A synthetic peptide corresponding to hFSH-β-(81–95) has thioredoxin-like activity

Molecular and Cellular Endocrinology, 78 (1991) 163-170 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50

MOLCEL

163

02521

A synthetic peptide corresponding to hFSH-P-( 81-95) has thioredoxin-like activity Patricia Grasso, Tomb A. Santa-Coloma,

J. Jay Boniface * and Leo E. Reichert, Jr.

Department of Biochemistry, Albany Medical College, Albany, NY 12208, U.S.A. (Received

Key words: Thioredoxin;

Synthetic

peptide;

18 January

hFSH-P-subunit;

1991; accepted

Disulfide

4 March

1991)

bond formation

Summary

The thioredoxin-like activity of human follicle stimulating hormone (hFSH), hFSH-P-(83-88) peptide amide (hFSH-P-(83-88) which has a sequence similar to the thioredoxin active center (-His-Cys-Gly-LysCys-Asp-)) and thioredoxin-(31-36)-peptide amide (TD-(31-36) which contains the redox-active dithiol of thioredoxin (-Trp-Cys-Gly-Pro-Cys-Lys-1) was characterized by their ability to reactivate reduced and denatured bovine pancreatic ribonuclease (RNase). This assay reflects the recently recognized ability of thioredoxin to catalyze disulfide bond formation in proteins. Compared to uncatalyzed refolding of reduced, denatured substrate, hFSH was approximately lo-fold more active than thioredoxin on a molar basis. The catalytic activity of hFSH-P-(83-88) and TD-(31-36) was equivalent to that of an equimolar concentration of thioredoxin. Screening of 11 overlapping peptide amides representing the entire primary structure of hFSH+subunit indicated that hFSH-P-(81-95), which contains the sequence similar to the thioredoxin active center within a receptor-binding region of the hFSH-P-subunit, possesses strong thioredoxin-like activity and was more active than an equimolar concentration of thioredoxin. In contrast, hFSH-P-(33-53), a thiol-containing peptide which corresponds to a second FSH receptor-binding domain but lacks the sequence similar to the thioredoxin active center, was inactive. Synthetic peptide amides corresponding to other regions of hFSH-P-subunit were less effective than hFSH-P-(81-95) in reactivating reduced and denatured RNase. Our data provide evidence that the recently reported thioredoxin-like catalytic activity of FSH may be due, at least in part, to the redox-active dithiol present within a receptor-binding domain of its /?-subunit, and thus may have a physiological role in receptor binding or signal transduction.

Introduction * Present

address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. This work was supported by NIH grant HD13938. Address for correspondence: Dr. Leo E. Reichert, Jr., Department of Biochemistry A-10, Albany Medical College, Albany, NY 12208, U.S.A. Tel. (5181 445-5365; Fax (518) 445-5365.

Thioredoxin is a small (M, 11,700) ubiquitous protein having two redox-active half-cystine residues located in a protruding segment of its three-dimensional structure (Holmgren, 1979a 1985). It exists in the oxidized form (thioredoxin-

164

S,) in which the active center residues, Cys-32 and Cys-35, form an intramolecular disulfide bridge (Holmgren, 1981), or in the reduced dithiol form (thioredoxin-(SH),). Although thioredoxins have been isolated from prokaryotic and eukaryotic species (reviewed in Holmgren, 19851, Escherichia coli thioredoxin, the first thioredoxin to be purified (Laurent et al., 1964) and sequenced (Holmgren, 19681, is best characterized. E. coli thioredoxin-S, contains 108 amino acid residues, has been crystallized (Holmgren and Soderberg, 1975), and it,” three-dimensional structure resolved to 2.8 A by X-ray crystallographic methods (Holmgren et al., 1975). Thioredoxin participates in redox reactions through reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol-disulfide interchange reactions (Holmgren, 1979b), and is a hydrogen donor for ribonucleotide reductase (Laurent et al., 1964). Thioredoxin has also been shown to act as a protein disulfide oxido-reductase (Moore et al., 1964; Holmgren, 1979b) and to have protein disulfide isomerase activity (Pigiet and Schuster, 1986). The evolving roles of thioredoxin in cellular differentiation and metabolism (Moore et al., 1964; Holmgren et al., 1975; Holmgren, 1979b; Mark and Richardson, 1979; Grippo et al., 1985; Lim et al., 1985; Pigiet and Schuster, 1986; Russel and Model, 1986; van Haarlem et al., 1987; Porter et al., 1988; Wiegand et al., 1989) strongly suggest that regulation of the redox state of proteins is critical in many physiological processes, and may include those influenced by gonadotropic hormones, as evidenced by our recent report of a novel thioredoxin-like catalytic property of both FSH and LH (Boniface and Reichert, 1990). Identification of a tetrapeptide of homology between the active center of thioredoxin and the P-subunit of LH and a similar tetrapeptide in the P-subunit of FSH, suggested that the thioredoxin-like activity of FSH may be due to the presence of the related tetrapeptide (Boniface and Reichert, 1990). In order to examine this hypothesis, peptides containing the active center of thioredoxin and the similar tetrapeptide in hFSH-P-subunit, as well as 11 overlapping peptides corresponding to the entire primary structure of hFSH-/?-subunit, were synthesized and characterized for their ability to

reactivate reduced and denatured bovine pancreatic ribonuclease (RNase). Our results support the notion that the thioredoxin-like tetrapeptide found within a receptor-binding region of the p-subunit of hFSH (hFSH-P-(81-95) (Santa Coloma and Reichert, 1990) contains a redox-active disulfide. Association of this redox-active center with a receptor-binding domain of hFSH suggests its possible involvement in FSH-receptor interaction or signal transduction. Materials

and methods

Materials E. coli thioredoxin

(Catalog No. 86-0830-00, Lot 141400) was purchased from Chemical Dynamics (South Plainfield, NJ, U.S.A.), and bovine pancreatic ribonuclease A and cytidine 2’,3’-cyclic monophosphate were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). 3-(N-Morpholinejpropanesulfonic acid (MOPS) was purchased from Research Organics (Cleveland, OH, U.S.A.) and 8 molar guanidine hydrochloride was Sequanal grade obtained from Pierce (Rockford, IL, U.S.A.). Sephadex G-25 was purchased from Pharmacia LKB (Uppsala, Sweden). All other chemicals were reagent grade. Human FSH was NIH-FSH-HSI (4990 IU/mg).

Peptide synthesis, purification and characterization Synthetic peptide amides corresponding to 11 overlapping regions of the hFSH-P-subunit (Watkins et al., 19871, hFSH-P-(33-53), hFSH-/?(83-88) and thioredoxin fragment (31-36) were prepared by Multiple Peptide Systems, San Diego, CA, U.S.A. Synthesis was by the solid-phase method (Merrifield, 1963) using the tert-butoxycarbonyl protection scheme. The resulting peptide amides were purified by preparative reversephase liquid chromatography 0,” octa-decyl-silica (Waters Delta-Pak C ,x, 100 A pore diameter) using a linear acetonitrile gradient (5-100%) in 0.1% trifluoroacetic acid at 30 o C. Homogeneity was verified by analytical high performance liquid chromatography. The amino acid compositions of the peptides were determined by amino acid analysis using the Waters Pica-Tag system (Bidlingmeyer et al., 1984).

165

Reduction and denaturation of RNase Reduced, denatured RNase was prepared prior to each experiment, as recommended by Pigiet and Schuster (1986). RNase (30 mg) was reduced and denatured by incubation overnight in 1.5 ml 0.1 mM Tris-HCl (pH 8.6) containing 0.15 M dithiothreitol and 6 M guanidine hydrochloride. Reduced, denatured RNase was separated from excess dithiothreitol and guanidine hydrochloride by gel filtration through Sephadex G-25 (1.0 X 30 cm column), equilibrated and eluted with degassed, nitrogen-sparged 0.01 M HCl at a flow rate of 22 ml/h. Fractions (1.2 ml> were collected under nitrogen and the peak fraction (determined spectrophotometrically at A & was quantified for RNase and used immediately in reactivation studies or stored frozen (- 70 o C) under nitrogen for 1 day. Determination of protein concentrations The concentration of RNase was determined spectrophotometrically using molar extinction coefficients of 9800 cm-’ M-’ at 277.5 nm for native RNase (Hantgan et al., 1974) and 9200 cm -’ M-’ at 275 nm for reduced, denatured RNase (Anfinsen et al., 1961). Reactkation of reduced, denatured RNase Reactivation of reduced, denatured RNase was initiated by diluting the enzyme to 400 pg/ml in 0.1 M Tris-HCl (pH 7.4) with 1 mM EDTA containing either hFSH, thioredoxin or peptide. Reactivation reactions (500 ~1 total volume) were carried out in 1.5 ml polypropylene microcentrifuge tubes at 25 o C. At various times, a 30 ~1 aliquot of each reaction mixture was removed and assayed for RNase activity. Activity was expressed as fold increase over uncatalyzed refolding of reduced, denatured RNase. RNase activity RNase activity was measured as described by Crook et al. (1960) with modifications. A 14 mM stock solution of cytidine 2’,3’-cyclic monophosphate was prepared just before each experiment and stored at 4’ C during the course of the reactivation. At each time point, an aliquot of the stock solution was diluted to 0.467 mM with 0.1 M MOPS (pH 7.0) for use. A 30 ~1 aliquot of the

reactivation mixture was pipetted into a quartz microcuvette and the reaction initiated by addition of 600 ~1 0.467 mM cytidine 2’,3’-cyclic monophosphate. Absorbance was measured within 10 s at 284 nm using a Gilford Model 250 spectrophotometer. The final assay mixture consisted of 7 PM RNase and 440 FM cytidine 2’,3’-cyclic monophosphate in 0.1 M MOPS (pH 7.0). Computer analyses Predictions of secondary structure, hydrophilicity, flexibility and surface probability were obtained using Genetic Computer Group programs (Devereux et al., 1984). Statistical analyses were performed using the BMDP program (BMDP Statistical Software, Los Angeles, CA, U.S.A.) on a VAX 8650 computer. Half-times for RNase reactivation were determined by fitting the data to a exponential model. Results Refolding of reduced, denatured RNase by TD(31-36) and hFSH-P-(83-88) Alignment of the redox-active Cys residues of thioredoxin, Cys-32 and Cys-35, with the ninth and tenth Cys residues of the P-subunit of FSH revealed a sequence similar to the active center of thioredoxin, but containing a Lys for Pro substitution (Boniface and Reichert, 1990). The vicinyl Cys residues of thioredoxin can be reversibly oxidized to form disulfide bonds and are responsible for its redox activity (Holmgren, 1985). To determine whether recently reported thioredoxin-like activity of FSH (Boniface and Reichert, 1990) could be attributed to the region of its P-subunit similar to the thioredoxin active center, synthetic peptide amides containing the redox-active dithiol of thioredoxin, TD-(31-361, and the region of similarity found in hFSH-P-subunit, hFSH-P-(83-88), were characterized by their ability to reactivate reduced, denatured RNase. As shown in our previous study in which ovine FSH was more effective than thioredoxin in reactivating reduced, denatured RNase (Boniface and Reichert, 1990), hFSH was also more active (approximately IO-fold) than thioredoxin on a molar basis (Fig. 1A). The catalytic activity of hFSH-P-

20

30 Time

40

5ff

(hours)

Time

(hours)

Fig. 1. Kinetics of the reactivation of reduced, denatured RNase in the presence of thior~doxin or hFSH (A) or synthetic peptide amides (B). Reactivation of reduced, denatured RNase was carried out as described in Materials and Methods in the absence or presence of 5 FM hFSH or 100 FM thioredoxin, hFSH-@-(83-88X hFSH-@X33-53) or TD-U-36). hFSH-P-033-88) contains a sequence similar to the thioredoxin active center sequence; hFSH-P-(33-53) does not. The final assay mixture consisted of 7 PM RNase and 440 PM cytidine 2’,3’-cyclic monophosphate in 0.1 M MOPS (pH 7.0). RNase activity is expressed as fold increase over uncatalyzed refolding of reduced, denatured RNase. The data shown are mean f SD of three duplicate experiments. I: uncatalyzed reaction.

(83-88) and TD-(31-36) was equivalent to that of an equimolar concentration of thioredoxin. hFSH-P-(33-53), a synthetic peptide containing a free sul~yd~1 group in Cys-51 but lacking the thiotedoxin-like active-center sequence, was unable to reactivate reduced, denatured RNase (Fig. 1B). Thus, it appears that peptides containing the thioredoxin active center have the ability to mimic the catalytic effect of the holoenzyme in reactivating reduced, denatured RNase.

than an equimolar concentration of thioredoxin. The thioredoxin-Iike activity of hFSH-/3-(81-95) was not unexpected due to the presence of residues 83-88 (similar to the active center residues of thioredoxin) within its structure. Thioi compounds such as cystine, glutathione and dithiothreitol also catalyze disulfide bond formation (Pigiet and Schuster, 1986). Each of the

Refolding of reduced, denatured RNase by hFSHp-(81-95)

Identification of thioredoxin-like catalytic activity in hFSH-~-(~3-88), a sequence contained within one of two regions of the hFSH-~-subunit reported to bind to receptor (Santa Coloma and Reichert, 1990; Santa Coloma et al., 1990), prompted us to test 11 overlapping peptide amides corresponding to the entire primary structure of hFSH+subunit for their ability to refold reduced, denatured RNase. The catalytic activity of equimolar concentrations of hFSH+subunit peptide amides on reactivation of reduced, denatured RNase is summarized in Fig. 2. hFSH-~-t81-9S) catalyzed the refolding of reduced, denatured substrate at a greater rate than any of the other peptide amides, and after 10 h of incubation, was more active

Fig. 2. Reactivation of reduced. denatured RNase by 1I overlapping peptidc amides corresponding to the primary structure of hFSH+-subunit. Reduced, denatured RNase was reactivated for 10 h as described in Materials and Methods in the absence or presence of 100 PM thioredoxin or synthetic peptide amides. The final assay mixture consisted of 7 ,uM RNase and 441) ,uM cytidine 2’,3’-cyclic monophosphate in 0.1 M MOPS (pH 7.0). RNase activity is expressed as fold increase over uncatalyzed refolding of reduced, denatured RNase. The data shown are meanISD of two duplicate experiments. I: uncatalyzed reaction.

167 TABLE

1

MULTIPLE

LINEAR

REGRESSION:

CORRELATION

MATRIX

Values for peptide parameters Hydrophilicity, Surface probability, Flexibility and percentage of Extended, Turn and Helix conformations were calculated as average values for each peptide and normalized to mean = 0 and SD = 1 [Value,,, = (Value,,, mean)/SD] to have comparable coefficients, and intercept = 0. The program P9R from BMDP software was used to calculate the multiple linear regression.

Hydrophilicity (Hy) a Surface probability (Sp) Flexibility(F) No. of Cys residues (Ncys) Helix (HI Extended (El a Turn (T) RNase activity (R.A.) a Variables

removed

during

HY

SP

F

Ncys

H

E

T

R.A.

1 .oo 0.63 0.76 0.07 _ 0.32 0.11 0.14 _ 0.22

1.oo 0.42 - 0.22 - 0.01 -0.12 0.08 - 0.06

1.00 0.25 - 0.28 0.15 0.17 -0.17

1 .oo -0.18 - 0.40 0.74 - 0.46

1.00 - 0.50 - 0.48 0.68

1.00 - 0.43 - 0.33

1.00 - 0.43

1.00

the calculation

of the best subset of predictors

overlapping synthetic peptide amides studied contained at least one Cys. It was not surprising, therefore, that several of the Cys-containing peptides other than hFSH-P-(81-95) were also able to reactivate reduced, denatured RNase. Noteworthy, however, is the observation that hFSH-/?(81-95), containing the thioredoxin-like active center sequence, was the most active.

TABLE

of the low significance.

Structure-activity peptide amides

relationships of hFSH-P-subunit

In an attempt to characterize the peptide amides possessing thioredoxin-like activity, several structural and physicochemical parameters (predicted from the primary structure of hFSHP-subunit) were analyzed by multiple linear regression, estimating the best subset of predictors.

2

MULTIPLE

LINEAR

REGRESSION:

STATISTICS

FOR

BEST SUBSET

Multiple correlation coefficient Squared multiple correlation coefficient Residual mean square Standard error of estimate F-statistic Numerator degrees of freedom Denominator degrees of freedom P Variable

because

a

Surface probability Flexibility No. of Cys residues Helix Turn

OF PREDICTORS 0.92 0.84 0.25 0.50 8.16 5 8 < 0.006

Regression coefficient

T-statistic

Two-tail

- 0.65 0.56 - 1.24 1.06 0.95

- 3.04 2.69 -4.14 5.36 3.09

< < < < <

k 0.21 & 0.21 + 0.30 +0.20 * 0.31

0.02 0.03 0.01 0.01 0.02

P

Contribution to Rzh 0.19 0.15 0.35 0.59 0.20

a Variables hydrophilicity and Extended conformation were removed because of low significance (P > 0.05). b The contribution to R2 for each variable is the amount by which R2 would be reduced if that variable were removed regression equation.

from the

Fig. 3. Multiple RNase

linear regression: estimation

reactivation

coefficients

derived

similarity between high correlation Region half-time

81-95,

by synthetic

peptide

nf half-times amides

from the best subset of predictors. observed and estimated

coefficient

obtained

for

using the The

values reflects the

(r = 0.84;

corresponds

Discussion

P < 0.006).

which possesses the highest activity (shortest

or reactivation),

of the regression coefficients pius the contribution to R’). The number of Cys residues, aithough an important parameter, is not sufficient by itself to explain the thioredoxin-like activity (r = -0.46, Table 1). Our analysis suggests that the important characteristics of the active peptide amides are a low (regression coefficient > 0) percentage of predicted a-helical (contribution to R2 = 0.59) and p-turn (contribution to R2 = 0.20) conformations, a high (regression coefficient < 0) number of Cys residues ~contribution to R’ = 0.351, a high surface probability ~~ontribution to R’ = 0.19) and low ~lex~b~l~ty~~ontribution to R” = 0.15).

to the thioredoxin

ac-

tive center.

The parameters considered were hydrophiIi~ity (Kyte and Doolittfe, 1982), surface probability (Emini et al., 1985f, flexibility (Karptus and Schulz, 19851, percentage of hefical, extended, and turn conformations (Gamier et al., 1978) and number of Cys residues. The matrix of correlation between variables (Table 1) indicates that some of these variables are highly correlated (i.e., hydrophi~icity, surface probability and flexibility) and are, therefore, likely to contribute with redundant information to the regression. Nevertheless, hydrophilicity (P > 0.8) was omitted from the regression when the best subset of predictors was chosen. The number of Cys residues and The percentage of p-turn also appeared to be highly correlated. When these parameters were considered together, however, they made a significant cantribution to the regression (Table 2). The regression was statistically significant (P < 0.0061 and a high coefficient of multiple correlation was obtained (r = 0.91). This is illustrated in Fig. 3 where the experimental values are plotted together with the values predicted by the multiple linear regression. The more important characteristics of the peptide amides possessing thioredoxin-like activity (Table 2) are a Iow percentage of predicted cu-hefix and a high number of Cys residues (as indicated by the sign and magnitude

The initial reaction in the refolding of reduced and denatured proteins is the formation of random disulfide bonds followed by rearrangement of the disulfides to the native conformation (Hantgan et al., 1974). Thioredoxin has been known to effectively reduce a number of protein disulfides (Holmgren, 1981, 1985f and to catalyze the formation and rearrangement of protein disulfide bonds in RNase (Pigiet and Schuster, 1986). As occurs for other catalysts of disulfide bond formation, this reaction probably includes the production of a mixed-disulfide intermediate. Data presented here demonstrate that a synthetic peptide amide, TD-(31-361, containing the redox-active dithiol of the holoenzyme is able to reactivate reduced, denatured RNase. The observation of simiiar activity in a synthetic peptide amide derived from the p-subunit of hFSH, hFSH-@-(83-B), containing a sequence similar to the thioredoxin active center, suggests that the thioredoxin-like activity of hFSH is due to the presence of this redox-active dithiol within the primary structure of its p-subunit. Additional evidence supporting this notion is provided by the results of our study with 11 synthetic overlapping peptide amides containing the entire primary structure of hFSH-p-subunit. Among these peptide amides, hFSH-@(81-95) which contains the region similar to the thioredoxin active center, was most active in regenerating activity in reduced, denatured RNase. Although other peptide amides were less effective

169

than hFSH-~-(81-95), the sum of their contributions may be important to the overall thioredoxin-like activity of FSH. The characteristics of the catalytically active peptide amides, derived from the multiple linear regression analysis, are in close agreement with those of the thioredoxin active center. The structure of E. coli thioredoxin-S, is known by X-ray crystallography to contain a core of five P-pleated sheets flanked by four a-helical segments (Holmgren et at., 1975). Amino acid residues 29-37, which inctude the active center, are uniquely located in a protrusion on the surface of the enzyme rather than in a cleft, as normally occurs with the active sites of enzymes. Thioredoxin’s active dithiol is formed between Cys-32 and Cys-35, and is located between the middle strand of a pleated sheet (Cys-32) and one of the first turns of an a-helix (Cys-3.5). The low pK of Cys-32 (p K = 6.35) compared to Cys-35 (p K > 9.0) suggests that Cys-32 is the more important residue for activity, since only the thiolate ion is known to participate in thiol-disulfid~ interchange (Edman et al., 1985). Interestingly~ the most active peptide amide, hFSH-~-(Bl-95), contains residues equivalent to Cys-32 and Cys-35 of thioredoxin, whereas the second most active peptide amide, hFSH-P(71~85), contains only the residue equivalent to Cys-32 of thioredoxin (possessing a low pK value) but not the residue equivalent to Cys-35 of thioredoxin. The multiple linear regression analysis of the experimental results suggests that properties which characterize the active peptide amides are low percentage of cu-helices or turns, a high number of Cys residues, low flexibiIity and high surface probabili~, These results are in agreement with the characteristics of the active center of thioredoxin, indicating that the best subset of predictors, identified by multiple linear regression, are appropriate to characterize these peptide amides. Further studies will be required to determine if the additional Cys residues needed to reduce the mixed disulfide and release the peptide following its reduction of the incorrect disuIfide in the substrate protein, are contributed by neighboring Cys residues in the peptide, by a second peptide molecule (as would be required for peptides having only one Cys residue), by the Cys residue which would form the correct disul-

fide bridge, or a combination of these possibilities. Thioredoxin-like active center domains have been identified in a number of proteins. Protein disulfide isomerase (PDI) contains two presumed active center sequences with strong homology to E. coli thioredoxin (Jocelyn, 1972; Edman et al., 1985) and is also involved in the formation of native disulfide bonds in proteins (Bulleid and Fredman, 1988). More recently, thioredoxin-like active center sequences have been localized in phosphoinositide-specific phosphoIipase C (Bennet et al., 1988), although the implications of this in signal transduction and/or cell metabolism remain to be determined. Since most thioredoxinlike activity was found in hFSH-P-(81-95), a region of hFSH+-subunit representing a receptorbinding domain (Santa Coloma and Reichert, 19901, our results suggest that this activity may have a physiological role, either in the anchoring of the hormone to the receptor or in signal transduction. In this regard, the binding of FSH to its receptor becomes increasingly irreversible as a function of time and temperature (Andersen et al., 1983; Sanborn et at., 1987) and such conversion from a low to high affinity state could be explained by covalent bond formation. Disulfidelinked FSH-receptor complex formation catalyzed by a thioredoxin-like redox-active center within the P-subunit of FSH could account for this observation. Recent elucidation of the primary structure of the rat testicular FSH receptor and identification of conserved Cys residues in its extracellular domain (Sprengel et al., 1990), suggests that the natural ‘substrate’ for FSH, i.e., its receptor, contains thiol groups that may be active in redox reactions. Such FSH-catalyzed dithioldisulfide redox reactions and disulfide isomerization may have a physiological role in receptor binding or signal transduction. References Andersen, T.A., Curatolo, L.M. and Reichert,

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Laurent, TX., Moore, E.C. and Reichatd, P. (1964) J. Biol. Chem. 259, 3436--3444. Lim, C.-J., Hailer, B. and Fuchs, J.A. (1985) J. Bacteriot. 161, 799-802. Mark, D.F. and Richardson, C.C. (1979) Proc. Natl. Acad. Sci. U.S.A. 73, 780-784. Merrifield, R.B. (1963) J. Am. Chem. Sot. 85, 2149-2154. Moore, E.C., Reichard, P. and Thelander, L. (1964) J. Biol. Chem. 239, 3445-3452. Pigiet, V.P. and Schuster, B.J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7643-7647. Porter, M.A., Stringer, C.D. and Hartman, F.L. (1988) J. Biol. Chem. 263, 123-129. Russel, M. and Model, P. (1986) J. Biol. Chem. 261, 1499715005. Sanborn, B.B., Andersen, T.A. and Reichert, Jr., L.E. (19871 Biochemistry 26, 8196-8200. Santa Coloma, T.A. and Reichert, Jr., L.E. (iY90) J. Biol. Chem. 265, 5037-5042. Santa Coloma, T.A., Dattatreyamurty, B. and Reichert, Jr., L.E. (1990) Biochemistry 29, 1194-1200. Sprengel, R., Braun, T., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1990) Mol. Endocrinol. 4, 525-530. Watkins, P.C., Eddy, R., Beck, A.K., Vellucci, V., Leverone, B., Tan& R.E., Gusella, J.F. and Shows, T.B. (1987) DNA 6, 205-212. Wiegand. M., Ofend~h-Hahnle, B. and Eisele, K. (1989) J. Steroid B&hem. 43, 53-58. van Haarlem, L.J.M., Saute, B.A.M. and Vermeer, C. (1987) FEBS Lett. 222, 353-357.