Expression of 11/?-Hydroxysteroid Dehydrogenase Using Recombinant Vaccinia Virus
Anil K. Agarwal, Maria-Teresa Tusie-Luna, Carl Monder, and Perrin C. White Division of Pediatric Endocrinology Cornell University Medical College New York, New York 10021 Population Council (CM.) New York, New York 10021
Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11/3-hydroxysteroid dehydrogenase. To determine the kinetic properties of this enzyme, rat liver cDNA was expressed in cultured cells using recombinant vaccinia virus. Although this enzyme catalyzes only dehydrogenation when purified from rat liver, the recombinant enzyme obtained from cell lysates catalyzed both 11/9-dehydrogenation of corticosterone to 11dehydrocorticosterone and the reverse 11-oxoreduction reaction. At pH 8.5, the first order rate constant Kc.,/Km for dehydrogenase activity exceeded that for reductase (63 vs. 38 min~1 x 10~4), whereas the rate constants for the two reactions were nearly equal (48 vs. 47 min"1 x 10~4) at pH 7.0. These results are consistent with the previously determined pH optima for these activities in liver microsomes. Removal (with glucose-6-phosphate dehydrogenase) of NADP* produced by the reductase reaction significantly increased reductase activity. Glycyrrhetinic acid, a known inhibitor of the dehydrogenase reaction, also inhibited the reductase reaction at slightly higher concentrations (50% inhibitory concentration, 0.05). Transforming the data obtained at pH 7.0 according to the Hill equation produced Hill coefficients of 1 for both 11-DH and 11-OR reactions, consistent with a single active site for each reaction. Inhibition by Glycyrrhetinic Acid 30
60
90
150
TIME (mln) Fig. 3. Time Curve for 11 -OR Activity (Represented as Percent Conversion of 2 ^ M 11-DH) at pH 8.5 in the Presence (•) or Absence (•) of Glucose-6-Phosphate Dehydrogenase and Glucose-6-Phosphate No 11 -DH activity was observed in the presence of glucose6-phosphate dehydrogenase.
When enzymatic activity was measured in intact infected cells, 500 nM glycyrrhetinic acid inhibited 11-DH activity by 50% (32% conversion of corticosterone to dehydrocorticosterone decreased to 16%), but had a minimal effect on 11 -OR activity (20% conversion decreased to 17%) over 2.5 h. This conforms with our previous observations made in Chinese hamster ovary
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MOL ENDO-1990 1830
cells transfected with a plasmid containing 11-HSD cDNA under the control of an SV40 promoter (10) and with the behavior of 11-HSD in rat kidney. Activities in cell lysates were affected at much lower concentrations of glycyrrhetinic acid. Dehydrogenase activity at pH 7.0 was inhibited 50% by less than 5 nwi glycyrrhetinic acid, and 11-OR activity was inhibited 50% by about 20 nM (Fig. 5).
DISCUSSION Recombinant 11 -HSD appears to be identical to purified rat liver 11-HSD in the size of the polypeptide and in kinetic properties of its 11-DH activity (Km at pH 8.5 of 2.14 MM for recombinant 11-HSD vs. 1.83 M M for the liver enzyme). The 11-OR activity of rat liver 11-HSD is lost during purification, and the recombinant enzyme also has relatively low 11-OR activity at pH 8.5, which appears to be reduced further during prolonged incubation with substrate and cofactor. This may be due in part to dehydrogenation of product by the much more active 11 -DH activity of the enzyme at this pH, because removal of all NADP+ (the cofactor for the 11-DH reaction) significantly increases 11-OR activity (Fig. 3). Alternatively, binding of NADP+ under the assay conditions may irreversibly inactivate 11-OR. In support of the latter explanation, it was not possible to reactivate 11 -OR activity of the rat liver enzyme once it had been lost even when NADP+ was removed using the same methods as those employed in the present study (Lakshmi, V., and C. M., unpublished observations). At pH 7.0, closer to the optimum for rat liver 11-OR activity, the 11-OR and 11-DH activities of recombinant 11-HSD have identical first order rate constants (Kcat/ Km) and similar Km values. Both activities are inhibited in vitro by relatively low concentrations of glycyrrhetinic acid, although 11-DH is more sensitive to it than 11-
OR. Hill coefficients of 1 for both reactions permit us to conclude that for each substrate the enzyme contains no noncatalytic regulatory sites, contains a single independent binding site, and undergoes no measurable conformational change that affects the velocity constants. Thus, the similarities in the kinetic properties of 11-DH and 11-OR are notable. The data are consistent with either topographically distinct 11 -OR and 11 -DH sites on a single polypeptide or a single site that undergoes conformational reordering to favor dehydrogenation or reduction. Further studies will be required to distinguish between these possibilities. The data are not consistent with the hypothesis that 11 -DH and 11OR are independent enzymes. There is at present no satisfactory explanation for the instability of 11-OR activity or for why, if it is indeed the reciprocal property of a unique active site, its stability is not equal to that of 11-DH activity. However, other examples exist of enzymes with multiple activities that may be differentially affected. For example, purified bovine or porcine steroid 11/3-hydroxylase (P450c11) also has 18-hydroxylase and 18-oxidase activities; the last activity is specifically inhibited by an unknown mechanism in the zona fasciculata of the adrenal cortex, but not in the zona glomerulosa (11). These studies do not rule out the existence of additional 11 -DH or 11 -OR isozymes. It is also possible that additional isoforms of 11-HSD are generated from the same gene. Because vaccinia replicates in the cytoplasm, only cDNA can be expressed, and so alternatively spliced mRNA species could not be identified in these experiments. However, 31- and 32-kDa polypeptides were observed in immunoprecipitates in addition to the major 34-kDa band. These might be unglycosylated or partially glycosylated products; 11-HSD is a glycoprotein, and the predicted size of the polypeptide alone is 31.7 kDa. The increased relative intensity of the 31-kDa band in lysates from cells treated with A t tunicamycin is consistent with this possibility. Inspection of the cDNA sequence suggests an alternative possibility: residue 27 is an additional in-frame methionine in a good context (GAAATGC) for initiation of translation (12), and if ribosomes occasionally initiated at this internal methionine, the resulting polypeptide would be about 3 kDa smaller than the full-length product. This methionine occurs before the predicted NADP+/NADPH-binding site, and so it is possible that such a truncated enzyme would still be active (Fig. 6).
11-HSD 17-HSD
5
10
15
20
100
GE(nM) Fig. 5. Inhibition of 11-DH (O) and 11-OR (•) Activities by Glycyrrhetinic Acid (GE) Activities are measured as the percent conversion of 2 HM substrate.
MKKYLLPVLVLCLGYYYSTNEEFRPE1ILQGKKVIVTGASKGIG MARTWLITGCSSGIG + *++** * ***
Fig. 6. Comparison between the Amino-Terminal Sequence of 11-HSD (9) and That of a Related Enzyme, 17/8-Hydroxysteroid Dehydrogenase (17-HSD) (21). Single letter amino acid codes are used. Amino acid residues that are identical in these two enzymes are indicated by asterisks, and functionally conserved residues by crosses. The second methionine residue in 11 -HSD is underlined; note that if translation were initiated at this methionine, the two enzymes would have very similar amino-termini.
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1831
Expression of 11-HSD
Tunicamycin-treated cells had a relative decrease in 11-DH activity, suggesting that glycosylation might be important for full 11 -DH activity. A more definitive way to determine whether unglycosylated or truncated enzymes differ from the normal enzyme in relative 11 -DH and 11 -OR activities is to study recombinant enzymes that have had potential glycosylation sites and/or the initial methionine modified by in vitro mutagenesis of the cDNA.
MATERIALS AND METHODS Construction of Recombinant Vaccinia Virus A 1.2-kb EcoRI fragment was isolated containing a nearly fulllength cDNA for rat 11 -HSD, including the entire coding region. The shuttle vector pTF7 (13) (obtained from B. Moss) was digested with BamH\. The vector and fragment were rendered blunt-ended with the Klenow fragment of DNA polymerase and ligated. E. coli transformants carrying the insert were selected by colony hybridization, and a clone with the insert in the correct orientation relative to the vector's T7 promoter was identified by restriction mapping. TK~143B human osteosarcoma cells were obtained from American Type Culture Collection and grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum and 25 (tg/m\ 5-bromodeoxyuridine. Cells were transfected with the pTF7/11-HSD construct and infected with wildtype vaccinia virus strain WR, as previously described (14). Recombinant viruses lacked thymidine kinase (i.e. they were tk~) and could form plaques in the presence of bromodeoxyuridine. The presence of 11-HSD cDNA in the viral genome was verified by dot blot hybridization. One clone was termed v11-HSD. Infection of Cells TK~143B cells were infected simultaneously with v11-HSD and with vTF7, a recombinant vaccinia virus expressing T7 RNA polymerase under the control of a vaccinia promoter. T7 polymerase synthesized under the direction of vTF7 transcribed 11-HSD cDNA from the T7 promoter included in the pTF7 shuttle vector used to generate v11-HSD. Viruses (10:10 multiplicities of infection of for vTF7 and v11 HSD) were adsorbed to cells at 37 C for 60 min in a minimal volume of serum-free medium with occasional rocking. A,Tunicamycin homolog(Boehringer-Mannheim, Indianapolis, IN) was added to some wells at 1, 10, or 50 ng/ml immediately after adsorption of virus. Control plates were infected with vTF7 alone. Cells were then fed with medium supplemented with 10% serum. Activity was assayed in intact cells or cell lysates 24 h after infection. Preparation of Cell Lysates Cells were washed once with PBS and once with ice-cold homogenizing buffer (0.1 M Tris-HCI, pH 8.5-0.1 mM phenylmethylsulfonylfluoride-10 mM EDTA). Cells were scraped off the culture dishes and homogenized in the same buffer using a Dounce homogenizer (Kontes Co., Vineland, NJ). The lysate was centrifuged at 1000 x g for 5 min. The supernatant was used directly for enzyme assays or frozen in aliquots at - 2 0 C and used within 1 week of preparation. Protein concentrations were determined according to the method of Bradford (15), using reagents from Bio-Rad (Richmond, CA). Assays of Enzymatic Activity in Cell Lysates Except where noted, lysates were treated with Triton DF-18 at a ratio of 0.15 mg detergent/mg protein for 60 min at 4 C
immediately before being assayed. Dehydrogenase activity was determined by measuring the conversion of corticosterone to 11-dehydrocorticosterone in the presence of NADP+. Assays were performed in 1 ml buffer containing 0.1 M TrisHCI (pH 8.5), 0.1 mM phenylmethylsulfonylfluoride, 10 mM EDTA, 250 fiM NADP\ approximately 20,000 cpm [1,2-3H] corticosterone (SA, 60 Ci/mmol), and 2 ^M unlabeled corticosterone (in kinetic studies 0.125-4.0 MM corticosterone was used). In some experiments 0.1 M Na2HPO4, pH 7.0, was substituted for 0.1 M Tris-HCI, pH 8.5. After 10 min of preincubation at 37 C, 50 M9 cell lysate protein were added, and incubation was continued for various times. Steroids were extracted into ethyl acetate, and unlabeled corticosterone and 11-dehydrocorticosterone were added as markers. Extracts were concentrated, and the steroids were separated by TLC on silica support in chloroform-methanol (95:5, vol/vol) (16). Spots corresponding to steroids were located under UV light, cut out, and counted by scintillation spectrophotometry. Reductase activity was assayed in a similar manner by measuring the conversion of 11 -dehydro-[1,2-3H]corticosterone [prepared as described previously (16)] to corticosterone in the presence of NADPH. Assays performed at pH 8.5 used 200 ng protein/reaction. Reductase activity was also determined at pH 8.5 in the presence of 2 U glucose-6-phosphate dehydrogenase purified from L. mesenteroides (Sigma, St. Louis, MO) and 1 mM glucose-6-phosphate. Kinetic measurements were performed using a 60-min incubation for 11-DH activity and a 30-min incubation for 11-OR activity. Reaction rates were linear with time within these intervals, and less than 25% of substrate was converted under these reaction conditions. Each experimental point was determined in duplicate, and the data were analyzed using double reciprocal plots. Data were also analyzed using an adaptation of the Hill equation (17). Inhibition of enzyme activity by glycyrrhetinic acid was determined at pH 7.0 using the assay conditions described above in the presence of 2 ^M steroid substrate. Reductase activity was assayed during a 15-min incubation using 300 ng protein/ reaction. All determinations were repeated using lysates prepared from cells infected with vTF7 helper virus alone. Background values were subtracted from experimental values before further analysis. Typical background conversions observed during 60-min incubations of lysates from vTF7-infected cells were approximately 2% for dehydrogenation of corticosterone to dehydrocorticosterone and approximately 8% for reduction of dehydrocorticosterone to corticosterone.
Statistical Analysis Experimental points were plotted using the Enzfit program. Enzyme kinetic data were fitted to K^/Km as proposed previously (18) for the two-step reaction: Ki
Ko
K_i
K_2
E + S ^ E S ^ E + P, where E is 11-DH or 11-OR, and S is the appropriate steroid substrate. It is assumed that the enzyme is saturated with cofactor (NADP+ or NADPH > 10 Km) and that the pyridine nucleotides are not dead-end inhibitors (Monder, C , and V. Lakshmi, unpublished observations). The Michaelis-Menten equation may be rewritten as: v = (Km x S x Kca^K^Kn, + S), where K^t/Kn, = k^zAkz + k_,). The data were analyzed by nonlinear regression. Parametric analysis was performed using a weighted least squares curvefitting program with iterative reweighting (19). Significance of differences between means was determined using two-tailed Student's f test for the appropriate degrees of freedom.
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MOL ENDO-1990 1832
Radiolabeling and Immunoprecipitation TK~143B cells were seeded in six-well plates and grown to 80% confluency. Cells were infected with vTF7 alone or vTF7 and v11-HSD together. At-Tunicamycin homolog (1-50 ng/ml) was added to some wells. After 24 h, the medium was changed to 2 ml medium (including tunicamycin if previously used) containing 150 fid [35S]methionine/cysteine (1000 Ci/mmol) without additional methionine and cysteine. After 1 h, cells in each well were washed once in PBS, collected, and lysed in 250 n\ 0.01 M Tris-HCI, pH 8.0-1% Triton X-100-0.1% sodium dodecyl sulfate. To 200 n\ lysate were added 300 n\ dilution buffer (0.1 M Tris-HCI, pH 8.0-0.14 M NaCI-0.1% Triton X100). Lysates were precleared by successive 1 -h incubations on ice with a 1:100 dilution of normal rabbit serum and a 1:10 volume of protein-A-Sepharose. After centrifugation, a 1:100 dilution of rabbit antirat 11-HSD serum was added to the supernatant and incubated for 2 h at 4 C. Antigen-antibody complexes were adsorbed to protein-A-Sepharose and washed twice in dilution buffer, once in 0.1 M Tris-HCI, pH 8.0-0.14 M NaCI, and once in 0.05 M Tris-HCI, pH 6.8 (20). Samples were boiled in Laemmli sample buffer and analyzed by electrophoresis in a 12.5% polyacrylamide gel. The gel was dried and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY) for 4 days.
Acknowledgments Received August 27, 1990. Revision received September 20, 1990. Accepted September 20, 1990. Address requests for reprints to: Dr. Perrin C. White, Division of Pediatric Endocrinology, Cornell University Medical College, New York, New York 10021. This work was supported by NIH Grants DK-37094 and DK37867.
6. 7.
8.
9.
10. 11. 12. 13. 14.
15.
16.
REFERENCES 1. Monder C, Shackleton CHL 1984 110-Hydroxysteroid dehydrogenase: fact or fancy? Steroids 44:383-417 2. Funder JW, Pearce PT, Smith R, Smith Al 1988 Mineralcorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242:583-585 3. Edwards CRW, Burt D, Mclntyre MA, deKloet ER, Stewart PM, Brett L, Sutanto WS, Monder C 1988 Localization of 11/3-hydroxysteroid dehdrogenase-tissue specific protector of the mineralocorticoid receptor. Lancet 2:986-989 4. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM 1987 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268-275 5. Ulick S, Levine LS, Gunczler P, Zanconato G, Ramirez
17. 18. 19. 20.
21.
LC, Rauh W, Rosier A, Bradlow HL, New Ml 1979 A syndrome of apparent mineralocorticoid excess associated with defects in the peripheral metabolism of cortisol. J Clin Endocrinol Metab 49:757-764 Phillipou G, Higgins BA 1985 A new defect in the peripheral conversion of cortisone to cortisol. J Steroid Biochem 22:435-437 Monder C, Stewart PM, Lakshmi V, Valentino R, Burt D, Edwards CRW 1989 Licorice inhibits corticosterond 110dehydrogenase of rat kidney and liver: in vivo and in vitro studies. Endocrinology 125:1046-1053 Lakshmi V, Monder C 1988 Purification and characterisation of the corticosteroid 110-dehydrogenase component of the rat liver 11 /3-hydroxysteroid dehydrogenase complex. Endocrinology 123:2390-2398 Agarwal AK, Monder C, Eckstein B, White PC 1989 Cloning and expression of rat cDNA encoding corticosteroid 11/S-dehydrogenase. J Biol Chem 264:1893918943 Lakshmi V, Monder C 1985 Extraction of 11 /3-hyroxysteroid dehydrogenase from rat liver microsomes by detergents. J Steroid Biochem 22:331 -340 Yanagibashi K, Haniu M, Shively JE, Shen WH, Hall P 1986 The synthesis of aldosterone by the adrenal cortex. J Biol Chem 261:3556-3562 Kozak M 1986 Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283-292 Fuerst TR, Earl PL, Moss B 1987 Use of a hybrid vaccinia virsu-T7 RNA polymerase system for expresssion of target genes. Mol Cell Biol 7:2538-2544 Fuerst TR, Niles EG, Studier FW, Moss B1986 Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 83:8122-8126 Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 Lakshmi V, Monder C 1985 Evidence for independent 11 oxidase and 11 -reductase activities of 11 /3-hydroxysteroid dehydrogenase: enzyme latency, phase transition, and lipid requirements. Endocrinology 116:552-560 Segal IH 1975 Enzyme Kinetics. Wiley and Sons, New York, pp 346-385 Northrop DB 1983 Fitting enzyme-kinetic data to V/K. Anal Biochem 132:457-461 Bevington P 1969 Data Reduction and Error Analysis for the Physical Sciences. MacGraw-Hill, New York Kessler SW 1975 Rapid isolation of antigens from cells with a staphylococcal protein A-antibody absorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol 115:1617-1624 The VL, Labrie C, Zhao HF, Couet J, Lachance Y, Simard J, Leblanc G, Cote J, Berube D, Gagne R, Labrie F 1989 Characterization of cDNAs for human estradiol 17/3-dehydrogenase and assignment of the gene to chromosome 17: evidence of two mRNA species with distinct 5'-termini in human placenta. Mol Endocrinol 3:1301-1309
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Anil K. Agarwal, Maria-Teresa Tusie-Luna, Carl Monder, and Perrin C. White Division of Pediatric Endocrinology Cornell University Medical College New York, New York 10021 Population Council (CM.) New York, New York 10021
Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11/3-hydroxysteroid dehydrogenase. To determine the kinetic properties of this enzyme, rat liver cDNA was expressed in cultured cells using recombinant vaccinia virus. Although this enzyme catalyzes only dehydrogenation when purified from rat liver, the recombinant enzyme obtained from cell lysates catalyzed both 11/9-dehydrogenation of corticosterone to 11dehydrocorticosterone and the reverse 11-oxoreduction reaction. At pH 8.5, the first order rate constant Kc.,/Km for dehydrogenase activity exceeded that for reductase (63 vs. 38 min~1 x 10~4), whereas the rate constants for the two reactions were nearly equal (48 vs. 47 min"1 x 10~4) at pH 7.0. These results are consistent with the previously determined pH optima for these activities in liver microsomes. Removal (with glucose-6-phosphate dehydrogenase) of NADP* produced by the reductase reaction significantly increased reductase activity. Glycyrrhetinic acid, a known inhibitor of the dehydrogenase reaction, also inhibited the reductase reaction at slightly higher concentrations (50% inhibitory concentration, 0.05). Transforming the data obtained at pH 7.0 according to the Hill equation produced Hill coefficients of 1 for both 11-DH and 11-OR reactions, consistent with a single active site for each reaction. Inhibition by Glycyrrhetinic Acid 30
60
90
150
TIME (mln) Fig. 3. Time Curve for 11 -OR Activity (Represented as Percent Conversion of 2 ^ M 11-DH) at pH 8.5 in the Presence (•) or Absence (•) of Glucose-6-Phosphate Dehydrogenase and Glucose-6-Phosphate No 11 -DH activity was observed in the presence of glucose6-phosphate dehydrogenase.
When enzymatic activity was measured in intact infected cells, 500 nM glycyrrhetinic acid inhibited 11-DH activity by 50% (32% conversion of corticosterone to dehydrocorticosterone decreased to 16%), but had a minimal effect on 11 -OR activity (20% conversion decreased to 17%) over 2.5 h. This conforms with our previous observations made in Chinese hamster ovary
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MOL ENDO-1990 1830
cells transfected with a plasmid containing 11-HSD cDNA under the control of an SV40 promoter (10) and with the behavior of 11-HSD in rat kidney. Activities in cell lysates were affected at much lower concentrations of glycyrrhetinic acid. Dehydrogenase activity at pH 7.0 was inhibited 50% by less than 5 nwi glycyrrhetinic acid, and 11-OR activity was inhibited 50% by about 20 nM (Fig. 5).
DISCUSSION Recombinant 11 -HSD appears to be identical to purified rat liver 11-HSD in the size of the polypeptide and in kinetic properties of its 11-DH activity (Km at pH 8.5 of 2.14 MM for recombinant 11-HSD vs. 1.83 M M for the liver enzyme). The 11-OR activity of rat liver 11-HSD is lost during purification, and the recombinant enzyme also has relatively low 11-OR activity at pH 8.5, which appears to be reduced further during prolonged incubation with substrate and cofactor. This may be due in part to dehydrogenation of product by the much more active 11 -DH activity of the enzyme at this pH, because removal of all NADP+ (the cofactor for the 11-DH reaction) significantly increases 11-OR activity (Fig. 3). Alternatively, binding of NADP+ under the assay conditions may irreversibly inactivate 11-OR. In support of the latter explanation, it was not possible to reactivate 11 -OR activity of the rat liver enzyme once it had been lost even when NADP+ was removed using the same methods as those employed in the present study (Lakshmi, V., and C. M., unpublished observations). At pH 7.0, closer to the optimum for rat liver 11-OR activity, the 11-OR and 11-DH activities of recombinant 11-HSD have identical first order rate constants (Kcat/ Km) and similar Km values. Both activities are inhibited in vitro by relatively low concentrations of glycyrrhetinic acid, although 11-DH is more sensitive to it than 11-
OR. Hill coefficients of 1 for both reactions permit us to conclude that for each substrate the enzyme contains no noncatalytic regulatory sites, contains a single independent binding site, and undergoes no measurable conformational change that affects the velocity constants. Thus, the similarities in the kinetic properties of 11-DH and 11-OR are notable. The data are consistent with either topographically distinct 11 -OR and 11 -DH sites on a single polypeptide or a single site that undergoes conformational reordering to favor dehydrogenation or reduction. Further studies will be required to distinguish between these possibilities. The data are not consistent with the hypothesis that 11 -DH and 11OR are independent enzymes. There is at present no satisfactory explanation for the instability of 11-OR activity or for why, if it is indeed the reciprocal property of a unique active site, its stability is not equal to that of 11-DH activity. However, other examples exist of enzymes with multiple activities that may be differentially affected. For example, purified bovine or porcine steroid 11/3-hydroxylase (P450c11) also has 18-hydroxylase and 18-oxidase activities; the last activity is specifically inhibited by an unknown mechanism in the zona fasciculata of the adrenal cortex, but not in the zona glomerulosa (11). These studies do not rule out the existence of additional 11 -DH or 11 -OR isozymes. It is also possible that additional isoforms of 11-HSD are generated from the same gene. Because vaccinia replicates in the cytoplasm, only cDNA can be expressed, and so alternatively spliced mRNA species could not be identified in these experiments. However, 31- and 32-kDa polypeptides were observed in immunoprecipitates in addition to the major 34-kDa band. These might be unglycosylated or partially glycosylated products; 11-HSD is a glycoprotein, and the predicted size of the polypeptide alone is 31.7 kDa. The increased relative intensity of the 31-kDa band in lysates from cells treated with A t tunicamycin is consistent with this possibility. Inspection of the cDNA sequence suggests an alternative possibility: residue 27 is an additional in-frame methionine in a good context (GAAATGC) for initiation of translation (12), and if ribosomes occasionally initiated at this internal methionine, the resulting polypeptide would be about 3 kDa smaller than the full-length product. This methionine occurs before the predicted NADP+/NADPH-binding site, and so it is possible that such a truncated enzyme would still be active (Fig. 6).
11-HSD 17-HSD
5
10
15
20
100
GE(nM) Fig. 5. Inhibition of 11-DH (O) and 11-OR (•) Activities by Glycyrrhetinic Acid (GE) Activities are measured as the percent conversion of 2 HM substrate.
MKKYLLPVLVLCLGYYYSTNEEFRPE1ILQGKKVIVTGASKGIG MARTWLITGCSSGIG + *++** * ***
Fig. 6. Comparison between the Amino-Terminal Sequence of 11-HSD (9) and That of a Related Enzyme, 17/8-Hydroxysteroid Dehydrogenase (17-HSD) (21). Single letter amino acid codes are used. Amino acid residues that are identical in these two enzymes are indicated by asterisks, and functionally conserved residues by crosses. The second methionine residue in 11 -HSD is underlined; note that if translation were initiated at this methionine, the two enzymes would have very similar amino-termini.
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1831
Expression of 11-HSD
Tunicamycin-treated cells had a relative decrease in 11-DH activity, suggesting that glycosylation might be important for full 11 -DH activity. A more definitive way to determine whether unglycosylated or truncated enzymes differ from the normal enzyme in relative 11 -DH and 11 -OR activities is to study recombinant enzymes that have had potential glycosylation sites and/or the initial methionine modified by in vitro mutagenesis of the cDNA.
MATERIALS AND METHODS Construction of Recombinant Vaccinia Virus A 1.2-kb EcoRI fragment was isolated containing a nearly fulllength cDNA for rat 11 -HSD, including the entire coding region. The shuttle vector pTF7 (13) (obtained from B. Moss) was digested with BamH\. The vector and fragment were rendered blunt-ended with the Klenow fragment of DNA polymerase and ligated. E. coli transformants carrying the insert were selected by colony hybridization, and a clone with the insert in the correct orientation relative to the vector's T7 promoter was identified by restriction mapping. TK~143B human osteosarcoma cells were obtained from American Type Culture Collection and grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum and 25 (tg/m\ 5-bromodeoxyuridine. Cells were transfected with the pTF7/11-HSD construct and infected with wildtype vaccinia virus strain WR, as previously described (14). Recombinant viruses lacked thymidine kinase (i.e. they were tk~) and could form plaques in the presence of bromodeoxyuridine. The presence of 11-HSD cDNA in the viral genome was verified by dot blot hybridization. One clone was termed v11-HSD. Infection of Cells TK~143B cells were infected simultaneously with v11-HSD and with vTF7, a recombinant vaccinia virus expressing T7 RNA polymerase under the control of a vaccinia promoter. T7 polymerase synthesized under the direction of vTF7 transcribed 11-HSD cDNA from the T7 promoter included in the pTF7 shuttle vector used to generate v11-HSD. Viruses (10:10 multiplicities of infection of for vTF7 and v11 HSD) were adsorbed to cells at 37 C for 60 min in a minimal volume of serum-free medium with occasional rocking. A,Tunicamycin homolog(Boehringer-Mannheim, Indianapolis, IN) was added to some wells at 1, 10, or 50 ng/ml immediately after adsorption of virus. Control plates were infected with vTF7 alone. Cells were then fed with medium supplemented with 10% serum. Activity was assayed in intact cells or cell lysates 24 h after infection. Preparation of Cell Lysates Cells were washed once with PBS and once with ice-cold homogenizing buffer (0.1 M Tris-HCI, pH 8.5-0.1 mM phenylmethylsulfonylfluoride-10 mM EDTA). Cells were scraped off the culture dishes and homogenized in the same buffer using a Dounce homogenizer (Kontes Co., Vineland, NJ). The lysate was centrifuged at 1000 x g for 5 min. The supernatant was used directly for enzyme assays or frozen in aliquots at - 2 0 C and used within 1 week of preparation. Protein concentrations were determined according to the method of Bradford (15), using reagents from Bio-Rad (Richmond, CA). Assays of Enzymatic Activity in Cell Lysates Except where noted, lysates were treated with Triton DF-18 at a ratio of 0.15 mg detergent/mg protein for 60 min at 4 C
immediately before being assayed. Dehydrogenase activity was determined by measuring the conversion of corticosterone to 11-dehydrocorticosterone in the presence of NADP+. Assays were performed in 1 ml buffer containing 0.1 M TrisHCI (pH 8.5), 0.1 mM phenylmethylsulfonylfluoride, 10 mM EDTA, 250 fiM NADP\ approximately 20,000 cpm [1,2-3H] corticosterone (SA, 60 Ci/mmol), and 2 ^M unlabeled corticosterone (in kinetic studies 0.125-4.0 MM corticosterone was used). In some experiments 0.1 M Na2HPO4, pH 7.0, was substituted for 0.1 M Tris-HCI, pH 8.5. After 10 min of preincubation at 37 C, 50 M9 cell lysate protein were added, and incubation was continued for various times. Steroids were extracted into ethyl acetate, and unlabeled corticosterone and 11-dehydrocorticosterone were added as markers. Extracts were concentrated, and the steroids were separated by TLC on silica support in chloroform-methanol (95:5, vol/vol) (16). Spots corresponding to steroids were located under UV light, cut out, and counted by scintillation spectrophotometry. Reductase activity was assayed in a similar manner by measuring the conversion of 11 -dehydro-[1,2-3H]corticosterone [prepared as described previously (16)] to corticosterone in the presence of NADPH. Assays performed at pH 8.5 used 200 ng protein/reaction. Reductase activity was also determined at pH 8.5 in the presence of 2 U glucose-6-phosphate dehydrogenase purified from L. mesenteroides (Sigma, St. Louis, MO) and 1 mM glucose-6-phosphate. Kinetic measurements were performed using a 60-min incubation for 11-DH activity and a 30-min incubation for 11-OR activity. Reaction rates were linear with time within these intervals, and less than 25% of substrate was converted under these reaction conditions. Each experimental point was determined in duplicate, and the data were analyzed using double reciprocal plots. Data were also analyzed using an adaptation of the Hill equation (17). Inhibition of enzyme activity by glycyrrhetinic acid was determined at pH 7.0 using the assay conditions described above in the presence of 2 ^M steroid substrate. Reductase activity was assayed during a 15-min incubation using 300 ng protein/ reaction. All determinations were repeated using lysates prepared from cells infected with vTF7 helper virus alone. Background values were subtracted from experimental values before further analysis. Typical background conversions observed during 60-min incubations of lysates from vTF7-infected cells were approximately 2% for dehydrogenation of corticosterone to dehydrocorticosterone and approximately 8% for reduction of dehydrocorticosterone to corticosterone.
Statistical Analysis Experimental points were plotted using the Enzfit program. Enzyme kinetic data were fitted to K^/Km as proposed previously (18) for the two-step reaction: Ki
Ko
K_i
K_2
E + S ^ E S ^ E + P, where E is 11-DH or 11-OR, and S is the appropriate steroid substrate. It is assumed that the enzyme is saturated with cofactor (NADP+ or NADPH > 10 Km) and that the pyridine nucleotides are not dead-end inhibitors (Monder, C , and V. Lakshmi, unpublished observations). The Michaelis-Menten equation may be rewritten as: v = (Km x S x Kca^K^Kn, + S), where K^t/Kn, = k^zAkz + k_,). The data were analyzed by nonlinear regression. Parametric analysis was performed using a weighted least squares curvefitting program with iterative reweighting (19). Significance of differences between means was determined using two-tailed Student's f test for the appropriate degrees of freedom.
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Vol4No. 12
MOL ENDO-1990 1832
Radiolabeling and Immunoprecipitation TK~143B cells were seeded in six-well plates and grown to 80% confluency. Cells were infected with vTF7 alone or vTF7 and v11-HSD together. At-Tunicamycin homolog (1-50 ng/ml) was added to some wells. After 24 h, the medium was changed to 2 ml medium (including tunicamycin if previously used) containing 150 fid [35S]methionine/cysteine (1000 Ci/mmol) without additional methionine and cysteine. After 1 h, cells in each well were washed once in PBS, collected, and lysed in 250 n\ 0.01 M Tris-HCI, pH 8.0-1% Triton X-100-0.1% sodium dodecyl sulfate. To 200 n\ lysate were added 300 n\ dilution buffer (0.1 M Tris-HCI, pH 8.0-0.14 M NaCI-0.1% Triton X100). Lysates were precleared by successive 1 -h incubations on ice with a 1:100 dilution of normal rabbit serum and a 1:10 volume of protein-A-Sepharose. After centrifugation, a 1:100 dilution of rabbit antirat 11-HSD serum was added to the supernatant and incubated for 2 h at 4 C. Antigen-antibody complexes were adsorbed to protein-A-Sepharose and washed twice in dilution buffer, once in 0.1 M Tris-HCI, pH 8.0-0.14 M NaCI, and once in 0.05 M Tris-HCI, pH 6.8 (20). Samples were boiled in Laemmli sample buffer and analyzed by electrophoresis in a 12.5% polyacrylamide gel. The gel was dried and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY) for 4 days.
Acknowledgments Received August 27, 1990. Revision received September 20, 1990. Accepted September 20, 1990. Address requests for reprints to: Dr. Perrin C. White, Division of Pediatric Endocrinology, Cornell University Medical College, New York, New York 10021. This work was supported by NIH Grants DK-37094 and DK37867.
6. 7.
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