[18] Peptide hormone receptors in intracellular structures from rat liver

[18]

PEPTIDE

HORMONE

RECEPTORS

219

[18] P e p t i d e H o r m o n e R e c e p t o r s in I n t r a c e l l u l a r S t r u c t u r e s from Rat Liver B y BARRY I. POSNER, MASOOD N . K H A N , a n d JOHN J. M . BERGERON

This chapter presents methods which have been used in our laboratories for the preparation and analysis of peptide hormone receptor-rich structures derived from within the rat hepatocyte. A new entity--the intermediate or "unique" vesicle--has been observed to contribute to the heterogeneity of Golgi fractions.1 Though we only present a method for resolving intermediate vesicles from Golgi elements we are confident that in the near future a more general quantitative scheme for preparing receptor-rich intracellular structures will be available. Sherman or Sprague-Dawley rats 150 to 250 g each have been used in these studies. The animals are fed ad libitum to the day before use and are fasted overnight before the experiment. They are killed by decapitation and exsanguinated before the livers are removed. All sucrose solutions (w/w solution) are prepared with reagent grade material (Fisher Scientific Limited). In earlier studies the rats received ethanol (0.6 g/100 g body wt) in a 50% (w/v) solution by stomach tube 90 min prior to sacrifice. 2-1° In more recent studies the use of ethanol has been discontinued. TM Golgi Subfractions by a Differential Centrifugation

P r o c e d u r e 1,2,11

Livers are collected individually or in batches in ice-cold 0.25 M sucrose (5 ml/g liver), weighed, minced with scissors, and drained. All sub1 M. N. Khan, B. I. Posner, R. J. Khan, and J. J. M. Bergeron, J. Biol. Chem. 257, 5969 (1982). 2 j. H. Ehrenreich, J. J. M. Bergeron, P. Siekevitz, and G. E. Palade, J. Cell Biol. 59, 45 (1973). 3 j. j. M. Bergeron, B. I. Posner, R. Sikstrom, and Z. Josefsberg, J. Biol. Chem. 253, 4058 (1978). 4 B. I. Posner, J. J. M. Bergeron, and Z. Josefsberg, J. Biol. Chem. 253, 4067 (1978). 5 B. I. Posner, Z. Josefsberg, D. Raquidan, and J. J. M. Bergeron, Proc. Natl. Acad. Sci. U.S.A. 75, 3302 (1978). 6 B. I. Posner, Z. Josefsberg, and J. J. M. Bergeron, J. Biol. Chem. 254, 12494 (1979). 7 B. I. Posner, R. M. Gonzalez, and H. J. Guyda, Can. J. Biochem. 58, 1075 (1980). 8 Z. Josefsberg, B. I. Posner, B. Patel, and J. J. M. Bergeron, J. Biol. Chem. 254, 209 (1979). 9 B. I. Posner, B. Patel, A. K. Verma, and J. J. M. Bergeron, J. Biol. Chem. 255, 735 (1980). "~ B. I. Posner, A. K. Verma, B. A. Patel, and J. J. M. Bergeron, J. Cell Biol. 93, 560 (1982). 11 B. I. Posner, B. Patel, M. N. Khan, and J. J. M. Bergeron, J. Biol. Chem. 257, 5789 (1982).

METHODS IN ENZYMOLOGY, VOL. 109

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182009-2

220

INTERNALIZED HORMONES AND RECEPTORS

[18]

sequent procedures are carried out in a cold room at 4°. The same volume of ice-cold 0.25 M sucrose is added to the minced tissue which is transferred to a Potter-Elvehjem glass-Teflon homogenizer. The mixture is homogenized with 6 full strokes of the motor-driven Teflon pestle at 1000 rpm. The homogenate is centrifuged at 4° at 8800 gmaxfor 10 min (8000 rpm in a Beckmann J2-21 using a JA-17 rotor). The supernatant is decanted into chilled 25 ml polycarbonate tubes and the pellet is discarded. The supernatant is centrifuged at 200,000 gav for 30 min (52,000 rpm in a Spinco L2-65 or L2-65B using a 60 Ti rotor). The supernatant is decanted and discarded and the pellet gently vortexed in a small vol of 0.25 M sucrose and then decanted into a Dounce homogenizer. The following final volumes of sucrose solutions are added in order to the pellet based on the wet weight of liver from which the pellet was derived: 0.25 M sucrose, 0.33 ml/g liver; 2.0 M sucrose, 0.85 ml/g liver; and distilled water, 0.33 ml/g liver. The pellet, in 1.5 ml/g liver of 1.15 M sucrose, is gently homogenized with a loose-fitting pestle to yield an even suspension. While the above centrifugation steps are proceeding the following sucrose gradient (see tabulation below) is being prepared in polyallomer tubes (for SW27 rotor), or cellulose nitrate tubes (for SW40 rotor). The sucrose solutions are prepared the day before and kept ice cold. The centrifuge tubes and sucrose solutions are embedded in crushed ice. Each succeeding sucrose layer is placed beneath the others using a syringe with a long needle.

Volume (ml)/tube Sucrose solution (M)

SW27 rotor tubes

SW40 rotor tubes

0.25 0.60 0.86 1.00 Microsome suspension

7.0 7.0 7.0 7.0 -10.0

2.4 2.4 2.4 2.4 -4.0

The microsome suspension (see above) is slowly injected beneath the four sucrose layers using a syringe and long needle so as to fill the tube. The approximate homogenate volumes required are noted above. Centrifugation is carried out at: 100,000 gay for 190 min (25,000 rpm, SW27 rotor), or 200,000 gay for 95 min (40,000 rpm, SW40 rotor). Each fraction is removed from the gradient (Fig. 1) with a 90° angle needle and syringe: Golgi light (GI) at 0.25/0.60 M sucrose interface Golgi intermediate (Gi) at 0.60/0.86 M sucrose interface

[18]

PEPTIDE HORMONE RECEPTORS

221

Golgi heavy (Gh) at 0.86/1.00 M sucrose interface Small vesicles (SV) at 1.00/1.15 M sucrose interface If it is desired that each fraction is maintained in isotonic conditions then each is placed in a separate cellulose nitrate tube and diluted to 0.25 M sucrose as follows: G1, fill with 0.25 M sucrose; Gi, add equal volume of distilled water then fill with 0.25 M sucrose; Gh, add 2 volumes of distilled water and fill with 0.25 M sucrose; and SV, add 2.5 volumes of distilled water and fill with 0.25 M sucrose. The samples are then centrifuged at 200,000 gav for 30 min (53,000 rpm, 60 Ti rotor). Corresponding pellets may be combined. For binding studies, all pellets are resuspended in 25 mM Tris-10 mM MgC12, pH 7.4.

Characterization Protein Recovery. The recovery of protein is depicted in Fig. 1. Morphology. The subfractions generated as depicted in the scheme of Fig. 1 have distinctive morphological features. GI consists largely of vesicles containing lipoprotein particles. Gi is similar, but is somewhat more heterogeneous. The vesicles in this fraction show a wider range of size and there are some tubular and flattened saccular elements as well. The Gh fraction is quite heterogeneous. Lipoprotein containing vesicles are minority elements. There is an abundance of flattened saccules and tubular elements and a significant proportion of small vesicles. SV was formerly part of our Gh fraction prepared without a 1.0 M sucrose layer. It consists largely of small vesicles. It is not clear to what extent these represent vesiculated plasmalemma or endocytotic vesicles. Enzyme Composition. It has been recently appreciated that various endoplasmic reticulum marker enzymes (viz. NADPH cytochrome c reductase and glucose-6-phosphatase) are present in bona fide Golgi compoFraction

Yield (mg /g liver)

~-

GI

0.13

-

Gi

0.29

_'003 _*0.07

~ G h ~ S V SM

0.48 1.71 1887

" 0,10 _':0.48 _'5.10

RM

4,17

-*0,75

FIG. 1. Schematic depiction of fractions generated from microsomes by centrifugation in the discontinuous sucrose gradient. Sucrose concentrations and the yield of each subfraction are noted. G1, Gi, and Gh refer to Golgi light, intermediate and heavy fractiohs respectively. SM refers to those smooth microsomal elements constituting the residual load zone. RM is the residual pellet containing predominantly rough microsomes. Each determination is the mean _+ standard deviation of 36 fractionations.

222

INTERNALIZED HORMONES AND RECEPTORS

[18]

nents 12 in addition to those regarded as plasma membrane markers. 13 However, progress in approaching the Golgi biochemically followed the recognition that the enzymes which characterize the Golgi and are especially enriched therein are various terminal glycosyltransferases. 14 We assay galactosyltransferase (EC 2.4. 1.38) by a modification of the method of Bretz and Staubli.t5

Reagents I. Sodium cacodylate buffer, 0.35 M MnC12, 0.32 M 2-Mercaptoethenol, 0.31 M (pH adjusted to 6.6) 2. UDP-[3H]Galactose, 10 ~Ci/ml (evaporate original material from New England Nuclear and redissolve in H20) 3. UDP-Galactose, 10 mM Adenosine triphosphate, 50 mM 4. Ovomucoid, 175 mg/ml 5. Triton X-100 in H20, 2% (w/v) 6. Phosphotungstic acid in 0.5 N HCI, !.0% (w/v) (Note: Solutions 1, 2, 4, and 5 can be stored frozen ( - 2 0 °) for several months; solution 3 must be freshly prepared; and solution 6 can be stored at 4° for an indefinite time).

Procedure The assay solution is prepared by mixing solutions 1, 2, 3, 4, and 5 in the ratio 5 : 1 : 4 : 4 : 4 (vol). Samples, containing 2 to 50 t~g protein in a volume up to 220/zl, are mixed with 180/zl of assay solution and assayed in triplicate. Duplicate controls in which sample is added after terminating the reaction are prepared in parallel. After incubating at 37° for 30 min the reaction is stopped by adding 2 ml of ice-cold phosphotungstic acid-HC1. The mixture is vortexed and the tubes are left on ice for 30 min before centrifuging at 1600 gay for 10 min. The pellet is washed 2 or 3 times with 2-ml aliquots of phosphotungstic acid and then once with 2 ml of ether: ethanol, 1 : 1 (v/v). The pellet is left to dry overnight at room temperature and dissolved in 0.5 ml Protosol (New England Nuclear) or I ml of 2 M 12 K. J3 M. ~4 R. i~ R.

E. Howell, A. Ito, and G. E. Palade, J. Cell Biol. 79, 581 (1978). G. Farquhar, J. J. M. Bergeron, and G. E. Palade, J. Cell Biol. 60, 8 (1974). Bretz, H. Bretz, and G. E. Palade, J. Cell Biol. 84, 87 (1980). Bretz and W. Staubli, Eur. J. Biochern. 77, 181 (1977).

[18]

PEPTIDE HORMONE RECEPTORS

223

NH4OH. The solution is neutralized with glacial acetic acid and counted in 12 ml of Aquasol-2 (New England Nuclear). Enzyme activity is calculated as pmol galactose incorporated into protein/30 min/mg protein. Sialyltransferase (EC 2.4.99.1) is assayed by a modification of the method of Bretz et al. 14 Reagents

1. Sodium cacodylate buffer, 0.4 M 2-Mercaptoethanol, 0.16 M 2. CMP-[3H]sialic acid, l0/zCi/ml (evaporate original material from New England Nuclear and redissolve in n 2 0 ) 3. CMP-N-acetylneuraminic acid, I0 mM (ammonium salt) 4. Asialofetuin, 175 mg/ml [Fetuin (Sigma Chem. MO) is acid hydrolyzed in 0.05 N H2SO4 by the method of Spiro, 16the lyophilized protein is dissolved in H20] 5. Triton X-100 in H 2 0 , 2% (w/v) 6. Phosphotungstic acid in 0.5 N HC1, 1% (w/v) 7. Trichloroacetic acid in H 2 0 (TCA), 10% (w/v) All solutions are stored frozen at - 2 0 ° except for solutions 5 and 6 which can be stored at 4 ° Procedure

The assay solution is prepared by mixing solutions 1 to 5 in the ratios 25 : 10 : I0 : 10 : 10 (vol). Samples containing 1 to 8/zg protein in a volume up to 35/xl are mixed with 65/xl of the assay solution. Duplicate controls in which sample is added after terminating the reaction are prepared in parallel. After incubating at 37° for 30 min the reaction is stopped by adding 1 ml of phosphotungstic acid. The mixture is vortexed and left on ice for 30 rain prior to centrifuging at 1600 gay for 10 rain. The pellet is washed 2 or 3 times with 2 ml ice-cold TCA and once with 1 ml ether: ethanol, 1 : 1 (v/v). The pellet is left overnight at room temperature to dry and is dissolved in 0.5 ml Protosol or 1 ml 2 M NH4OH before neutralizing with glacial acetic acid and counting in Aquasol-2. Enzyme activity is calculated as nmol of CMP-neuraminic acid incorporated into protein/30 min/mg protein. In distinguishing between various vesicle populations it is often useful ~6R. G. Spiro, J. Biol. Chem. 235, 2860 (1960).

224

INTERNALIZED HORMONES AND RECEPTORS

[18]

to measure acid phosphatase (EC 3.1.3.2). Our procedure is based on the method of Gianetto and de Duve 17 with minor modifications.

Reagents 1. /3-Glycerophosphate, disodium salt, 0.5 M Sodium acetate buffer,pH 5.0, 0.6 M 2. Triton X-100 in H20, 1% (w/v) 3. Trichloroacetic acid (TCA), 16% (w/v)

Procedure The reaction mixture is prepared in phosphorus-free, clean glass tubes by mixing 50/zl of solution 1 and 50/xl of solution 2 with sample containing 2 to 25/zg of protein. The volume is adjusted to 500/zl with distilled water. Control tubes are those without added sample. After 60 min at 37° the reaction is stopped by adding 500/xl of 16% TCA. The clear supernatant is collected after centrifugation at 2400 gav for 15 min and inorganic phosphorus is determined by the method of Chen et al.18 The results are corrected for readings of the control incubations. A unit of enzyme is /zmol of P~ released/hr/mg protein. Hormone Binding. The Golgi fractions are enriched in binding sites for insulin, insulin-like growth factors, lactogen, and growth hormone) 9 In order to maximize binding to these fractions they must be frozen and thawed 4 times or incubated in the presence of 0.1% Triton X-100 to permeabilize the vesicles. This latency is explained as due to the presence of receptors on the inner aspect of the vesicles. Hormone binding is measured essentially as previously described. 4-6

Reagents 1. 125 mM Tris-HC1, 50 mM MgCl2, 0.5% bovine serum albumin (BSA), pH 7.4 2. 125I-labeled hormone (prepared by chloramine-T procedure as described elsewhere 2°) 3. Unlabeled hormone prepared in solution 1 (insulin, oPRL, and bGH, 50/zg/ml; hGH, 20/xg/ml) 4. Glass fiber filters (2.4 cm, Reeve Angel or Whatman 934-AH) pre-soaked for 24 hr in 25 mM sodium acetate buffer, pH 5.0, containing 0.1% BSA 17 R. Gianetto and C. de Duve, Biochem. J. 59, 432 (1955). 18 p. S. Chen, Jr., T. Y. Toribara, and H. Warner, Anal. Chem. 28, 1756 (1956). 19 B. I. Posner, J. J. M. Bergeron, Z. Josefsberg, M. N. Khan, R. J. Khan, B. A. Patel, R. A. Sikstrom, and A. K. Verma, Recent Prog. Horm. Res. 37, 539 (1981). 20 B. I. Posner, Diabetes 23, 209 (1974).

[18]

PEPTIDE HORMONE RECEPTORS

225

Procedure Solution 2 (100/xl) is incubated in triplicate with 800/~1 of sample (20 to 100/zg protein) and 100/xl of solution 1 (final vol of 1.0 ml) for 24 to 30 hr at 4 ° with constant shaking. Duplicate control tubes for determining nonspecific binding are run under identical conditions except that 100/zl of solution 1 is replaced by 100/xl of solution 3. The incubations are terminated by adding 3 ml of an ice cold 1 : 5 dilution (with H20) of solution 1. Bound and free radioactivity are separated by filtration on presoaked glass fiber filters using a Millipore manifold assembly. After filtration the filters are washed with 20 ml of a 1 : 5 dilution of solution 1. Radioactivity retained on the filters is counted in a gamma-scintillation counter as described elsewhere. Specific binding is the difference between radioactivity bound in the absence (total binding) and in the presence (nonspecific binding) of excess unlabeled hormone and is expressed as a percentage of total radioactivity in the incubation. In circumstances where the sample is suspended in 25 mM Tris-HCl-10 mM MgCl2-0.1% BSA a I :5 dilution of solution l rather than solution 1 itself is added to the incubation.

Intact Golgi by a Single Step Gradient Procedure 21 The following solutions are prepared in advance: 5 mM MgClz, 25 mM KCI, 50 mM Tris, pH 7.4 (TKM); 0.25 M sucrose TKM (0.25 M STKM); and 2.3 M sucrose TKM (2.3 M STKM). Excised livers are placed in icecold 0.25 M STKM, weighed, and adjusted to 6 ml buffer per g liver. The tissue is minced, rinsed, and homogenized as above, in a Potter-Elvehjem homogenizer with 4 full strokes of the motor-driven Teflon pestle. Twenty-five milliliters of homogenate is mixed with 13 ml of 2.3 M STKM to give a final concentration of 1.02 M STKM and 6 ml is pipetted into each of six 14 ml cellulose nitrate tubes (for SW40 rotor). In preparation for gradient formation rinse the gradient maker with 0.25 M STKM and allow this to fill the valve space before emptying both chambers. Then add 4 ml of 0.25 M STKM to the mixing chamber. An equal volume of 1.02 M STKM solution, made by mixing 8 volumes of 2.3 M STKM with 10 volumes of TKM, is added to the reservoir chamber of the gradient maker. With the stirrer moving the valve is opened and the outlet tube set against the wall of the SW40 tube just above the homogenate. On completing the overlying gradient the tubes are centrifuged at 200,000 gav for 90 min (40,000 rpm, SW40 rotor). The appearance of the centrifuge tube 2~ j. j. M. Bergeron, R. A. Rachubinski, R. A. Sikstrom, B. !. Posner, and J. Paiement, J. Cell Biol. 92, 139 (1982).

226

INTERNALIZED

HORMONES

AND RECEPTORS

[18]

contents after centrifugation is depicted in Fig. 2. The material from the Golgi region is removed with a 90° angle needle and syringe. The suspension is diluted in 0.25 M STKM if isotonicity must be preserved as for morphologic studies or in TKM or 25 mM Tris-10 mM MgCI2, pH 7.4 for binding studies.

Characterization Protein Recovery. This is depicted in Fig. 2. The recovery is similar to that of the Golgi intermediate fraction (Fig. 1). Morphology. The intact Golgi preparation is morphologically quite different from the Golgi fractions prepared above. In the intact preparation one sees oriented stacks of fenestrated saccules with associated vesicles containing lipoprotein particles. These vesicles are often seen to have continuity with the fenestrated tubules. 2~ Presumably, in the course of preparing G1, Gi, and Gh the individual components (vesicles and saccules) become separated from one another.

Fraction

Yield (mg / g liver )

0,2,5 M - -

Gradient

~.~'~

-

o

o,2

.oo,

--

c

2,76

*-0,24

--

b Golgi

0.24

~'0.02

64.80

*-0.98

49.00

~-I.10

--C 1.02 M

I

--

d

Load

1.02 M -

e

Pellet

FIG. 2. Diagrammatic representation of continuous gradient used to isolate intact Golgi fraction. The homogenate is made to 1.02 M STKM and a linear gradient generated above the load zone (vol of gradient : vol of load zone; 1 : 1). After 1.5 hr of centrifugation at 190,000 gay a prominent band (b) is observed 1/3 of the distance up the density gradient overlay with another band noted at the air/0.25 M STKM interface (fraction a). Five fractions are removed from the gradient with a bent blunt needle (#16, Becton Dickenson and Co., Rutherford, N.J.) and syringe. Fraction a, air:0.25 M STKM interface; fraction b, intact Golgi fraction; fraction c, remainder of gradient after removal of fraction b; fraction d, residual load zone; fraction e, pellet. On the right is indicated the mean --- SEM of protein determinations carded out on 12 fractionations.

[18]

PEPTIDE HORMONE RECEPTORS

227

Enzyme Composition. There are few noteworthy differences between the intact Golgi preparation and the other Golgi fractions. The intact Golgi fraction contains a high concentration of galactosyltransferase and detectable, though much lower, levels of 5'-nucleotidase, acid phosphatase, and various endoplasmic reticulum enzymes (viz. glucose-6-phosphatase). Hormone Binding. The pattern and extent of ~25I-labeled hormone binding closely resembles that seen in the other Golgi fractions. Comment The intact Golgi fraction is morphologically different but biochemically similar to the other Golgi fractions. It is more easily prepared in a one-step procedure. Of particular interest is the demonstration that it is more potent than the other Golgi fractions in effecting endogenous glycosylation. 21 Isolation of Nonlysosornal, Non-Golgi Vesicles (i.e., Intermediate or "Unique" Vesicles) by Percoll Density Gradient Centrifugation Nonlysosomal, non-Golgi vesicles, highly enriched in peptide hormone receptors, are isolated by further fractionation of Golgi intermediate or heavy fractions on an isoosmotic, continuous, self-generating Percoil gradient. 1,22

Solutions 1. 2. 3. 4.

0.25 M sucrose solution (8.55 g sucrose/100 ml distilled water) 62% sucrose solution (62 g sucrose/100 ml distilled water) 2.5 M sucrose solution. Percoll (Pharmacia Fine Chemicals, Sweden), 100% solution in H20 5. Density marker beads (Pharmacia Fine Chemicals, Sweden) 6. 25 mM Tris-HCl, 10 mM MgCI2, pH 7.4

Percoll Gradient Fractionation The density of a freshly prepared Golgi intermediate (or heavy) fraction is determined by weighing 100-/A aliquots in triplicate. The sucrose concentration in the sample is computed from tabulated specific gravities of sucrose solutions. 23 The Golgi fractions (5-10 ml; 1 mg/ml protein) is 22 M. N. Khan, B. I. Posner, A. K. Verma, R. J. Khan, and J. J. M. Bergeron, Proc. Natl. Acad. Sci. U.S.A. 78, 4980 (1981). 23 Scientific Tables, in "Documents Geigy" (K. Diem, ed.), p. 320.

228

INTERNALIZED HORMONES AND RECEPTORS

[18]

diluted to 24 m! (the volume of the centrifuge tube) with a 100% Percoll solution (4.13 ml) and water or 2.5 M sucrose as required to yield a final solution of 0.25 M sucrose-17.2% (v/v) Percoll at a final density of

1.05 g/ml. A continuous gradient is generated in situ by centrifuging at 60,000 gay for 30 min at 4 ° (Spinco Ti 60, fixed angle rotor). Twelve 2 ml fractions are collected sequentially from the top of the centrifuge tube by pumping a 62% sucrose solution into the bottom of the centrifuge tube. The density of individual fractions is determined by weighing 100-/zl aliquots in triplicate, or by running an additional tube containing identical Percoll and sucrose concentrations and density marker beads as described by Mickelson et al. 24 Most of the assays for marker enzymes, protein, and hormone binding can be done in off-the-gradient fractions. Where it is desirable to remove Percoll the fraction is diluted 1 : 3 with 25 mM Tris-HCl-10 mM MgCI2 (pH 7.4) and centrifuged at 82,000 gay for 120 min at 4° (SW27 rotor). The supernatant is removed with a Pasteur pipet and the membranous material resting on the Percoll pellet is resuspended in a small volume ( - 2 ml) of Tris-HCl, MgCI2 buffer, and either assayed immediately or frozen at - 2 0 °. Characterization

With Percoll gradient centrifugation two populations of receptor-rich structures can be resolved from the Golgi intermediate and heavy fractions. Those entities which have a density greater than 1.055 are vesicles containing lipoprotein particles which are devoid of both galactosyl- and sialyltransferases. These structures morphologically resemble the lipoprotein-containing lighter structures which cosediment with glycosyltransferases. In addition the heavier vesicles contain modest levels of acid phosphatase and other lysosomal enzymes (approx. 1/4 to 1/3 the concentration in lysosomes). Because these structures appear to share features of both Golgi vesicles and lysosomes we have referred to them as intermediate or "unique" vesicles to emphasize their apparent distinctiveness. 1,22 The analysis of fractions off the Percoll gradients is complicated by the interference of Percoll with the Lowry method of protein estimation. We have devised a modified Lowry procedure for protein estimation in which absorbance at 750 and 420 nm of unknowns, bovine serum albumin standards in 0.25 M sucrose and Percoll solutions is determined. 25 In this procedure Percoll-containing samples develop a thick white precipitate on 24 j. R. Mickelson, M. L. Breaser, and B. B. Marsh, Anal. Biochem. 1119, 255 (1980). 25 M. N. Khan, R. J. Khan, and B. I. Posner, Anal. Biochem. 117, 108 (1981).

[18]

PEPTIDE HORMONE RECEPTORS

229

the addition of 1.2 N NaOH which disappears after 20 min in a boiling water bath. Since the relation between the concentration of protein or Percoll and the absorbance values at the above two wavelengths is linear one can devise a simple equation for computing protein concentrations. The method is suitable for samples containing as much as 30% Percoll. 25 Methods for Quantitative Autoradiography of Subcellular Fractions

Subcellular fractions are routinely processed for random sampling by the filtration protocol outlined by Baudhuin et al. 26as modified by Wibo et al. 27 Briefly, after fixation in 2.5% glutaraldehyde in 0.05 M sodium cacodylate, buffer pH 7.4, Golgi fractions are recovered onto MiUipore membranes (Type HA, 0.45/xm pore size), using the automated filtration apparatus of Baudhuin et al. 26 then postfixed in 1% OsO4 and block-stained in uranyl acetate. Membrane pellicles are then dehydrated, embedded in Epon 812, and thin sections cut with block face orientated in order to assure a cross-section through the depth of the filtered pellicle. These thin sections can then be viewed directly in the electron microscope following grid staining in uranyl acetate and lead citrate. This method which has been standardized by Dr. B. Kopriwa 28 is as follows. Reagents

Celloidon (Randolph Finishing Products, Carlstadt, N.J.) Isoamylacetate (Tousimis, New Jersey) Ilford L-4 nuclear emulsion (Ilford Canada, Markham, Ont.) Gold chloride (Canlab, Montreal, Canada) Potassium thiocyanate (Fisher Scientific Limited, Canada) Potassium bromide (Fisher Scientific Limited, Canada) Elon (p-methylaminophenol sulfate) (Kodak, New Jersey) Anhydrous sodium sulfite (Fisher Scientific Limited, Canada) Sodium thiosulfate (Fixer) (Fisher Scientific Limited, Canada) Equipment

Semiautomatic coating apparatus (Averlaid, Toronto). Procedures

Precleaned microscope slides are further cleaned by wiping with lens tissue. The slides are dipped into a solution of 1% celloidon in isoamylacetate and dried vertically in a dust-free cabinet overnight. Thin sections 26 p. Baudhuin, P. Evrard, and J. Berthet, J. Cell Biol. 32, 181 (1967). 27 M. Wibo, A. Amar-Costesec, J. Berthet, and H. Beaufay, J. Cell Biol. 51, 52 (1971). 28 B. M. Kopriwa, Histochemie 37, 1 (1973).

230

INTERNALIZED HORMONES AND RECEPTORS

[18]

Fro. 3. Electron microscope autoradiography of tzsI-labeled insulin in Golgi preparations isolated 5 rain after ~25I-labeled insulin injection in vivo. The starting material was a Golgi intermediate fraction which was further subfractionated on Percoll gradients into subfractions of light (a, b), intermediate (c), or heavy density (d). The autoradiographic "fine" silver grains overlie lipoprotein-containing vesicular structures in all regions of the Percoll gradient. Taken from Khan et al) a, ×23,000: b, c, d, ×34,000.

[18]

PEPTIDE HORMONE RECEPTORS

231

( - 5 0 nm in thickness) are placed on the celloidon-covered glass slides. These sections are further covered with 5 nm of carbon using an Edwards carbon evaporator especially modified to assure a uniform application of carbon. The slides are subsequently dipped in Ilford L-4 nuclear emulsion (diluted - 1 : 2 : 5 in distilled water) at 40 ° using the semiautomatic dipping device to ensure covering by a monolayer of emulsion. The slides are dried vertically, then stored in lightproof boxes at 8° with added Drierite in each box. After suitable exposure ( - 6 0 days) the emulsion is developed for "fine grain" development by the "solution-physical" procedure after prior treatment with gold in order to stabilize the exposed latent images. Thus slides are dipped vertically into distilled water (1 min) followed by a freshly prepared gold thiocyanate solution (0.4 mg of gold chloride in 100 ml of 0.5% potassium thiocyanate, 0.6% potassium bromide), for 1 min followed by 7 min development in freshly prepared Agfa-Gevaert developer (0.75 g Elon, 0.5 g anhydrous sodium sulfite, 0.2 g potassium thiocyanate in 100 ml distilled water). 28 Slides are subsequently placed in distilled water for 30 sec followed by fixer (2 min in 24% sodium thiosulfate) and washed in five changes of distilled water. Sections are finally transferred to EM grids by flotation of the cut celloidin-emulsion couplet onto the surface of distilled water. The key feature of the above technique involves the use of gold deposition about the exposed latent images. This treatment ensures the symmetrical deposition of silver about the exposed latent images by the AgfaGavaert developer. The final visualized micrograph is therefore one of exposed latent images and not, as for all other autoradiographic methods 29 a micrograph showing developed silver crystals where the latent images have been randomly developed. An example for the autoradiographic analysis of 125I-labeled insulin distribution of Golgi fractions is illustrated in Fig. 3.

29 B. M. Kopriwa,

Histochemistry 44,

201 (1975).