteristics to those in mammals. In this report we describe the hydrodynamic characteristics of the solubilized lactogenic h o r m o n e receptor from tadpole tail fin membranes.
MATERIALS AND METHODS
Materials ~zsI for iodination of human growth hormone was obtained from Amersham Corp. Human growth hormone (hGH; HS2243E) and ovine prolactin (oPRL; PS-14) were gifts from the NIAMDD, NIH. Triton X-100 was purchased from RPI International. Sepharose 6B, Sephadex G-50, Blue Dextran 2000, and protein standards for the calibration of columns were obtained from Pharmacia Fine Chemicals. The proteins used as standards for the density gradient sedimentation experiments were purchased from Sigma, and 2H20 was obtained from Aldrich. All other
chemicals were obtained from commercial suppliers. Rana catesbeiana tadpoles, 8-10cm in length, were purchased from Lemberger-Kons Scientific and maintained at 22°C on a diet of canned spinach until used.
Preparation of plasma membranes Tadpole tail fin membranes were prepared as described previously (Carr and Jaffe, 1980). In brief, the excised tissue was homogenized in a 1 mM sodium bicarbonate0.5 mM calcium chloride buffer, pH 7.0, filtered through cheesecloth and centrifuged at 900g for 20 min at 4°C. The resultant supernatant was then centrifuged at 20,000g for 20 min at 4°C. The 20,000g pellet was resuspended in 25 mM Tris-0.1% bovine serum albumin (BSA) buffer, pH 7.4, and frozen until used.
Preparation of labeled prolactin Iodinated hGH was prepared by the lactoperoxidase method of Thorell and Johannson (1971) and purified by chromatography on Sephadex G-50. The specific activity, determined as described by Shiu and Friesen (1974a), ranged from 90 to 120 Ci/g.
Binding reaction Following resuspension of the frozen membranes by homogenization, aliquots containing 0.15-0.30 mg of membrane protein were incubated with t25I-hGH (-600,000 cpm) in 25 mM Tris-10 mM MgCI2-0.1% BSA, pH 7.4, in a total volume of 1.5 ml. To an identical set of tubes an excess of unlabeled oPRL (6p.g) was added to determine the extent of nonspecific binding. After incubation for 20 hr at 19°C the tubes were cooled on ice and 2 ml of 10mM sodium phosphate, pH 7.0, was added. The tubes were then centrifuged at 20,000g for 15 min at 05°C, the supernatant was removed, and the pellets counted. Total binding was generally between 10 and 25% of the counts added of which approximately 40-50% represented specifically bound hormone.
Solubilization Solubilization was performed by adding 0.5 or 1 ml of 1% Triton X-100 in 10mM Tris, pH 7.4 (made up in 2H20 when the material was to be applied to sucrose/ZH20 gradients), to the membrane pellets. After homogenization and incubation for 1 hr at 20°C, the mixtures were 529
centrifuged at 100,000g for 1 hr at 0-4°C. The supernatant was removed and used for subsequent experiments. About 50% of the counts bound to the membrane were solubilized by this procedure. There was no difference between the amount solubilized using 0.5 and 1 ml of 1% Triton X-100-10 mM Tris.
measuring the radioactivity in a 7-counter. The refractive index was used to derive the density and viscosity of the individual fractions of the sucross/HzO gradients from standard tables while the density and viscosity of the individual fractions of the sucrose/~H20 gradients were derived as described by O'Brien et al. (1978).
Gel filtration Sepharose 6B chromatography was performed at 16ml/ hr in an ascending direction in a 1.6 x 80cm column equipped with flow adaptors. The column was equilibrated and eluted with 0.1% Triton X-100-10mM Tris, pH7.4. The column was calibrated by the method of Porath (1963) using the following proteins of known Stokes' radius: thyroglobulin, 8.50nm; ferritin, 6.10nm; catalase, 5.22 nm; aldolase, 4.81 nm; ovalbumin, 3.05 rim. The void volume (Vo) was determined by using Blue Dextran 2000 and the total volume (V,) with ~z~I. The distribution coefficient, Ka, was calculated from the elution volume (V¢) of a peak by:
Calculation o f sedimentation constants The sedimentation coefficients were calculated from the equation:
1-- 9pm.~ ( . c02trl...... J
r(t) ~](~)dr r(o) r[1
(2) 9plr) ]
where ? is the partial specific volume, c0 is the angular velocity in radians/sec, t is the sedimentation time in seconds, and r(o) to r(t) are the distances in centimeters from the rotor center through a gradient described by density p(,~ and viscosity ]](r)" P20.wand rho,. are the density and viscosity of water at 20°C (Tanford, 1961). Equation 2 was integrated using Simpsons' rule for various values of 9 as described by Smigel and Fleischer (1977). The calculated s2o,w values for both the sucrose/H20 and sucrose/ 2H:O gradients were then plotted vs 9. The point of intersection of the two curves defines the s2~,.~and ~, of the macromolecule.
V~-Vo Density gradient centrifugation Linear sucrose density gradients (4.6 ml) of 5-20% (w/v) sucrose in 0.1% Triton X-100-10mM Tris, pH 7.4, were prepared in H:O or ZH20. Aliquots (0.2 ml) of samples or standards were applied to the tops of the gradients. The standards were: ribonuclease, 1.78S, 0.703cm3/g (Richards and Wycoff, 1971); carbonic anhydrase, 2.75 S, 0.731 cm3/g (Armstrong et al., 1966); lactate dehydrogenase, 6.95 S, 0.730cm3/g (Schwert and Winer, 1963); aldolase, 7.70S, 0.742cm3/g (Taylor and Lowry, 1965; Sober, 1970). Centrifugation was carried out at 55,000rpm for 2.5 hr for sucrose/H:O gradients and for 5 hr for sucrose/ ~H:O at 4°C in a VTi 65 rotor. The bottoms of the tubes were then pierced and ~0.15 ml fractions collected. The position of the standards was determined by measuring the A~s0, and the position of the ~z5I-hGH was ascertained by
Other analytical methods Protein was determined by the method of Lowry et al. (1951) using BSA as the standard. All values represent the mean + standard deviation of three determinations. RESULTS
G e l filtration
T h e r e were t h r e e p e a k s of radioactivity w h e n the l : S I - h G H - r e c e p t o r - d e t e r g e n t c o m p l e x was fractionated o n S e p h a r o s e 6B (Fig. 1); o n e just prior to the
6' / I ,-J
Fig. 1. Sepharose 6B chromatography of ~25I-hGH-receptor-detergent complex solubilized with Triton X-100. ~25I-hGH (total binding, o) or 1251-hGHplus excess unlabeled oPRL (nonspecific binding, A) was incubated with tadpole tail fin membranes as described in the Materials and Methods. The lzSI-hGHreceptor complex was solubilized by resuspending the membrane pellets in 1 ml of 1% Triton X-10010 mM Tris, pH 7.4. After 30 min at 20°C the suspension was centrifuged at 100,000g for 1 hr at 4°C. The supernatant was applied to a 1.6 x 80cm Sepharose 6B column and eluted at a flow rate of 16 ml/hr. The solid line represents the difference between the total and nonspecific binding. Arrows indicate the elution position of (1) thyroglobulin, (2) ferritin, (3) catalase, (4) aldolase, (5) ovalbumin, and free hGH.
Tadpole lactogenic hormone receptor
Kd of 0.46+0.01. The smaller second peak had the same distribution coefficient as ~25IhGH. From a plot of the k~v3 of the proteins used as standards vs their Stokes' radii as described by Porath (1963) the Stokes' radius of the ~2SI-hGH-receptor-detergent complex was calculated to be 5.03+0.11 nm.
Sucrose density gradients The receptor bound radioactivity sedimented slightly faster than carbonic anhydrase in sucrose/ H20 gradients (Fig. 2A) while in sucrose/2H20 gradients (Fig. 2B) the peak of radioactivity was between ribonuclease and carbonic anhydrase. This suggests that the complex has a partial specific volume which is greater than that of the protein standards• oPRL displaced much of the labeled material from the peaks indicating that the hormone in those regions was specifically bound• Figure 3 presents the S2o,wvalues for the l~SI-hGH-receptordetergent complex as a function of the partial specific volume determined using equation 2 as described in the Materials and Methods. From the intersection of the sucrose/HzO and sucrose/ZH20 curves a sedimentation coefficient of 4.05 S and a partial specific volume of 0.810cm3/g can be assigned to the ~zsI-hGH-receptor-detergent complex.
Calculation of the molecular weight and amount of detergent bound The molar mass (M) of the 125I-hGH-receptordetergent complex was calculated by the formula (Siegel and Monty, 1966):
1 - ~P2o,. where r120,, is the viscosity of the medium, N is Avogadros' number, Rs is Stokes' radius, sz0.wis the sedimentation coefficient, ÷ is the partial specific volume, and P20.wis the density of the media. Using the values obtained above, the molecular weight of the ~zSI-hGH-receptor-detergent complex was calculated to be 121,000, 7"
Fig. 2. Sucrose/H20 (k) and sucrose/2H20 (B) gradients of solubilized ~2Sl-hHG receptor-detergentcomplex. Sucrose/ H20 and sucrose/2HzO gradients prepared as described in the Materials and Methods were centrifuged for 2.5 and 5 hr, respectively,at 55,000rpm in a Beckman TVi 65 rotor at 4~C. (O) Total binding; (e) nonspecific binding. Arrows indicate the position of(l) ribonuclease, (2) carbonic anhydrase, (3) lactate dehydrogenase, (4) aldolase and free hGH.
Z ,( tOr) :z
I~ Z W r~ UJ U~
position where catalase elutes, the second corresponds to the position of h G H and the most retarded peak at the total volume. If the membranes were incubated with ]2S-hGH plus unlabeled oPRL, there was a drastic decrease in the first peak and small reductions in the other two areas. The difference between the amount of radioactivity present when membranes were incubated with labeled hormone and labeled hormone plus excess unlabeled hormone, represented by the solid line in Fig. 1, is the specifically bound hormone. The major peak had a
° 4' N
PARTIAL SPECIFIC VOLUME g (cn~/g)
Fig. 3. Determination of szo., and 9 from sedimentation in sucrose/HzO and sucrose/2H20 gradients. The curves of the results from sucrose/H20 (o) and sucrose/ZH20 (e) gradients were calculated using equation 2 in the Materials and Methods for values of ~? from 0.70 to 0.84cm3/g with p(,) and ~1(~being derived from the refractive index. Error bars represent +1 SD from three separate experiments. The horizontal end vertical arrows indicate the szo., and ~,value, respectively, of the lzSI-hGH-receptor-detergent complex.
RANDAL C. JAFFE
The partial specific volume of the complex is the result of the contribution of the weight fraction of Triton X-100 (Xd) and its partial specific volume (~'d) and the contribution of the weight fraction of protein (Xp) and the partial specific volume of the protein (~,p) (Smigel and Fleischer, 1977): "~= Xp% + X~% If a value of 0.736cm3/g for the partial specific volume of a typical nonglycosylated protein and a value of 0.908 cm3/g for the partial specific volume of Triton X-100 above its critical micellar concentration (Tanford et al., 1974) are used, the weight fraction of Triton X-100 in the 125I-hGH-receptordetergent complex is 0.430g of Triton X-100/g complex. The molecular weight of the 125I-hGHreceptor complex is, therefore, 69,100 and based on a molecular weight of 22,000 for hGH (Li and Dixon, 1971) the molecular weight of the receptor is 47,100. Based on a molecular weight of 636 for Triton X-100 (Makino et al., 1973) there are 82 mol of Triton X-100/mol receptor. The frictional ratio OC/fo) of the lz~I-hGH-receptor-detergent complex, using the formula (Tanford, 1961):
R~L3M(') + 6/p20.w)J was calculated to be 1.38. The axial ratio of the complex is thus 7 if it is a prolate elipsoid and 8 if it is a oblate elipsoid (Svedberg and Perdesen, 1940). DISCUSSION
The lactogenic hormone receptor is an integral part of the plasma membrane. In order to study its characteristics it must be solubilized. We have previously shown that lactogenic hormone receptors solubilized with Triton X-100 retain their binding characteristics (Carr and Jaffe, 1982). In this study we have used 12SI-hGH rather than 12SI-oPRL as the labeled ligand. It appears to interact with the same set of receptors as prolactin in the tadpole (Carr and Jaffe, 1980) which allows the circumvention of the problems associated with the anomalous behaviour of labeled oPRL in Triton X100 (Shiu and Friesen, 1974b; Cart and Jaffe, 1981). The molecular weight of the 12SI-hGH-receptordetergent complex determined in this study, 121,000, is greater than that based on Ferguson plots of the mobility of the receptor during polyacrylamide disc gel electrophoresis in gels of different acrylamide concentration (Carr and Jaffe, 1981), 103,000. If we were to use 0.810 cm3/g for the partial specific volume of the 125I-hGH-receptor-detergent complex as determined in this study, the molecular weight of the complex from our previous study would be 93,000. The reason for this discrepancy is unclear as the molecular weight of the rat liver lactogenic hormone-receptor-detergent complex determined using the two different methodologies agree quite well (Jaffe, 1982). The results of this study indicate that more detergent on a mol/mol basis (82 vs 76) binds to the tadpole tail fin than rat liver lactogenic hormone
receptor (Jaffe, 1982). The calculated molecular weight of the tail fin lactogenic hormone receptor, 47,100, is much smaller than that of the rat liver lactogenic hormone receptor, 77,800 (Jaffe, 1982). Studies on purified receptor from both the tadpole and rat are necessary before conclusions can be reached as to whether the receptors are completely different or that the receptor has retained the same central core. This work was supported in part by National Institutes of Health Grant AM 18985.
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