R. Weishaar of Warner Lambert Co., Ann Arbor, MI; amrinone and milrinone by Dr. A. Soria of Sterling Winthrop Research Institute,. Rensselaer, NY; the starting ...
THE JOURNALOF BIOLOGICAL CHEMISTRY
Val. 262, No. 12, Issue of April 25, pp. 5797-5607,1987 Printed in U.S.A.
Purification of the PutativeHormone-sensitive Cyclic AMP Phosphodiesterase from RatAdipose Tissue Usinga Derivative of Cilostamide as a Novel Affinity Ligand* (Received for publication, October 21,1986)
Eva Degerman and Per Belfrage From the Department of Physiological Chemistry, University of Lund, Sweden
Amy Hauok Newman$#,Kenner C. Rice$, and Vincent C. Manganiellon[( From the $Laboratory of Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases and the TLaboratory of Cellular M e t ~ l i s mNational , Heart, Lung, and Blood Institute, National Inst~tutesof Health, Rethesda, M a r y 20892 ~ ~
. enzyme was also inhibited by several A “low K,” CAMPphosphodiesterase with properties -0.15 p ~ The of a peripheral membrane proteinaccounts for -90% OPC derivatives and “cardiotonic” drugs, but not by of total cAMP phosphodiesterase activity in particulateRO 20-1724. It was very sensitive to inhibition by (100,000 X g) fractions from rat fat cells. Incubation agents which covalently modify protein sulfhydryls, of fat cells with insulin for 10 min increased particu- but not by diisopropyl fluorophosphate. late (but not soluble) cAMP phosphodiesterase activity, The activationby insulin and other findings indicate with a maximum increase (-100%) at 1 nM insulin. that the purified enzyme, which seems to belong to a Most of the increase in activity was retained after subtype of low K,,, cAMP phosphodiesterases that is solubilization (with non-ionic detergent and NaBr) andspecifically and potently inhibited by cGMP, cilostampartial purification (-20-fold) on DEAE-Sephacel.The ide, other OPC derivatives, and certain cardiotonic solubilized enzyme from adipose tissue was purified drugs, is likely to account for the hormone-sensitive -65,000-fold to apparent homogeneity (yield -20%) particulate low KmcAMP p h ~ p ~ o d i e s t e activity r~e by chromatography on DEAE-Sephacel and Sephadex of rat adipocytes. on aminoethyl G-200 andaffinitychromatography agarose conjugated with the N-(Z-isothiocyanato)ethyl derivative of the phosphodiesterase inhibitor cilostamCyclic nucleotide phosphodiesterases constitute a complex irJ, (OPC 3689). A 63,800 f 200-Da polypeptide (acgroup of enzymes, multiple forms of which are found in most counting for >90%of the protein eluted from the affinity column) was identifiedby polyacrylamide gel elec- mammalian tissues and cells (1-4). These enzymes differ in trophoresis in sodium dodecyl sulfate (withor without amount and proportions in different cells, subcellular localreduction). Enzyme activity was associated with the ization, substrate affinities, kinetic characteristics, physisingle protein band after electrophoresis under non- ochemical properties, responsiveness to various effectors or was drugs (especially phosphodiesterase inhibitors), and mechadenaturing conditions. On gel permeation, Mrfapp> 100,000-1 10,000, suggesting that the holoenzyme is nisms of regulation (1-4). a dimer. A PI of 4.9-5.0 was estimated by isoelectric In adipose tissue, lipolysis is promoted by hormones which focusing. At 30 “C, the purified enzyme hydrolyzed increase cAMP content and activate CAMP-dependent proboth cAMP and cGMP with normal Michaelis-Menten for cAMP tein kinase, with subsequent phosphorylation and activation kinetics; the pH optimum was 7.5. The Km(app) 8.5 pmol/min/mg: for cGMP, of the hormone-sensitive lipase (5-7). By catalyzing hydrolywas 0.38 p~ and V,,, Km(app) was 0.28 p~ and V,,, 2.0 pmol/min/mg. cGMP sis of CAMP, phosphodiesterases may regulateeffects of competitively inhibited cAMP hydrolysis with a Kt of agents that increase cAMP and stimulate lipolysis. Activation of the hormone-sensitive lipase canbe completely or partially *This work was supported in part by grants from A Pahlsson’s prevented or reversed by insulin and otherantilipolytic agents Foundation, Helsingborg; Nordic Insulin Foundation, Copenhagen; (7). Although the precise mechanism(s) of the antilipolytic Swedish Diabetes Association and KabiVitrum AB, Stockholm; The effect of insulin is unknown (6), with low or moderate stimMedical Faculty, University of Lund and the Swedish Medical Re- ulation of lipolysis, it correlates well with a reduction in search Council (Project No. 3362) (all to P. B.). Some of the work done by E. D. was carried out asa guest worker at theNational Heart, “activated” CAMP-dependent kinase (8) which presumably content (9, Lung, and Blood Institute, NationalInstitutes of Health. Travel costs results from an insulin-induced decrease in cAMP 10). and living expenses during the work of E. D. a t the National Heart, Lung, and Blood Institute were paid by the Medical Faculty, UniverInsulin could reduce cAMP content by either inhibition of sity of Lund and The Swedish Institute, Stockholm. The costs of adenylate cyclase, stimulation of cAMP phosph~esterase, publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- or both. A number of workers have, in fact, demonstrated tisement” in accordance with 18U.S.C. Section 1734 solely to indicate that incubation of intact rat fat cells and hepatocytes or cultured 3T3-Ll adipocytes with insulin results in activation this fact. Q Recipient of a National Research Service Award from the Na- of particulate low K,,, cAMP phosphodiesterase (11-18). Retional Institute of Drug Abuse and also received partial support from cent studies employing specific cAMP analogues or phosphoKey Pharmaceuticals, Inc. diesterase inhibitors further support an important role forthe 11 To whom correspondence should be addressed Laboratory of Cellular M e ~ ~ ~ i Bldg. s m ,10, Rm, 5N-307, NHLBI, NIH, Bethesda, activation of particulate low Km p h o s p h o ~ e s ~ r a sine the antilipolytic action of insulin (19, 20). Of a series of cAMP MD 20892.
5797
5798
Purification of Particulate “Low K,” CAMPPhosphodiesterase
EXPERIMENTAL PROCEDURES~ analogues which activated protein kinase and stimulated lipolysis, insulin inhibited the action of only those that were Sephadex G-200 superfine (size graded according to a described substrates for the particulatelow K, cAMP phosphodiester- procedure (36))and DEAE-Sephacel were fromPharmacia, Uppsala, ase (19). In 3T3-Ll adipocytes, OPC 3689l (cilostamide), a Sweden. The heterogeneous non-ionic alkyl polyoxyethylene glycol relatively specificinhibitor of the particulate insulin-sensitive detergent CI3E12 (abbreviated as C,,Ez,from the generalformula C,HZ,+~(OCH&HZ),OH) was obtained from Berol Kemi AB, Stenphosphodiesterase, prevented insulin inhibition of lipolysis ungsund, Sweden. Crotalus atrox venom, CAMP, cGMP, AMP, GMP, whereas RO 20-1724, a relatively specific inhibitor of soluble insulin, bovine serum albumin (fatty acid free), transferrin, 8-galaccAMP phosphodiesterase,did not (20). tosidase,phosphorylase,catalase,myoglobin,NaBr,Hepes,octylglucoside, dithioerythritol,and aminoethylagarosewere Particulate low K, cAMP phosphodiesterase activity inrat methyl ~ P Ci/mmol) and [8,5-3H]cGMP (-30 fat cells or hepatocytes and 3T3-Ll adipocytes is also in- from Sigma. [ 2 , 8 - 3 ~ ] c (36.4 Ci/mmol) were from New England Nuclear, Dreieich, Federal Repubcreased by agents such as catecholamines that activate ade- lic of Germany and purified by thin-layer chromatographyon cellunylate cyclase and increase intracellular cAMP content(13lose with 0.5M ammonium acetate:ethanol,2.5 (v/v) and on columns 18).Whereas activationof particulate cAMP phosphodiester- of DEAE-Sephadex (from PharmaciaP-L Biochemicals) before use. ase is thought to be important in the antilipolytic action of Acrylamide and bisacrylamide were from Serva, Heidelberg, Federal insulin (19, 201, activation of the phosphodiesterase by cate- Republic of Germany; ~‘,~’-methylenebisacrylamide and N,N,N‘,N‘-tetramethylethylenediamine from Bio-Rad; and sodium cholamines may be secondary to changes in cAMP CAMPand dodecyl sulfate (SDS), from Merck, Darmstadt, Federal Republic of dependentproteinkinaseactivity,representing a type of Germany. Riboflavin 5’-phosphate and carrier a m p h o l ~were s from “feedback” mechanism to terminate thecAMP signal (13, 14, LKB, Bromma, Sweden. OPC 3689 (cilostamide),OPC 13135, OPC 13013, and OPC 3911 were generously supplied by Dr. H. Hidaka of 18, 20-22). Indeed, cAMP analogues that activate CAMPdependent protein kinasein vitro and increase lipolysis in fat Mie University and Otsuka Pharmaceuticals,Japan; RO 20-1724 by Dr. M. Lin, National Institutes of Health; CI 914 and CI 930 by Dr. cells or glycogen phosphorylase activityin intact hepatocytes R. Weishaar of Warner LambertCo., Ann Arbor, MI; amrinone and increase particulatelow K,,, cAMP phosphodiesterase activity milrinone by Dr. A. Soria of Sterling Winthrop Research Institute, in fatcells and hepatocytes(13-16,22) and actually decrease Rensselaer, NY; the starting compound forthe affinity ligand,4-(1,2dihydro-2-oxo-6 quinoly1oxy)-butyric acid (Fig.4A, compound I ) , by cAMP content of rat hepatocytes (23). Drs.AkioSonoda and S. Ayukawafrom Otsuka Pharmaceuticals, Apparently soluble low K,,, CAMP phosphodiesterases have Osaka, Japan and Rockville, MD. been isolated from several sources (24-28), extensively puriBufferSolutions-Allbuffers and solutionsexcept the Krebsfied from human platelets (29) and bovine myocardium (301, RingerHepesbufferusedfor fat cell incubationscontained the and characterizedas regards their responses to various phos- protease inhibitors leupeptin (10 gg/ml), antipain (10 rg/ml), and phodiesterase inhibitors (24, 27-33). Yamamoto et al. (32) pepstatin (1 pg/ml).Unless stated otherwise,pH of buffers was determined at 4 “C. BufferA contained 50 m M Tris-HCI, pH7.50, 5 separated from calf liversupernatant two distinct subtypesof mM MgClz, 1 mM EDTA, 20% (w/v) glycerol, and, unless otherwise low Km cAMP phosphodiesterase that differed in responses stated, 0.03% (w/v) C13EX2. Krebs-Ringer Hepes, pH 7.40, contained to cGMP and specific phosphodiesterase inhibitors. Onesub- 119 mM NaCl, 4.96 mM KCl, 2.54 mM CaC12, 1.19 mM KH2P04,1.9 type was very sensitive to inhibition by cGMP and cilostam- mM MgSO4, and 24 mM Hepes.After the DEAEchromatography step, all buffers contained1mM ~ t h i o e r y t ~ i t o l . ide; the other, to RO 20-1724. In differentiated 3T3-Ll adiPreparation and Incubation of Isolated Fat Cells; Preparation of pocytes, these two subtypes differed in subcellular localiza- Particulate Fraetions-Isolated fat cells were prepared from epididytion. The hormone-sensitive low K , particulate cAMP phos- mal fat pads of Sprague-Dawley rats (150-160 g) hy digestion with phodiesterase form was rather sensitive to cGMP and cilos- collagenase as described (37). Small samplesof the final cell suspentamide, whereasRO 20-1724-sensitive cAMP phosphodiester- sion were aspirated into capillary hematocrit tubesand centrifuged for 3 min in a microhematocrit centrifuge in order to estimate the ase activity was essentially confinedto the supernatant frac- packed cell volume. Adipocytes, usually a 2% (v/v) suspension, were tion (20). incubated with or without insulin for 10 min. An equal volume of Since, as reported here, particulate low K, cAMP phospho- cold 50 mM TES buffer,pH6.7, containing 250 mM sucrosewas diesterase fromrat adipose tissue belongs to the subtype that added (final pH 7.2) before homogenization and centrifugation at 100,000 X g for 60 min. Portions of particulate fractions, suspended is very sensitive to inhibitionby cilostamide and several other in buffer A without C1sEl2,and supernatant fractions were assayed OPC derivatives, as well as cGMP and certain new “cardi- for cAMP phosphodiesterase activity whichwas related to packed otonic” drugs(33-35), we prepared a derivative of c i l o s ~ m i d e cell volume. (CIT) coupled t o aminoethyl agarose (CIT-agarose) and used Polyacrylamide Stob Gel Etectrophoresis in Sodium DodecylSulfate under Nondenaturing Conditions-Proteins were incubated at it to purify the putative hormone-sensitive particulate low K,,, and 96 “c for 3 min in 50 mM Tris, 5% glycerol, 1%SDS,100 mM cAMP phosphodiesterase from the rat adipose tissue to ap- dithioerythritoland subjected to electrophoresis in8%polyacrylamparent homogeneity. Some of the properties of the purified ide slab gels accordingto the method of Laemmli(38).Mobilities and apparent Mr of the ~ l ~ e p t i d (stained es withsilver (39)) were phosphodiesterase arealso described in this report. calculated from comparison withthe mobilities of reference proteins * The abbreviations used are: OPC 3689, N-cyclohexyl-N-methyl- (&galactosidase, M. 116,000; phosphorylaseb, Mr 97,000; transferrin, 4-(1,2-dihydro-2-oxo-6-quinolyloxy)butyramide (cilostamide); PDE, Mr 76,600;bovineserum albumin, M , 67,000; and ovalbumin, M, phosphodiesterase;SDS,sodiumdodecyl sulfatq ClaElZ, non-ionic 43,000). Purified enzyme was also subjected to electrophoresis under nonalkyl polyoxyethylene detergent; Hepes,4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid;SDS,sodium dodecyl sulfate; TES, N- denaturing conditions with modification of the procedure described by Lovell-Smith et al. (40). 5 or 6.5% polyacrylamide slab gels (1.5 tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid;OPC3911,
N-cyclohexyl-N-(2-hydroxyethyl)-4-(6-(1,2-dihydro-2-oxoquinolyPortions of this paper (including part of “Materialsand Methods,” 1oxy))butyramide; 13013, OPC 6-4-(l-cyclohexyl-5-tetrazopart of “Results,” part of “Discussion,”and Refs. Sl-Sl6) are prelyl)butoxyl)-1,2,3,4-tetrahydro-2-oxoquinolone; OPC13135,N-cyclohexyl-N-(2-hydroxybutyl)-5-(6-1,2,3,4-tetrahydro-2-oxoquinoly- sented in miniprintat the end of this paper. Miniprintis easily read with the aid of a standard magnifying glass. Full size photocopiesare 1oxy)-butyramide; milrinone, 1,6-dihydro-2-methyl-6-0~0-[3,4’-bipyridine]-5-carbonitrile; Amrinone, 5-amino-3,4’-bipyridin-6(lH)-available from the Journal of Biological Chemistry, 9650 Rockville one;RO20-1724,4-(3-butoxy-4 methoxybenzyl)-2-imidazolidone; Pike, Bethesda, MD 20814. Request Document No. 86M-3656, cite CIT, N-(2-isothiocyanato)ethylderivative of cilostamide, ie. cilos- the authors, and include a check or money order for $2.00 perset of
in microfilm tamide isothiocyanate;N,inhibitory guanyl nucleotide-binding reg- photocopies. Full size photocopies are also includedthe edition of the Journal that is available from Waverly Press. ulatory subunit.
Purification of Particulate "Low K," CAMPPhosphodiesterase
5799
mm) were polymerized in buffer containing150 mM Tris (pH 9.0), 3 mM MgSO, 30 mM NaBr, 5% glycerol, and 0.03% ClaEI2 and incubated overnight at 5 "C in the same buffer containing5 mM dithioerythritol. The electrode buffer contained 20 mM Tris (pH 9.0), 200 mM glycine, 5 mM dithi~rythritol,3 mM MgSO,, 5 mM NaBr, 5% glycerol, 0.03% C13E12. Slab gels were exposed to 100 V for 45 min at 5 "C prior to addition ofsample, after which electrophoresis was continued for2 h at 100 V. Lanes were delineated and pieces(0.5 cm) cut out and assayed(20 min, 20 "C) for phosphodiesterase activityas described below in 0.3 ml of substrate-elution solution which contained 0.5 PM [3H]cAMP,50 mM Tris (pH 7.5), 5 mM MgClz, 1 mM EDTA, 1 mM dithioerythritol,20% glycerol, 0.015% CISE12. Phosphodiesterase Assay-Activity was assayed by a modification of a previously described procedure (12). Samples were incubated at 30 'c usually for 8 min in a total volume of 0.3 ml containing 50 mM Hepes, pH 7.40, 0.1 mM EDTA, 8.3 mM M&l*, 0.5 p~ ['HjcAMP (-15,000 cpm). Hydrolysis of substrate did not exceed 20%; under these conditions phosphodiesterase activitywas proportional to time and enzyme concentration. Protein-Except after the final purification step protein was measured using Coomassie Brilliant Blue G-250 (Bio-Rad) with bovine serum albuminas a standard (41). To measure submicrogram amounts of pure enzyme proteinafter affinity chromatography,various amounts of reference proteins (4-300 ng of phosphorylase 6, bovine serum albumin, ovalbumin, trypsin inhibitor) and samplesof the affinity-purified phosphodiesterase were subjected to SDS-polyacrylamide gel electrophoresis (see above) on the same 8%slab gel. The polypeptide bands were silver stained (39) and peak areasquantified by scanning densitometry (436 nm) with a Joyce-Loebl Chromoscan 3 densitometer. Approximately linear relationships wereobtained between the amountsof protein and the densitometric values for 4-60 ng of each of the reference proteins, with iess than 20% difference in the relative silver staining. The average curve was used for estimating phosphodiesterase protein which was proportional to sample size within the absorbance range employed.
The suspension was further manually homogenized (10 strokes) in a tight-fitting Potter-Elvehjem glass homogenizer. Homogenates from 144 rats were pooled and centrifuged for 10 min at 2,800 X g in a Beckman JA-20 rotor. The infranatant below the floating fat layer was drawn off with a syringe and long needle and centrifuged a t 100,000 x g for 45 min in a Beckman SW 28 swinging bucket rotor at 4 "C. The supernatant was used for preparation of hormone-sensitive lipase (5); pellets (from 144 rats) which contained low K,,, cAMP phosphodies~rasewere pooled and suspended in 15 ml of buffer A without C,,E,, (4 "C). The particulate suspensions could be stored at -80 "C for severai months with little loss or release of enzyme activity from the particulate fractionand then used to purify the phosphodies~rase. Step 2. Solubilization of the Phosphodiesterase ActivityS o l u b i l i ~ t ~conditions on were chosen to optimize the amount of cAMP phosphodiesterase activity extracted from the particulate fractions, the extentof purification at this step, and the stability of enzyme activity at this and subsequent steps (Table 11). In the absence of detergents, with 500 mM NaBr, more cAMP phosphodiesterase activity was extracted from the particulate fractions by freeze-thaw then by sonication (Table 11). Enzyme solubilized by freeze-thaw with 500 mM NaBr, however, was rather unstable, and extensive dilution or dialysis was also required prior to ion-exchange chromatography. Sonication in the presence of a combination of a low concentration of C13E12(0.03%),150 mM NaBr, and 20% glycerol resulted in therelease of >70% of particulate cAMP phosphodiesterase activity, which exceeded that released with detergent alone (Table 11).Higher concentrations of detergent in the same buffer solution sometimes extracted up to 90% of RESULTS the cAMP phosphodiesterase activity, but also solubilized much more membraneprotein, resulting in lower specific P u r i f ~ c aoft ~the ~ P u r t i c u Low ~ ~ K, cAMP activity. Inclusion of glycerol and detergent was essential to Phosphodiesterase maintain enzyme stabilityduringsubsequentpurification Data from a representativepreparation (epididymal fat steps. Based on these results(Table 11),particulate cAMP phospads from 432 rats) are presented in Table I. Similar results were obtained with six preparations from similar numbers of phodiesterase was solubilized on a larger scale by sonication rats. All steps were performed a t 4 "C, and all solutions (7 x 30 s on ice) and centrifugation of a suspension of 100,000 contained the protease inhibitors described under "Experi- X g pellets from 432 rats in buffer A (120 ml) containing 0.03% CI3El2,150 mM NaBr, and 20% glycerol. More than mental Procedures" unless otherwise stated. Step 1. Preparation of a Particulate Phosphodiesterasefrom 70% of cAMP phosphodiesterase activity was solubilized with Adipose Tissue-All procedures in this step,unless otherwise -70% increase in activity and a %fold purification (Table I). Step 3. DEAE Chromatography-The solubilized enzyme noted, were performed at 10 "C in order to avoid trapping of enzyme by solidified fat. Fat pads (removed from animals was diluted 1.5-fold in buffer A and applied to a column of within a few minutes after death, immediately frozen in liquid DEAE-Sephacel equilibrated and washed with buffer A connitrogen, and then maintained at -80 "C for up to several taining 100 mM NaBr (Fig. 1). As judged from the AZm,more months before use) from 72 rats (-72 g) in 200 ml of 250 mM than 90% of the applied protein did not adsorb to thecolumn. sucrose, 1 mM EDTA, pH 7.4, were minced to a thick slurry CAMP phospho~esteraseactivity in the nonadsorbed fracwith a pair of scissors; the suspension was homogenized with tions (Fig. 1) was identified as a Ca2+/calmodulin-stimulated 1 0 up-and-down strokes ina large engine-driven Teflon-pestle p h o s p h ~ e s t e r a s ecapable of hydrolyzing both cAMP and Potter-Elvehjem-type homogenizer. Most of the connective cGMP (data not shown). The main peak of cAMP phosphotissue was removed by vacuum suction through a nylon net. diesterase activity, which accounted for 7545% of the re-
1 0 0 , O ~X g "Results." step
I__-
TABLEI Purification of particulate low K,,, phosphodiesterasefrom adipose tissue pellets were prepared from the epididymal fat pads (about 475 g) of 432 rats as described under Fraction
1 Pellet, 100O , OOX g pellet 2 Solubilized DEAE-Sephacel 3 4 Sephadex G-200 5 DEAE-Sephacel CIT-agarose, Dialysis,
Total protein
Yield
Total activity Purification activity" Specific
mg
nmollmin
nmol/min/mg
-fold
%
720 391 22 0.9 0.0017
59.0 98.5 42.1 17.0 9.1
0.082 0.25 1.9 18.9 5,353
1 3 23 230 65,280 .-
100
Substrate concentration,0.5 PM [3H]cAMP.
167 71 29 18
Purification of Particulate “Low Km”cAMP P h o s p h ~ ~ e s t e r ~ e
5800
TABLE I1 S o ~ ~ i ~ i zofa the t ~ nparticulateCAMP p h o s p ~ ~ s t e r ~ e Particulate fractions (12 mg of protein/ml) were prepared as described under “Experimental Procedures” and suspended in 50 mM Tris (pH7.50), with 5 mM M&lz, 1 mM EDTA, 20% (w/v) glycerol, 10 pg/ml antipain and leupeptin, and 1 rg/ml pepstatin at 4 “C, then treated with additions as indicated, and centrifuged at 100,000 X g for 30 min. The percent p h o s p h ~ i e s t e r a ~ s o l u b was i l i ~calculated d as phosphodiesterase activity in the supernatant (100,000 X g)/total phosphodiesterase activity X 100. ~Solubilization conditions Treatment
Incubation, 4 h Sonication, 3 X 30 s Sonicat~on Freeze-thaw, sonication
NaBr
CI3El2
PM
%
500 125 250 500
Sonication 59
Sonication 81
150 150
0.03 1.0 3.0 0.03 1.0
Solubilized phosphodiesterase %
12 30 40 34 69 86 49 57
TABLE 111 Effectsof j n ~ ~ ~ tono CAMP rs ~ y d r o l y by s ~low X, CAMP phosphodiesterase The CAMP phosphodiesterase, obtained by DEAE chromatography, was assayed without or with >5 concentrations of the indicated inhibitors. IC, values were determined graphically. Activity in the absence of inhibitors was 2.1 nmol/min/mg of protein. Compound
IC, FM
Phosphodiesterse inhibitors OPC 3911 OPC 3689 OPC 13135 CI 930 . Milrinone OPC 13013 GI 914 Amrinone RO 20-1724 SH modifiers p-chloromercuribenzoate Iodoacetamide
0.042 0.070 0.185 0.45 0.63 0.730 39.8 52.5 190 0.190 0.75
72
0.8
0.6
0
co
2 0.2
Fraction
FIG. 1. DEAE-cellulosechromatography of solubilized particulate cAMP phosphodiesterase. Material from Step 2 was applied to a DEAE-cellulose column (2.6 X 20 em) equilibrated and washed with buffer A containing 100 mM NaBr (flow rate, 38 ml/h). Phosphodiesterase (PDE) activity was eluted in a gradient from 100 to 400 mM NaBr. Fractions (7.5 ml) were collected, and portions were assayed for phosphodiesterase activity. Shaded area indicates enzyme fractions pooled for the next purification step.
covered cAMP phospho~esteraseactivity (6 preparations), eluted with 170 mM NaBr and was pooled as indicated for further purification. An 8-fold increase of the specific activity with a total recovery of more than 70% was obtained in this step. The enzyme obtained by chromatographyon DEAE-Sephacel was potently inhibited by agents such as p-chloromercuribenzoate (ICso -0.19 p ~ ) which , modify protein sulfhydryl groups (Table III), but was not inhibited by millimolar concentrations of diisopropyl fluorophosphate (data not shown). Although functional sulfhydryl groups may be important for maintenance and/or expression of catalytic activity, dithioerythritol was not included in homogenization or solubilization buffers or during chromatography on DEAE-Sephacel. Inclusion of dithioer~hritolat these early steps was associated with a decreased yieldof phosphodiesterase activity, perhaps secondary to activation of thiol-sensitive and other proteases (21,22,40,42-44).
10
Fraction FIG.2. Chromatography on Sephadex G-200. Material from Step 3 was concentrated to 6.5 ml by dialyzing for 20 h against 1 liter of buffer A containing 20%polyethylene glycol (M*= 1 5 , ~ 2 0 , 0 0 0 ) and 200 mM NaBr and applied to a column (5.3 X 88 em) of sizegraded Sephadex G-200superfine equilibrated and eluted with buffer A containing 10% glycerol and 200 mM NaBr (flow rate, 4.2 ml/h). Fractions (6.2 mi) were collected and portions assayed for phosphodiesterase (PDE) activity. Shaded area indicates enzyme fractions pooled for the next purification step. Inset, plot of K,, versus log M , of standard proteins and phosphodie~terase. I&, 150 kDa; lactic dehydrogenase (LDH), 140 kDa; bovine serum albumin (BSA), 67 kDa; ovalbumin (OV), 43 kDa; soybean trypsin inhibitor (STZ),21 kDa.
Step 4. Gel Filtration on Sephadex G-200-The material from Step 3 was concentrated about 15-foldby dialysis against Buffer A containing 20% polyethylene glycol and 200 mM NaBr andthen applied to and eluted from a column of Sephadex G-200 (Fig. 2). A minor and variable peak of cAMP phosphodiesterase activity, constituting 9-15%of the recovered cAMP phosphodiesterase activity, was eluted at the
Purification of Particulate “Low K,” CAMP Phosphodiesterase void volume of the column and presumably represented an aggregated form of the enzyme. The main peak of enzyme activity, which eluted at a Mr(spp) of100-110 kDa (Fig. 2, inset), was well separated from other proteins with a total yield (including the concentration step) of about 60% and a 10-fold purification (Fig. 2, Table I). Step 5. Affinity Chromatography-In order to identify the compound(s) appropriatefor use as an affinity ligand in purifying the phosphodiesterase, a number of inhibitors (Fig. 3) were tested for their effects on the particulate phosphodiesterase. Cilostamide (OPC 3689) and otherOPC derivatives, i.e. OPC 3911, OPC 13135, and OPC 13013 (Fig. 3), were potent inhibitors of solubilized particulate cAMP phosphodiesterase activity (data notshown). IC, values for inhibition of phosphodiesterase activity after DEAE chromatography ranged from 40 to 730 nM (Table 111). The cardiotonic drugs (40-42) CI 930 and milrinone were also potent inhibitors, whereas the closely related compounds CI 914 and amrinone were less effective (Table 111). RO 20-1724was a rather ineffective inhibitor of enzyme activity (ICw -190 pM). Examination of the structure of the OPC inhibitors, especially cilostamide, OPC 3911, and OPC 13135 (Fig. 3 and Table 111),indicated that, of the 2 N-linked substitutions in the amide moiety of the derivatives, the cyclohexyl substituent was more important to the drugs’ inhibitory action than the second N-linked side chain, which differed in several derivatives. As described in the Miniprint Section and outlined in Fig. 4A, an OPC derivative was, therefore, synthesized in which the N-(2-methyl) substituent of cilostamide was replaced with an N-(2-amino)ethyl side chain (compound 4 in Fig. 4A). The terminalfreeamine was converted to the
II
/
-
5801
isothiocyanate (N=C=S) function which is highly reactive toward primary amines (cf. Miniprint Section). The isothiocyanate-modified derivative (CIT) (compound 5 in Fig. 4A) was coupled to aminoethyl agarose via formation of the thiourea bond (Fig. 4B).The thiourea function is quite stable, allowing storage of the CIT-agarose columns for an extended period of time, as well as repeated use of the same column without significant loss of binding capacity or selectivity for the phosphodiesterase. The CIT-agarose column was usedto furtherpurify enzyme from Step 4 (Sephadex G-200). Before chromato~aphyon the affinity column, pooled material from Step 4 was dialyzed against buffer A without CI3El2 (2 x 1 liter, 4 and 20 h, respectively) (for details cf. Fig. 5 ) . Step 5 resulted in a 300-fold increase of specific enzyme activity with a total yield (including the dialysis and the removal of cAMP by DEAE chromatography) of about 60%. The overall recovery was -18% with a -65,000-fold purification (Table I).Fig. 6 shows an SDS-polyacrylamide gel (silver stained) of the proteins present after steps 2-5 of purification. After affinity chromatography ( E a n e d), a single predominant protein band with an apparent M , = 63,800 & 200 (mean & S.E. from 4 independent d e ~ ~ i n a t i o nwas s ) observed after polyacrylamide gel electrophoresis in SDS under reducing (Fig. 6) or nonreducing conditions (data not shown). This band accounted for >90% of the protein in each of six preparations of purified enzyme, as judged from scanning densitograms of silver-stained gels similar to that seen in Fig. 6, Eane d (see “Experimental Procedures”). It is possible that some minor bands with apparent M , lower than the purified phosphodiesterase (not obvious in Fig. 6, lane d ) represented
OPC 3689 (Cilostarnide)
CH3
81
82
NH2
H
Amrinone
Milrinone
CN
0 H
OH
I
1-METHYL,
,CH~CHCH~CH~
J-ISOBUTYU(ANTHINE (IBMXI
3OPC 5 131
-O-{CH,I~-C-N
-0-ICH2).-C‘ \N-N
i
OPC 1 3 0 1 3
W HJ”&m3
0
CI 9 1 4
CI 930
FIG. 3. Structure of phosphodiesterase inhibitors.
JL
ROLIPRAM (2K62.7111
A
0.04
-3 0.03
I
TMSI I
T v
0
: co
0.02 5OmM CAMP
I H
0.01
-5
0
G o N - c p,c.C,H,, p ~ : W
Agarore
II
H
20
40
60 80 100 120 1 4 0 Volume/ml
FIG. 5. Affinity chromatography on CIT-agarose. Pooled and dialyzed material from Step 4 was applied to anaffinity column (4 ml) (prepared as described in the legend to Fig. 4B)equilibrated in buffer A. The column was washed with 6 volumes of 2 M NaBr in buffer A until Am was undetectable and then with 8 volumes of 100 mM NaBr in buffer A. T o elute enzyme activity, the gel was removed and incubated with 2 volumes of 100 mM NaBr and 50 mM cAMP in buffer A at 4 “C for 5 h. The eluate was collected by transferring the gel back to thecolumn and washing with 3 volumes of elution buffer. To remove CAMP,the eluate was applied to a small (0.4ml) DEAESephacel column, equilibrated with 100 mM NaBr in buffer A. After extensive washing the enzyme activity was eluted with the same buffer containing 300 mM NaBr. The total amount of cAMP phosphodiesterase (PDE) activity eluted with cAMP is represented by the shuded bar.
FIG. 4. A, scheme for the synthesis of the N-(2-isothiocyanato)ethyl derivative of cilostamide (CIT). CIT (compound 5; Nconditions (in slabgels incubated overnight with dithioerythcyclohexyl-l\”[2-(isothiocyanato)ethyl]-4-(1,2-dihydro-2-oxo-6-quinoly1oxy)butyramide)was prepared in three steps from 4-(1,2-dihydro- ritoland subjected to electrophoresis for 45 min prior to 2-oxo-6-quinolyloxy)butyricacid (compound I ) . The carboxylic acid addition of sample in order to remove persulfate and other (compound I ) was condensed with ethylchloroformate to form the oxidants which totally inactivated phosphodiesterase), all mixed anhydride which wasdirectly reacted with N-carbobenzoxy-2- phosphodiesterase activity recovered (-30% of that applied) cyclohexylaminoethane (compound 2, which was synthesized from N- was associated with the single protein-staining band (which carbobenzoxybromoethane and cyclohexylamine) to give the pro- accounted for >90% of the total silver-stained material) (data tected intermediate compound 3 in 50% yield. Deprotection of comnot shown). pound 3 with iodotrimethylsilane in acetonitrile followed by treatment with ethanolic HCl afforded the dihydr~hloridesalt of compound 4 in 72% yield. Treatment of compound 4 with freshly distilled Properties of the Purified Enzyme thiophosgene in a two-phase chloroform aqueous sodium bicarbonate The purified enzyme exhibited normal Michaelis-Menten system resulted in the isothiocyanate (compound 5) in 92% yield. B, scheme for the coupling reaction between CIT ( c o m ~ u n d5) and kinetics with an apparent Km for cAMP of 0.38 ptM and an aminoethyl agarose to form the thiourea linkage with aminoethyl estimated VmaXof 8.5 pmol/min/mg at 30 “C. Corresponding agarose. 40 mg of CIT (compound 5 in A ) , synthesized as shown in A values for cGMP hydrolysis were 0.28 PM and 2.0 pmol/min/ and described in theMiniprint, was dissolved in 20 ml of 50%dioxane at pH 9.0 and incubated with 10 ml of aminoethyl agarose for 18h a t mg, respectively. The pHoptimum for cAMP hydrolysis was room temperature. Excess ligand was removed by washing, and the 7.5. Lineweaver-Burk analysis indicated that cGMP inhibited cAMP hydrolysis in a competitive manner with a Ki of -0.15 gel was equilibrated with buffer A. After use, the affinity gel was extensively washed with 8 M urea, 1M NaBr, and thenwashed further PM (data notshown). The purified enzyme was also sensitive and stored at 4°C in sterile-filtered 50 mM Tris-HC1, pH 7.5, 5 mM to inhibition by eilostamide (ICso,-0.07 PM) andp-chloromerMgC12, 1 mM EDTA. curibenzoate (IC5,,, -0.19 PM) (cf. Table 111). The activity of
proteolyzed fragments of the phosph~esterasesince they were found in variable amounts in many other preparations. In gels stained with Coomassie Blue, the 64-kDa polypeptide was the only visible band. When subjected to polyacrylamide slab gel electrophoresis ( 5 or 6.5%) under nondenaturing
the purified enzyme was not stimulated by Ca2+and calmodulin. The purified phosphodiesterase was stable, retaining>90% activity for at least 2 months in buffer A containing 20% glycerol, 0.03%CI3Ex2, 1 mM d i t h ~ o e ~ h r i t o l -80 a t “C. Glycerol and detergent notonly apparently increased stability but
Purification of Particulate “Low K,” c A M P Phosphodiesterase
5803
220
116977766-
* al
180
0
u
- PDE
al Y
a
140
-
. . E
.-c
43-
E - 100 0
E a
b
c
d
FIG.6. Protein composition of the preparations obtained after the various purification steps.Samples of pooled material from different steps (10-40 pg of protein for lanes a-c; 1 pg for lane d) were subjected to SDS-polyacrylamide gel electrophoresis and thereafter silver stained; lane a, solubilized 100,000X g pellet; lane 6, pooled DEAE-Sephacel enzyme; lane c, pooled Sephadex G-200 enzyme; and lane d, enzyme after the affinity chromatography. Reference proteins indicated by the kDa values are: p-galactosidase, glycogen phosphorylase, transferrin, bovine serum albumin. and ovalbumin. PDE, phosphodiesterase.
>.
c
’5 .-c u m
60
W
n
a
20
0
ins
con -
-
J
Particulate Supernatant Fractions
FIG.7. Insulin stimulationof particulate cAMP phosphodiesterase (PDE)activity. Fat cells were incubated with or without 1or 3 nM insulin for 10 min. Particulate fractions and supernatants were prepared and assayed for cAMP phosphodiesterase activity as described under “Experimental Procedures.” Data are the mean -+ S.E. ( n= 7 experiments inwhich fat cells wereincubated in duplicate or triplicate).
also seemed to prevent or reduce nonspecific binding of dilute solutions of purified enzyme to column matrices or other surfaces. Enzyme activity was stable for several days at 4°C and at least for 1 day at 22 “C in the samebuffer. At 37 “C, however, 40% of enzyme activity was lost after 1 h and 70% after 15 h. TABLE IV The purified cAMP phosphodiesterase (as did phosphodiChromatography on DEAE-Sephucel of solubilized particulate fractions from control and insulin-treated fat cells esterase activity after Step3 (DEAE)) eluted from Sephadex G-200 with a n M,(app) of 100,000-110,000 (cf. Fig. 2). The PI, Particulate fractions were prepared from fat cells incubated withdetermined by isoelectric focusing for the enzyme after chro- out orwith 1 nM insulinfor 10 min, homogenized in buffer A containing 20mM NaBr and 1%C13E12, and centrifuged (100,000 X matography on DEAE or for the purified enzyme, was 4.9-5.0 g, 30 min). Supernatants wereapplied to small (1.0 ml) DEAE(data not shown). Sephacel columns equilibrated in buffer A containing 100 mM NaBr and eluted with 300 mM NaBr in the same buffer. Morethan 90% of Actiuation of the Particulate CAMPPhosphodiesterase during the applied phosphodiesterase activity bound to the DEAE column. Recovery of activities was similarforcontrol and insulin-treated Incubation of Fat Cells with Insulin; Maintenanceof preparations in both the detergent-solubilizedsupernatant fractions Activation throughPartial Purification and in fractions from DEAE-Sephacel. Resultsare presented as total Particulate cAMP phosphodiesterase activity from isolatedpmol/min/ml of packed cells; values in parentheses represent the rat fatcells was solubilizedwith C13E12and NaBr and applied ratio of activity in fractions from insulin-treated cells/control cells. to DEAE-Sephacel. The main peak of cAMP phosphodiester- Similar resultswere obtained in another experiment. cAMP phosphodiesterase ase activity, which accounted for -90% of the total cAMP activity phosphodiesterase activity recovered after DEAE, was the No insulin 1 nM insulin same enzyme that was purified from adipose tissue,as indicated by its identical elution profile from DEAE-Sephacel pmollminlml packedcella ( i e . at -170 mM NaBr(datanotshown, cf. Fig. 1)) and Particulate fraction 96 157 (1.6) inhibition by cilostamidebutnot RO 20-1724 (datanot Detergent-treatedparticulate fraction 126 171 (1.4) Solubilized supernatant 79 119 (1.5) shown). The remaining activity was attributable to a Ca2+/ DEAE eluate 71 102 (1.4) calmodulin-stimulated phosphodiesterase (cf. Fig. 1). Exposure of adipocytesfor10min toinsulin (1-3nM) increasedparticulatecAMPphosphodiesteraseactivity by on the methods of preparing, manipulating, and homogenizing -80% but did not significantly altersoluble activity (Fig. 7). fat cells, effects of insulin were essentially confined to the In three experiments, activation was maximal in cells incu- particulate fraction. The activation by insulin was maintained during solubilibated with1nM insulin (i.e. 64,110,70,58, and40% activation with 0.3, 1.0,3.0,10.0, and 30.0nM insulin). In the experimentzation of the particulate cAMP phosphodiesterase with NaBr presented in Fig. 7, about 40% of the total fat cell cAMP and C13E12 (Table IV) and also, to a large extent, through phosphodiesterase activity (assayed with 0.5 PM [3H]cAMP) partial purification(-2O-fold, in good yield) of the enzymeby was associatedwith the particulate fraction from control cells. chromatography on DEAE-Sephacel (TableIV). As has been Although proportions of cAMP phosphodiesterase activity in previously reported (13-15), incubation of rat fat cells with the supernatant and particulate fraction depended somewhat catecholamines as well as insulin increased particulate cAMP
Pur~ficationof P a r t ~ c u ~“Low t e X,,,c”A ~ ~P ~ s ~ ~ d i e s t e r ~ e
5804
phosphodiesterase activity. Activated particulate cAMP phosphodiesterase from cells incubated with insulin or catecholamines or control particulate preparations was inhibited by cilostamide and cGMP.3
of phosphodiesterase. Since cilostamide, recently found to be a selective and potent inhibitor of type “111-C” or %GMPinhibited” subclass of low K, cAMP phosphodiesterases (20, 24,27,32), was a potent inhibitorof the hormone-responsive particulate low K,,, cAMP phosphodiesterase in 3T3-Ll adipocytes and ratfat cells, we prepared the N-(2-isothiocyanato) DISCUSSION ethyl derivative of cilostamide for use as an affinity ligand to Incubation of intact rat fat cells (12-15) or 3T3-Ll adipo- purify the particulate phosphodiesterase. Umekawa et al. (27) cytes (18) with insulin or agents that increase CAMP (i.e. described an attempt to purify a platelet cAMP pbosphodicatecholamines) results inincreased particulate low K, cAMP esterase with OPC 13135 directly coupled to epoxy-Sepharose, phosphodiesterase activity. These agentspresumably activate but recovery was
