[29]
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Viuo
[29] P 4 5 0 P h o s p h o r y l a t i o n
in Isolated Hepatocytes
305
and
in Vivo
By JOHN A. KOCH and DAVID J. WAXMAN Introduction Many P450 enzymes are regulated at the m R N A level, in response to changes in circulating levels of drugs and hormones. Foreign compounds such as phenobarbital and 3-methylcholanthrene can elicit large increases in select P450 activities by transcriptional activation of individual P450 genes.~ In addition to these well-studied examples of pretranslational regulation, phosphorylation of P450 has emerged as a potentially significant posttranslational regulatory event. Studies in vitro have demonstrated that a n u m b e r of hepatic P450s can be phosphorylated by purified protein kinases.Z Support for the o c c u r r e n c e o f P450 phosphorylation under physiological conditions is provided by studies of P450 phosphorylation in isolated rat hepatocytes 3-5 and in vivo. 3 Several reports point to the potential importance of these phosphorylation reactions for the control of P450 activity, and perhaps also the regulation of P450 degradation. 3,4,6-9 This chapter describes both in vitro and in v i v o experimental protocols that have been developed for studies of P450 phosphorylation.
I F. J. Gonzalez, Pharmacol. Rev. 40, 243 (1988). 2 W. Pyerin, H. Taniguchi, F. Horn, F. Oesch, Z. Amelizad, and T. Friedberg, Biochem. Biophys. Res. Commun. 142, 885 (1987). 3j. A. Koch and D. J. Waxman, Biochemistry 28, 3145 (1989). 4 W. Pyerin and H. Taniguchi, EMBO J. 8, 3003 (1989). 5 B. Bartlornowicz, D. J. Waxman, D. Utesch, F. Oesch, and T. Friedberg, Carcinogenesis 10, 225 (1989). 6 E. Eliasson, I. Johansson, and M. Ingelman-Sundberg,Proc. Natl. Acad. Sci. U.S.A. 87, 3225 (1990). 7 W. Pyerin, H. Taniguchi, A. Stier, F. Oesch, and C. R. Wolf, Biochem. Biophys. Res. Commun. 122, 620 (1984). 8 I. Jansson, P. M. Epstein, S. Bains, and J. B. Schenkman, Arch. Biochem. Biophys. 259, 441 (1987). 9 B. Bartlomowicz, T. Friedberg, D. Utesch, E. Molitor, K. Platt, and F. Oesch, Biochem. Biophys. Res, Commun. 160, 46 (1989).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, lnc. All rights of reproduction in any form reserved.
306
REGULATORYPARADIGMS
[29]
Phosphorylation of Hepatic Cytochromes P450
P450 Phosphorylation in Isolated Hepatocytes Hepatocyte Isolation. Hepatocytes are isolated from untreated rats or from rats induced with drugs such as phenobarbital (four daily injections of phenobarbital at 80 mg/kg, intraperitoneally) by perfusion of liver tissue with buffers containing collagenase using the nonrecirculating system described by Seglen.a° Rats are anesthetized with ether for 3 min and prepared for hepatocyte isolation. A midsagittal incision is made from the pelvic region to the xiphoid process and lateral incisions from the base of the rib cage to expose the peritoneal cavity. The hepatic portal vein is located and connective tissue surrounding the vessel removed. The vein is clamped distal to the proposed site of eannulation to prevent significant blood flow through the vein during subsequent procedures. The vein is opened and a cannula inserted (0.020-gauge Tygon tubing), which is held in place with two lengths of suture. The cannula is then connected to a peristaltic pump and the liver perfused with calcium-free bicarbonate buffer (142 mM NaC1, 4.37 mM KC1, 1.24 mM KH2PO 4, 24 mM NaHCO3, 0.62 mM MgSO4, 0.62 mM MgCI2, bubbled with 95% 02/5% CO2 and maintained at 37°), at a flow rate of 25-30 ml/min. The liver should soon swell, at which time the inferior vena cava is severed, leading to rapid blanching of the liver. After 20 min of perfusion, the liver is perfused for an additional 20 rain with membrane-filtered (0.45/zm) bicarbonate buffer, as above, but containing 5.0 mM CaCI2 and 0.05% coUagenase (Clostridium histolyticum, type IV; Sigma, St. Louis, MO). The liver is then removed to a beaker containing Krebs-Ringer bicarbonate medium (119 mM NaC1, 4.7 mM KCI, 24 mM NaHCO3, 2.4 mM MgSO4, 2.3 mM CaC12), taking care to avoid rupture of the capsule. Hepatocytes are released by disruption of the capsule with a pair of scissors and gentle shaking. Large fragments of hepatic connective tissue are removed by filtration through nylon mesh (297/zm, Spectrum Medical Industries, Los Angeles, CA). The released cells are washed twice with Krebs-Ringer bicarbonate medium by centrifugation at 70 g, then resuspended in Leibovitz L-15 medium (GIBCO, Grand Island, NY) to a density of 2 x 106 cells/ml. Typical yield is 2-4 x 108 hepatocytes consisting of greater than 90% viable cells (as judged by trypan blue exclusion). Incubation with Ortho[3ZP]phosphate. Ortho[3Ep]phosphate is added to the cell suspension to give a concentration of 0.5-I mCi/ml, and the cells 10 p. O. Seglen, Exp. Cell. Res. 82, 391 (1973).
[29]
P450 PHOSPHORYLATION IN HEPATOCYTE$ AND in Vivo
307
are then incubated at 37° for 40 min to equilibrate the radiolabel with cellular ATP pools. Hormones are added to stimulate phosphorylation by one or more protein kinases (see Comments, below) and incubation is continued for an additional 10 rain. Hepatocytes are then collected by centrifugation, resuspended in homogenization buffer (120 mM Tris-HCl, pH 7.4, 100 mM KC1, 1 mM EDTA, 50 mM NaF, 5/.~M sodium orthovanadate, 500 txM molybdic acid, and 6 mM Na2ATP) and frozen in a dry ice/ ethanol bath. The hepatocytes are thawed to room temperature, homogenized with a Potter-Elvehjem homogenizer, and centrifuged for 5 min at 6000 g, then 15 min at 12,500 g. The supernatant is centrifuged at 105,000 g for 1 hr, and the resulting microsomal pellet is resuspended in microsome storage buffer (100 mM KPi, pH 7.4, 20% glycerol, 1 mM EDTA, 5 mM NaF, 50 IzM sodium orthovanadate, 500 t~M molybdic acid) by brief homogenization. Comments. L-15 medium is recommended for these short-term hepatocyte incubations, because the cells can be kept in air at 37°, without the need for a tissue culture incubator. Other media that can support hepatocyte phosphorylation, including Dulbecco's modified minimal essential medium, Krebs-Henseleit medium, and low phosphate (0.1 mM) Krebs-Ringer bicarbonate medium, 11 must be maintained in an atmosphere of 95% O1/5% COL. The choice of media might conceivably influence the P450 form specificity or hormone responsiveness of P450 phosphorylation, but this has not been examined in a systematic fashion. When using Krebs-Ringer medium, equilibration of ortho[32p]phosphate with cellular ATP pools occurs after 40 min of incubation, lz at which time hormones or other protein kinase activators are added. Many hormones rapidly stimulate phorphorylation, often within 3-10 min. These include glucagon (0.1 tzM), norepinephrine (10/~M), and angiotensin II (10 riM). ~3 Phosphatase inhibitors (NaF, sodium orthovanadate, and molybdic acid) are included in the homogenization and microsome storage buffers to inhibit enzymatic dephosphorylation during microsome isolation, whereas unlabeled ATP is added to block further incorporation of radioactivity. Control experiments should be carried out to determine the stability of the P450 under study (e.g., assessed by Western blotting analysis), as some P450s are readily lost in the early stages of hepatocyte culture. 14 P450 u W. W. Umbreit, R. H. Burris, and J. F. Stauffer, in "Manometric Techniques," 4th ed., p. 132. Burgess, Minneapolis, Minnesota, in press. 12 K. Yamada, K. E. Lipson, M. W. Marino, and D. B. Donner, Biochemistry 26, 715 (1987). t3 j. C. Garrison, M. K. Borland, V. A. Florio, and D. W. Twible, J. Biol. Chem. 254, 7147 (1979). 14 A. R. Steward, S. A. Wrighton, D. S. Pasco, J. B. Fagan, D. Li, and P. S. Guzelian, Arch. Biochem. Biophys. 241, 494 (1985).
308
REGULATORYPARADIGMS
[29]
inducers or ligands (e.g., 0.6 mM phenobarbital or 0.5 mM metyrapone) can be included in the isolation and incubation media to stabilize P450.15,16 Stimulation ofhepatocyte phosphorylation catalyzed by cAMP-dependent protein kinase is achieved using the membrane-permeable, cAMP analog dibutyryl-cAMP (But2cAMP). Concentrations as low as 5 /~M But2cAMP can markedly stimulate phosphorylation of P450IIB 1 when added to isolated hepatocytes as described above) A combination of But2cAMP with 8-thiomethyl-cAMP, which act synergistically on cAMPdependent protein kinase, 17can also be used to stimulate P450IIB1 phosphorylation in rat hepatocytes. 5 Control incubations using cAMP, which does not readily cross membranes, should be performed to ensure that P450 phosphorylation is occurring in intact cells, and not during the course of microsome isolation. The integrity of hepatocyte membranes may be assessed by incubation with glucagon, which interacts with membrane receptors to elevate intracellular levels of cAMP. Although cellular studies on P450 phosphorylation have thus far used only isolated hepatocytes, other cellular systems, such as primary hepatocytes cultured under conditions where they retain liver-specific functions (e.g., phenobarbital induction of P450IIB 1)18,19or vaccinia virus-infected Hep G2 cells that selectively express a single P450 form, 2° may be useful if carrying out longer term experiments on P450 phosphorylation. In Vivo Phosphorylation o f C y t o c h r o m e P450 c A M P Stimulation o f P450 Phosphorylation in Vivo. Uninduced or drug-induced rats are transferred to disposable cages where they are given a total of three intraperitoneal injections of But2cAMP and theophylline (2 nag each compound/100 g body weight/injection and suspended in 0.5 ml of 0.9% NaC1) at 90-min intervals. A single intraperitoneal injection of ortho[32p]phosphate (6 mCi/100 g body weight) is administered to the rats 2 hr prior to sacrifice. Livers are removed and minced in homogenization buffer (see above), and microsomes then prepared as already described for isolated hepatocytes. Microsomes obtained in this manner may have specific activities ranging from I000 to 3000 counts per minute (cpm) 32p//zg protein.
15E. T. Canepa, E. B. C. Llambias,and M. Grinstein,Biochim. Biophys. Acta 841, 186 (1985). 1~A. J. Paine, Chem.-Biol. Interact. 74, 1 (1990). 17S. J. Beebe,R. Holloway,S. R. Rannels,and J. D. Corbin,J. Biol. Chem. 259, 3539(1984). is E. G. Schuetz,D. Li, C. J. Omiecinski,U. Muller-Eberhard,and H. K. Kleinman,J. Cell. Physiol. 134, 309 (1988). 19D. J. Waxman,J. J. Morrissey,S. Naik, and H. O. Jauregui,Biochem. J. 271, 113 (1990). 2oF. J. Gonzalez, T. Aoyama,and H. V. Gelboin, this volume [8].
[29]
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Vivo
309
In order to facilitate electrophoretic analysis of in vivo phosphorylated hepatic P450s, partial P450 purification by cholate solubilization, followed by 8-16% polyethylene glycol 8000 precipitation 21 may be carried out in buffers containing phosphatase inhibitors (see above). The crude P450 fraction precipitating between 8 and 16% polyethylene glycol can be analyzed for specific phosphorylated microsomal proteins by immuneprecipitation or by two-dimensional gel electrophoresis as described below. Comments. Moderate to high levels of ortho[32p]phosphate are required in order to phosphorylate hepatic P450 to a specific activity that is high enough for detection by autoradiography. Dissection of rats and removal of livers is best carried out in plastic biological containment bags to minimize contamination with radiolabeled blood and other body fluids. Theophylline (or other methylxanthines) can be included in the protocol to inhibit phosphodiesterase activity in vivo. 22 The protocol provided above appears to be effective for stimulating phosphorylation of P450IIB 1 and P450IIB2 in vivo. 3 However, other hormone/drug combinations or time courses may be required for optimal phosphorylation in vivo of other hepatic P450 forms (e.g., P450 forms IIC6 and IIIA1). 3 In Vitro Phosphorylation o f Purified Cytochrome P450 General Strategy. Several purified hepatic P450s can be phosphorylated in vitro in reactions catalyzed by purified protein kinases. These include cAMP-dependent protein kinase and protein kinase C. 2,23Purified P450s are incubated with purified kinases in the presence of [y-32p]ATP, and the incorporation of phosphate is then monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Filter-binding assays can be used to quantitate, on a molar basis, the extent of phosphate incorporation. The effects of phosphorylation on P450 activity and protein chemical analysis of the site ofphosphorylation can also be examined. P450 Phosphorylation by cAMP-Dependent Protein Kinase. Bovine heart cAMP-dependent protein kinase, catalytic subunit (10 units; Sigma) is dissolved in 14/zl of an aqueous solution of dithiothreitol (50 mg/ml) and incubated on ice for 10 min. Purified P450 (75 pmol), magnesium acetate, pH 6.5 (15 mM final concentration), and [7-32p]ATP (1-3/xCi; 100 /zM ATP, final concentration) are added to give a total volume of 25/zl. Samples are then incubated at 37° for times up to 90 min. Samples are
21S. B. West, M.-T. Huang, G. T. Miwa, and A. Y. H. Lu, Arch. Biochem. Biophys. 193, 42 (1979). 22j. N. Wells and J. R. Miller, this series, Vol. 159, p. 489. 2t R. Muller, W. E. Schmidt, and A. Stier, FEBS Lett. 187, 21 (1985).
310
REGULATORYPARADIGMS
[29]
analyzed for 32p incorporation by SDS-PAGE followed by autoradiograincorporation is quantitated by precipitation with 10% trichloroacetic acid followed by trapping on glass fiber filters, s The effects of phosphorylation on P450 activity may be assessed after reconstitution of the cytochrome with purified NADPH-cytochrome-P450 reductase using any one of several P450 substrates (e.g., 7-ethoxycoumarin24) and any changes in P450 activity then correlated to phosphate incorporation on a molar basis. Comments. The efficiencyand P450 form specificity ofphosphorylation can vary widely among different protein kinases 2 and may also be influenced by the presence of P450 substrates and heme-binding ligands, or by the presence of interacting electron-transfer components such as cytochrome b5.6.s When attempting to relate changes in P450 activity or P450 stability to associated changes in P450 phosphorylation, it is essential that the stoichiometry of phosphate incorporation be monitored, but in general this has not been done. 6'9 p h y . 32p
Methods for Analysis of Phosphorylated P450s Immunoprecipitation Analysis Method. Microsomal protein (200/zg) prepared from 32p-labeled isolated hepatocytes or from in vivo labeling experiments are diluted in 1.5ml microcentrifuge tubes with 50 mM Tris-HC1, pH 7.8, 1 mM MgC12, 10 mM dithiothreitol to give 2 mg protein/ml. Samples are adjusted to I% SDS, boiled for 3 min, cooled to room temperature, and then diluted with 3 volumes of buffer B (50 mM Tris-HCl, pH 7.5, 190 mM NaC1, 6 mM EDTA, and 2.5% Triton X-100). Rabbit anti-P450 antiserum (10/xl) is then added, and the samples are incubated at 4° overnight. Protein A-bearing Staphylococcus aureus cells (40/xl of a 10% suspension; Sigma P-7155) are added, and the samples are then incubated at room temperature for 60 min with continuous mixing. The S. aureus cells are collected by brief centrifugation (30 sec) and washed 5 times (1 ml/wash) with buffer B containing 0.25% SDS. The final cell pellets are resuspended in 75/zl of SDS-PAGE sample buffer (62 mM Tris-HCl, 1% SDS, 0.001% bromphenol blue, 10% glycerol, 5% 2-mercaptoethanol), heated to 100° for 3 min, and electrophoresed on SDS-polyacrylamide gels (7.5% gel) according to Laemmli. 25Gels are stained, destained, and dried onto Whatman (Clifton, NJ) 3MM filter paper. Acrylaid (12.4% gel; FMC Corp., Princeton, NJ), 24R. A. Prough, M. D. Burke, and R. T. Mayer, this series, Vol. 52, p. 372. 25 U. K. Laemmli, Nature (London) 227, 680 (1970).
[29]
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Vivo
311
which can be covalently bonded onto Gel Bond PAG film (FMC Corp., Princeton, N J), can be used in place of polyacrylamide. After destaining, the PAG film with the attached gel is soaked in 3% glycerol to prevent cracking, then oven dried at 70° for 4-8 hr. Gels are autoradiographed at - 7 0 ° for 2-5 days using Cronex (Du Pont-NEN, Boston, MA), intensifying screens and Kodak (Rochester, NY) XAR-5 film. Comments. The concentration of SDS used in the immuneprecipitation step may be critical. SDS concentrations lower than 0.25% can lead to significant nonspecific contamination of the immunoprecipitates with other phosphorylated microsomal proteins, whereas greater than 0.5% SDS may disrupt the antigen-antibody complexes and give a low yield of immunoprecipitate. Monoclonal anti-P450 antibodies may be used for immuneprecipitation. However, pilot experiments should be conducted to determine the binding efficiency of a given monoclonal antibody to S. aureus cells, since many mouse monoclonals do not have a high affinity for S. aureus protein A. The use of Acrylaid allows one to dry relatively thick gels (1.5 mm) in a standard oven with little risk of cracking. Two-Dimensional Gel Electrophoresis of Microsomal Protein General Strategy. Phosphorylated liver microsomes and purified P450s are analyzed by two-dimensional gel electrophoresis, which is useful for monitoring charge changes induced by phosphorylation. Peptides of interest can be detected by a combination of Coomassie blue staining, Western blotting using P450 form-specific antibodies, and autoradiography. These methods, when applied to both kinased and phosphatased P450 samples, can be useful in identifying endogenously phosphorylated P450 polypeptides, which can subsequently be monitored without the need to employ 32p tracers. Method. Liver microsomes (100-200 p,g) or purified P450s (30-60 pmol) are analyzed by two-dimensional (2D) gel electrophoresis. 26 All procedures are carried out at 37° to prevent crystallization of urea from the gel solutions. Isoelectric focusing gels contain 10 M urea (UltraPure, Bethesda Research Laboratories, Gaithersburg, MD), 4% gel (acrylamide/ bisacrylamide, 17.5 : 1), 2% Nonidet P-40 (NP-40) detergent, 1.5% ampholytes pH 3-10, 0.5% ampholytes pH 6-8 (Sigma), 0.01% ammonium persulfate, and 0.05% Temed. Isoelectric focusing gels (14.5 cm long) are polymerized in siliconized glass tubes (16 cm long, 3 mm i.d.) sealed at the bottom with 1-cm Parafilm plugs and overlaid with 20/xl of 8 M urea. Gels are prepared for preelectrophoresis by adding 40 /zl of 2-D gel sample
26G. P. Vlasuk and F. G. Walz, Jr., Anal. Biochem. 105, 112 (1980).
312
REGULATORYPARADIGMS
[29]
buffer (3% NP-40, 10 M urea, 1.5% ampholytes pH 3-10, 0.5% ampholytes pH 6-8, and 5% 2-mercaptoethanol; titrated to pH 4.5 with 3 N HCI) to the anodic end of the gels. The bottom space created by the Parafilm plug is filled with bottom gel buffer (3% NP-40, 10 M urea, 1.5% ampholytes pH 3-10, 0.5% ampholytes pH 6-8, and 5% 2-mercaptoethanol) held in place with a section of dialysis membrane secured with cross sections of Tygon tubing. Gels are prefocused (cathode solution, 0.02 N NaOH; anode solution, 0.0057 N H3PO4) at constant voltages of 200, 300, and 400 V for 15 min and two 30-min intervals, respectively. Protein samples are solubilized by the addition of 2 volumes of 2-D gel sample buffer followed by a few crystals of solid urea. Solubilized microsomal protein is applied to the anodic end of each gel in a total volume of 75/zl, then overlaid with 10 ttl of sample overlay buffer (2% NP-40, 0.75% ampholytes pH 3-10, 0.25% ampholytes pH 6-8, 8 M urea, and 5% 2-mercaptoethanol; titrated to pH 4.3 with 3 N HCI). Electrophoresis is carried out at 400 V (constant voltage) for 12 hr. Gels are removed to test tubes containing 5 ml of gel equilibration buffer (10% glycerol, 2.3% SDS, 62.5 mM Tris-HC1, pH 6.8, and 5% 2-mercaptoethanol) and gently shaken for 15 min. The gels can be stored at - 70° for several days or used directly for electrophoresis in the second dimension. Isoelectric focusing tube gels are thawed, washed briefly with doubly distilled water, and then applied to the top of 7.5% polyacrylamide (3% stacking) SDS gel. The tube is sealed in place with 1% agarose dissolved in gel equilibration buffer. The SDS gels are electrophoresed, 25 then stained with Coomassie blue R-250, dried onto Whatman 3MM filter paper, and exposed to X-ray film at - 8 0 °. Alternatively, gels may be analyzed immunochemicallyfor specific P450 forms and their phosphorylated derivatives by Western blotting using standard methods. 27 Comments. Thorough solubilization of samples with detergent and urea is important for the resolution of microsomal P450s and their phosphorylated derivatives in isoelectric focusing gels. Incomplete solubilization will impede the entry of P450 proteins into the isoelectric focusing gel and may contribute to streaking. Solubilization is facilitated by the addition of a few crystals of solid urea, until the point where the sample is supersaturated. Excessive vortexing should be avoided since the frictional heat may lead to dissolution of excessive amounts of urea which can recrystallize and interfere with the loading of the isoelectric focusing gel. Ultrapure urea is recommended to minimize carbamoylation of protein amino groups and the resultant introduction of charge heterogeneity. Removal of gels is
27
D. J. Waxman, this volume [24].
[29]
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Vivo
313
facilitated by applying air pressure to one end of the glass tubes with a Pasteur pipette bulb. Phosphorylation of P450 is indicated by an acidic shift in the mobility of the P450 in the isoelectric focusing dimension that is associated with 32p incorporation. In the case of some hepatic P450s, however, charge heterogeneity apparently unrelated to phosphorylation may be present, and this can complicate the analysis. Thus, rat hepatic P450IIB1 and P450IIB2 are each comprised of one major and one minor (more acidic) charge variant that appears to be nonphosphorylated. In addition, a second, even more acidic polypeptide that corresponds to an endogenously phosphorylated derivative that comprises approximately 15% of total microsomal P450IIB1 plus P450IIB2 is present)
Phosphoamino Acid Analysis and Tryptic Peptide Mapping of 32p-Labeled P450 General Strategy. Immunoprecipitated 32p-labeled P450s derived from hepatocytes in oivo or purified P450 phosphorylation reactions are analyzed to identify the amino acid that is phosphorylated. Peptide mapping is performed to ascertain whether there are multiple phosphorylation sites, and whether the P450 phosphorylation site recognized by a purified protein kinase is the same as that which is phosphorylated in whole cells. Phosphoamino Acid Analysis. 32p-Labeled liver microsomes or purified P450s are immunoprecipitated then electrophoresed on 0.75-mm thick SDS gels using the methods described above. Gels are fixed in 10% acetic acid/45% ethanol for 60 min, then incubated at 20-22 ° for 24 hr with two changes of 10% acetic acid/15% ethanol (200 ml per wash). Gels are then washed 3 times in 200 ml of doubly distilled water (30 min/wash) and dried onto Whatman 3MM filter paper, which is marked with radiolabeled ink then autoradiographed. Radiolabeled P450s are located by aligning the autoradiograph with the dried gals. The P450-containing bands are then excised with the adhering filter paper using a scalpel blade. The 32p-labeled dried gel slice is incubated with L-l-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (50 /~g/ml; Worthington Biochemical Corp., Freehold, N J) in 0.4 ml of 50 mM ammonium bicarbonate buffer, pH 8, for 5 hr at 37°. An additional 0.2-ml aliquot of TPCK trypsin (50/zg/ml) is then added and the incubation continued an additional 19 hr. Particulate matter is removed in a microcentrifuge, and the supernatant is then lyophilized repeatedly to remove residual ammonium bicarbonate. Samples are resuspended in 6/.d of 0.033% 2-mercaptoethanol, and a 2-/~1 aliquot is then removed for tryptic peptide analysis as described below. The remaining material is lyophilized, resuspended in 0.1 ml of doubly
314
REGULATORYPARADIGMS
[29l
distilled water then relyophilized. HCI (5.7 N; Pierce Chemical Co., Rockford, IL) (0.1 ml) is then added, and the sample is heated in a sealed Eppendorf tube at 100° under N2 for 90 rain to effect partial acid hydrolysis. The samples are lyophilized repeatedly to remove residual HCI, then dissolved in double distilled water (10/xl/sample) containing 8/zg each of phosphoserine, phosphothreonine, and phosphotyrosine standards (Sigma) and a 1:250 dilution of dye mix (0.3% each of xylene cyanole FF, Orange G, and Acid fushin; Eastman-Kodak, Rochester, NY) to standardize migration distances. Aliquots (5/xl) are spotted onto 20 x 20 cm thin-layer cellulose chromatography plates (Eastman-Kodak 13255) which are then carefully moistened with pH 3.5 electrophoresis buffer (pyridine/acetic acid/water, 5 : 50 : 945). Plates are electrophoresed for 75 min at 800 V in a Shandon, Inc. (Sewickley, PA) or equivalent thin-layer electrophoresis apparatus. Phosphorylated amino acids and dye markers migrate toward the cathode. 32p-Labeled phosphoamino acids and partial hydrolysis products and released phosphate are detected by autoradiography (typical ratio of 32plabel in these fractions 1 : 1 : 1). Migration distances (relative to free orthophosphate) of 0.28, 0.52, 0.61, and 0.72 are observed for partial peptide hydrolysis products and for phosphotyrosine, phosphothreonine, and phosphoserine, respectively. Phosphorylated amino acid standards are visualized by treatment with 0.25% ninhydrin dissolved in acetone followed by heating at I00° for 3 min. Tryptic Mapping of Phosphopeptides. Samples for tryptic peptide analysis, prepared as described in the previous section, are transferred to drawn-out 100-/~1capillary tubes and then applied to 10 x 10 cm cellulosecoated thin-layer plates (EM Science, Cherry Hill, NJ, #5577) on areas previously spotted with 0.5-1.0/~1 dye mix to give spots about 4 mm in diameter. Electrophoresis is performed with pH 1.9 buffer (formic acid/ acetic acid/water, 1 : 3 : 16) in a Shandon thin-layer electrophoresis apparatus for 20 min at I000 V. Tryptic peptides migrate toward the anode while the dye markers migrate toward the cathode. Plates are air dried for 60 min then chromatographed in a second dimension at 200-22 ° by development with n-butanol/pyridine/acetic acid/water (6.5 : 5 : 1 : 4). Plates are air dried and autoradiographed. Comments. Immunoprecipitated P450s that can be detected by autoradiography within 12-24 hr will generally provide sufficient material for phosphoamino acid analysis and tryptic peptide mapping. Excessive spreading of samples should be avoided while wetting the thin-layer plates with buffer. Sample diffusion can be minimized by coveting the area of sample application with a cap from a screw-top Eppendorf tube and applying the buffer evenly around the samples using a compressed air sprayer (Chromist Spray Unit; Gelman Instrument Co., Ann Arbor, MI).
[30]
CYTOCHROMEP450 TURNOVER
315
Buffer will enter the sites of application by capillary action and focus the samples. As an alternative to thin-layer electrophoresis, thin-layer chromatography methods can be applied to resolve and identify phosphoamino acids. 28 Trypsin digestion of P450 should be carried out under two or three conditions (e.g., varying the protease-to-P450 ratio) to ensure complete digestion and thereby avoid misinterpretation of the tryptic maps. Peptide maps of samples prepared in parallel may be compared to ascertain whether the site on the P450 that is phosphorylated in vitro is the same as that which is phosphorylated in intact hepatocytes. As an alternative to thin-layer electrophoresis, phosphorylated P450 peptides may be resolved by high-performance liquid chromatography (HPLC) using standard methods. 29 Acid hydrolysis of tryptic digests under the recommended conditions will typically yield 32p-labeled hydrolyzed peptides, phosphoamino acids, and free phosphate in a ratio of 1 : 1 : 1. P450 phosphorylations characterized to date include serine (rat P450IIB 1 and rabbit P450IIB4) 4,23 and threonine residues 3° as the site of phosphorylation. A cAMP-dependent protein kinase recognition sequence Arg-Arg-X-Ser is present in the region of amino acids 125-130 in many members of P450 gene family II, but it is recognized as a substrate for cAMP-dependent kinases in only some of the P450s that contain this conserved sequence) Acknowledgments Supported in part by Grant DK-33765from the National Institutes of Health (D.J.W.) E. Neufeld, H. J. Goren, and D. Boland, Anal. Biochem. 177, 138 (1989). J. E. Shively, "Methods of Protein Microcharactedzation." Humana Press, Clifton, New Jersey, 1986. 3oI. Vilgrain, G. DeFaye, and E. M. Chambaz, Biochem. Biophys. Res. Commun. 125, 554 (1984).
[30] C y t o c h r o m e P 4 5 0 T u r n o v e r B y MARIA ALMIRA CORREIA
Introduction The hepatic microsomal hemoproteins, collectively termed as cytochrome P450, consist of a family of multiple isozymes, all of which recruit heme (iron-protoporphyrin IX), as their essential prosthetic moiety. METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Viuo
[29] P 4 5 0 P h o s p h o r y l a t i o n
in Isolated Hepatocytes
305
and
in Vivo
By JOHN A. KOCH and DAVID J. WAXMAN Introduction Many P450 enzymes are regulated at the m R N A level, in response to changes in circulating levels of drugs and hormones. Foreign compounds such as phenobarbital and 3-methylcholanthrene can elicit large increases in select P450 activities by transcriptional activation of individual P450 genes.~ In addition to these well-studied examples of pretranslational regulation, phosphorylation of P450 has emerged as a potentially significant posttranslational regulatory event. Studies in vitro have demonstrated that a n u m b e r of hepatic P450s can be phosphorylated by purified protein kinases.Z Support for the o c c u r r e n c e o f P450 phosphorylation under physiological conditions is provided by studies of P450 phosphorylation in isolated rat hepatocytes 3-5 and in vivo. 3 Several reports point to the potential importance of these phosphorylation reactions for the control of P450 activity, and perhaps also the regulation of P450 degradation. 3,4,6-9 This chapter describes both in vitro and in v i v o experimental protocols that have been developed for studies of P450 phosphorylation.
I F. J. Gonzalez, Pharmacol. Rev. 40, 243 (1988). 2 W. Pyerin, H. Taniguchi, F. Horn, F. Oesch, Z. Amelizad, and T. Friedberg, Biochem. Biophys. Res. Commun. 142, 885 (1987). 3j. A. Koch and D. J. Waxman, Biochemistry 28, 3145 (1989). 4 W. Pyerin and H. Taniguchi, EMBO J. 8, 3003 (1989). 5 B. Bartlornowicz, D. J. Waxman, D. Utesch, F. Oesch, and T. Friedberg, Carcinogenesis 10, 225 (1989). 6 E. Eliasson, I. Johansson, and M. Ingelman-Sundberg,Proc. Natl. Acad. Sci. U.S.A. 87, 3225 (1990). 7 W. Pyerin, H. Taniguchi, A. Stier, F. Oesch, and C. R. Wolf, Biochem. Biophys. Res. Commun. 122, 620 (1984). 8 I. Jansson, P. M. Epstein, S. Bains, and J. B. Schenkman, Arch. Biochem. Biophys. 259, 441 (1987). 9 B. Bartlomowicz, T. Friedberg, D. Utesch, E. Molitor, K. Platt, and F. Oesch, Biochem. Biophys. Res, Commun. 160, 46 (1989).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, lnc. All rights of reproduction in any form reserved.
306
REGULATORYPARADIGMS
[29]
Phosphorylation of Hepatic Cytochromes P450
P450 Phosphorylation in Isolated Hepatocytes Hepatocyte Isolation. Hepatocytes are isolated from untreated rats or from rats induced with drugs such as phenobarbital (four daily injections of phenobarbital at 80 mg/kg, intraperitoneally) by perfusion of liver tissue with buffers containing collagenase using the nonrecirculating system described by Seglen.a° Rats are anesthetized with ether for 3 min and prepared for hepatocyte isolation. A midsagittal incision is made from the pelvic region to the xiphoid process and lateral incisions from the base of the rib cage to expose the peritoneal cavity. The hepatic portal vein is located and connective tissue surrounding the vessel removed. The vein is clamped distal to the proposed site of eannulation to prevent significant blood flow through the vein during subsequent procedures. The vein is opened and a cannula inserted (0.020-gauge Tygon tubing), which is held in place with two lengths of suture. The cannula is then connected to a peristaltic pump and the liver perfused with calcium-free bicarbonate buffer (142 mM NaC1, 4.37 mM KC1, 1.24 mM KH2PO 4, 24 mM NaHCO3, 0.62 mM MgSO4, 0.62 mM MgCI2, bubbled with 95% 02/5% CO2 and maintained at 37°), at a flow rate of 25-30 ml/min. The liver should soon swell, at which time the inferior vena cava is severed, leading to rapid blanching of the liver. After 20 min of perfusion, the liver is perfused for an additional 20 rain with membrane-filtered (0.45/zm) bicarbonate buffer, as above, but containing 5.0 mM CaCI2 and 0.05% coUagenase (Clostridium histolyticum, type IV; Sigma, St. Louis, MO). The liver is then removed to a beaker containing Krebs-Ringer bicarbonate medium (119 mM NaC1, 4.7 mM KCI, 24 mM NaHCO3, 2.4 mM MgSO4, 2.3 mM CaC12), taking care to avoid rupture of the capsule. Hepatocytes are released by disruption of the capsule with a pair of scissors and gentle shaking. Large fragments of hepatic connective tissue are removed by filtration through nylon mesh (297/zm, Spectrum Medical Industries, Los Angeles, CA). The released cells are washed twice with Krebs-Ringer bicarbonate medium by centrifugation at 70 g, then resuspended in Leibovitz L-15 medium (GIBCO, Grand Island, NY) to a density of 2 x 106 cells/ml. Typical yield is 2-4 x 108 hepatocytes consisting of greater than 90% viable cells (as judged by trypan blue exclusion). Incubation with Ortho[3ZP]phosphate. Ortho[3Ep]phosphate is added to the cell suspension to give a concentration of 0.5-I mCi/ml, and the cells 10 p. O. Seglen, Exp. Cell. Res. 82, 391 (1973).
[29]
P450 PHOSPHORYLATION IN HEPATOCYTE$ AND in Vivo
307
are then incubated at 37° for 40 min to equilibrate the radiolabel with cellular ATP pools. Hormones are added to stimulate phosphorylation by one or more protein kinases (see Comments, below) and incubation is continued for an additional 10 rain. Hepatocytes are then collected by centrifugation, resuspended in homogenization buffer (120 mM Tris-HCl, pH 7.4, 100 mM KC1, 1 mM EDTA, 50 mM NaF, 5/.~M sodium orthovanadate, 500 txM molybdic acid, and 6 mM Na2ATP) and frozen in a dry ice/ ethanol bath. The hepatocytes are thawed to room temperature, homogenized with a Potter-Elvehjem homogenizer, and centrifuged for 5 min at 6000 g, then 15 min at 12,500 g. The supernatant is centrifuged at 105,000 g for 1 hr, and the resulting microsomal pellet is resuspended in microsome storage buffer (100 mM KPi, pH 7.4, 20% glycerol, 1 mM EDTA, 5 mM NaF, 50 IzM sodium orthovanadate, 500 t~M molybdic acid) by brief homogenization. Comments. L-15 medium is recommended for these short-term hepatocyte incubations, because the cells can be kept in air at 37°, without the need for a tissue culture incubator. Other media that can support hepatocyte phosphorylation, including Dulbecco's modified minimal essential medium, Krebs-Henseleit medium, and low phosphate (0.1 mM) Krebs-Ringer bicarbonate medium, 11 must be maintained in an atmosphere of 95% O1/5% COL. The choice of media might conceivably influence the P450 form specificity or hormone responsiveness of P450 phosphorylation, but this has not been examined in a systematic fashion. When using Krebs-Ringer medium, equilibration of ortho[32p]phosphate with cellular ATP pools occurs after 40 min of incubation, lz at which time hormones or other protein kinase activators are added. Many hormones rapidly stimulate phorphorylation, often within 3-10 min. These include glucagon (0.1 tzM), norepinephrine (10/~M), and angiotensin II (10 riM). ~3 Phosphatase inhibitors (NaF, sodium orthovanadate, and molybdic acid) are included in the homogenization and microsome storage buffers to inhibit enzymatic dephosphorylation during microsome isolation, whereas unlabeled ATP is added to block further incorporation of radioactivity. Control experiments should be carried out to determine the stability of the P450 under study (e.g., assessed by Western blotting analysis), as some P450s are readily lost in the early stages of hepatocyte culture. 14 P450 u W. W. Umbreit, R. H. Burris, and J. F. Stauffer, in "Manometric Techniques," 4th ed., p. 132. Burgess, Minneapolis, Minnesota, in press. 12 K. Yamada, K. E. Lipson, M. W. Marino, and D. B. Donner, Biochemistry 26, 715 (1987). t3 j. C. Garrison, M. K. Borland, V. A. Florio, and D. W. Twible, J. Biol. Chem. 254, 7147 (1979). 14 A. R. Steward, S. A. Wrighton, D. S. Pasco, J. B. Fagan, D. Li, and P. S. Guzelian, Arch. Biochem. Biophys. 241, 494 (1985).
308
REGULATORYPARADIGMS
[29]
inducers or ligands (e.g., 0.6 mM phenobarbital or 0.5 mM metyrapone) can be included in the isolation and incubation media to stabilize P450.15,16 Stimulation ofhepatocyte phosphorylation catalyzed by cAMP-dependent protein kinase is achieved using the membrane-permeable, cAMP analog dibutyryl-cAMP (But2cAMP). Concentrations as low as 5 /~M But2cAMP can markedly stimulate phosphorylation of P450IIB 1 when added to isolated hepatocytes as described above) A combination of But2cAMP with 8-thiomethyl-cAMP, which act synergistically on cAMPdependent protein kinase, 17can also be used to stimulate P450IIB1 phosphorylation in rat hepatocytes. 5 Control incubations using cAMP, which does not readily cross membranes, should be performed to ensure that P450 phosphorylation is occurring in intact cells, and not during the course of microsome isolation. The integrity of hepatocyte membranes may be assessed by incubation with glucagon, which interacts with membrane receptors to elevate intracellular levels of cAMP. Although cellular studies on P450 phosphorylation have thus far used only isolated hepatocytes, other cellular systems, such as primary hepatocytes cultured under conditions where they retain liver-specific functions (e.g., phenobarbital induction of P450IIB 1)18,19or vaccinia virus-infected Hep G2 cells that selectively express a single P450 form, 2° may be useful if carrying out longer term experiments on P450 phosphorylation. In Vivo Phosphorylation o f C y t o c h r o m e P450 c A M P Stimulation o f P450 Phosphorylation in Vivo. Uninduced or drug-induced rats are transferred to disposable cages where they are given a total of three intraperitoneal injections of But2cAMP and theophylline (2 nag each compound/100 g body weight/injection and suspended in 0.5 ml of 0.9% NaC1) at 90-min intervals. A single intraperitoneal injection of ortho[32p]phosphate (6 mCi/100 g body weight) is administered to the rats 2 hr prior to sacrifice. Livers are removed and minced in homogenization buffer (see above), and microsomes then prepared as already described for isolated hepatocytes. Microsomes obtained in this manner may have specific activities ranging from I000 to 3000 counts per minute (cpm) 32p//zg protein.
15E. T. Canepa, E. B. C. Llambias,and M. Grinstein,Biochim. Biophys. Acta 841, 186 (1985). 1~A. J. Paine, Chem.-Biol. Interact. 74, 1 (1990). 17S. J. Beebe,R. Holloway,S. R. Rannels,and J. D. Corbin,J. Biol. Chem. 259, 3539(1984). is E. G. Schuetz,D. Li, C. J. Omiecinski,U. Muller-Eberhard,and H. K. Kleinman,J. Cell. Physiol. 134, 309 (1988). 19D. J. Waxman,J. J. Morrissey,S. Naik, and H. O. Jauregui,Biochem. J. 271, 113 (1990). 2oF. J. Gonzalez, T. Aoyama,and H. V. Gelboin, this volume [8].
[29]
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Vivo
309
In order to facilitate electrophoretic analysis of in vivo phosphorylated hepatic P450s, partial P450 purification by cholate solubilization, followed by 8-16% polyethylene glycol 8000 precipitation 21 may be carried out in buffers containing phosphatase inhibitors (see above). The crude P450 fraction precipitating between 8 and 16% polyethylene glycol can be analyzed for specific phosphorylated microsomal proteins by immuneprecipitation or by two-dimensional gel electrophoresis as described below. Comments. Moderate to high levels of ortho[32p]phosphate are required in order to phosphorylate hepatic P450 to a specific activity that is high enough for detection by autoradiography. Dissection of rats and removal of livers is best carried out in plastic biological containment bags to minimize contamination with radiolabeled blood and other body fluids. Theophylline (or other methylxanthines) can be included in the protocol to inhibit phosphodiesterase activity in vivo. 22 The protocol provided above appears to be effective for stimulating phosphorylation of P450IIB 1 and P450IIB2 in vivo. 3 However, other hormone/drug combinations or time courses may be required for optimal phosphorylation in vivo of other hepatic P450 forms (e.g., P450 forms IIC6 and IIIA1). 3 In Vitro Phosphorylation o f Purified Cytochrome P450 General Strategy. Several purified hepatic P450s can be phosphorylated in vitro in reactions catalyzed by purified protein kinases. These include cAMP-dependent protein kinase and protein kinase C. 2,23Purified P450s are incubated with purified kinases in the presence of [y-32p]ATP, and the incorporation of phosphate is then monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Filter-binding assays can be used to quantitate, on a molar basis, the extent of phosphate incorporation. The effects of phosphorylation on P450 activity and protein chemical analysis of the site ofphosphorylation can also be examined. P450 Phosphorylation by cAMP-Dependent Protein Kinase. Bovine heart cAMP-dependent protein kinase, catalytic subunit (10 units; Sigma) is dissolved in 14/zl of an aqueous solution of dithiothreitol (50 mg/ml) and incubated on ice for 10 min. Purified P450 (75 pmol), magnesium acetate, pH 6.5 (15 mM final concentration), and [7-32p]ATP (1-3/xCi; 100 /zM ATP, final concentration) are added to give a total volume of 25/zl. Samples are then incubated at 37° for times up to 90 min. Samples are
21S. B. West, M.-T. Huang, G. T. Miwa, and A. Y. H. Lu, Arch. Biochem. Biophys. 193, 42 (1979). 22j. N. Wells and J. R. Miller, this series, Vol. 159, p. 489. 2t R. Muller, W. E. Schmidt, and A. Stier, FEBS Lett. 187, 21 (1985).
310
REGULATORYPARADIGMS
[29]
analyzed for 32p incorporation by SDS-PAGE followed by autoradiograincorporation is quantitated by precipitation with 10% trichloroacetic acid followed by trapping on glass fiber filters, s The effects of phosphorylation on P450 activity may be assessed after reconstitution of the cytochrome with purified NADPH-cytochrome-P450 reductase using any one of several P450 substrates (e.g., 7-ethoxycoumarin24) and any changes in P450 activity then correlated to phosphate incorporation on a molar basis. Comments. The efficiencyand P450 form specificity ofphosphorylation can vary widely among different protein kinases 2 and may also be influenced by the presence of P450 substrates and heme-binding ligands, or by the presence of interacting electron-transfer components such as cytochrome b5.6.s When attempting to relate changes in P450 activity or P450 stability to associated changes in P450 phosphorylation, it is essential that the stoichiometry of phosphate incorporation be monitored, but in general this has not been done. 6'9 p h y . 32p
Methods for Analysis of Phosphorylated P450s Immunoprecipitation Analysis Method. Microsomal protein (200/zg) prepared from 32p-labeled isolated hepatocytes or from in vivo labeling experiments are diluted in 1.5ml microcentrifuge tubes with 50 mM Tris-HC1, pH 7.8, 1 mM MgC12, 10 mM dithiothreitol to give 2 mg protein/ml. Samples are adjusted to I% SDS, boiled for 3 min, cooled to room temperature, and then diluted with 3 volumes of buffer B (50 mM Tris-HCl, pH 7.5, 190 mM NaC1, 6 mM EDTA, and 2.5% Triton X-100). Rabbit anti-P450 antiserum (10/xl) is then added, and the samples are incubated at 4° overnight. Protein A-bearing Staphylococcus aureus cells (40/xl of a 10% suspension; Sigma P-7155) are added, and the samples are then incubated at room temperature for 60 min with continuous mixing. The S. aureus cells are collected by brief centrifugation (30 sec) and washed 5 times (1 ml/wash) with buffer B containing 0.25% SDS. The final cell pellets are resuspended in 75/zl of SDS-PAGE sample buffer (62 mM Tris-HCl, 1% SDS, 0.001% bromphenol blue, 10% glycerol, 5% 2-mercaptoethanol), heated to 100° for 3 min, and electrophoresed on SDS-polyacrylamide gels (7.5% gel) according to Laemmli. 25Gels are stained, destained, and dried onto Whatman (Clifton, NJ) 3MM filter paper. Acrylaid (12.4% gel; FMC Corp., Princeton, NJ), 24R. A. Prough, M. D. Burke, and R. T. Mayer, this series, Vol. 52, p. 372. 25 U. K. Laemmli, Nature (London) 227, 680 (1970).
[29]
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Vivo
311
which can be covalently bonded onto Gel Bond PAG film (FMC Corp., Princeton, N J), can be used in place of polyacrylamide. After destaining, the PAG film with the attached gel is soaked in 3% glycerol to prevent cracking, then oven dried at 70° for 4-8 hr. Gels are autoradiographed at - 7 0 ° for 2-5 days using Cronex (Du Pont-NEN, Boston, MA), intensifying screens and Kodak (Rochester, NY) XAR-5 film. Comments. The concentration of SDS used in the immuneprecipitation step may be critical. SDS concentrations lower than 0.25% can lead to significant nonspecific contamination of the immunoprecipitates with other phosphorylated microsomal proteins, whereas greater than 0.5% SDS may disrupt the antigen-antibody complexes and give a low yield of immunoprecipitate. Monoclonal anti-P450 antibodies may be used for immuneprecipitation. However, pilot experiments should be conducted to determine the binding efficiency of a given monoclonal antibody to S. aureus cells, since many mouse monoclonals do not have a high affinity for S. aureus protein A. The use of Acrylaid allows one to dry relatively thick gels (1.5 mm) in a standard oven with little risk of cracking. Two-Dimensional Gel Electrophoresis of Microsomal Protein General Strategy. Phosphorylated liver microsomes and purified P450s are analyzed by two-dimensional gel electrophoresis, which is useful for monitoring charge changes induced by phosphorylation. Peptides of interest can be detected by a combination of Coomassie blue staining, Western blotting using P450 form-specific antibodies, and autoradiography. These methods, when applied to both kinased and phosphatased P450 samples, can be useful in identifying endogenously phosphorylated P450 polypeptides, which can subsequently be monitored without the need to employ 32p tracers. Method. Liver microsomes (100-200 p,g) or purified P450s (30-60 pmol) are analyzed by two-dimensional (2D) gel electrophoresis. 26 All procedures are carried out at 37° to prevent crystallization of urea from the gel solutions. Isoelectric focusing gels contain 10 M urea (UltraPure, Bethesda Research Laboratories, Gaithersburg, MD), 4% gel (acrylamide/ bisacrylamide, 17.5 : 1), 2% Nonidet P-40 (NP-40) detergent, 1.5% ampholytes pH 3-10, 0.5% ampholytes pH 6-8 (Sigma), 0.01% ammonium persulfate, and 0.05% Temed. Isoelectric focusing gels (14.5 cm long) are polymerized in siliconized glass tubes (16 cm long, 3 mm i.d.) sealed at the bottom with 1-cm Parafilm plugs and overlaid with 20/xl of 8 M urea. Gels are prepared for preelectrophoresis by adding 40 /zl of 2-D gel sample
26G. P. Vlasuk and F. G. Walz, Jr., Anal. Biochem. 105, 112 (1980).
312
REGULATORYPARADIGMS
[29]
buffer (3% NP-40, 10 M urea, 1.5% ampholytes pH 3-10, 0.5% ampholytes pH 6-8, and 5% 2-mercaptoethanol; titrated to pH 4.5 with 3 N HCI) to the anodic end of the gels. The bottom space created by the Parafilm plug is filled with bottom gel buffer (3% NP-40, 10 M urea, 1.5% ampholytes pH 3-10, 0.5% ampholytes pH 6-8, and 5% 2-mercaptoethanol) held in place with a section of dialysis membrane secured with cross sections of Tygon tubing. Gels are prefocused (cathode solution, 0.02 N NaOH; anode solution, 0.0057 N H3PO4) at constant voltages of 200, 300, and 400 V for 15 min and two 30-min intervals, respectively. Protein samples are solubilized by the addition of 2 volumes of 2-D gel sample buffer followed by a few crystals of solid urea. Solubilized microsomal protein is applied to the anodic end of each gel in a total volume of 75/zl, then overlaid with 10 ttl of sample overlay buffer (2% NP-40, 0.75% ampholytes pH 3-10, 0.25% ampholytes pH 6-8, 8 M urea, and 5% 2-mercaptoethanol; titrated to pH 4.3 with 3 N HCI). Electrophoresis is carried out at 400 V (constant voltage) for 12 hr. Gels are removed to test tubes containing 5 ml of gel equilibration buffer (10% glycerol, 2.3% SDS, 62.5 mM Tris-HC1, pH 6.8, and 5% 2-mercaptoethanol) and gently shaken for 15 min. The gels can be stored at - 70° for several days or used directly for electrophoresis in the second dimension. Isoelectric focusing tube gels are thawed, washed briefly with doubly distilled water, and then applied to the top of 7.5% polyacrylamide (3% stacking) SDS gel. The tube is sealed in place with 1% agarose dissolved in gel equilibration buffer. The SDS gels are electrophoresed, 25 then stained with Coomassie blue R-250, dried onto Whatman 3MM filter paper, and exposed to X-ray film at - 8 0 °. Alternatively, gels may be analyzed immunochemicallyfor specific P450 forms and their phosphorylated derivatives by Western blotting using standard methods. 27 Comments. Thorough solubilization of samples with detergent and urea is important for the resolution of microsomal P450s and their phosphorylated derivatives in isoelectric focusing gels. Incomplete solubilization will impede the entry of P450 proteins into the isoelectric focusing gel and may contribute to streaking. Solubilization is facilitated by the addition of a few crystals of solid urea, until the point where the sample is supersaturated. Excessive vortexing should be avoided since the frictional heat may lead to dissolution of excessive amounts of urea which can recrystallize and interfere with the loading of the isoelectric focusing gel. Ultrapure urea is recommended to minimize carbamoylation of protein amino groups and the resultant introduction of charge heterogeneity. Removal of gels is
27
D. J. Waxman, this volume [24].
[29]
P450 PHOSPHORYLATIONIN HEPATOCYTESAND in Vivo
313
facilitated by applying air pressure to one end of the glass tubes with a Pasteur pipette bulb. Phosphorylation of P450 is indicated by an acidic shift in the mobility of the P450 in the isoelectric focusing dimension that is associated with 32p incorporation. In the case of some hepatic P450s, however, charge heterogeneity apparently unrelated to phosphorylation may be present, and this can complicate the analysis. Thus, rat hepatic P450IIB1 and P450IIB2 are each comprised of one major and one minor (more acidic) charge variant that appears to be nonphosphorylated. In addition, a second, even more acidic polypeptide that corresponds to an endogenously phosphorylated derivative that comprises approximately 15% of total microsomal P450IIB1 plus P450IIB2 is present)
Phosphoamino Acid Analysis and Tryptic Peptide Mapping of 32p-Labeled P450 General Strategy. Immunoprecipitated 32p-labeled P450s derived from hepatocytes in oivo or purified P450 phosphorylation reactions are analyzed to identify the amino acid that is phosphorylated. Peptide mapping is performed to ascertain whether there are multiple phosphorylation sites, and whether the P450 phosphorylation site recognized by a purified protein kinase is the same as that which is phosphorylated in whole cells. Phosphoamino Acid Analysis. 32p-Labeled liver microsomes or purified P450s are immunoprecipitated then electrophoresed on 0.75-mm thick SDS gels using the methods described above. Gels are fixed in 10% acetic acid/45% ethanol for 60 min, then incubated at 20-22 ° for 24 hr with two changes of 10% acetic acid/15% ethanol (200 ml per wash). Gels are then washed 3 times in 200 ml of doubly distilled water (30 min/wash) and dried onto Whatman 3MM filter paper, which is marked with radiolabeled ink then autoradiographed. Radiolabeled P450s are located by aligning the autoradiograph with the dried gals. The P450-containing bands are then excised with the adhering filter paper using a scalpel blade. The 32p-labeled dried gel slice is incubated with L-l-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (50 /~g/ml; Worthington Biochemical Corp., Freehold, N J) in 0.4 ml of 50 mM ammonium bicarbonate buffer, pH 8, for 5 hr at 37°. An additional 0.2-ml aliquot of TPCK trypsin (50/zg/ml) is then added and the incubation continued an additional 19 hr. Particulate matter is removed in a microcentrifuge, and the supernatant is then lyophilized repeatedly to remove residual ammonium bicarbonate. Samples are resuspended in 6/.d of 0.033% 2-mercaptoethanol, and a 2-/~1 aliquot is then removed for tryptic peptide analysis as described below. The remaining material is lyophilized, resuspended in 0.1 ml of doubly
314
REGULATORYPARADIGMS
[29l
distilled water then relyophilized. HCI (5.7 N; Pierce Chemical Co., Rockford, IL) (0.1 ml) is then added, and the sample is heated in a sealed Eppendorf tube at 100° under N2 for 90 rain to effect partial acid hydrolysis. The samples are lyophilized repeatedly to remove residual HCI, then dissolved in double distilled water (10/xl/sample) containing 8/zg each of phosphoserine, phosphothreonine, and phosphotyrosine standards (Sigma) and a 1:250 dilution of dye mix (0.3% each of xylene cyanole FF, Orange G, and Acid fushin; Eastman-Kodak, Rochester, NY) to standardize migration distances. Aliquots (5/xl) are spotted onto 20 x 20 cm thin-layer cellulose chromatography plates (Eastman-Kodak 13255) which are then carefully moistened with pH 3.5 electrophoresis buffer (pyridine/acetic acid/water, 5 : 50 : 945). Plates are electrophoresed for 75 min at 800 V in a Shandon, Inc. (Sewickley, PA) or equivalent thin-layer electrophoresis apparatus. Phosphorylated amino acids and dye markers migrate toward the cathode. 32p-Labeled phosphoamino acids and partial hydrolysis products and released phosphate are detected by autoradiography (typical ratio of 32plabel in these fractions 1 : 1 : 1). Migration distances (relative to free orthophosphate) of 0.28, 0.52, 0.61, and 0.72 are observed for partial peptide hydrolysis products and for phosphotyrosine, phosphothreonine, and phosphoserine, respectively. Phosphorylated amino acid standards are visualized by treatment with 0.25% ninhydrin dissolved in acetone followed by heating at I00° for 3 min. Tryptic Mapping of Phosphopeptides. Samples for tryptic peptide analysis, prepared as described in the previous section, are transferred to drawn-out 100-/~1capillary tubes and then applied to 10 x 10 cm cellulosecoated thin-layer plates (EM Science, Cherry Hill, NJ, #5577) on areas previously spotted with 0.5-1.0/~1 dye mix to give spots about 4 mm in diameter. Electrophoresis is performed with pH 1.9 buffer (formic acid/ acetic acid/water, 1 : 3 : 16) in a Shandon thin-layer electrophoresis apparatus for 20 min at I000 V. Tryptic peptides migrate toward the anode while the dye markers migrate toward the cathode. Plates are air dried for 60 min then chromatographed in a second dimension at 200-22 ° by development with n-butanol/pyridine/acetic acid/water (6.5 : 5 : 1 : 4). Plates are air dried and autoradiographed. Comments. Immunoprecipitated P450s that can be detected by autoradiography within 12-24 hr will generally provide sufficient material for phosphoamino acid analysis and tryptic peptide mapping. Excessive spreading of samples should be avoided while wetting the thin-layer plates with buffer. Sample diffusion can be minimized by coveting the area of sample application with a cap from a screw-top Eppendorf tube and applying the buffer evenly around the samples using a compressed air sprayer (Chromist Spray Unit; Gelman Instrument Co., Ann Arbor, MI).
[30]
CYTOCHROMEP450 TURNOVER
315
Buffer will enter the sites of application by capillary action and focus the samples. As an alternative to thin-layer electrophoresis, thin-layer chromatography methods can be applied to resolve and identify phosphoamino acids. 28 Trypsin digestion of P450 should be carried out under two or three conditions (e.g., varying the protease-to-P450 ratio) to ensure complete digestion and thereby avoid misinterpretation of the tryptic maps. Peptide maps of samples prepared in parallel may be compared to ascertain whether the site on the P450 that is phosphorylated in vitro is the same as that which is phosphorylated in intact hepatocytes. As an alternative to thin-layer electrophoresis, phosphorylated P450 peptides may be resolved by high-performance liquid chromatography (HPLC) using standard methods. 29 Acid hydrolysis of tryptic digests under the recommended conditions will typically yield 32p-labeled hydrolyzed peptides, phosphoamino acids, and free phosphate in a ratio of 1 : 1 : 1. P450 phosphorylations characterized to date include serine (rat P450IIB 1 and rabbit P450IIB4) 4,23 and threonine residues 3° as the site of phosphorylation. A cAMP-dependent protein kinase recognition sequence Arg-Arg-X-Ser is present in the region of amino acids 125-130 in many members of P450 gene family II, but it is recognized as a substrate for cAMP-dependent kinases in only some of the P450s that contain this conserved sequence) Acknowledgments Supported in part by Grant DK-33765from the National Institutes of Health (D.J.W.) E. Neufeld, H. J. Goren, and D. Boland, Anal. Biochem. 177, 138 (1989). J. E. Shively, "Methods of Protein Microcharactedzation." Humana Press, Clifton, New Jersey, 1986. 3oI. Vilgrain, G. DeFaye, and E. M. Chambaz, Biochem. Biophys. Res. Commun. 125, 554 (1984).
[30] C y t o c h r o m e P 4 5 0 T u r n o v e r B y MARIA ALMIRA CORREIA
Introduction The hepatic microsomal hemoproteins, collectively termed as cytochrome P450, consist of a family of multiple isozymes, all of which recruit heme (iron-protoporphyrin IX), as their essential prosthetic moiety. METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
