Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme

Molecular and Cellular Endocrinology, 0 1991 Elsevier Scientific Publishers MOLCEL

79 (1991) 13-20 Ireland, Ltd. 0303.7207/91/$03SOa

13

02542

Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme Katherine F. Roby *, Douglas Larsen, Santanu Deb and Michael J. Soares Department of Physiology, Ralph L. Smith Mental Retardation Research Center, lJnir:ersity of Kansas Medical Center, Kansas City, KS 66103, U.S.A. (Received

Key words: Antipeptide

antibody;

Cytochrome

24 December

P-450,,;

1990; accepted

5 April 1991)

(Rat)

Summary In this report, we describe the generation of immunologic probes to rat P-450,,,. Two regions of the P-450,,, amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-5261, chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450,,, regions that recognized the entire 49 kDa rat P-450,,, protein. Antiserum directed to the internal domain of P-450,,, showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450,,, crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P-450,,, expression. In addition to recognizing the major 49 kDa P-450,,, protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450,, specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450,,, between the internal and carboxy terminal epitopes.

Introduction The formation of biologically active steroid hormones is significantly influenced by the mitochondrial cytochrome P-450 enzyme, cholesterol side-chain cleavage (P-450,,,, see Waterman and Simpson, 1989, for a review). Cells from a variety

Address for correspondence: Dr. Michael J. Soares, Department of Physiology, University of Kansas Medical Center, Kansas City, KS 66103, U.S.A. Supported by a grant from the National Institute of Child Health and Human Development, ND 20676. * Recipient of a Wesley Foundation postdoctoral fellowship.

of different tissues involved in steroidogenesis, including the ovary, testis, adrenal gland, and placenta express P-450,,, (see Waterman and Simpson, 1989). Expression of P-450,,, has been a useful tool for monitoring the differentiation state of cells from various endocrine tissues (see Erickson, 1983; Waterman and Simpson, 1989; Ringler and Strauss, 1990). Identification and characterization of P-450,,, expression has been facilitated by the generation of antibodies to P450,,, (Watanuki et al., 1978; DuBois et al., 1981; Farkash et al., 1986; Anakwe and Payne, 1987). In most instances, P-450,,, purified from bovine adrenal glands has been used as an antigen due

14

to its relative abundance. The isolation of homogeneous P-450,,, is not a simple procedure. In this report, we present an alternative procedure for the development of highly specific antibodies to rat P-450,,,. The availability of amino acid sequence information for rat P-450,,, (Oonk et al., 1989) permitted the development of immunologic probes to specific regions of rat P450,,, (see Walter, 1986 for a review of the technique). The development of ‘antipeptide’ antibodies or ‘site-directed’ antibodies to specific domains of a protein has two important advantages: (1) large quantities of homogeneous antigen free of contamination with other tissue proteins can be obtained by chemical synthesis, and (2) antibodies can be generated to different and potentially significant regions of a protein. These features provide the researcher with tools of potentially greater specificity and reliability, especially when investigating heterogeneous proteins. We have generated antipeptide antibodies to two regions of rat P-450,,,. The antibodies are highly specific probes useful for Western blot and immunocytochemical analyses. Furthermore, generation of antibodies to the two different P-450,,, domains has enabled us to identify a mechanism for generation of different immunoreactive P450,,, species. Materials and methods Reagents

Reagents for sodium dodecyl sulphate (SDS)polyacrylamide gel electrophoresis were purchased from Bio-Rad Chemicals (Richmond, CA, U.S.A.). Nitrocellulose was purchased from Schleicher and Schuell (Keene, NH, U.S.A.). Streptavidin-biotin immunoperoxidase kits for rabbit immunoglobulin G (IgG) were obtained from Zymed Laboratories (South San Francisco, CA, U.S.A.). Unless otherwise noted all other chemicals and reagents were purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). Animals, tissues, and tissue preparation

Adrenal glands were obtained from Holtzman rats (Sasco, NE, U.S.A.), golden hamsters

(Harlan, Indianapolis, IN, U.S.A.), CD-1 mice (Charles River Laboratories, Wilmington, MA, U.S.A.), and New Zealand white rabbits (Myrtle’s Rabbitry, Thompson Station, TN, U.S.A.). The animals were sacrificed, adrenal glands dissected and immediately frozen in liquid nitrogen then stored at - 70 a C until subsequent analysis. Porcine and bovine adrenal glands were obtained from a local abbatoir (Bichelmeyer Meat Company, Kansas City, KS, U.S.A.), snap frozen on dry ice, transported to the laboratory and stored at - 70 o C until further analysis. Additional adrenal glands and ovaries from Holtzman rats were dissected, immediately submerged in freshly prepared Bouin’s fixative for approximately 24 h, and then used for immunocytochemical analysis of P-450,,, Enriched mitochondrial fractions were prepared from adrenal tissues utilizing modifications of previously described methods (Farkash et al., 1986; Goldring et al., 1987). Briefly, tissues were homogenized in 0.25 M sucrose containing 0.5 mM phenylmethylsulfonic fluoride and then centrifuged at 900 X g for 10 min. The resulting supernatant was centrifuged at 16,000 X g for 10 min and the supernatant removed. The pellet, defined as the enriched mitochondrial fraction, was resuspended in 10 mM potassium phosphate, pH 6.8, containing 1 mM ethylenediaminetetraacetic acid and 10 mM 3-[(3-cholamidopropyl)dimethyl-ammoniol-1-propanesulfonate, sonicated and then centrifuged at 10,000 x g for 5 min. Protein present in the supernatant was estimated by the method of Bradford (1976) and used as enriched preparations of P-450,,, for Western blot analysis. Generation of antipeptide antibodies Selection and synthesis of oligopeptides.

Two regions of the rat P-450,,, sequence (Oonk et al., 1989) were selected for use as antigens based on previously described criteria (Deb et al., 1989a, b). The peptides represented an internal domain (amino acids 421-441) located between the putative steroid and heme binding domains and a carboxyl-terminal domain (amino acids 509-526; see Fig. 1). The peptides were synthesized by Multiple Peptide Systems (San Diego, CA, U.S.A.). A cysteine residue was added to the

15

amino-terminus of each peptide to facilitate coupling to a carrier protein. Coupling and immunization. Each peptide was coupled to keyhole limpet hemocyanin (KLH) as previously described (Deb et al., 1989a). Three adult New Zealand white rabbits (Myrtle’s Rabbitry) were immunized with each oligopeptide-KLH preparation. Immunization and blood collection schedules were as previously described (Deb et al., 1989a). Characterization of antibodies Enzyme-linked immunoassay

(EL4).

The reactivity of the antipeptide antisera with their corresponding peptide antigens was determined by EIA as previously described (Deb et al., 1989a). Western blot analysis. P-450,,, protein species present in enriched adrenal mitochondrial preparations were characterized by Western blot analysis. Samples were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Processing and de-

bovine human

P-450s~~ P-45oscc

rat

P-450

rat

P-45oscc

Ill3

R-D-P-A-~-S-S-P-D-K-F-D-P-T-R-W-L-S-X_D ~-P-T-~-F-D-E-E-N-F-D-P-T-R-“-L-S-X-D B-N-P-A-V-E-P-R-P-E-R-Y-M-e-Q-R-W-L-E-R-~

Q-P-L-K-Q-D-L-G-S-T-M-P-II-K-G-D-T-V

bovine P-450s~~

R-pF-N-QrB-P-P-Q-*

human

W-pF-N-Q-E-A-T-cj-*

rat

P-45oscc P-450

Ill3

R-E-V-S

Fig. 1. Rat P-450,,, sequences selected for synthesis and used as antigens. An internal region (amino acids 421-441) located between the putative steroid and heme binding domains and a sequence representing the carboxy terminal domain (amino acids 509-526, see Oonk et al., 1989) were selected for synthesis. A cysteine residue was added to the amino-terminus of each peptide to facilitate coupling to the carrier protein. We have included homologous amino acid sequences from bovine and human P-4.50,,, and rat P-450,tp for comparisons (see Morohashi et al., 1984; Chung et al., 1986; Nonaka et al., 1989). Single letter abbreviations for amino acids are used (alanine = A, aspartic acid = D, threonine = T, serine = S, glutamic acid = E, proline = P, glycine = G, valine = V, methionine = M, leucine = L, phenylalanine = F, tyrosine = Y, lysine = K, arginine = R, tryptophan = W, glutamine = Q, asparagine = N).

TABLE

1

ANTIPEPTIDE ANTISERUM TIVE RAT P-450,,, PEPTIDE

TITER FOR THE RESPECANTIGEN a

Antigen (peptides)

Rabbit number

Titer h

421-441 (internal)

45 46 47

500 000 100 000 nd ’

51 52 53

305 000 2115000 2300000

509-526 (carboxy

terminal)

a Polystyrene 96-well microtiter plates were coated with the respective peptides at a concentration of 1 pg/ml in a volume of 100 ~1. h Values are expressed as the reciprocal of the endpoint dilution. ’ Not determined, preimmune serum for this rabbit contained antibodies that reacted with other tissue proteins.

velopment of the nitrocellulose filters for reactivity with various antisera was performed as previously described (Soares et al., 1988). Specificity of immunoblot analyses was determined by comparing reactivities of each antiserum with the same antiserum absorbed with excess antigen. Immunocytochemistry. Ovarian and adrenal tissues fixed in Bouin’s fixative were dehydrated, embedded in paraffin, sectioned at 8 pm and mounted on poly-L-lysine-coated microscope slides. Localization of P-450,,, was determined using antipeptide antibodies and a streptavidinbiotin immunostaining kit. Antisera were used at a final dilution of 1: 200 and all immunocytochemical procedures were performed according to the manufacturer’s instructions. Specificity of the immunoreactions was determined by comparing the reactivity of the antisera with antisera absorbed with excess antigen (100 pg antigen/ml of antiserum incubated overnight at 4 o 0. Results Biochemical characterization and specificity

The reactivities of each antiserum with its respective antigen are presented in Table 1. Serum from rabbit number 45 possessed the highest titer for the internal domain (amino acids 421-441) and serum from rabbit number 53 possessed the

highest titer for the carboxy terminal domain (amino acids 509-526). These two antisera also exhibited the greatest reactivity with P-450,,, in Western blot analysis and were used for ail subsequent analyses. A major 49 kDa species from rat adrenal mitochondria was specifically recognized by antibod-

CD

A

6

rt

fn

rt

-

_...

+

9767-

30-

Species Excess

Species Excess Peptfde

rt

m

tl

_

-

-

A

f3

ttmfl i-i-+

p -

c

D

E

p c F

B

Species Excess Peptfde

rt -

rb _

b _

rt

rb

b

f

f

4”

Fig. 2. immunor~activit~~s of antipeptjde antibodies directed to the internal domain of rat P-450,,, (amino acids 421-441) with P-450,, isolated from rat (rt), mouse (m), hamster (h), porcine (p), rabbit trb). and bovine fb) adrenal glands. Mitochondrial extracts (20 +qIlane) were separated by SDS-polyac~iamide gel eiectrophoresis in 10% gels, elcctrophoretically transferred to nitrocellulose and probed with the rat P-450, antipeptide antiserum at a final dilution of 1: 1000 (top panel, lanes A-D and bottom panel, lanes A-C) or with the antipeptide antiserum saturated with the peptide antigen (top panel, lanes E-H and bottom panel, lanes D-F). The location of molecular weight standards (x10-“) is indicated at the left.

Peptide

m +

Fig. 3. Immunoreactivities of antipeptide antibodies directed to the carboxy terminal domain of rat F-450,, (amino acids 509-526) with P-450, isolated from rat (rt) and mouse fm> adrenal glands. ~itochondriai extracts (20 ,~g/lanc) were separated by SDS-polyacrylamide gel electrophoresis in 10% gels, electrophorctica~iy transferred to nitrocellulosc and probed with the rat P-450, antipeptide antiserum at a final dilution of 1: 1000 (lanes A and f3) or with the antip~ptid~ antiserum saturated with the peptide antigen (lanes C and D). The location of molecular weight standards (X lo-‘) is indicated at the left.

ies directed to either the internal or carboxy terminal domains of P-450,,, (see Figs. 2 and 3). The 49 kDa rat adrenal immunoreactive species was also recognized by an antiserum directed to bovine P-450,,, kindly provided by Dr. Michael Waterman of University of Texas Southwestern Medical Center, Dallas, TX (data not shown). In addition to the major 49 kDa species, each antip~ptide antiserum also specifically recognized different lower molecular weight species. Antibodies directed to the internal domain specifically recognized a 42 kDa species (Fig. 4, lanes A and S) while antibodies directed to the carboxy terminal domain specifically recognized a 8 kDa species (Fig. 4, lanes C and D). An additional experiment was performed to decrease the potential of proteolysis during mitochondrial isolation. Adrenal tissues directly extracted also showed the lower moi~cular weight immunorea~tive species.

17

Species crossreactivity

Adrenal P-450,,, from several species exhibited crossreactivity with the antipeptide antiserum generated to rat P-450,,,. Antibodies directed to the internal domain of rat P-450,,, specifically recognized P-450,,, extracted from rat, mouse, hamster, rabbit, porcine and bovine adrenal glands, while antibodies directed to the carboxy terminal domain specifically recognized only rat and mouse P-450,,, species (Fig. 3).

stained with antisera to both P-450,,, domains, whereas cells of the adrenal medulla exhibited no detectable immunostaining with either antiserum (Fig. 5). Immunocytochemical localization of P450,,, in the rat ovary was performed using antisera directed to the carboxy terminus (Fig. 6). Immunostaining was specifically localized to the theta interna, interstitial and luteal tissues; granulosa cells exhibited no detectable immunostaining (Fig. 6).

Immunolocalization of P-450,,,

Discussion

The usefulness of each antiserum for examining cell-specific P-450,,, protein expression in the rat adrenal gland and ovary was determined. Adrenal cortical cells were specifically immunoA

B

C

D

21 14 -

-

6Excess Peptide Antiserum

-

+

421-441

-

+

509-526

Fig. 4. Immunoreactivity of lower molecular weight P-450,,, species. Rat adrenal mitochondrial extracts (20 fig) were separated by SDS-polyacrylamide gel electrophoresis in 15% gels, electrophoretically transferred to nitrocellulose and antipeptide antisera at a final probed with the rat P-450, dilution of 1: 1000 (lanes A and C) or with antipeptide antisera saturated with the respective peptide antigen (lanes B and D). Lanes A and B were probed with antisera directed to the internal domain of rat P-450,,,, whereas lanes C and D were probed with antisera directed to the carboxy terminal domain of rat P-450,,,. Note the specific detection of a 49 kDa species with both antisera, the specific detection of a 42 kDa species with antibodies directed to the internal domain and the specific detection of an 8 kDa species of P-450,,,, with antibodies directed to the carboxy terminal domain of P-4.50,,. The location of molecular weight standards (X 10-s) is indicated at the left.

In this report we have described the generation of antisera to two different regions of the rat P-450,,, amino acid sequence, an internal sequence and a carboxy terminal sequence. The antisera were useful for Western blot and immunocytochemical characterization of P-450,,,. Both antisera specifically recognized 49 kDa protein species from the rat adrenal gland, similar to the molecular weight for rat P-450,, previously reported by other laboratories (Farkash et al., 1986; Trzeciak et al., 1986; Goldring et al., 1987). The antipeptide antisera have proven useful for the examination of cell-specific localization of methods. The P-450,,, by immunocytochemical specific localizations of P-450,,, to adrenocortical cells of the rat adrenal gland and to the theta interna, interstitium, and corpus luteum of the rat ovary are consistent with previous reports using antibodies generated against P-450,,, isolated from tissues (Mitani et al., 1982; Farkash et al., 1986; Le Goascogne et al., 1989). An important consideration regarding the utility of the P-450,,, antipeptide antibodies generated in this report is related to their potential to crossreact with other related mitochondrial cytochrome P-450 proteins. Of particular interest is the relationship of P-450,,, with cytochrome P450 lip-hydroxylase (P-450,,,). Both proteins are expressed by the adrenocortical cells and possess similar molecular weights (Ogishima et al., 1989; present report). Although, we have not directly tested the crossreactivity of the P-450,,, antipeptide antisera against purified P-450,,,, the P-450,,, peptide domains used for antibody generation in our report possess limited sequence identity with homologous domains of P-450,,,

in rat adrenal gland. Sections of the rat adrenal gland were stained for the Fig. 5. lmmunocytochemical localization of P-450,,, presence of P-450,., using a streptavidin-biotin immunoperoxidase kit for rabbit IgG. P-450,,, was detected with antibodies directed to the internal domain of P-450,,, (amino acids 421-441, micrograph A), with antibodies directed to the carboxy terminal domain of P-450,,, (amino acids 509-526, micrograph C), or with the antibodies saturated with their respective peptide antigen (micrograph B is the control for micrograph A; micrograph D is the control for micrograph C). The antisera were used at a final dilution of 1: 200. Specific immunostaining was localized to the adrenocortical cells (abbreviated: c), whereas the adrenomedullary cells (abbreviated: m) lacked detectable P-450,,,. Absorption of the antisera with their respective peptide antigens resulted in loss of immunostaining (micrographs B and D). Magnification, x 100.

(approximately 38% for the internal domain and 5% for the carboxy terminal domain, see Nonaka et al., 1989; Oonk et al., 1989; Fig. 1). In addition to developing specific immunologic probes for monitoring the expression of rat P450,,,, we have also made two interesting discoveries: (1) we have identified an epitope within the P-450,,, sequence that is shared by several species, and (2) we have gained insight into the mechanism responsible for the generation of low molecular weight P-450,,, protein species. Antibodies directed to the internal domain of rat P-450,,,, located between the putative steroid and heme binding domains (Oonk et al., 1989), showed a relatively broad crossreactivity with P450,,, from a number of different species, whereas crossreactivity of antibodies directed to the car-

boxy terminal domain of rat P-450,,, was limited to P-450,,, proteins from the rat and mouse. Amino acid sequence comparison of rat, human and bovine P-450,,, proteins provides an explanation for the differences in crossreactivity of the antibodies directed to the two different rat P450,,, domains (see Fig. 1). The internal rat P450,,, domain shows considerable sequence identity with similar domains located in human and bovine P-450,,, proteins (approximately 62%), whereas the carboxy terminal domain of rat P450,,, shows marked differences when compared to the carboxy terminus of human and bovine P-450,,, (approximately 15%; see Morohashi et al., 1984; Chung et al., 1986; Oonk et al., 1989). extends eight The rat P-450,,, carboxy terminus amino acids beyond the termini of bovine and

Fig. 6. Immunocytochemical localization of rat P-450,,, in the rat ovary. Sections of the rat ovary were stained for the presence of P-450,,, using a streptavidin-biotin immunoperoxidase kit for rabbit IgG. P-450,,, was detected with antibodies directed to the carboxy terminal domain of P-450,,, (amino acids 5099526, micrographs A and C), or with the antibodies saturated with peptide antigen (micrograph B is the control for micrograph A; micrograph D is the control for micrograph C). The antisera were used at a final dilution of 1:200. Specific immunostaining was localized to the theta interna (micrograph A, see arrow), interstitium (micrograph A, abbreviated: i), and corpus luteum (micrograph C, abbreviated: CL). Granulosa cells did not show detectable gc). Absorption of the antisera with peptide antigen resulted in loss of immunoreactive P-450,,, (micrograph A, abbreviated: immunostaining (micrographs B and D). Micrographs A and C, magnification X 100; micrographs B and D, magnification x 200.

human P-450,,, proteins. Based on reactivities of antibodies directed to the rat P-450,,, carboxy terminal domain with P-450,,, proteins, we hypothesize that mouse P-450,,, may also possess the extended carboxy terminus, while the hamster, rabbit, and pig may possess truncated carboxy termini. In summary, the internal domain located between amino acids 421 and 441 of rat P-450,,, represents a well-conserved epitope. It has been repeatedly observed that protein species possessing molecular masses less than the accepted size of P-450,,, (49 kDa) are detected by Western blot analysis of P-450, (Rodgers et al., 1986, 1988; Trzeciak et al., 1986). Furthermore, it has been speculated that the lower molecular weight species may be generated by proteolysis and possibly representing a physiolog-

ical mechanism for inactivating P-450,,, (Rodgers et al., 1988). Examination of the reactivities of antibodies directed to the internal and carboxy terminal domains of rat P-450,,, provides some insight into the ontogeny of the low molecular weight immunoreactive P-450,,, species. Antibodies directed to the internal domain specifically recognize a 42 kDa species, similar in size to that previously reported (Rodgers et al., 1986; Trzeciak et al., 1986). Antibodies to the carboxy terminal domain do not recognize the 42 kDa domain but do specifically recognize a species approximately 8 kDa. We hypothesize that the 42 kDa species is generated by proteolytic cleavage of a 7-8 kDa peptide from the P-450,,, protein. Confirmation of this hypothesis will require isolation and amino-terminal sequence analysis of the

20

42 and 8 kDa immunoreactive species. Cleaving P-450,, in the hypothesized location is very intriguing in that it effectively separates the steroid and heme binding domains and generates apparently stable species. The 42 kDa species would possess the steroid binding domain, while the 8 kDa species would possess the heme binding domain. Whether these proteolytically generated P-450,,, species have any physiological significance remains to be determined. Acknowledgements

The authors gratefully acknowledge Dr. JoAnne Richards (Baylor College of Medicine, Houston, TX) for sharing the amino acid sequence of rat P-450,,, prior to its publication, Dr. Michael Waterman (University of Texas Southwestern Medical Center, Dallas, TX) for providing our laboratory with antibodies to bovine P-450,,,, and Dr. Donald C. Johnson (University of Kansas Medical Center, Kansas City, KS) for providing valuable discussions and insights throughout the course of these studies. The authors would also like to acknowledge Linda Carr for her assistance with the preparation of the manuscript. References Anakwe. 0.0. and Payne, A.H. (1987) Mol. Endocrinol. 1, 595-603. Bradford, M. (1976) Anal. Biochem. 72, 248-254. Chung, B.C., Matteson, K.J., Voutilainen, R., Mohandas, T.K. and Miller, W.L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8962-8966.

Deb, S., Hashizume, K., Boone, K., Southard, J.N., Talamantes, F., Rawitch, A. and Soares, M.J. (1989a) Mol. Cell. Endocrinol. 63, 45-56. Deb, S., Youngblood, T., Rawitch, A. and Soares, M.J. (1989b) J. Biol. Chem. 264, 14348-14353. DuBois, R.N., Simpson, E.R., Tuckey, .I., Lambeth, J.D. and Waterman, M.R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1028-1032. Erickson, G.F. (1983) Mol. Cell. Endocrinol. 29, 21-49. Farkash, Y., Timberg, R. and Orly, J. (1986) Endocrinology 118, 1353-1365. Goldring, N.B., Durica, J.M., Lifka, J., Hedin, L., Ratoosh. S.L., Miller, W.L., Orly, J. and Richards, J.S. (1987) Endocrinology 120, 1942-1950. Le Goascogne, C., Sananes, N., Gouezou, M. and Baulieu, E.E (1989) J. Reprod. Fertil. 85, 61-72. Mitani, F., Shimizu, T., Ueno, R., Ishimura, Y., Izumi, S.. Kumatsu, N. and Watanabe, K. (1982) J. Histochem. Cytochem. 30, 1066-1074. Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4647-4651. Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. and Okamoto, M. (1989) FEBS Lett. 255, 21-26. Ogishima, T., Mitani, F. and Ishimura, Y. (1989) J. Biol. Chem. 264, 10935-10938. Oonk, R.B., Krasnow, J.S., Beattie, W.G. and Richards, J.S. (1989) J. Biol. Chem. 264, 21934-21942. Ringler, G.E. and Strauss, J.F. (1990) Endocr. Rev. Il. IO5123. Rodgers, R.J., Waterman, M.R. and Simpson, E.R. (1986) Endocrinology 118, 1366-1374. Rodgers, R.J., Waterman, M.R., Simpson, E.R. and Magness, R.R. (1988) J. Reprod. Fertil. 83, 843-850. Soares, M.J., De, SK., Foster, B.A., Julian, J.A. and Glasser, S.R. (1988) J. Endocrinol. 116, 101-106. Trzeciak, W.H., Waterman, M.R. and Simpson, E.R. (1986) Endocrinology 119, 323-330. Walter. G. (1986) J. Immunol. Methods 88, 148-161. Watanuki, M., Granger, G.A. and Hall, P.F. (1978) J. Biol. Chem. 253, 2927-2931. Waterman, M.R. and Simpson, E.R. (1989) Recent Prog. Horm. Res. 45, 533-563.