BIOLOGICAL THE OF CHEMISTRY No. Vol. 10, 1981 rJ. in ... · 23, hue of December 10, pp. 12118-12126, 1981 Printed in rJ. S A. Characterization of Detergent-solubilized Membrane Proteins

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256. No. 23, h u e of December 10, pp. 12118-12126, 1981 Printed in rJ. S A.

Characterization of Detergent-solubilized Membrane Proteins HYDRODYNAMIC AND SEDIMENTATION EQUILlBRIUM PROPERTIES OF THE INSULIN RECEPTOR OF THE CULTURED HUMAN LYMPHOBLASTOID CELL*

(Received for publication, March 4, 1981)

Robert J. Pollet, Barry A. Haase, and Mary L. Standaert From the Departments of Medicine and Biochemistry, University of South Florida and Veterans Administration Medical Centers, Tampa, Florida 33612

The high affinity insulin receptor from the plasma membrane of the cultured human lymphoblastoid cell IM-9 was solubilized with the nonionic detergent Triton X-100. Scatchard analysis of insulin binding revealed a homogeneous class of noncooperative binding sites with K , = 2 X lo8 M-’, corresponding to the high affinity component of cellular insulin binding. The solubilized receptor and insulin-receptor complex sedimented in sucrose gradients as single species at positions corresponding to 10.5 and 11 S, respectively, and co-chromatographed on agarose gel filtration columns at a position corresponding to a Stokes radius of 81 A. On isoelectric focusing, the insulin binding activity was described by a single peak with an isoelectric point of 4.5. To circumvent the difficulties of unambiguously interpreting empirically derived hydrodynamic data, the sedimentation equilibrium behavior of the detergent-solubilized receptor covalently cross-linked to ‘251-insulin by disuccinimidyl suberate was studied, employing the nonempirical methods we previously described (Pollet, R. J., Haase, B. A., and Standaert, M. L. (1979) J. Biol. Chem 254, 30-33). Following centrifugation in a fixed angle preparative air-turbine rotor at 5 “C for 36 h and fractionation of the reoriented solution, the slope of the natural logarithm of the ‘“I-insulin-receptor concentration (In c ) as a function of the square of the radial distance (9) yielded M(l - Vp) = 102,000, in agreement with the value independently derived from the hydrodynamic data.

We have extended the applicability of this thermo- dynamically based method to the determination of the partial specific volume (V) of detergent-protein complexes and the molecular weights of their protein components by studying their sedimentation equilibrium profiles in buffers with varying concentrations of DzO and D2‘‘O. Without the use of empirical molecular weight standards or assumptions regarding molecular shape, analysis of (2RT/w:)(d In c /d f l ) as a function of buffer density resulted in V = 0.73 for human y-globulin and 0.91 for Triton X-100, in good agreement with their known values. For the detellgent-insulin-receptor COM- plex, this method yielded V = 0.78 ml/g, indicating a molecular weight of 310,000 for the receptor protein alone and 0.5 g of Triton X-100 bound per g of protein.

Furthermore, while the high frictional ratio (f/A = 1.6) derived from the hydrodynamic properties of this

Institutes of Health (AM 186081, the Veterans Administration (MRIS * This study was supported by research grants from the National

7129), and the American Diabetes Association. The cask of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- merit" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

receptor and many other detergent-membrane protein complexes has been previously interpreted as reflecting a high degree of molecular asymmetry (elongation), confirmation of the high level of hydration of the bound detergent indicates that the asymmetry component of the frictional ratio ( ( f / f o ) ~ = 1.2) for these complexes actually approaches the range characteristic of oligomeric globular proteins.

The characterization of small quantities of biological macromolecules having a specific measurable biochemical activity or derivative thereof has been somewhat limited by the empirical bases of available techniques, including sedimentation through sucrose gradients (1-3), gel fitration chromatography (4, 5 ) , and a sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis ( 6 4 , by anomalous behavior (6, 8, 9), and by assumptions regarding molecular shape required for interpretation (10). For intrinsic membrane proteins, the problem is further compounded by the requirement for detergent solubilization and the consequent uncertainties with respect to partial specific volume and possible anomalous behavior of the protein-detergent complex. We have developed a method for the characterization of macromolecules by sedimentation equilibrium in a small preparative air-turbine ultracentrifuge (11) which has an entirely thermodynamic and nonempirical basis, and which therefore avoids these difficulties. Further- more, we have presently extended the method to a detenni- nation of the partial specific volume of protein-detergent complexes by examining their sedimentation equilibrium profile in buffers with varying concentrations of D20 and D2’’O. The resulting information permits a determination of the molecular weight of the protein component alone and an estimate of the degree of detergent, binding.

These methods are applied in the present report to the characterization of the high affinity insulin receptor of the cultured human lymphoblastoid cell IM-9 solubilized with Triton X-100. Considerable interest has centered on this receptor as a model for the investigation of insulin-receptor interactions (12-14) and for the cellular regulation of insulin receptor number in response to hormones and metabolites (15, 16). In addition, the binding characteristics of this receptor are very similar to those of the circulating human mono- nuclear leukocyte receptor which has demonstrated changes in receptor number and occasionally affinity in states of al- tered insulin sensitivity in man (16, 17).

MATERIALS AND METHODS

Crystalline zinc porcine insulin was purchased from Elanco Prod- ucts Co. (Indianapolis, IN). Carrier-free NaI2‘I and Na“”1 were obtained from Union Carbide. L-C’HILeucine (57 Ci/mmol), Cphenyl-

12118

Characterization of the Insulin Receptor 12119

'HITriton X-100 (1.5 mCi/mg), and [methyl-"C]human y-globulin (20 pCi/mg) were obtained from New England Nuclear. Triton X-100 was obtained from Baker Chemicals. Suberic acid, N-hydroxysuccinimide, and N,N'-dicyclohexylcarbodiimide were obtained from Ald- rich Chemical Co. D,O was purchased from Sigma and Dz"0 was obtained from Alfa Products. All other chemicals were reagent grade.

Radioiodination of Insulin-Iodination of porcine insulin with "'1 or '"1 was carried out by a modification (18) of the chloramine-T method. This method yielded '251-insulin specific activities of 150-200 pCi/,ug and '"I-insulin specific activities of 1000-1500 pCi/pg, corresponding to 0.4-0.6 iodine atom/insulin molecule.

Cell Culture-IM-9 cultured human lymphocytes were cultured a t 37 "C in Spinner minimal essential medium supplemented with 10% heat-inactivated fetal calf serum, 125 units/ml of penicillin G, and 125 pg/ml of streptomycin sulfate (Grand Island Biological Co.). Stock cultures were transferred every 2-3 days into 2 volumes of fresh media. Viability as determined by the trypan blue exclusion method was routinely greater than 90%. Hemocytometer counts were used to determine cell density.

Membrane Preparation and Detergent Solubilization-Following the method of Cuatrecasas and co-workers (19), cells were collected and washed three times by centrifugation in cold phosphate-buffered saline a t 600 X g and resuspended at 4 "C in 10 m Tris buffer, pH 7.4, containing 0.2 m MgCl:! and 0.2 mM CaC12, to a concentration of 3 X 10" cells/ml. Five-milliliter aliquots were homogenized with a Brinkmann Polytron, Model PCU-2 for 30 s a t setting 2.5. Nuclei and whole cells were removed by centrifugation at 600 X g for 5 min a t 4 "C. The supernatant was centrifuged at 40,000 X g for 30 min at 4 "C and the pellet was suspended in %o of the volume of cold PBS' and stored at -70 "C. After thawing, membrane preparations were centrifuged for 10 min a t 600 X g a t 4 "C. Membranes in the supernatant were pelleted by centrifugation a t 40,000 X g for 30 min a t 4 "C, resuspended in PBS or RRA buffer (100 mM 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid, 120 mM NaC1, 1.2 mM MgS04, 2.5 mM KCI, 15 mM Na acetate, 10 mM glucose, 1 mM EDTA, pH 7.6) containing 1% Triton X-100, and solubilized for 30 min on ice. The solubilized receptor preparation was diluted 10-fold with cold buffer and the remaining particulate material was removed by centrifugation at 40,000 X g for 30 min a t 4 "C. Digitonin-solubilized receptors were prepared in the same manner utilizing 0.8% digitonin for solubilization (20).

Insulin Binding-Steady state binding of "'I-insulin to soluble receptors was performed at 15 "C in RRA buffer containing 0.1% Triton X-100 and 1% bovine serum albumin for 90 min in a shaking water bath. Binding was determined by precipitation of the insulin- receptor complex with polyethylene glycol (Sigma), essentially following the method of Cuatrecasas (21). All assays were corrected for nonspecific binding by subtraction of the "'I-insulin binding remaining in the presence of 2 X 10.' M unlabeled insulin. The nonspecific binding was less than 1% of the total radiolabeled insulin. Under these conditions, degradation of '"I-insulin was less than 3%, as measured by precipitation of intact insulin by 5% trichloroacetic acid, In addition, specific binding of "'I-insulin was stable for a t least 2 h, indicating that this steady state was a reasonable reflection of equilibrium binding of hormone to receptor in this system (14,22).

Gel Filtration-Solubilized receptor preparations were chromatographed on a Sepharose 6B (Pharmacia) column (1.6 x 59 cm) equilibrated with RRA buffer containing 0.1% Triton X-100 a t 2 "C. The excluded ( Vu) and included ( V,) volumes were determined under these conditions using Blue Dextran 2000 and JH20, respectively. The column was standardized with marker proteins under these conditions using porcine thyroglobulin, Escherichia coli /3-galactosidase, equine apoferritin, bovine y-globulin, and bovine serum albumin, Free solubilized receptors and/or receptor-"'I-insulin complexes in a volume of 1 ml were loaded on the column and eluted with RRA buffer with 0.1% Triton X-100 at a flow rate of 6 ml/h. Free receptors were detected in the eluted fractions by determination of specific binding of 1 X lo-" M '."I-insulin added to each fraction. The insulin-receptor complex was detected in the eluted fractions by determination of specifically bound "'1-insulin radioactivity. The distribution coefficient, K, was calculated from the relation K = ( V, - XI)/( V, - V,,), where V, is the elution volume.

Sedimentation through Sucrose Gradients-The free solubilized receptors and/or '251-insulin-receptor complexes in a volume of 0.2 ml of RRA buffer with 0.1% Triton X-100 were layered onto 5 - d 5.20%

The abbreviation used is: PBS, phosphate-buffered saline.

linear sucrose density gradients in RRA buffer containing 0.1% Triton X-100. The gradients were centrifuged for 8 h a t 4 "C in a Spinco SW-65 rotor a t 63,000 rpm in a Beckman Model L5-75 ultracentrifuge. The gradients were standardized with marker proteins under these conditions using bovine liver catalase, bovine y-globulin, and bovine serum albumin. The insulin-receptor complex was detected in the gradient fractions by determining specifically bound "'I-insulin. Free receptors were detected in the gradient fractions by incubation with ""I-insulin followed by determination of specifically bound "I radioactivity. Catalase activity, as an internal marker, was measured by the reduction of hydrogen peroxide (23). An approximate estimate of the partial specific volume of the detergent-solubilized insulin-receptor complex was obtained by sedimentation through identical sucrose gradients prepared in D 2 0 (24, 25) and analyzed employing Equation 9 of OBrien et al. (26).

Analysis of Hydrodynamic Data-As suggested by Siege1 and Monty (lo), the empirically determined Stokes radius (a) and sedimentation coefficient ( s ) may sufficiently approximate the analyti- cally determined parameters to allow estimation of the molecular weight (M) and molecular asymmetry component of the frictional ratio ( f / f i , ) , , of a macromolecule of partial specific volume V , employing the classical equations

M(l - V p ) = 677 NUS (1)

( f / f i , ) A = u ( ~ T T N / ~ M [ O + (S/p)])"' (2)

where N is Avogodro's number, S is the degree of hydration (w/w), and p and 9 refer to the density and viscosity of the solvent under the conditions employed for the analytic determination of the standard protein parameters (water, 20 "C). The best conservative estimate of the hydration factor, 8, for the protein component is 0.3 g/g, as summarized in Table XXIV of Kuntz and Kauzmann (27). The hydration factor for the Triton X-100 component of the complex was estimated to be 1.75 g of HsO/g of detergent (28) (see "Discussion").

Isoelectric Focusing-The isoelectric point for the solubilized lymphocyte insulin receptor was determined using an LKB 8100 Ampho- line electrofusing column (110 ml) a t 4 "C. A pH gradient of 2.5-8 was developed in a 5-5076 sucrose gradient containing 0.1% Triton X-100. Catalase was added as a marker (PI 6.1 (29)) to the detergent- solubilized receptors after which the preparation was dialyzed against three changes of 9% sucrose with 0.1% Triton X-100. Equal volumes of the samples were added to both the dense and light sucrose solutions. Three hundred volts were applied for 30 min followed by 550 V Cor 30 min, after which 800 V were applied for the duration of the column focusing. Equilibrium was assured after 41 h when 2-ml fractions were collected and the pH w-as determined at 4 "C. Each fraction was dialyzed against two changes of PBS (pH 7.4) with 0.147. Triton X-100 and one change of RRA buffer with 0.1% Triton X-100. The final pH of each fraction was confirmed a t 7.6. Aliquots of 500 pl were taken from each fraction for the determination of "'I-insulin binding activity and catalase activity.

Covalent Cross-linking of the Insulin Receptor to '2"I-Insulin- The cross-linking agent, disuccinimidyl suberate, was synthesized from suberic acid and N-hydroxysuccinimide utilizing N, N-dicyclohexylcarbodiimide in dioxane and was recrystallized from acetone/ diethyl ether, following the method of Anderson et al. (30) as modified by Pilch and Czech (31). IM-9 lymphoblastoid cell membranes (3 mg of membrane proteins/ml) were incubated with '2511-insulin (1.5 x M) a t 0 "C for 16 h in RRA buffer. The free "'I-insulin was removed by centrifugation and the pellet was resuspended in 500 pl of 0.02 M phosphate buffer, pH 7.3, containing 0.1% bovine serum albumin, Five pl of 0.1 M disuccinimidyl suberate in dimethyl sulfoxide was added and the suspension was incubated at 4 "C for 20 min, after which the reaction was terminated with 5 pl of 1 M ammonium acetate. The suspension was then brought to 20 "C for 30 min and unreacted l2'1-

insulin was removed by centrifugation. The membrane pellet was solubilized with 1% Triton X-100 as described above. The resulting preparation was partially purified by gel filtration on Sepharose 6B equilibrated with 0.2 M phosphate buffer containing 0.1% Triton X- 100. The hydrodynamic properties of the material were identical with the non-cross-linked "'I-insulin-receptor complex as determined by gel filtration and sedimentation through sucrose density gradients.

Sedimentation Equilibrium-The macromolecule to be studied was dissolved in 100 p1 of PBS containing 0.5% bovine serum albumin for density stabilization and centrifuged for a t least 24 h in an air turbine tabletop centrifuge (Beckman Spinco Airfuge) as previously described (11). Briefly, the 3.8-cm diameter fixed angle turbine rotor is lifted and driven by internally pressure-regulated compressed air a t

12120 Characterization of the Insulin Receptor

a rotational velocity (up to 100,OOO rpm) which is determined stro- boscopically. All procedures were carried out in the cold and the rotor temperature at the end of centrifugation was 5 "C. Following centrifugation, the tubes were oriented vertically and successive 1o-pl fractions were withdrawn from the meniscus of the reoriented solution, utilizing disposable 10-pl capillary pipettes which were stabilized and guided by a micromanipulator. Each fraction was analyzed for mat- romolecular concentrations by the determination of "'I, I4C, or 3H radioactivity. Corrections were made for small amounts of residual nonsedimentable material (less than 10%) by subtracting from every data point the macromolecular concentration remaining at the meniscus of a parallel tube after further sedimentation at high speed (11). The omission of this correction may lead to substantial underestimation (-20%) of molecular weights. The radial distance of each fraction from the center of rotation during centrifugation was determined by numerical integration of exact equations describing the tube geometry and orientation (11).

For sedimentation equilibrium at a given temperature ( T ) , angular velocity ( w ) , and solution density ( p ) of a macromolecu!e with a given molecular weight ( M ) and partial specific volume ( V ) , the macromolecular concentration ( c ) as a function of radial distance (r) from the center of rotation under ideal conditions is determined by the fact that throughout the gradient

d(lnc)/dr' = Mw'(1 - Vp)/2RT (3)

irrespective of the tube geometry. Achievement of sedimentation equilibrium employing this method was confirmed by the experimental finding that Inc is a linear function of r2 and that the slope yields through Equation 3 molecular weights which are in agreement with known values and are independent of the angular velocity used for the determination (11).

Sedimentation Equilibrium in DzO and h"0"For the determination of macromolecular partial specific volumes, the above procedures were performed in buffers with varying concentrations of D20 and D?'"O, analogous to the method employed in the analytical ultracentrifuge (32). Buffer densities were measured pycnometrically. Minor corrections for the slight increase in molecular weight and corresponding decrease in partial specific volume of proteins in buffers containing D2O or D2"O due to exchangeable hydrogen atoms were taken as 1.5% (33) multiplied by the mole fraction deuterium content of the solution. The extremely small error resulting from the single exchangeable hydrogen atom in the Triton X-100 molecule was ne- glected. All tubes sealed within the rotor contained the same D20 or D2l8O concentration, since cross-evaporation between tubes containing different D20 concentrations within the same rotor caused density inversion and mixing near the meniscus of those tubes with less DzO. To ensure achievement of equilibrium despite the increased viscosity of D20 and Dr''0, centrifugation was prolonged to 48 and 60 h, respectively (1 1, 34).

Protein was determined by the method of Lowry et al. (35) using bovine serum albumin as a standard. "'I and '"'I radioactivities were determined with a Packard Auto-Gamma counter. 'Hz0 and L-["H] leucine radioactivity was determined with a Packard Tri-Carb scin- tillation counter.

In several of these studies, the cells were grown in the presence of ~['Hlleucine (168 mCi/mmol) for 48 h before harvest, resulting in protein-specific "H radioactivity of 20 pCi/mg. Protein content could then be monitored by determining ~['H]leucine radioactivity.

RESULTS

Incubation of the membranes of the cultured human lymphoblastoid cell IM-9 with 1% Triton X-100 for 30 min a t 4 "C resulted in the solubilization of approximately 30% of the membrane protein and virtually complete recovery of insulin binding activity. Negligible binding activity remained in the undissolved pellet recovered by centrifugation at 40,000 X g for 30 min. The binding of '251-insulin (5 X 1 0 - l ' ~ ) to the solubilized receptor preparation in RRA buffer at 15 "C reached equilibrium within 60 min and remained stable for a t least 2 h, suggesting the absence of significant ~'cgradation of insulin or receptors. Insulin binding activity wa ; stable for a t least 1 week at 4 "C. The specificity of the ceILular insulin receptor was retained by the solubilized preparation, as shown by the fact that 1251-insulin binding was not inhihit ?d by other polypeptide hormones (1 pg/ml), including hu, ;,in growth

hormone, bovine thyrotropin, human chorionic gonadotropin, follicle stimulating hormone, prolactin, and corticotropin. As reported for other hormone receptors (19), the dissociation a t 4 "C of bound 1251-insulin from the particulate and solubilized membrane receptor occws at a very slow rate, allowing the study of the hydrodynamic properties of the insulin-receptor complex at low temperature.

Equilibrium Binding-1251-insulin binding to the solubilized insulin receptor of the cultured human lymphocyte in RRA buffer containing 0.1% Triton X-100 in the presence of increasing concentrations of native insulin is shown in Fig. 1. The linear Scatchard analysis, displaying bound/free '251-insulin as a function of the total insulin bound, indicates the presence of a single homogeneous class of independent binding sites in agreement with results for the soluble rat hepatocyte and adipocyte receptor. The slope yields an affinity constant of 2.0 X lo9 M", consistent with the high affinity component of the curvilinear binding plots for the lymphoblastoid cellular receptor (13). This value is inconsistent with that predicted by the negative cooperativity model (12) and provides confirmation of kinetic data indicating that negative cooperativity is not a significant factor for insulin binding to the cellular receptor (13). The abscissa intercept yields an insulin binding capacity of 1 pmol/mg of protein.

Gel Filtration Chromatography-Chromatography of the free solubilized receptor and the insulin-receptor complex on a Sepharose 6B column in the presence of 0.1% Triton X-100 is shown in Fig. 2. The column was calibrated by determining the distribution coefficient ( K ) for globular proteins of known Stokes radii, yielding a linear empirical relationship (inset) between (-ln K)"? and Stokes radius (36). The free insulin receptor (solid line), as measured by the binding activity of each fraction for '"I-insulin, chromatographed as a single peak with a position corresponding to a Stokes radius of 81 A. The insulin-receptor complex (broken line), as detected by the simultaneous chromatography of solubilized membranes which had previously been incubated with '251-insulin, also

r I I I 1 I 1

I I I I I 05 1 0 1 5 2 0 2 5

BOUND INSULIN Ing/ml)

FIG. 1. Equilibrium binding of 1251-insulin to the insulin receptor of the IM-9 lymphoblastoid cell solubilized in Triton X- 100. '"I-insulin (5 X lo-" M ) was incubated with the solubilized receptor preparation (80 pg of membrane protein/ml) at 15 "C for 90 min in RRA buffer containing 1% bovine serum albumin, 0.1% Triton X-100, and varying concentrations of unlabeled insulin (0-1 pg/ml). Bound hormone was determined by precipitation with 108 polyethylene glycol. The data are presented by Scatchard analysis, displaying specifically bound/free "'I-insulin as a function of specifically bound insulin. The slope of the plot yields an affinity constant of 2.0 X 10' M" and the abscissa intercept indicates a binding capacity of 1 pmol of insulin/mg of protein.


' i ?I 40 60 80 c

a I .

40 60 80 100

FRACTlON NUMBER

b - FIG. 2. Gel filtration chromatography of the detergent-sol-

ubilized insulin receptor. Solubilized "'I-insulin-receptor complexes were prepared by incubation of '*'I-insulin (5 X 10"' M) with IM-9 lymphoblastoid cell membranes (10 mg of membrane protein/ ml) for 18 h at 4 "C, followed by a 3-fold dilution with cold buffer and removal of free "'I-insulin by centrifugation. The membranes (3 mg of protein/ml) were solubilized with Triton X-100 as described under "Materials and Methods." These complexes were mixed with solubilized membrane receptors (3 mg of membrane protein/ml) and chromatographed on a Sepharose 6B column in RRA buffer containing 0.1% Triton X-100 at 4 "C. The insulin-receptor complex (0- - -0) was detected by the polyethylene glycol-precipitable "'I-insulin in aliquots of each fraction. The free insulin receptor (W) was determined by the binding activity of each fraction for ""I-insulin (1 X lo-" M). The elution position for blue dextran ( VO), thyroglobulin ( T G ) , apoferritin ( A F ) , y-globulin ( yG) , bovine serum albumin (BSA ), and JH20 ( V,) are given in the figure. The inset displays (-ln K)lr2 as a function of the Stokes radius (in Angstrom units) for the protein standards, the insulin-receptor complex (O), and the free receptors (0).

yielded a single peak in a position corresponding to a Stokes radius of 81 A. No significant insulin binding activity was detected on this column in the absence of detergent-solubilized membranes or in the presence of unlabeled insulin at a concentration of 2 X M. Solubilized membrane protein, as measured by ~-['H]leucine activity, chromatographed as a very broad peak with an average Stokes radius of 60 A (data not shown).

Sedimentation through Sucrose Gradients-The sedimentation behavior of the solubilized free insulin receptor and the insulin-receptor complex was examined simultaneously in 5- 20% sucrose gradients containing 0.1% Triton X-100, as shown in Fig. 3. Calibration of these gradients with globular protein markers yielded a nearly linear relationship between sedimentation coefficient and distance from the meniscus as shown in the inset. The free insulin receptor (solid line), as measured by the binding activity of each fraction for 1311-insulin, sedimented as a single peak with position corresponding to a sedimentation coefficient of 10.5. In the same gradients, the insulin-receptor complex (broken line), as detected by the behavior of solubilized membranes to which 1251-insulin had been bound, sedimented slightly more rapidly as a single peak with a position corresponding to a sedimentation coefficient of 11. In the absence of solubilized membranes, or in the presence of native insulin at a concentration of 2 X M, no significant binding activity was detected on these gradients. Solubilized membrane protein, as measured by ~['Hlleucine radioactivity, sedimented as a very broad peak with an average sedimentation coefficient of 5 (data not shown). The sedimen-

tation behavior of the detergent-solubilized "'1-insulin receptor complex relative to the globular protein standards was nearly identical in 540% (w/v) sucrose-DZO gradients containing 0.1% Triton X-100 (data not shown).

Isoelectric Focusing-The behavior of the free solubilized insulin receptor on a 110-ml isoelectric focusing column over a wide pH range in the presence of 0.1% Triton X-100 is shown in Fig. 4. Catalase, which was included as an internal control protein under these conditions, appeared as a narrow peak with an isoelectric point of 6.1 in good agreement with previously published results (29). The free insulin receptor, as measured by the binding activity of each dialyzed fraction for

I-insulin, appeared as a single broader peak with an isoelectric point of 4.8.

Sedimentation Equilibrium of the Detergent-solibilized Receptor Cross-linked to "51-Insulin-In order to eliminate the multiple ambiguities inherent in the determination of the Stokes radius by gel filtration chromatography, in the estimation of the sedimentation coefficient by centrifugation through sucrose density gradients, and in the use of these parameters to estimate the molecular weight and axial ratio of the macromolecules, we examined the sedimentation equilibrium profile of the detergent-solubilized insulin-receptor complex. This complex was stabilized for the prolonged centrifugation required (at least 24 h) by covalent cross-linking with disuccinimidyl suberate following the method of Pilch and Czech (31). Although this material had identical hydrodynamic properties as the non-cross-linked "'I-insulin-receptor complex, retention of the native conformation after cross- linking is not a requirement for the sedimentation equilibrium method. Fig. 5A shows the sedimentation equilibrium profile of the cross-linked 1251-insulin-receptor complex in PBS containing 0.1% Triton X-100 and 0.5% bovine serum albumin for

125

' 21 E 3 7

FRACTION NUMBER

FIG. 3. Sedimentation through sucrose gradients. The l"1- insulin-receptor complex (0- - -0) and free solubilized receptors (M), prepared as outlined in the legend to Fig. 2 in a volume of 200 ml were sedimented through a 5-ml 5-20% sucrose density gradient in RRA buffer containing 0.1% Triton X-100 by centrifugation for 8 h at 4 "C at 63,000 rpm in a Spinco SW-65 rotor. The insulin receptor complex was detected in aliquots of each fraction by determination of the specifically bound '"I-insulin. Free receptors were detected in each fraction by incubation with ',"I-insulin (1 X 10"' M), followed by determination of specifically bound ''''I radioactivity. The sedimentation position for the internal standard protein catalase (Cat) in addition to y-globulin (yClob) and bovine serum albumin (BSA) are shown. The inset shows the sedimentation coefficient ( s ) as a linear function of the distance from the meniscus for the protein standards, insulin-receptor complex (O), and the free insulin receptor (0).


FRACTION NUMBER

FIG. 4. Isoelectric focusing of the solubilized insulin receptor. The detergent-solubilized insulin receptor (1.3 mg of protein) and the catalase standard (3 mg) were dialyzed against 9% sucrose containing 0.1% Triton X-100 and subjected to isoelectric focusing (800 V, 41 h) at 4 "C, during which a pH gradient of 2.5-8 was developed in a 5-5096 sucrose gradient containing 0.1% Triton X-100. After extensive dialysis of each fraction, specific binding activity for 1251- insulin ( 5 X 10"' M) (."--.) and catalase activity (m) were determined. The pH of each fraction before dialysis (.-.-) is also shown.

V O l U ~ (# I )

FIG. 5. Sedimentation equilibrium of the cross-linked '*'I- insulin-receptor complex. The covalently cross-linked '*'I-insulin- receptor complex (200 pg/100 pl) in PBS containing 0.1% Triton X- 100 and 0.5% bovine serum albumin was centrifuged at 5 "C for 36 h at 18,OOO rpm in the Airfuge. After deceleration and reorientation of the solution, 10-pl fractions were collected from the meniscus and 12'1-

insulin-receptor content was determined. All data were corrected for a small amount (less than 10%) of nonsedimenting material, by subtracting the Iy5I radioactivity remaining at the meniscus of a parallel tube after further sedimentation at 70,000 rpm for 6 h. A , the -,'I-insulin-receptor content is shown as a function of the radial

distance from the center of rotation. B, the natural logarithm of the I-insulin-receptor concentration (In c) is shown as a function of the

square of the radial distance (r') .

1 . v

125

density stabilization following centrifugation at 5 "C for 24 h a t 18,000 rpm. The concentration of the insulin-receptor complex increases monotonically as a function of distance from the center of rotation. As expected for sedimentation equilibrium, the natural logarithm of the concentration is a linear function of the square of the radial distance as shown in Fig. 5B. Under these conditions, the slope of this line yields through Equation 3 a value for M(l - v p ) of 102,000. This result is in excellent agreement with the value of 100,000

derived from the hydrodynamic properties of the insulin-receptor complex employing Equation 1.

Determination of Molecular Weight and Par t ia l Specific Volume by Sedimentation Equilibrium of Macromolecules in Buffers of Varying Density-For ideal solutions, sedimentation equilibrium of a macromolecular complex yields (2RTl w' ) (d lncldr') = M,(1 - pep) where M, and q. are the molecular weight and partial specific volume of the complex, respectively (8,38). Therefore, knowledge of (2RT/w2)(d lnc/ d?) as a function of solution density determines M, and vc. Since this approach has been employed successfully in the analytical ultracentrifuge with H20/D20 solutions of varying densities (32), we have developed the method for sedimentation equilibrium in the air-turbine preparative ultracentrifuge. The use of DzO and Dzl'O rather than high solute concentrations to produce changes in solution density minimizes the effects of preferential interactions of the macromolecule with solute or solvent.

To accommodate substitution of deuterium for exchangeable hydrogen atoms in the macromolecule (32), the above equation may be modified to yield

( 2 R T / k w 2 ) ( d Incldr') = Mc[l - q ( p / k ) ] (4)

where k is the ratio of the molecular weight of the substituted complex to that of the unsubstituted complex and is depend- ent on the mole fraction of deuterium in the solvent and the nature of the complex. For proteins (33), k at each solvent density was taken as 1.015 multipled by the mole fraction of deuterium in the solvent. For Triton X-100, deuterium substitution for the single exchangeable hydrogen atom was ne- glected and k was taken as 1.00. For the insulin-receptor- Triton X-100 complex, the protein was initially estimated to comprise approximately two-thirds of the complex and k was taken as 1.01 multiplied by the deuterium mole fraction in the solvent.

Sedimentation equilibrium was studied at 5 "C in PBS containing 0.5% bovine serum albumin and varying concentrations of D20 or D2I80, which resulted in a density range of approximately 1.0 to 1.2 g/cm3. (2RT/kw2)(d1nc/dr2) for I4C- methylated human y-globulin as a function of p lk is shown in Fig. 6. The linear character of this experimental relation c o n f i i s t h e applicability of the method to the characterization of proteins by demonstrating that v is independent of solution density. The abscissa intercept (where M[l - v(p/ k ) ] = 0 and P = k / p ) yields v = 0.73, in agreement with known values (37 ) . A similar study for 1% [ phenyl-"]Triton X-100 in these buffers is also shown in Fig. 6. These data are likewise linear and the abscissa intercept yields v = 0.91 for this detergent at a concentration well above its critical micelle concentration, in good agreement with other methods (38). These results are summarized in Table 11.

The results of sedimentation equilibrium determinations of M,[1 - v J p / k ) ] as a function of p / k for the cross-linked lZ5I- insulin-receptor complex in PBS containing 0.1% Triton X- 100, 0.5% bovine serum albumin, and varying concentrations of D20 and Dzl"O are given in Fig. 7. The linear relationship c o n f i i s t h a t vc is independent of density, and the abscissa intercept yields v, = 0.78 for the detergent-receptor complex. Utilizing this value, the molecular weight of the complex (M,) from these data is 475,000.

For sedimentation equilibrium of multicomponent systems, including a macromolecular protein and detergent or other components which interact with the protein, the system may be described by (8, 38)

M,. = Mp( l + 6, + X,&) (5)

q = ( V p + S d V d + Zz&Vz)/( l + &I + ZJJ (6)


p: CY 1.10 1.20 1.30 1.40

yk FIG. 6. Density dependence of the sedimentation equilib-

rium behavior of human 7-globulin and Triton X-100. [phenyl- 3H]Triton X-100 (10,000 cpm) was added to 1% Triton X-100 and 0.5% bovine serum albumin in PBS containing varying concentrations of D20 and Dz''0. The solutions were centrifuged at 5 "C for 24 h at 30,000 rpm. [ rnethyl-14C]human y-globulin (10,000 cpm) was added to 0.5% bovine serum albumin in PBS containing varying concentrations of D20 and D2"0 and centrifuged at 5 "C for 24 h at 50,000 rpm. For each buffer density, the data were analyzed as in Fig. 5. (2RT/ ko2)[d(ln c)/dr2] = Mc[l - V c ( p / k ) ] is plotted as a function of p / k in the above figure. The reciprocal of the abscissa intercept of the linear extrapolation of the data yields a partial specific volume of 0.91 for Triton X-100 and 0.73 for human y-globulin.

I , 1

110 1.20 1.30 1.40

c/1, FIG. 7. Density dependence of the sedimentation equilib-

rium behavior for the cross-linked '2SI-insulin-receptor in 0.1% Triton X-100 and 0.5% bovine serum albumin. Covalently cross- linked '"I-insulin-receptor complexes (0.2 mg of protein/100 pl) (M) were centrifuged for at least 36 h at 5 "C in PBS containing varying concentrations of D20 and D2'*0. The sedimentation equilibrium results are analyzed and presented as described in the legend to Fig. 6. The reciprocal of the abscissa intercept of linear extrapolation for (RT/kw2)[d(ln c)/dr2] = Mc[l - V,(p/k)] as a function of p/k yields a partial specific volume of 0.78 for the detergent-receptor- hormone complex. The contributions of the receptor protein ( vp = 0.71) (- -) and Triton X-100 ( V , = 0.91) (. . . . ) to the sedimentation equilibrium behavior of the complex are shown schematically in the figure as a function of buffer density.

where S represents the grams of detergent or other component bound per g of protein and the subscripts p , d, and i refer to the protein, bound detegent, and other bound components, respectively. Several reasonable approximations are necessary to make this expression most useful. We assume that Sd and 6, are independent of D20 concentration and that during detergent extraction and solubilization membrane lipids have largely been replaced by detergent molecules (39), reducing the 6, of the most likely non-detergent component to negligible

levels. We also approximate v d of the bound detergent by the partial specific volume of the detergent alone above its critical micelle concentration (38), which for Triton X-100 was found to be 0.91 cm3/g from the data in Fig. 6.

Assuming that the insulin receptor is a membrane receptor glycoprotein with a carbohydrate content of approximately 5%,' its partial specific volume at 5 "C is approximately 0.71 cm3/g. Equations 5 and 6 then yield Mp = 310,000 for the molecular weight of the receptor glycoprotein alone and & = 0.54 g of Triton X-100 bound per g of receptor protein. These results are listed in Table 11. Furthermore, the separate contributions of the protein (- - -) and detergent (. . - - - ) to Mc[ 1 - v c ( p / k ) ] are also shown schematically in Fig. 7 as a function of p l k . At buffer densities near 1.1, the detergent contribution to sedimentation equilibrium is negligible and the behavior of the system is determined by Mp[l - v J p / k ) ] . This fact has formed the basis for an alternative method of determining Mp in the analytical ultracentrifuge (41). In buffers having densities greater than 1.1, the detergent makes an increasingly negative contribution, so that if p = 1.29 were achievable in this buffer system, the negative contribution of the detergent would be equal to the positive contribution of the protein and the complex would not sediment.

DISCUSSION

The insulin receptor of the cultured human lymphoblastoid cell IM-9, solubilized with the nonionic detergent Triton X- 100, behaves as a single class of high affinity binding sites ( K , = 2 X lo9 M-') as shown by the linear Scatchard analysis in Fig. 1. This receptor corresponds to the high affinity component of cellular insulin binding to IM-9 lymphoblastoid cells and independently confirms earlier work from our laboratory employing equilibrium and kinetic methods to demonstrate the non-cooperative character of insulin-receptor interactions (13).

The detergent-solubilized insulin receptor also behaves as a single species when analyzed by gel filtration chromatography, sedimentation through sucrose gradients, and isoelectric focusing. As summarized in Table I, these methods indicate that the free insulin receptor has a Stokes radius of 81 A, a sedimentation coefficient of 10.5 S, and an isoelectric point of 4.8. The slightly broad peak at pH 4.8 upon isoelectric focusing of the insulin receptor may reflect its glycoprotein nature, as indicated by its binding to multiple lectins (42), with a variable number of sialic acid residues contributing to the low PI and to microheterogeneity (43). For the insulin-receptor complex, the data in Figs. 2 and 3 indicate a Stokes radius of 81 A and a sedimentation coefficient of 11 S. Substitution of these hydrodynamic properties into Equation 1 results in a value for Mc(l - vcp) of 100,000.

The above indirect estimate of the physical parameters which characterize the insulin receptor is subject to several major limitations. I t is difficult to theoretically relate the Stokes radius of globular proteins to their behavior on analytical gel filtration chromatography ( 5 ) and large deviations have been observed for asymmetric molecules which are anomalously retarded (9). Furthermore, the estimation of true sedimentation coefficients by comparison with the sedimentation of standard proteins through sucrose density gradients is questionable when the partial specific volume of the protein- detergent complex differs significantly from that of the protein standards (3) and is further complicated by the preferential hydration of proteins in concentrated sucrose solutions (2).

The most extensively characterized membrane receptor, the acetylcholine receptor (Torpedo calzfornica), contains 75 carbohydrate residues (40) representing approximately 3-4% by weight.


The method of sedimentation equilibrium in the air-turbine preparative ultracentrifuge eliminates many of these prob- lems. Interpretation of results has an entirely theoretical basis in the thermodynamic description of a system in sedimentation-diffusion equilibrium, thus eliminating the requirement for empirical molecular weight standards. In addition, since the system is in equilibrium, the assumptions regarding macromolecular shape inherent in the interpretation of standard hydrodynamic techniques are unnecessary. Furthermore, the preferential interactions which complicate the interpretation of sedimentation data in concentrated sucrose solutions and the possibility of anomalous macromolecular interactions with a gel matrix are absent in the sedimentation equilibrium method. Lastly, the accuracy of this method has been con- f i i e d by other investigators (44). The single major assump- tion required is the absence of interaction between the macromolecule of interest and the 0.5% bovine serum albumin which is present for density stabilization. The sedimentation equilibrium results in Fig. 5B for the Triton X-100-solubilized insulin receptor covalently cross-linked to "51-insulin confirm that the natural logarithm of the concentration is a linear function of the square of the radial distance from the center of rotation. From Equation 3, the slope of this line gives a value for M,(1 - vc) of 102,000. The extreme degree of agreement of this value with that derived by substitution of the hydrodynamic parameters into Equation 1 may be somewhat fortuitous.

The most important remaining problem for the characterization of protein-detergent complexes concerns _determination of the partial specific volume of the complex, V,, which then yields through Equations 5 and 6 an estimation of the molecular weight of the protein component alone, M,, and the degree of detergent binding, 6d. We have performed this determination by extending the sedimentation equilibrium technique in the air-turbine preparative ultracentrifuge through the use of high density buffers containing D20 and D2180 as previously utilized in the analytical ultracentrifuge (32). This technique avoids the preferential interactions in- volved in the use of high solute concentrations to increase density. We have applied this method to a determination of

TABLE I Hydrodynamic properties of the insulin receptor

Receptor 81 10.5 96,000 1.6 (1.5) 1.2 (Triton)

Receptor-insulin 81 11 100,000 1.5 (1.4) 1.2 complex (Triton)

Receptor-insulin 84 16 153,000 1.5 (1.4) complex (digitonin)

Triton X-100 39 0.85 3,800 1.6 1.1 (I%, micellar)

The frictional ratios were calculated from Equation 2 utilizing partial specific volumes of 0.78,0.73, and 0.91 cm3/g for the receptor- Triton, receptor-digitonin, and micellar Triton X-I00 complexes, respectively.

*The values in parentheses have been partially corrected for hydration employing 0.3 g of HrO/g of protein component.

The asymmetry component of the frictional ratio has been estimated from Equation 2 by fully correcting for hydration using 0.3 g of H,O/g of protein component and 1.75 g of HzO/g of detergent component.

the partial specific volumes of human y-globulin, micellar Triton X-100, and the Triton X-100-''51-insulin-receptor complex. The data for each of these macromolecules in Figs. 6 and 7 show that M(1 - v ( p / k ) ] is indeed a linear function of p / k , as predicted from theoretical considerations and the assump- tion that the binding of a given component is independent of density. The abscissa intercepts extrapolated from these data yield partial specific volumes of 0.73 for human y-globulin and 0.91 for Triton X-100 as summarized in Table 11. The agreement of these values with those in the literature (37, 38) provides c o n f i a t i o n of the validity of this technique for the determination of partial specific volumes. While the required extrapolation from the data is considerable, the close linearity of the data lends confidence to the values obtained.

The sedimentation equilibrium results as a function of p / k for the Triton X-100-'"I-insulin-receptor complex, as shown in Fig. 7, are likewise linear and yield a partial specific volume of 0.78 cm3/g. Assuming that the receptor is a membrane receptor glycoprotein with a carbohydrate content of approximately 5%' having a partial specific volume of 0.71, that the partial specific volume for bound Triton X-100 is 0.91 as determined for the detergent above its critical micelle concentration, and that only negligible levels of membrane lipids remain after detergent extraction of the insulin-receptor (39), Equations 5 and 6 yield a molecular weight of 310,000 for the receptor protein alone and a level of Triton X-100 binding of 0.54 g/g of protein. These results are summarized in Table 11. This molecular weight is in reasonable agreement with pre- vious estimates for the insulin receptor of the hepatocyte and placenta (45) and corresponds closely to that of the (a)2(p1)2

form of the insulin receptor reported by Massague et al. (46) for the rat adipocyte. I t should be noted that we can estimate the possible error in the determination of M , resulting from the uncertainty in the determination of vc, employing the relation (1/Mp)(dMp/dV,) = ( V d p - 1)/(1 - Vcp)(Vd - V<). Assuming that Vc = 0.78 f 0.02, the uncertainty in M p is at an acceptable level of +-6.3%. The partial specific volume of the detergent-solubilized '"I-insulin-receptor complex, as estimated from its largely unchanged sedimentation behavior relative to globular protein standards in 5-20% (w/v) sucrose- H 2 0 and sucrose-D20 gradients containing 0.1% Triton X-100, would be 0.74 cm3/g (26,39). However, this method is limited by assumptions concerning unchanged preferential hydration, hydrogen bonding, and conformational structure in sucrose- D20 and is quite approximate if the protein studied is highly asymmetric and/or binds significant amounts of detergent of low density (8, 38).

The determination of the partial specific volume of the complex by sedimentation equilibrium in buffers of varying density permits an estimation of the molecular asymmetry of the complex from the hydrodynamic properties determined by gel filtration and sedimentatiol through sucrose gradients. Substitution into Equation 2 of V, = 0.78, the Stokes radius of 81 A, the sedimentation coefficient of 11 S, and an estimated

TABLE 2 Smfimentation eauilibrium results "." ~.

M(1 - V p ) v M M P a d

Receptor-insulin 102,000 0.78 475,000 310,000 0.54 complex (Triton X-100)

Triton X-100 4,000 0.91 47,000 (micellar)

7 S y-globulin 42,000 0.73 158,000 (human)


hydration of 0.3 g of H20/g of protein, results in a partidly corrected frictional ratio ( f / f , ) of 1.4 as summarized in Table I. This corresponds to an axial ratio for the detergent-receptor complex of 8:1 for an equivalent prolate ellipsoid (47). AI- though this degree of molecular asymmetry is reportedly characteristic of many detergent-solubilized membrane receptors, several factors which may be responsible for apparent molecular asymmetry must be considered. The likelihood of significant underestimation of the level of detergent binding to the membrane protein has been minimized in the present study by an independent determination of the partial specific volume of the complex by sedimentation equilibrium. Fur- thermore, when ambiguities concerning the partial specific volume of the complex are avoided by solubilization with digitonin which has a partial specific volume ( vd = 0.738) (48) nearly identical with that of proteins, the hydrodynamic properties of the resulting complex (Table I) continue to indicate a frictional ratio ( f / f o ) of 1.4 corresponding to an axial ratio of 8:l.

The other major factor to be considered concerns the contribution of the high level of detergent hydration to the apparent asymmetry of the complex. The hydrodynamic properties of Triton X-100 micelles and other considerations sug- gest the presence of significant hydration of the oxygen atoms of the polyoxyethylene chains and hydrodynamically en- trapped water, resulting in estimates of 1.1-1.9 g of water bound/g of detergent (49, 50) at 25 "C. The Triton X-100 detergent micelle at 25 "C (Mr = 90,000) is an oblate ellipsoid containing 1.1 g of HzO/g of Triton X-100 (50). Employing the same structural considerations for the smaller 47,000-dalton Triton X-100 micelle at 5 "C (Table II), the hydration factor becomes 1.75 g of H,O/g of Triton for this mildly oblate ellipsoid (axial ratio 1.5). Equations 1 and 2 predict a Stokes radius of 37 A and a sedimentation coefficient of 0.95 S for this hydrated detergent micelle. As shown in Table I, this degree of hydration at 5 "C was confirmed by our experimental finding for [phenyL3H]Triton X-100 in 1% Triton X-100 of a = 39 A by gel filtration chromatography and s = 0.85 S by sucrose gradient centrifugation corrected to water density by Equation 14 of O'Brien et al. (26).

While hydration need not be considered in the determination of molecular weight or partial specific volume of the detergent-receptor complex as formulated in this paper, its effect on the hydrodynamic properties of the complex should be emphasized. Utilizing the above hydration value of 1.75 g of H20/g of detergent for the Triton X-100 component of the detergent-receptor complex at 5 "C, the overall hydration factor (6) becomes 0.81 g of HzO/g of complex and Equation 2 yields ( f / f o ) ~ = 1.2. This value approaches the range characteristic of oligomeric globular proteins (51).

Since a high level of hydration is tautologically inherent in the detergent function of these solubilizing agents, the high degree of asymmetry generally reported for detergent-solubilized membrane proteins has been systematically overesti- mated. This is further aggravated when sedimentation coefficients are estimated in 5-20% sucrose gradients, since the greater hydration of the detergent-protein complex relative to the protein standards which bind little or no detergent (39) preferentially retards its sedimentation rate by buoyancy effects in the more dense bulk solution as well as by frictional effects.

The contribution of detergent hydration to the overestimation of membrane protein asymmetry is underscored by results for the detergent-solubilized acetylcholine receptor (Torpedo californica) which has been extensively purified and characterized in several laboratories. While analytical (52) and empirical (53, 54) hydrodynamic data in Triton X-

100 and other nonionic detergents are consistent in yielding a frictional ratio (f/fo) of 1.5-1.6 corresponding to an equivalent prolate ellipsoid with an axial ratio of approximately lO:l, electronmicroscopic and x-ray diffraction analysis of this func- tional 250,000-dalton protein shows it to be globular, with approximate molecular dimensions of 85 x 85 X 100 A and a small central channel (55). Therefore, neglecting to consider hydration of the detergent and to a lesser extent water en- trapped within the central channel (approximately 0.1 g of HsO/g of protein) in the interpretation of the hydrodynamic data results in serious overestimation of the actual molecular asymmetry.

In addition, although frictional coefficients for proteins have classically been separated into components reflecting their degree of hydration and molecular asymmetry (56), it has become clear that additional factors such as rugosity of the protein surface may also contribute significantly (51). There- fore, the possibility remains that radially oriented detergent molecules on the surface of the complex further increase the frictional coefficient of solubilized membrane proteins through this mechanism and that the shape of these proteins is even closer to the globular range.

Note Added in Proof-The reciprocal (0.94 ml/g) of the density of pure Triton X-100, as determined by Greenwald and Brown (57), has in several instances been substituted by others for its partial specific volume (v,) in H20, leading to 20-25% underestimation of the levels of detergent binding to solubilized proteins and 5-10% overestimation of their molecular weights by hydrodynamic methods. Actually, the vd of Triton X-100 from the data of Greenwald and Brown (57) at 25 "C is 0.908 in H20 and 0.915 in 1 M NaC1, in good agreement with subsequent determinations of 0.908 in H20 at 25 "C (38) , 0.915 in 0.1 M sodium acetate at 20 "C (49), and with the present results of 0.91 by sedimentation equilibrium and approximately 0.90 gravimetrically at 5 "C.

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BIOLOGICAL THE OF CHEMISTRY No. Vol. 10, 1981 rJ. in ... · 23, hue of December 10, pp. 12118-12126, 1981 Printed in rJ. S A. Characterization of Detergent-solubilized Membrane Proteins