The Biochemical Characterization of Detergent-solubilized Insulin-like Growth Factor I1 Receptors from Rat Placenta*

A membrane preparation, the Rat obtained by differ- ential centrifugation of rat placental homogenates is enriched in receptors that bind insulin-like growth factor I1 (IGF-11) preferentially and with avidity 282-288). When this preparation was incubated with 2% (w/v) octyl-B-D- glucopyranoside for 60 min at 0-4 OC, 60% of the membrane protein was solubilized without loss of bind- ing activity. The 12SI-IGF-II binding properties of the detergent-solubilized receptors were found to be similar to those of the membrane-associated receptor. The rate constants for association, k,, and dissociation, k,, and equilibrium dissociation constant, KD, were 8.5 X 10' M-' min", 7.5 X min", and 1.3 nM for the detergent-solubilized receptors and 5.3 X 10' M" min-l, 4.2 X min", and 0.6 nM for the membrane receptors.

and equilibrium dissociation constant, KD, were 8.5 X 10' M-' min", 7.5 X min", and 1.3 nM for the detergent-solubilized receptors and 5.3 X 10' M" min-l, 4.2 X min", and 0.6 nM for the membrane receptors.
Gel chromatography on Sephacryl 5-300 concentrated the solubilized receptors into a major peak of binding activity with a Stokes radius of 7.2 nm; a second peak of less specific binding had a Stokes radius of 4.3 nm. The receptors in the major peak bound 12'1-IGF-I1 with a KO of 0.6 nM; the total binding capacity, Ro, was 21.6 pmol mg of protein" compared to 1.6 pmol mg of protein" for the membrane-associated receptor. Centrifugation of the receptors on 5-20% (w/ v) gradients of sucrose in H 2 0 or D 2 0 disclosed a heterogeneous pattern of receptor distribution. When they were labeled with 12SI-IGF-II prior to centrifugation, a major form of the receptor with a sedimentation constant, s2,,+,, of 9.9 X l O l 3 s and other, possibly smaller, forms of the receptor were observed. However, only the 9.9 s20,w form of the receptor was observed if it was labeled with 1261-IGF-II subsequent to centrifugation. Based on these hydrodynamic measurements and a partial specific volume of 0.72 cm3/g, the IGF-I1 receptor was calculated to have a M, of 290,000 and frictional ratio, f/fo, of 1.6. This value for the M, is similar to the mass of 220,000 or 250,000 Dal de-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. termined by cross-linking '2SI-IGF-II to the membrane-or detergent-solubilized receptors with disuccimidyl suberate and separating the complex by electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels in the absence or presence of dithiothreitol, respectively.
Purification of serum Sm' and NSILA from human serum have established two classes of growth factors (1) with similar molecular weights (2), i.e. 7,500 and biological properties (3)(4)(5) but with different amino acid sequences and PI values. One class of somatomedins has alkaline PI values and includes the peptide IGF-I whose structure has been established. Somatomedin C ( 6 ) and basic somatomedin (7) appear to be similar if not identical in structure. The rat also contains an analogous basic somatomedin of very similar structure (8). The second class of somatomedins are neutral to acidic in PI and is represented in human serum by IGF-11, a peptide of defined structure (1,2), and ILAs, a peptide of slightly more acidic PI (9). MSA is the name of a mixture of peptides with somatomedin activity contained in the conditioned medium of cultured Buffalo rat liver 3A cells (10); one component of this mixture is a peptide with 93% sequence homology with human IGF-I1 (11). IGF-11-like radioreceptor detectable material is present in rat serum (8).
Due to the presence of a high degree of structural homology between insulin and Sm/IGF (12), Sm/IGFs are thought to stimulate the uptake and metabolism of hexose in adipocytes (3) and human fibroblasts (13) by interacting with the insulin receptor. However, evidence based on their effect on muscle metabolism, on their stimulation of DNA synthesis by avian fibroblasts, and on competitive RRAs are consistent with the hypothesis that Sm/IGF binds to receptors that are distinct from the insulin receptor and that those receptors that preferentially bind the alkaline class of Sm/IGF are different than those that bind the neutral-acidic growth factors (3)(4)(5). This conclusion has been confirmed recently; the receptors and their presumptive subunits have been identified by SDS-PAGE and radioautography in experiments employing isolated receptors for insulin (14) and basic Sm/IGF (15) and membrane-associated receptors that were cross-linked with iodinated ligands by photoactivated mechanisms (15,16) or by the bifunctional reagent DSS. The general picture that is emerging indicates that the insulin and basic Sm/IGF receptors are immunologically and structurally related. Antibodies to the insulin receptors recognize determinants on the receptor that bind IGF-I (17) and both are formed from 140-and possibly 95and 45-kDa subunits that are disulfide-linked to form protoaggregates of 300-400 kDa (18)(19)(20)(21). As predicted from the results of competitive binding studies (3)(4)(5), the receptors which bind the neutral-acidic Sm/IGFs are structurally very different from the basic Sm/IGF receptor. They appear to be single chained polypeptides of about 220 kDa that are maintained by internal disulfide bonds (18,19). This was concluded from cross-linking studies with DSS, but to date no one has reported on the structural composition of the isolated receptor. Therefore, we have undertaken this task and have chosen as our tissue source the rat placenta since it has been shown by Daughaday et al. (22) that it is enriched in receptors which bind IGF-11, and deficient of those which bind IGF-I. In this paper, we present our initial results on the characterization of detergent-solubilized IGF-I1 receptors.

MATERIALS AND METHODS
Aldolase, catalase, ferritin, thyroglobulin, and Sephacryl S-300 were purchased from Pharmacia; the "Good Buffers" including Bicine, HEPES, and MES, bovine serum albumin, myosin, ovalbumin, PMSF, Tris, and DzO were from Sigma; 8-galactosidase, lactate dehydrogenase, and phosphorylase a were from Boehringer-Mannheim; filamin was from Transformation Research, Farmington, MA; sodium dodecyl sulfate and the chemicals and equipment used for electrophoresis were from Bio-Rad; n-octylglucoside was from Calbiochem-Behring; DSS was from Pierce Chemical Co.; Cronex x-ray film was from DuPont Chemical Co.; [12SI]NaI was from Amersham; and other chemicals were from local sources. Highly purified IGF-I (16 S P 11), IGF-I1 (10 SE IV), and an impure IGF preparation were generously provided by Dr. Ren6 Humbel of Zurich, Switzerland. The impure preparation had a specific activity of 36 milliunits/mg of protein and was stated to contain 44 pg of IGF-I and 82 pg of IGF-II/mg of protein. It and a partially purified preparation of MSA were used as the competing ligands for certain of the experiments. The MSA was prepared by concentrating and washing conditioned medium from cultured rat liver cells (BRL-3A) on an Amicon ultrafiltration system using a hollow fiber cartridge with a 5-kDa cutoff. Following lyophilization and resuspension in 0.2 M acetic acid, the soluble peptides were resolved by chromatography in acetic acid on a column (2.5 X 100 cm) containing Sephadex G-75. The peak of activity, as determined in an RRA (22) was lyophilized and stored at -70 "C. Ten pg of protein was estimated to contain 100 ng of IGF-11-like activity when it was compared with the pure standard in an RRA. Finally, '"I-transferrin and rat placentae were the generous gifts of Drs. H. Schulman of Montreal and W. A. Peck of St. Louis, Mo, respectively.
Preparation of Rat Placental Membrane-Rat placentae that had been obtained from animals pregnant for 18-19 days and stored at -70 "C were thawed, cut into small pieces, diluted 1:l (w/v) with 250 mM sucrose, 1 mM PMSF and dissociated with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 2,000 rpm for 20 min (9,600 X g.min) in a Sorval SS-34 rotor. The pellet (R,) was discarded and the supernatant centrifuged at 11,500 rpm for 30 min (480,000 X g.min). The supernatant was decanted and centrifuged at 6 X lo6 X g.min to yield a pellet, the RS, and a particulate-free supernatant which was discarded. The R3 and the pellet obtained during the 480,000 X g . min centrifugation, the Rzr were resuspended in 50 mM Na2HP04, 1 mM PMSF and washed one time with 100 mM NaCI, 1 mM MgS04. The washed and unwashed preparations were analyzed for protein by the procedure of Lowry et al. (23), used in I-IGF-I1 binding experiments, or extracted with n-octylglucoside. Detergent Extraction of Rat Placental Membrane-Numerous detergents and extraction regimens were evaluated for solubilizing rat placental IGF-I1 receptors. Some of our observations will be discussed 12s under "Results." The procedure currently in use is the following. Rs, at a protein concentration of 5 mg/ml was stirred for 2 h at 0-4 "C in 50 mM NaZHP04, 1 mM PMSF, pH 7.4, containing 20 mg/ml of noctylglucoside. Following incubation, the detergent-treated suspension was centrifuged 6 X lo9 X g. min and the supernatant decanted and dialyzed for 4 h against 10 volumes of buffered 0.2% (w/v) noctylglucoside. Following centrifugation at 6 X lo9 X g.min, the clarified extract was divided into aliquots and stored frozen at -70 "C or evaluated for IGF-I1 binding.
Iodination of Insulin-like Growth Factors-IGF-I and IGF-I1 were iodinated by the lactoperoxidase procedure and free iodine separated from proteins by Sephadex G-50 gel filtration in 100 mM acetic acid as previously described (22). Specific activities of 40-100 pCi/pg were obtained for the various ligands.
Membrane and Detergent-solubilized Radioreceptor Assay for Znsulin-like Growth Factors-The methods employed to measure Iz5I-IGF-I1 binding to the placental membranes, i.e. R1, Rz, or RB, or to receptors in detergent-solubilized extracts of RS were essentially as described by Li and Perdue (24). In brief, 50-350 pg of membrane protein was incubated for 0-180 min (in most experiments it was 60 min) at 22 "C in 350 p1 of 50 mM Na2HP04, 1 mM PMSF, pH 7.4, containing 1 mg/ml of bovine serum albumin, 0.1-0.4 ng of '2sI-IGF-I or -11 (-14,000-70,000 cpm) and varying concentrations of an impure preparation of IGF or MSA. Following incubation, three 100pl aliquots of each suspension were centrifuged 800,000 X g.min on the Beckman Airfuge, unsedimented material removed by suction, and that portion of the nitrocellulose tube containing the pellets was cut and counted for radioactivity on a Nuclear-Chicago y-counter at 40% efficiency. Detergent-solubilized protein (10-100 pg) was incubated with Iz5I-IGF-II * unlabeled IGF-I1 or MSA in a final volume of 224 p1 of buffer as described above. Following incubation, 126 p1 of ice-cold 50% polyethylene glycol was added to a final concentration of 18% (w/v) to precipitate the ligand-receptor complex. After standing for 10 min at 0-4 "C, bound ligand was separated from free by sedimenting three 100-pl aliquots of the mixture on the Beckman Airfuge. The data are expressed as (a) specific binding, i.e. the difference between '251-labeled ligand binding to membranes or solu- impure IGF or MSA, as described above, resuspending the sedimented pellets in 20 p1 of 50 mM Na2HP04, pH 7.4, and incubating them fr lr 15 min on ice with 2 p1 of 1 mM DSS in dimethylsulfoxide essent.n! y as described by Pilch and Czech (25). After quenching the react:t'!l with 11 p1 of 1 M Tris, 0.2 M EDTA, the membranes were dissol. and heated in SDS-containing sample buffer with or without 100 mP.1 DTT. Polyacrylamide slab gels 1.5-mm thick, were prepared in tlw buffer system of Laemmli (26). The running gel was formed from 2.. ml of 5% acrylamide, 0.3% DATD and 12.5 ml of 15% acrylamide, 7% DATD. The stacking gel contained 3% acrylamide, 0.02% bisacrylamide. Standard M , markers of filamin (250,000), @-galactosidase (116,000), phosphorylase a (95,000), bovine serum albumin (66,200), and ovalbumin (45,000) were run with each series. Certain of these had been radioactively iodinated. Following electrophoresis, the gels were fixed and stained with Coomassie blue, destained in 7.5% acetic acid, dried, and the regions containing radioactivity identified by autoradiography at -70 "C using Cronex x-ray film and Cronex Lighting Plus intensifying screens.
Sucrose Density Gradient Ultracentrifugation-Detergent-solubilized IGF-I1 binding proteins resolved during gel chromatography by guest on March 23, 2020 http://www.jbc.org/ Downloaded from were concentrated against Carbowax and either stored on ice or incubated with 12SI-IGF-II in the absence or presence of unlabeled MSA. The protein, in a final volume of 0.2 ml, was layered on 4.7 ml of 5-20% (w/v) linear sucrose gradients containing 100 mM NaC1,50 mM Na2HP04, pH 7.4,0.2% n-octylglucoside prepared in either H20 or 99% D,O. The samples were centrifuged 15 (HzO) or 25 (DzO) h at 4 "C in a Beckman SW 50.1 rotor at 42,000 rpm according to the procedure of Martin and Ames (28). After centrifugation, approximately forty-seven 0.1-ml fractions were collected and their refractive index and distribution of calibrating proteins and 1251-IGF-II-binding activities determined. The Stokes radius, and partial specific volume of the calibrating proteins are: for catalase, 52.2 A, 11.3 s, and 0.73 ml/g; for lactate dehydrogenase, 49.2 A, 7.3 s, and 0.75 mg/ 9; for fumerase, 52.9 A, 8.9 s, and 0.74 mg/g; and for transferrin, 40 A, 5.5 s and 0.73 mg/g. Catalase activity was determined by following the consumption of H20, at 240 nM (29), lactate dehydrogenase by the decreases in absorbance at 340 nm of 0.24 nM NADH in the presence of 0.7 mM pyruvate (29), and fumerase by an increase in absorption at 240 nM in the presence of 50 mM L-malate (29).
Calculations-The molecular weight of the detergent-solubilized IGF-I1 receptor was determined by the formula (27) where N is Avogadro's number, qzo,w and ~2 0 ,~ are the viscosity and density of water at 20 "C, a is the Stokes radius, the sedimentation coefficient, and i the partial specific volume. ~2 0 .~ and b were calculated using Equations 13 and 14 of Clarke (30), respectively, and the experimentally determined sedimentation constant (s), viscosity ( q ) , and density ( p ) at rav. The distance from the center of rotation to a point midway between the miniscus and the proteins in the sucrose gradient is defined as the rav. Orav is a linear function of sucrose concentration and could be determined graphically. However, r., is not linear with sucrose concentration and the value for the IGF-I1 receptor was determined by extrapolation from the q rav of standard proteins according to Equation 12 of Clarke (30). For a more extensive description of the methods employed to calculate 7, p , and s, the reader should consult the recent publication by Costrini et al. (31) on the physical properties of the Triton X-100 solubilized nerve growth factor receptor. To estimate the asymmetry of the IGF-I1 receptor, the frictional ratio, f/fo, was calculated by the equation The solvation factor, 6, was taken to be 0.2 g of solvent/g of protein (32).

Distribution of Protein and Receptors that Bind IGF-II in Differentially Centrifuged Hornogenates of Rat Placenta
The R, fraction, obtained by centrifuging the homogenate at 9600 X g . min, is made up of partially dissociated tissue and intact cells (data not presented). Over 50% of the homogenate's protein is sedimented with this fraction (Table I). The R2 fraction contains broken cells, nuclei, mitochondria, and membrane fragments of varying sizes while the RB has large fragments of smooth membrane, some mitochondria, ribosome-free and ribosome-bearing vesicles of varying size, free ribosomes, and cellular debris. Eighteen to 23% of the 1251-IGF-I1 available for binding became associated with the protein in all of these fractions. Approximately 68-77% of this binding could be displaced with an excess of unlabeled ligand (Table I)  binding to fractions of rat placenta separated by differential centrifugation or extracted with n-octylglucoside Rat placenta was homogenized and differentially centrifuged at 0.0096, 0.48, and 6 X lo6 X g.min to give particulate fractions designated as R1, R2, and Rt, respectively. The R3 fraction was treated with 2% (w/v) n-octylglucoside in 50 mM Na2P04, 1 mM PMSF, pH 7.4, for 60 min at 0-4 "C as described under "Materials and Methods." The nonextractable material, designated the residue, was removed during 6 X lo6 X g.min of centrifugation and the soluble protein, i.e. the extract, was dialyzed against 0.2% detergent in the phosphate buffer for 0.5 to 4 h. Twenty-nine pg of protein and 3.8 fmol of '*'I-IGF-I1 * 50 pg of MSA were used to characterize binding properties of each fraction. The results are expressed as the mean f S.E. of protein content, Bo/T, or specific binding with the number of experiments indicated in parentheses.

Fraction designation distribution Protein
&IT Specific binding

Characterization of IGF-11 Binding
Although one of us (22) had previously reported that the IGF receptors on rat placental membrane preferentially bind IGF-I1 and with high affinity, these observations were repeated and extended because (a) the methods employed to prepare the membranes and quantitate ligand binding used previously were different from those we use now; and ( b ) it was essential that the membrane-associated receptor be characterized more completely so that valid comparisons can be made between it and the detergent-solubilized receptors.
lz5I-IGF-II binding to the membranes in the R3 was linear with increasing concentrations of growth factor (Fig. 1A) and, in part, membrane protein concentration ( Fig. 1B). In the majority of our studies, we have used 0.1 ng (13.3 fmol) of lZ5I-IGF-II and 100 pg of protein/350 pl of incubation medium. Under these conditions, 20 to 25% of the available ligand is bound and >80% of it could be displaced with 50 pg of a preparation of MSA. 1251-IGF-II binds specifically over a broad range of p H from 6.5 to 10 ( Fig. 2). More ligand could be bound a t p H 5 and 6 than in the neutral pH. However, it could not be displaced by MSA. At a p H greater than 10, IGF-I1 was not bound. These results were obtained using monoand dibasic sodium phosphate to buffer the reaction. Tris and certain of the other Good Buffers were also evaluated for their effect on the binding of lZ5I-IGF-II to membranes that were either prepared in, washed with, and resuspended in 50 mM phosphate buffer, or prepared in phosphate buffer but washed with and resuspended in 50 mM of the respective buffers.
When Tris was used as the buffer to prepare the membranes or the binding medium, more '251-IGF-II was bound than was observed in the phosphate buffer, e.g. a Bo/T of 0.42 was obtained with Tris at pH 7.4. However, over 40% of this binding was nonspecific. Greater specificity of Iz5I-IGF-II binding, i.e. between 70 and 75%, was obtained when R3 membranes were incubated in medium containing 50 mM Bis, HEPES, or MES but the Bo/T values were equal to or less than what was obtained when the binding was carried out in Extraction of the IGF-II Receptor Early in this work, several nonionic and ionic detergents were evaluated for their ability to extract the IGF-I1 receptor. As little as 0.02% Triton X-100, which solubilizes insulin (14) and basic Sm/IGF-I (15) receptors of human placenta a t concentrations of 0.5-1%, reduced the binding of lZ51-IGF-II to rat placental membrane from a control value of 35-23%; nonspecific binding increased from 26 to 64%. At a detergent concentration of 0.5%, little specific binding could be demonstrated. The inhibition of binding was not due to the presence of oxidizing impurities (33). Triton X-100 that had been treated with NaHS03 and repurified still inhibited IGF-I1 binding to the placenta. Similar adverse effects on lz5I-IGF-I1 binding were observed with 0.02-0.1% NIKKOL-BL-8SY and the zwitterionic detergent, Zwittergent (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). n-Octylglucoside a t a concentration of 0.1% (w/v) had no effect on either total or specific IGF-I1 binding and, thus, became the detergent of choice to attempt to solubilize the receptors in the Ra. Incubation of these membranes for 60 min at 0-4 "c with 2% (w/v) n-octylglucoside at a protein:detergent weight ratio of 1:4 solubilized 60% of the protein (Table I). These were the optimum conditions since slightly less protein was solubilized when 1% detergent was used and incubations at room temperature resulted in the preparation of extracts that bound greater quantities of '251-IGF-II nonspecifically (data not presented). Extraction of the receptor from the RB was incomplete as evidenced by Bo/T and specific binding values of 0.26 and 70%, respectively, for lZ5I-IGF-II binding to the residue ( Table I).
Dialysis of the extract for 0.5 or 4 h against 0.2% noctylglucoside resulted in only a small loss in protein, i.e. about 5%, with no effect on '251-IGF-II binding. It has previously been established that 4 h of dialysis adequately decreases the level of detergent from above its critical micellar concentration of 25 to 1.5 mM (34). Thus, under conditions of assay, gel chromatography, and sucrose gradient centrifugation, the soluble receptor exists in equilibrium with the nonmicellar form of n-octylglucoside.
Polyethylene glycol combined with centrifugation was used initially to evaluate ligand-soluble receptor interactions. Based o n our earlier study with the insulin receptor (24), we used a final concentration of this reagent of 6% (w/v). On reexamining the concentration dependency for precipitating the '251-IGF-II-soluble receptor complex, we observed that t,wice as much radiolabeled ligand was associated specifically with the receptor protein when 18% polyethylene glycol was used compared with that obtained using 6% (Fig. 3). At higher concentrations of polyethylene glycol, nonspecific binding of '251-IGF-II increased. A concentration of 18% was used in subsequent work.
Comparison of IGF-II Binding to Detergent-solubilized and Membrane Receptors '251-IGF-II binding to the placental membrane and detergent solubilized extract at 22 "C reached equilibrium by 60-90 min and was constant to 180 min (Fig. 4A). For the purposes of kinetic analysis, we have assumed the bound IGF-I1 is intact, that the binding is reversible, and that there is no cooperativity between receptors. Under these conditions, the formation of the receptor-ligand complex (RI) with time (t) can be described by the second order differential equation where RI(t) is the concentration of hormone-receptor complex at time (t), IO and RO the total hormone and receptor concentrations, and k, and kd the association and dissociation rate constants, respectively. Differentiation of Equation l a less the dissociation term (it was assumed the initial binding is Methods." Total binding (0) and nonspecific binding (0) for the membrane fraction (---) and extract (-) are presented as the mean of 3-9 determinations. B, the second order rate constant of association, &, was determined for IGF-I1 binding to the membrane and extract by plotting time against loglo (free IGF-I1 concentration/free receptor concentration). It was assumed the latter was equal to the quantity of '2sI-IGF-II bound specifically a t equilibrium, i.e. 0.26 fmol for the membrane and 0.94 fmol for the extract. The k,, for the IGF-I1 receptor in the membrane and the extract was 5.3 and 8.5 X 1O8/M/min, respectively. irreversible) results in the following formula: By plotting the left term of this expression as a function of time, a straight line was obtained with a slope of k,. The k, for the membranes and soluble receptors were 5.3 and 8.5 x 10' M" min", respectively (Fig. 4B).
Specifically bound ligands dissociate from their receptors with first order kinetics as indicated by the second term, i.e.
One-half dissociation rates (tlp) of the RI complex were determined by plotting the logarithm of bound radioactivity as a function of time following removal of free '251-IGF-II with dextran-coated charcoal and the addition of MSA (Fig. 5). The rate constant for dissociation, k d , for the soluble receptor was 7.5 X min" based on a one-half rate of dissociation of 93 min. The k d for the membrane receptor was 4.2 X min-'. As documented in Table I and previous figures, approximately 25 and 60% of the 3.8 fmol of 1251-IGF-II available for binding became associated with the 29 pg of membrane or detergent-soluble protein, respectively (Fig. 6). This binding was inhibited by unlabeled IGF-I1 and IGF-11-like peptide (11) present in MSA in proportion to their concentrations. Ten ng of unlabeled IGF-I1 inhibited 1251-IGF-II binding to the membrane by 50% and 40 ng inhibited binding to the soluble receptors by the same extent. The curves of MSA inhibition of 1251-IGF-II binding were parallel with those obtained with IGF-I1 but the former was one one-hundredth as potent as the latter. Nevertheless, because of its ease of preparation and availability, it has been employed extensively in this and in our current studies to measure nonspecific "' 1-IGF-I1 binding.
The data from the competition by unlabeled IGF-I1 were analyzed by the method of Scatchard after correcting for nonspecific binding (Fig. 7). As evidenced by the linear slope ~-  Fig. 6 of 12'I-IGF-II binding to the membranes and soluble receptors as competed for by unlabeled IGF-I1 were converted into pmol of bound growth factor after subtracting 4.8 and 12% for nonspecific binding to R3 and extract, respectively. These data were then plotted by the method of Scatchard and subjected to linear regression analysis to calculate the equilibrium dissociation constant, K d , and the total binding capacity, Ro. The binding of '"I-IGF-II to 10 pg of peak 1 protein and 50 pg of peak 2 protein as competed for by 0.16-85 ng of unlabeled IGF-I1 was also determined in two experiments (data not shown). The mean Bo/T and BIB0 values for peak 1 were 0.37 and 0.06 and those for peak 2 were 0.14 and 0.16, respectively. After correcting for nonspecific binding, the data was recalculated and plotted by the method of Scatchard as described above for the membrane and unchromatographed soluble receptor. of the regression line, IGF-I1 binds to a homogeneous class of membrane receptors with an equilibrium dissociation constant, Kd, of 0.6 nM. For each mg of protein, 1.6 pmol of IGF-I1 were bound (the correlation coefficient of this analysis was 0.978). Extraction of the membrane with n-octylglucoside also apparently solubilized a single class of receptors. However, they bind the ligand with less affinity, i.e. a Kd of 1.3 nM was obtained and the total binding capacity of the extract was 6fold greater than was observed for the membrane.

Hydrodynamic Properties of the IGF-II Receptor Gel Chromatography-n-Octylglucoside-solubilized IGF-I1
binding proteins can be resolved into two components on Sephacryl S-300 (Fig. 8). Those eluting between fractions 42 and 50, and designated peak 1, bind the greatest quantity of labeled ligand. This binding was also the most specific. The Stokes radius of the peak 1 receptors was determined to be 7.2 nm (Fig. 9). Proteins eluting between fractions 55 and 60 also bind IGF-I1 but with less specificity than the receptors in peak 1. Proteins present in peaks 1 and 2 were concentrated 4-to 5-fold with Carbowax (Union Carbide), and '251-IGF-II binding, as competed for by an impure preparation of unlabeled IGF-11, was determined and the data analyzed by the method of Scatchard. As illustrated in Fig. 7, the soluble receptor in peak 1 binds IGF-I1 with the same affinity as that associated with the intact membrane, i.e. they both have Kd values of 0.6 nM. The chromatographed preparation was also enriched 2-fold in specific IGF-I1 binding capacity when compared with the extract, e.g. the Ro of the former was 21.8 fmol/ mg of protein; for the latter it was 10.4. Peak 2 bound about one-fourth as much '251-IGF-II as peak I and also with a lower affinity. It is possible that this binding is not to a membrane receptor; rather, it may be to a somatomedin binding protein that was either present in the placenta or introduced as a contaminant with bovine serum albumin during the labeling of the receptors with 1251-IGF-II. These possibilities must be considered since the Stokes radii of peak 2 proteins (Fig. 9)

FRACTION NUMBER
FIG. 8. Gel filtration of the detergent-solubilized IGF-I1 receptor. One mg of extracted protein that had been dialyzed 4 h was incubated with 1.5 ng of lZ5I-IGF-II (150,000 cpm) & 500 pg of MSA for 0.5 h at 22 "C and chromatographed at 0-4 "C on Sephacryl S300 in buffer containing 0.2% n-octylglucoside, 100 mM NaCl, 50 mM Na2HP04, pH 7.4. The flow rate was 9.6 ml/h. The 1.6-ml fractions were analyzed for the binding of 12'I-IGF-II in the absence (0) and presence (0) of MSA and for the absorption at 206 nm (-).
The void volume of the column, Vo, and total volume of the gel bed, V,, were 56 and 144 ml, respectively. A major peak of specific IGF-I1 binding occurred at an elution volume, V., of 71 ml, while a minor one was found at a V, of 91 ml. The latter region also contains the majority of 206 absorbing material.
Characterization of Soluble IGF-I1 Receptors and one of the somatomedin c binding proteins of human serum are both 4.3 nm (35). We evaluated these, in part, by (a) washing the R3 membrane preparation two additional times prior to detergent solubilization; and (b) leaving out bovine serum albumin that might be contaminated with binding protein during the labeling procedure. Neither of these treatments had any effect on the magnitude of lZ5I-IGF-11 labeling of peak 2 proteins (data not presented).
Sucrose Gradient Centrifugution-The calculation of 7.2 nm for the Stokes radius of the Sm/IGF receptor in peak 1 provided no information on its molecular size and shape. In order to assign values to these parameters, we determined the sedimentation coefficient of the "'I-IGF-11 receptor complex on linear gradients of sucrose prepared in H 2 0 or DzO. The latter medium was employed since the determination of the positions of the IGF-I1 binding components in this gradient and in gradients prepared in H 2 0 when compared to the position of standard proteins allowed the calculation of the receptor's partial specific voume, V (30). Specific lZ5I-IGF-II binding was maximum in fractions 36.5 f 1.5 (S.E.) or 37.5 f 0.2 when peak 1 was incubated with lZ5I-IGF-II prior to centrifugation in the HzOor DzO-containing gradients, respectively (Fig. 10, A and B ) . If the distribution of the receptors in the two gradients was determined by measuring ' T -IGF-I1 binding to eluted fractions, then it was maximum in fraction 39 k 0.6. A small quantity of specific IGF-I1 binding was found consistently in fractions 24-30 if the proteins had been prelabeled with lZ5I-IGF-II prior to centrifugation, but specific binding could not be detected when comparable fractions were incubated with "'1-IGF-II after centrifugation. As evidenced by its rightward displacement in sucrose gradients prepared in D 2 0 relative to that of catalase, e.g. the enzyme activity was maximum in fractions 41.4 f 0.2 and 39.7 f 0.4 from the H20and D20-containing gradients, respectively,   Frictional ratio. f/fn 1.6 the i of the IGF-I1 receptor and enzyme must be similar. Using the formulation of Clarke (30) to estimate viscosity, a i of 0.72 cm3/g was calculated for the IGF-I1 receptor (Table  11). Since this value is within the range of values reported for the standard proteins, we plotted their experimentally determined and known szo,w values versus one another, and from this relationship (Fig. ll), derived a s20,w value of 9.9 s for the receptor. From this value, a Stokes radius of 7.2 nm and V of 0.72 cm3/g, an M , of 290,000 and a frictional ratio of 1.6 were calculated for the IGF-I1 receptor (Table 11).

Identification of ICF-II Receptors
Receptors in the placental membranes which were crosslinked to "'II-IGF-II subsequent to binding have M , values of -250,000 when SDS-polyacrylamide gel electrophoresis was carried out in the presence of 100 mM DTT, and 200,000-220,000 in its absence (Fig. 12, A , A', C, and C'). The binding of the 1251-labeled ligand to these polypeptides was specific since unlabeled MSA blocked it completely (Fig. 12, B, B', D, and D'). Under comparable conditions of labeling, crosslinking, period of autoradiography, etc., "'I-IGF-I could not be cross-linked to the 250-kDa receptor or to other polypeptides (data not presented).
Treatment of the RR with 2% (w/v) n-octylglucoside, followed by centrifugation, selectively enriched the extracts and residue fraction in specific polypeptides (Fig. 13). Although all three fractions bound '2sI-IGF-II specifically (Table I), cross-linked polypeptides were only identified in the extract and dialyzed extract. The M , of the soluble receptor was 250,000 in the presence of 100 mM DTT (Fig. 13, B, B', D, and D'). We have no explanation for our inability to crosslink 12sII-IGF-II to the presumed receptors in the residue (Fig.  13, F and F'). As evidenced by counting the fractions prior to solubilization and electrophoresis and the presence of a differential in the number of silver grains in the region of the dye front, we believe "'I-IGF-II binds to proteins in the residue, and this binding was specific. This anomaly is being evaluated further.
As described in the previous section, Sephacryl S-300 gel chromatography separated the detergent-solubilized Sm/IGF receptor into peaks 1 and 2 with Stokes radii of 7.2 and 4.3 nm, respectively. Cross-linking '"I-IGF-I1 to the proteins in peak 1 indicated that they are "250 kDa (Fig. 14, C and C'). Iodinated IGF-I1 was also cross-linked to a small number of polypeptides of MI -70 kDa (arrow in Fig. 14, C and C').

Characterization of Soluble ZGF-II Receptors
Although significantly less, '=I-IGF-II was bound by the proteins present in peak 2, the cross-linked polypeptide also had a molecular weight of 250,000 (Fig. 14, E and E').

DISCUSSION
Studies by Daughaday et al. (22) have shown that human placental membranes bind Sm/IGF through a minimum of two receptor systems. IGF-I binds to a low capacity, high affinity system with a Kd of 1.5 nM while IGF-11 binds to both high affinity, e.g. 1.8 nM, low capacity and low affinity, e.g. 20 nM, high capacity receptors. By contrast, rat placental membranes have a single clearly demonstrable Sm/IGF receptor system that binds up to 5 pmol of IGF-II/mg of protein with a Kd of 3.5 nM (22). Although a small quantity of IGF-I was also bound, it was more easily displaced by unlabeled IGF-I1 than it was by IGF-I (22). We have, therefore, chosen the rat placenta as our source of tissue to attempt to isolate the Sm/ IGF receptors.
Differential centrifugation of placental homogenates or centrifugation of the membranes in the R3 fraction on 5-30% (w/v) discontinuous gradients of dextran did not enrich any fraction in specific IGF-I1 binding activity (Table I) we must entertain the possibility that the '"I-IGF-I1 is binding to receptors on intracellular membranes as well as those associated with the plasma membrane. Hence, because of two possible sources of receptors, some variation in their properties, e.g. the degree of glycosylation, might be anticipated.
n-Octylglucoside became the detergent of choice to solubilize the receptors that bound '251-IGF-II. Of the detergents evaluated, e.g. Triton X-100, NIKKOL-BL-8SY, and Zwittergent (3-12), it was the only one that did not affect total or specific ligand binding to the intact membrane when present at concentrations up to 0.1% (w/v). These results are consistent with previous studies on the effect of different detergents on the solubilization and chemical and enzymatic properties of ATPase and NADH dehydrogenase from Streptococcus fwcalk (34), and rhodopsin from bovine retina (38). In addition to its mild effect on protein structure, this compound has several properties which made its use advantageous in our attempts to isolate the Sm/IGF receptor including (a) a definable structure, (b) minimal absorption in the ultraviolet, (c) a very high CMC of 20-25 mM, and ( d ) a 5 of 0.8197 (36).
Approximately 60% of the placenta's protein was solubilized with 2% (w/v), i.e. 67.9 mM, detergent during 60 min of incubation at 0-4 "C. The unextracted residue bound lZ5I-IGF-I1 and the binding was inhibited by an excess of unlabeled MSA (Table I). However, because of our inability to identify the binding unit by cross-linking it with iodinated growth factor, we cannot state with certainty what the nature of this binding material is. Dialysis of the extract for 4 h against 0.2% (w/v) (16.8 mM) detergent did not precipitate protein or alter the magnitude or specificity of '251-IGF-II binding (Table  I and Fig. 6). The result is consistent with the observation of Gould et al. (39) that 1Z51-insulin binding to solubilized turkey erythrocyte membrane receptors was unaffected by n-octylglucoside below its CMC of 0.63% (w/v); at concentrations greater than the CMC, the total binding capacity decreased.
The solubilized '251-IGF-II-receptor complex identified by cross-linking studies had a MI of 250,000, when reduced with DTT ( Fig. 13) that was identical to that observed in the intact membrane. The receptor also bound the ligand with rate constants of association and dissociation that were similar to those determined for the membrane-associated receptor, e.g. the k,, and k,, for the extract were 8.5 X 10s M" min" and 7.5 X min", while those for the membrane were 5.3 x lo* M" min" and 4.2 X min", respectively. The equilibrium dissociation constants, Kd, for the membrane and extract differed by a factor of two, e.g. 0.6 and 1.3 nM, respectively; the partially enriched receptor present in peak 1 following chromatography of the extract on Sephacryl S-300, however, had a Kd of 0.6 nM. These results are consistent with the premise that detergent-solubilization does not modify the binding properties of the IGF-I1 receptor. However, because of a discrepancy between the determination of Kd from measurements of the rate constants of association and dissociation, i.e. Kd = kd/k. = 8 pM, and the binding of '"I-IGF-11 at equilibrium in the presence of competing concentrations of unlabeled ligand, e.g. the Kd = 600 PM, it is probable that the value for k,, may be erroneously high. Alternatively, the interaction of IGF-I1 with its receptor may be more complicated than the reaction R + H c) RH implies.
The Kd of 0.6 nM, determined from equilibrium binding studies, is within the range of values reported for detergentsolubilized insulin receptors (14) and IGF-I receptors from human placenta (15), and for IGF binding to adipocytes (3) and cultured cells (3, 40). The kd values of 4-8 X min" for the IGF-I1 receptor are smaller than has been reported for the insulin receptor (41,42) and infers that the former binds its ligand with a greater affinity than the latter. Since we had no difficulty in measuring the dissociation of the IGF-IIreceptor complex, the discrepancy between the values for K d determined by the equilibrium uersus the kinetic method can be explained, in part, by our calculating a large k, for this receptor system. The k, for the binding of IGF-I1 to the placental receptors was about one log higher than has been reported for insulin's binding to its receptor (41) or for IGF binding to cultured chick embryo fibroblasts (40). We have ruled out methodological problems as accounting for these results and conclude that the high k, reflects our failure to measure association under pseudo first order conditions, i.e. the concentration of the ligand is assumed to remain essentially constant over the period of time binding to the receptor is determined with k, depending on receptor concentration alone (43). In these studies, we incubated 3.82 fmol of lz5I-IGF-I1 (-4300 cpm) with 6 or 1 fmol of receptor calculated to be present in 0.6 pg of extract protein or membrane protein, respectively. A plot of IGF-I1 binding with time ( Fig. 4) indicates that 6 and 16% of the free '251-IGF-II bound to the membranes and soluble receptor, respectively, by 30 min of incubation. If one-half of the '251-IGF-II was biologically active (this is a reasonable assumption since the maximum Bo/T that could be obtained with soluble receptor was 0.6, and 20% of this binding was nonspecific), then with increasing periods of incubation, less of it would be available for binding and the reaction of the receptor with the ligand would shift from pseudo first order to second order. This explanation assumes that the interaction of IGF-I1 with its receptor is a reversible reaction. However, if the receptor is modified concomitant with occupancy or has cooperative binding sites, then a simple, biomolecular reaction mechanism will be inadequate to explain our results. This possibility is supported by a study of progesterone binding to cytosol receptors from chick oviduct by Hansen et al. (44). These investigators observed a 180-fold difference between the & determined from a kinetic analysis and that from equilibrium binding experiments, e.g. 0.027 and 5 nM, respectively, when the incubations were carried out at 0 "C. Raising the temperature to 15 "C increased the difference by more than three logs. The Kd from the equilibrium binding measurements did not change with temperature and was similar to the concentration of steroid that evoked 50% of a maximum physiological response. Since the k d changed only slightly while the k, increased 100-fold with a 15 "C increase in temperature, they concluded that measurements of the latter parameter are most susceptible to error. This is particularly so, if the association of ligands with receptors are complex reactions involving more than one state of the receptor.
Based on cross-linking with '251-IGF-II (18), or 12sII"SA (19), or 'ZsII-ILAs3 the IGF-I1 receptor is thought to be a 220-kDa monomeric protein, i.e. it is not associated with other subunits. This interpretation must be viewed with caution, since we have observed that: 1) chromatography of detergentsolubilized extracts on Sephacryl S-300 resolved the binding components into two forms with Stokes radii of 7.2 and 4.3 nm; 2) cross-linking the IGF-I1 receptor with '251-IGF-II revealed a 250-kDa component (reduced) in the 7.2 and 3.8 nm forms and a lightly labeled 70-kDa component (reduced) in the former; 3) centrifugation of peak 1 proteins prelabeled with '251-IGF-II suggested multiple forms of the receptor; and 4) the molecular weight of this receptor was calculated from the Stokes radius, partial specific volume, and sedimentation constant to be 290,000.
The existence of more than one form of the insulin receptor C. Thibault and J . F. Perdue, unpublished results.
was observed by Ginsberg et al. (45). These investigators noted that the addition of small quantities of insulin to detergentsolubilized turkey erythrocyte receptors proportionally increased the amount of a receptor with a Stokes radius of 3.8 nm and concomitantly reduced the amount of the 7.2 nm form. Using similar methodology, Maturo and Hollenberg resolved the receptors from the membranes of rat liver (46) and adipocytes and cultured fibroblasts (47) into two insulin binding regions with Stokes radii of 7.2 and 3.8 nm. The 7.2 nm form of the receptor bound insulin with 10-fold greater affinity than did the 3.8 nm form. Krupp and Livingston identified two insulin binding components in Triton X-100 extracts of rat adipocytes (48) and liver plasma membrane (49) when the extracts were electrophoresed in polyacrylamide gels under nondenaturing conditions. They too found that small quantities of insulin converted some of the larger receptors present in peak 1 into a smaller form, designated peak 2. These bound 1251-insulin with different affinities. For example, Scatchard plots of insulin binding to peak 1 were curvilinear while those to peak 2 were linear.
The insulin binding component with a Stokes radius of 7. This form of the receptor has recently been shown to be functional, e.g. it binds 1251-insulin as well as the unreduced form of the receptor, and its occupancy stimulates hexose uptake by rat adipocytes (51).
The IGF-I receptor extracted from human placenta has a Stokes radius of 7.2 nm (15) and a sedimentation coefficient of 11 s (21). A molecular weight of 402,000 was calculated for the detergent-receptor complex. This receptor and that present in cultured cells contain 130-140-, and 95-kDa subunits (15,(18)(19)(20), and others of still smaller size (21) that are disulfide bonded to form 300-350-kDa complexes similar to the insulin receptor. However, unlike the latter, an IGF-I binding species with a Stokes radius of 3.8 nm has not been observed (15).
Two IGF-I1 binding species were detected in detergent extracts of rat placenta (Fig. 8). Peak 1 bound the greatest quantity of '251-IGF-II, e.g. the Ro = 21.8 pmol/mg of protein, and with the highest affinity. It had a Stokes radius of 7.2 nm, which is identical with that of the insulin (45)(46)(47)(48)(49) and the IGF-I receptor (15). A minor peak of IGF-I1 binding with a Stokes radius of 4.3 nm was consistently observed in the detergent extracts. The possibility was considered that this peak reflected the presence of a contaminating somatomedin binding protein but repeated washing of the membrane prior to solubilization or the use of bovine serum albumin-free elution buffer (this proteia is frequently contaminated with binding protein) did not alter the magnitude of the 4.3 nm peak. The affinity of '2sI-IGF-II for the binding components on peak 2 was much less than it was for those in peak 1; e.g. the Kd values were 2.6 and 0.6 nm, respectively, and numerous attempts to identify the '251-IGF-II protein complex following centrifugation in gradients of sucrose or electrophoresis in polyacrylamide gels under nondenaturing conditions3 were unsuccessful. 1z511-IGF-II could be cross-linked to a 250-kDa protein present in peak 2, but the quantity labeled was so small in comparison to the proteins labeled in peak 1 (Fig.  14) that the results could be accounted for by the carry over of a small amount of the latter into peak 1. Alternatively, the cross-linking of lZ51-IGF-II to a putative 4.3 nm receptor by disuccinimidyl suberate could be very inefficient. A precedent for this phenomenon was our inability to cross-link lZ51-IGF-I1 to binding components remaining in the residue following detergent extraction of the R3, even though the magnitude of this binding was significant and it was specific (Table I).
Consistent with the observations of others (18,19,52), lZ51-IGF-I1 could be cross-linked specifically to a receptor of 220-kDa (or 250-kDa when reduced) that was present in rat placental membrane (Fig. 12), a detergent extract of these membranes (Fig. 13), and peak 1 of the chromatographed extract (Fig. 14). However, we have also identified a radioactively labeled 50-or 75-kDa (when reduced with DTT) protein in the R3 and peak 1 (Fig. 14). Although the quantity of crosslinked IGF-I1 in this region of the gel was very small, the labeling was specific since it could be blocked completely by MSA. We also have observed specific cross-linking of lZ5I-IGF-I1 to a similar molecular weight protein on Swarm rat chondrosarcoma1 membranes (52). The possibility exists, therefore, that the 75-kDa protein is a poorly labeled subunit of the IGF-I1 receptor. Alternatively, it could be a fragment from a degraded 220-kDa receptor.
These possibilities are being examined.
The sedimentation behavior of the lZ5I-IGF-II binding components in peak 1 also indicates that there may be more than one form of this receptor. Sucrose density gradient centrifugation of these components, subsequent to their incubation with '2sI-IGF-II f MSA, disclosed specific ligand binding between fractions 24-42 (Fig. 10). The greatest quantity of label was found in fraction 38. Receptors with similar sedimentation properties also were identified by incubating the fractions from the gradients with 'ZsI-IGF-II and precipitating the complex with polyethylene glycol. The latter method did not disclose, however, the small but specific region of binding between fractions 24-29 and the shoulder around fraction 33 that had been observed for the prelabeled receptors. This reproducible pattern could represent IGF-11-receptor complexes that are either dissociating or are being degraded as a consequence of receptor occupancy. The latter effect has been reported to occur when the insulin receptor is occupied (50). Finally, the calculation of a molecular weight of 290,000 for the IGF-I1 receptor (Table 11) is consistent with the suggestion that the -220-kDa component may be associated with other proteins. However, this method to determine molecular size is fraught with potential errors, e.g. estimating i of a receptor-detergent complex from extrapolated p and 9 values, or the sZo,w from an experimentally determined s value. In addition, the presumed covalent linkage of heterosaccharides with the protein of the receptor would also influence the electrophoretic and diffusional mobilities in polyacrylamide and Sephacryl gels and lead to anomolous estimates of its molecular size. Therefore, it is premature to speculate about the possible structure of the IGF-I1 receptor without more information. Its eventual purification to homogeneity from the rat placenta or Swarm rat chondrosarcoma (53) and subsequent chemical and physical characterization should satisfy this requirement.