Tyrosine Hydroxylase Purification from Rat PC1 2 Ceils

PROTEIN EXPRESSION AND PURIFICATION 2, 10-14 (19%)

Laura G. Gahn and Robert Roskoski, Jr. Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70119

Received January 25,199l

of tyrosine hydroxylase and has been used to purify Tyrosine hydroxylase was purified in high yield from small amounts (240-300 pug) of the enzyme (12,13). We rat PC12 cells. This three-day procedure consisted of report here a protocol for the purification of about 15 differential ammonium sulfate precipitation, anion-ex- mg of homogeneous, stable rat tyrosine hydroxylase in change chromatography, and heparin-Sepharose affin- high yield from PC12 cells in culture. ity chromatography. It yielded an average of I5 mg of purified protein from 100 flasks of PC12 cells, with MATERIALS AND METHODS greater than 40% recovery of tyrosine hydroxylase. So- dium dodecyl sulfate-polyacrylamide gel electrophore- Materials sis yielded a single protein band with a molecular Catalase, tyrosine, piperazine-2\r,iV’-bis(2-ethanesul- weight of approximately 60,000. The protein had a spe- fonic acid) (Pipes), tris(hydroxymethyl)aminomethane, cific activity of 670 nmollminlmg and had a K,,, for its KCl, and dithiothreitol were obtained from Sigma reducing .cofactor tetrahydrobiopterin of 1.8 mM. The Chemical Co. (St. Louis, MO). Ammonium sulfate, po- purified protein can be phosphorylated and activated tassium phosphate, NaCl, HCl, MgCl,, and decolorizing by cyclic AMP-dependent protein kinase. o 1991 Academic carbon (Darco G-60) were obtained from J. T. Baker Press, Inc. (Jackson, TN). RPM1 1640 and gentamicin were from GIBCO (Grand Island, NY). Fetal calf serum and heat- inactivated horse serum were from Hazleton (Lenexa, Tyrosine hydroxylase (EC 1.14.16.2) catalyzes the KS). Sucrose was obtained from Bio-Rad (Richmond, rate-limiting step in catecholamine biosynthesis (1). It CA). L-[3,5-3H]Tyrosine was obtained from NEN Re- catalyzes the hydroxylation of tyrosine to form 3,4-di- search Products (Boston, MA). Diethylaminoethyl-cel- hydroxyphenylalanine (Dopa).’ The activity of this en- lulose (DE 52) was obtained from Whatman Biosystems zyme is regulated by many factors, including phosphor- (England) and heparin-Sepharose CL-6B was from ylation (2), salts (3), andpolyanions such as heparin (4). Pharmacia, Inc. (Piscataway, NJ). (6R)-5,6,7,8-Tetra- Studies of purified tyrosine hydroxylase have been hin- hydro-L-biopterin was obtained from Dr. B. Shircks dered by the difficulty in obtaining adequate amounts of Laboratories (Switzerland). purified enzyme. Published purification procedures report a yield of 0.2-2 mg of purified enzyme (5,6). Fur- Tyrosine Hydroxyluse Activity Assay thermore, purified tyrosine hydroxylase is reported to Tyrosine hydroxylase activity was assessed by mea- be unstable and to lose substantial activity in a matter suring the release of 3H,0 in the conversion of L-[3,5- of hours (7-9). The only large-scale purification of tyro- 3H]tyrosine to Dopa by the method of Reinhard et al. sine hydroxylase reported to date is that of the bovine (14). Enzyme (5-15 fig/ml) was incubated at 37°C with adrenal enzyme (lo), in which 17 mg of tyrosine hydrox- 100 PM L-[3,5-3H]tyrosine (0.5 &i per assay), 1 mM ylase was obtained from 2.5 kg of bovine adrenal me- (6R)-5,6,7,8-tetrahydro-L-biopterin, 1500 U/ml cata- dulla. The rat pheochromocytoma PC12 cell line estab- lase, 5 mM dithiothreitol, and 50 mM Pipes buffer (pH lished by Greene and Tischler (11) expresses high levels 6.0) in a total volume of 30 ~1. Samples were incubated for 10 min and the reaction was stopped by the addition of 300 ~17.5% charcoal (Darco G-60) in 1 N HCl. Char- * Abbreviations used: Dopa, 3,4-dihydroxyphenylalanine; Pipes, piperazine-N,N’-bis(2-ethanesulfonic acid); PMSF, phenylmethylsul- coal with adsorbed tyrosine and catechols was sedi- fonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide mented by centrifugation at 13,000g for 2 min, aliquots gel electrophoresis. (100 ~1) of supernatant were removed, and 3H,0 radioac-

10 1046.5928/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. TYROSINEHYDROXYLASE FROM PC12CELLS 11 tivity was determined by liquid scintillation spectrome- at 40,OOOg for 60 min. To the supernatant, solid ammo- try. Samples were assayed in triplicate unless otherwise nium sulfate (30% saturation) was added over 20 min noted. The blank radioactivity (about 300 cpm) from with gentle stirring and the solution was stirred an addi- parallel samples containing water in place of enzyme tional 20 min. After centrifugation at 15,000g for 15 min, was subtracted from the sample values to yield net ac- the supernatant was collected. Additional ammonium tivity. sulfate was added to 42% saturation as described above and the precipitate was collected. The precipitate was then dissolved in buffer A and dialyzed against 100 vol Phosphorylation of Tyrosine Hydroxylase by Cyclic of the buffer overnight with one change of solution. The AMP-Dependent Protein Kinase Catalytic Subunit dialysate was then applied to a DEAE-cellulose column The catalytic subunit of cyclic AMP-dependent pro- (1.5 X 18 cm) equilibrated with Buffer A. The column tein kinase was purified by the method of Hart1 and was washed with 1 column vol of Buffer A, and tyrosine Roskoski (15). For phosphorylation, tyrosine hydroxy- hydroxylase was eluted with a linear gradient of 40-300 lase (250-500 pg/ml) was incubated in the presence of 1 mM NaCl in Buffer A (Fig. 1). The enzyme eluted at pM purified catalytic subunit (40,000 Da), 100 pM ATP, about 100 mM NaCl. The tyrosine hydroxylase-contain- and 1 mM MgCl, in 50 mM Pipes buffer (pH 6.0) at 30°C ing fractions were then pooled, ammonium sulfate was for 20 min. This procedure leads to the phosphorylation added to 44% saturation, and the precipitate was col- and activation of tyrosine hydroxylase (16). Control lected as described above. samples were incubated with MgCl, and buffer only. This precipitate was dissolved in 20 mM potassium The samples were diluted to provide a tyrosine hydroxy- phosphate (pH 7.4), 1 mM dithiothreitol, 5 pg/ml leu- lase concentration of 15 pg/ml during the assay. peptin, and 10 pM PMSF (Buffer B). Following overnight dialysis against 100 vol of Buffer B with one RESULTS change of solution, the dialysate was applied to a heparin-Sepharose column (1 X 18 cm) equilibrated with Purification of Tyrosine Hydroxylase Buffer B. The column was then washed with Buffer B, Vulliet and co-workers (17) reported a procedure for 350 mM Tris-HCl in Buffer B, and Buffer B again. Tyro- the purification of 0.6 mg tyrosine hydroxylase from rat sine hydroxylase was not eluted from the column by pheochromocytoma. We have used a modification of Tris-HCl while other proteins were, so that the Tris- this procedure to purify tyrosine hydroxylase from HCl wash enhanced purification at this step (5). Tyro- PC12 cells. The cells were maintained in RPM1 1640 sine hydroxylase was eluted from the column with a lin- medium with 10% horse serum (heat inactivated), 5% ear gradient of 100-500 mM KC1 in Buffer B (Fig. l), fetal calf serum, and 50 pg/ml gentamicin in Nunc T175 with the enzyme eluting at approximately 250 mM KCl. flasks. Cells were kept at 37°C in a water-saturated, 5% The tyrosine hydroxylase-containing fractions were CO, atmosphere. Greene et al. (18) reported that the then pooled and concentrated by pressure dialysis cells attach poorly unless the culture flasks are colla- (Amicon stirred cell with Diaflo PM10 ultrafilter). For gen-coated. We found, however, that PC12 cells ad- PC12 enzyme preparations, the sample was stored in hered well to the Nunc flasks under these conditions. 0.5-ml aliquots of 0.4-2.5 mg/ml protein at -80°C in 5 Cultures were fed twice weekly by completely exchang- mM Tris-acetate (pH 8.2), 2 mM dithiothreitol, and 10% ing the medium. When confluent, the cells were either (w/v) sucrose until use. The resulting enzyme was stable subcultured or harvested by trituration, pelleted, and under these conditions for more than 2 years. Enzyme frozen at -80°C until use for tyrosine hydroxylase puri- was thawed prior to use and could be stored up to 6 fication. Cells were stored for 1 year with no loss of weeks at 2°C without appreciable loss of activity. There tyrosine hydroxylase activity. It is advisable to harvest was no loss of activity with up to four freeze-thaws, but confluent or near confluent cells for purification, since additional freeze-thaws were avoided. These prepara- cell-cell contact increases tyrosine hydroxylase expres- tions appear to be homogeneous as judged by visualiza- sion in PC12 cells (19). tion on sodium dodecyl sulfate-polyacrylamide gel elec- During the purification, all steps were performed at trophoresis (SDS-PAGE) with Coomassie blue staining 0-4°C. PMSF and leupeptin, protease inhibitors, were (Fig. 2). added to all solutions just before use. PC12 cells from Results from a typical purification cells are summa- 100 T175 flasks (30 ml of partially thawed, packed cells rized in Table 1. Total protein was assessed by the containing 1$2 g of protein, approximately 1 X lOlo method of Bradford (20) with bovine y-globulin as a cells) were homogenized with a polytron in 250 ml of 50 standard. Relative amounts of tyrosine hydroxylase mM potassium phosphate (pH 7.0), 7.5 mM mercap- were determined immunochemically by the method of toethanol, 1 mM EDTA, 5 pg/ml leupeptin, and 10 pM Haycock (21), using affinity-purified polyclonal rabbit phenylmethylsulfonyl fluoride (PMSF) (Buffer A) with antibodies to rat tyrosine hydroxylase and the purified 0.27 M sucrose. The resulting solution was centrifuged enzyme as the standard. We used the immunochemical 12 GAHN AND ROSKOSKI

I i

0 0 10 20 30 40 50 60 0 20 40 60 Fraction Fraction

FIG. 1. Tyrosine hydroxylase elution profiles from (A) DEAE-cellulose and (B) heparin-Sepharose. Tyrosine hydroxylase activity was measured in fractions as described under Materials and Methods, and protein was measured by the method of Bradford (20).

quantification of tyrosine hydroxylase to determine the We purified tyrosine hydroxylase from a radiation- amount of tyrosine hydroxylase in the starting material, induced rat pheochromocytoma by the same procedure which, in the purification shown in Table 1, was 3% of with the following additional manipulations (16). After the total protein in the high-speed supernatant. This frozen tumor (30 g) extract was carried through the hep- quantification indicated that the homospecific activity arin-Sepharose step, tyrosine hydroxylase-containing (activity per milligram tyrosine hydroxylase) decreased fractions were pooled and concentrated by pressure dial- somewhat during the purification but increased in the ysis to about 1 mg protein/ml. The sample was then final step. The basis for this change is unknown. The subjected to 52% saturation ammonium sulfate precipi- yield based on total activity of the dialyzed fractions and tation as described above and resolubilized in 5 mM immunoreactivity agreed within 10%. Tris-acetate (pH 8.2), 2 mM dithiothreitol. The sample This purification procedure yielded 9-25 mg (an aver- (about 25 mg protein) was centrifuged at 100,OOOg in a age of 15 mg from four preparations) of tyrosine hydrox- Beckman ultracentrifuge (SW27 rotor) for 40 h in a 35 ylase from PC12 cells. The recovery was consistently ml linear 5-20% sucrose gradient in 5 mM Tris-acetate 4050%. The variability in the quantity of purified pro- (pH 8.2) and 2 mM dithiothreitol. Tyrosine hydroxy- tein obtained from the PC12 cells was related to vari- lase-containing fractions were pooled and stored at able expression of enzyme (22). -80°C as previously described. This procedure yielded about 5 mg purified protein. The enzyme from these two sources was indistinguishable with the following excep- tion. The enzyme prepared from the transplantable tu- kDa ’ * mor migrated as a doublet in SDS-PAGE (23) while the enzyme prepared from the PC12 cells migrated as a sin- 116+ gle band (Fig. 2). The doublet was identified in SDS extracts of the tumor following Western blot immunochemical localization (not shown). Only one band was observed following SDS extraction of the PC12 cells.

Characterization of Purified Tyrosine Hydroxylase As noted above, the purified PC12 cell tyrosine hydroxylase appeared to be homogeneous, with a subunit molecular weight of approximately 60,000 by SDS- PAGE. Reports of the specific activity of tyrosine hydroxylase vary depending on assay conditions, but the specific activity of this enzyme (0.67 pmol/min/mg at 37°C 1 mM tetrahydrobiopterin, pH 6.0) was similar to that reported by others for purified rat tyrosine hydroxy- FIG. 2. SDS-PAGE of fractions from the purification of tyrosine mM hydroxylase. Equal amounts of total protein (10 rg) from lane 1, the lase (5,12,17,23). The enzyme had a K,,, of 1.8 + 0.5 high-speed supernatant, and lane 2, the final purified protein, were (n = 2) for tetrahydrobiopterin and a V,,, of 860 & 70 subjected to electrophoresis on an 8% acrylamide gel. nmol/min/mg (50 mM Pipes, pH 6,100 pM tyrosine). TYROSINE HYDROXYLASE FROM PC12 CELLS 13

TABLE 1 Purification of Tyrosine Hydroxylase from PC12 Cells

Total Specific Immunoreactive Homospecific Protein activity activity TH activity Yield Step (md (nmol/min) (nmol/min/mg) (md (nmol/min/mg TH) (SD)

High-speed supernatant 645 14,500 22.5 19.7 736 100 Applied to DEAE 218 10,300 47.5 15.7 656 80 Applied to heparin-Sepharose 25.1 6,300 250 10.6 594 54 Purified product 9.17 6,440 664 9.17 702 46

Note. Enzyme activity was assessed in the presence of 100 pM tryosine and 1 mM tetrahydrobiopterin at pH 6.0 as described under Materials and Methods. All samples, which were stored at -8O”C, were dialyzed for 4 h against 10 mM phosphate buffer (pH 6.8) prior to assay to avoid salt effects on activity (3). Specific activity refers to activity per milligram of total protein while homospecific activity refers to activity per milligram of immunoreactive tyrosine hydroxylase. Yield is based upon relative amount of tyrosine hydroxylase immunoreactive material. Similar results were obtained in five other preparations with the yield ranging from 9 to 25 mg.

Tyrosine hydroxylase is phosphorylated and acti- ture is similar, the preparation of PC12 cells was much vated by cyclic AMP-dependent protein kinase in puri- less labor-intensive. Moreover, both of these sources fied rat pheochromocytoma tyrosine hydroxylase (17) were less costly than the 360 rats required for preparing and in intact PC12 cells (24). Our purified tyrosine hy- 200 pg from rat brain (5). Haavik and co-workers (10) droxylase was phosphorylated by the kinase, with a stoi- have purified bovine adrenal tyrosine hydroxylase with chiometry of 0.7 mol phosphate/m01 subunit and acti- a yield of 17 mg enzyme from 2.5 kg of adrenal medulla vated about 20-fold (at pH 7.2 and 125 PM tetrahydro- (this corresponds to about 500 bovine adrenal glands). biopterin). As observed with tyrosine hydroxylase The procedure of Haavik et al. takes 6 days to complete, purified from rat pheochromocytoma (16), the stoichi- as compared to the 3-day procedure reported here. ometry varied from preparation to preparation presum- Another major problem frequently encountered with ably due to variation in the endogenous phosphate con- tyrosine hydroxylase is the extreme lability of the puri- tent in the different preparations of the enzyme. fied enzyme. For example, Togari and co-workers (8) purified tyrosine hydroxylase from rat adrenal and rat DISCUSSION striatum, but they reported that the enzyme lost 50% of We developed a procedure for the purification of rat its activity in 5 h at 4°C. Similar problems were reported tyrosine hydroxylase from cultured PC12 cells which for purified bovine and human tyrosine hydroxylase yielded 9-25 mg of purified protein from 30 ml of packed (7,9). The enzyme purified by our procedure, from ei- cells. Previously reported purifications of rat tyrosine ther tumor or PC12 cells, is quite stable and can be hydroxylase result in less enzyme, typically 0.2 mg from stored at -80°C for over 2 years with no loss of activity. 360 rat brains (5) to 2 mg from 20 g of rat pheochromo- Richtand et al. (5) emphasized the importance of in- cytoma (6). Our greater yield compared to that from cluding protease inhibitors to increase the stability of other PC12 cell preparations (12,13) is probably due to rat brain tyrosine hydroxylase during purification and two factors. First, our PC12 cell cultures express a high storage. We point out, furthermore, that tyrosine hy- level of tyrosine hydroxylase, typically 35% of the total droxylase is unstable during storage at pH 6.0 and is cellular protein. Second, the high purification afforded much more stable at higher pH. We store our enzyme at by the heparin-Sepharose chromatography (with the pH 8.2 as noted previously. Storage at 2°C overnight at Tris wash) allowed us to minimize purification steps pH 6.0 leads to a complete loss of activity, while at pH and maximize yield. The transplantable tumor used in 8.2 the enzyme can be stored for up to 6 weeks at 2°C our procedure yielded 5 mg of tyrosine hydroxylase with little or no decrease in activity. from 30 g of tumor (30 rats). Use of the transplantable ACKNOWLEDGMENTS rat pheochromocytoma necessitates maintenance of an The authors thank Ms. Josephine Roussell for maintenance of the inbred New England Deaconess rat colony for tumor PC12 cells, Mr. Jeffrey Kubinek for purification of the catalytic sub- transplantation and growth. We found maintenance of unit of cyclic AMP-dependent protein kinase, Dr. Harvey Wilgus for the colony to be costly, time-consuming, and labor-in- rat pheochromocytoma samples, and Dr. John W. Haycock for pro- tensive. Purification from PC12 cells required approxi- viding reagents for the tyrosine hydroxylase immunoassay. This work was supported by USPHS Grant NS 15994. mately 100 T175 (cm2) flasks of PC12 cells and yielded greater quantities of the enzyme. Our procedure can be REFERENCES scaled up or down readily. Although the cost of mate- 1. Levitt, M., Spector, S., Sjoerdsma, A., and Udenfriend, S. (1965) rials for the transplantable tumor and PC12 cells in cul- Elucidation of the rate-limiting step in norepinephrine biosynthe- 14 GAHN AND ROSKOSKI

sis in the perfused guinea pig heart. J. Pharmacol. Exp. Ther. 13. Kuhn, D. M., and Billingsley, M. L. (1987) Tyrosine hydroxylase: 14&l-8. Purification from PC12 cells, characterization and production of 2. Zigmond, R. E., Schwarzchild, M. A., and Rittenhouse, A. R. antibodies. Neurochem. Int. 11, 463-475. (1989) Acute regulation of tyrosine hydroxylase by nerve activity 14. Reinhard, J. F., Jr., Smith, G. K., and Nichol, C. A. (1986) A rapid and by neurotransmitters via phosphorylation. Annu. Reu. Neu- and sensitive assay for tyrosine-3-monooxygenase based upon rosci. 12, 415-461. the release of ‘H,O and adsorption of [3H]tyrosine by charcoal. 3. Katz, I. R., Yamauchi, T., and Kaufman, S. (1976) Activation of Life Sci. 39,2185-2189. tyrosine hydroxylase by polyanions and salts: An electrostatic 15. Hartl, T., and Roskoski, R., Jr. (1982) Adenosine 3’,5’-cyclic mono- effect. Biochim. Biophys. Acta 429,84-85. phosphate protein kinase from bovine brain: Inactivation of the catalytic subunit and holoenzyme by 7-chloro-4-nitro-2,1,3-ben- 4. Kuczenski, R. T., and Mandell, A. J. (1972) Allosteric activation zoxadiazole. Biochemistry 21,5175-5183. of hypothalamic tyrosine hydroxylase by ions and sulphated mucopolysaccharides. J. Neurochem. 19, 131-137. 16. Roskoski, R., Jr., Vulliet, P. R., andGlass, D. B. (1987) Phosphor- ylation of tyrosine hydroxylase by cyclic GMP-dependent protein 5. Richtand, N. M., Inagami, T., Misono, K., and Kuczenski, R. kinase. J. Neurochem. 48,840-845. (1985) Purification ahd characterization of rat striatal tyrosine hydroxylase. J. Biol. Chem. 260, 8465-8473. 17. Vulliet, P. R., Langan, T., and Weiner, N. (1980) Tyrosine hydroxylase: A substrate of cyclic AMP-dependent protein kinase. Proc. 6. Tank, A. W., and Weiner, N. (1987) Tyrosine 3-monooxygenase N&l. Acad. Sci. USA 77,92-96. from rat pheochromocytoma, in “Methods in Enzymology” 18. Greene, L. A., Aletta, J. M., Rukenstein, A., and Green, S. H. (Kaufman, S., Ed.), Vol. 142, pp. 71-82, Academic Press, San (1987) PC12 pheochromocytoma cells: Culture, nerve growth fac- Diego. tor treatment, and experimental exploitation, in “Methods in En- 7. Lazar, M. A., Truscott, R. J. W., Raese, J. D., and Barchas, J. D. zymology” (Barnes, D., and Sirbasku, D. A., Eds.), Vol. 147, pp. (1981) Dephosphorylation of purified brain tyrosine hydroxylase 207-216, Academic Press, San Diego. by rat striatal extracts. J. Neurochem. 36,677-682. 19. Saadat, S., and Thoenen, H. (1988) Regulation of tyrosine hydrox- 8. Togari, A., Kano, H., Oka, K., and Nagatsu, T. (1983) Simulta- ylase mRNA levels in rat pheochromocytoma PC12 cells by cell- neous simple purification of tyrosine hydroxylase and dihydrop- cell contact. Exp. Cell Res. 176, 187-193. teridine reductase. Anal. Biochem. 132, 183-189. 20. Bradford, M. M. (1976) Rapid and sensitive method for quantita- 9. Kojima, K., Mogi, M., Oka, K., and Nagatsu, T. (1984) Purifica- tion of microgram quantities of protein utilizing the principle of tion and immunochemical characterization of human adrenal ty- protein-dye binding. Nature 72, 248-254. rosine hydroxylase. Neurochem. Int. 6,475-480. 21. Haycock, J. W. (1989) Quantitation of tyrosine hydroxylase pro- 10. Haavik, J., Andersson, K. K., Petersson, L., and Flatmark, T. tein levels: Spot immunolabeling with an affinity-purified anti- (1988) Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) body. Anal. Biochem. 181, 259-266. from bovine adrenal medulla: Large scale purification and physi- 22. Lucas, C. A., Edgar, D., and Thoenen, H. (1979) Regulation of cochemical properties. Biochim. Biophys. Acta 963,142-156. tyrosine hydroxylase and choline acetyltransferase activities by 11. Greene, L. A., andTischler, A. S. (1976) Establishment of anorad- cell density in the PC12 rat pheochromocytoma clonal cell line. renergic clonal line of rat adrenal pheochromocytoma cells which Exp. Cell Res. 121, 79-86. respond to nerve growth factor. Proc. Natl. Acad. Ski. USA 73, 23. Wilgus, H., and Roskoski, R., Jr. (1988) Inactivation of tyrosine 2424-2428. hydroxylase activity by ascorbate in vitro and in rat PC12 cells. J. 12. Markey, K. A., Kondo, S., Shenkman, L., and Goldstein, M. Neurochem. 51, 1232-1239. (1980) Purification and characterization of tyrosine hydroxylase 24. Roskoski, R., Jr., and Roskoski, L. M. (1987) Activation of tyro- from a clonal pheochromocytoma cell line. Mol. Phurmacol. 17, sine hydroxylase in PC12 cells by the cyclic GMP and cyclic AMP 79-85. second messenger systems. J. iVeurochem. 48,236-242.