Proc. Nati. Acad. Sci. USA Vol. 76, No. 10, pp. 4903-4907, October 1979 Biochemistry Sequential adsorption-electrophoresis: Combined procedure for purification of calcium-dependent cyclic nucleotide phosphodiesterase (affinity chromatography/calcium-dependent regulatory protein/calmodulin) RANDALL L. KINCAID AND MARTHA VAUGHAN Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20205 Communicated by C. B. Anfinsen, July 5, 1979 ABSTRACT A procedure for combined sequential affinity EXPERIMENTAL PROCEDURES adsorption-electrophoresis has been devised. Its use for the Preparation of the Phosphodiesterase Fractions. Fresh rapid purification of a calcium-dependent cyclic nucleotide in 2 vol phosphodiesterase from bovine brain in high yield is described. bovine brain was homogenized in a Waring Blendor In this procedure, proteins bound to a solid phase of calcium- of 20 mM potassium phosphate, pH 7.0/1 mM EDTA/1 mM dependent regulatory protein (CDR) linked to Sepharose 4B ethylene glycol bis(3-aminoethyl ether)-N,N,N'N'-tetraacetic were electrophoretically eluted, concentrated, and separated, acid (EGTA)/1 mM NaN3/phenylmethylsulfonyl fluoride thus avoiding the large losses in activity incurred during at- (PMSF) at 75 ,g/ml. For the experiments shown in Table 1, tempts to purify further the phosphodiesterase eluted by con- the homogenate was centrifuged at 10,000 X g for 20 min. The ventional means. The highly purified phosphodiesterase pre- supernatant was made 50% saturated with solid (NH4)2SO4 and pared by this method was stable for months at -60'C in the brought to pH 7.0 with NH40H. After 4 hr at 4°C, the pre- presence of glycerol. It has a higher affinity for cyclic GMP than X for cyclic AMP, and hydrolysis of both substrates is stimulated cipitate was collected by centrifugation (10,000 g, 20 min) 5- to 6-fold by calcium plus CDR. Factors that influence ad- and suspended in 50 mM Tris acetate, pH 6.0/1 mM EGTA/1 sorption of the enzyme to CDR-Sepharose and selection of op- mM MgCl2/1 mM NaN3/PMSF (75 ,g/ml), and the suspension timal conditions for electrophoresis were investigated. Se- was centrifuged (10,000 X g, 20 min). The supernatant was quential adsorption-electrophoresis should be generally useful dialyzed against 10 vol of the Tris acetate buffer (10 hr, two in the purification of macromolecules for which affinity ad- changes of buffer) and was applied to a column of DEAE- sorbents are available. The procedures described here could be Sephadex or QAE-Sephadex A-25 equilibrated in the Tris ac- directly applicable to the purification of proteins that, like the etate buffer. The column was eluted with a linear gradient phosphodiesterase, interact with CDR. (0-0.6 M) of NaCl or with 0.25 M NaCl in the same buffer. The first introduced Cuatrecasas et enzyme was concentrated [Amicon UM-2 membrane, 60 psi Affinity chromatography, by (414 kPa)] before application to a column of Ultrogel AcA34 al. (1), offers a means of isolating macromolecules by virtue of equilibrated with 25 mM 2-[bis(2-hydroxyethyl)amino]eth- their biospecific interactions. Despite the potential of this anesulfonic acid (BES), pH 7.0/1 mM EGTA/1 mM MgCl2/1 technique, however, problems such as stability of the matrix- mM NaN3/PMSF (75 mg/ml). Fractions representing the peak bound ligand (2), nonspecific adsorption of proteins on the of phosphodiesterase activity (t65% of that applied) were matrix (3), variable recovery, and instability of isolated proteins pooled and concentrated by ultrafiltration (Amicon UM-2, 60 have in some instances limited its usefulness. Calcium-depen- psi). dent regulator protein (CDR) linked to Sepharose has been used For the experiments shown in Fig. 1, the 10,000 X g super- to purify several proteins that participate in calcium-regulated natant was centrifuged at 100,000 X g for 1 hr, the supernatant cellular processes (4-9), including a cyclic nucleotide phos- made 0.25 M in NaCl was applied to a column of QAE-Seph- phodiesterase (10-12). In all cases, the phosphodiesterase ac- adex, and the effluent containing the enzyme was collected. For tivity was extremely labile after the CDR-related step. We and the experiments in Table 2 and Fig. 2, the 100,000 X g super- others (4, 11) have found that, after CDR-affinity chromatog- natant was applied to a column (5 X 92 cm) of Ultrogel AcA34. raphy, phosphodiesterase preparations still contain several The eluted enzyme was concentrated (:5 mg of protein per contaminating proteins. We describe here a combined proce- ml), made to 0.25 M NaCl, and applied to a column (bed vol- dure termed "sequential adsorption-electrophoresis," in which ume equal to sample volume) of QAE-Sephadex equilibrated adsorption of phosphodiesterase to CDR-Sepharose is followed with the gel filtration buffer containing 0.25 M NaCl. The by direct electrophoretic elution and separation of the proteins column was washed with 1.5 vol of the same buffer. The com- bound to the affinity gel. The combined approach results in bined eluate and wash contained 90-95% of the applied phos- substantial purification of a high-affinity cyclic GMP (cGMP) phodiesterase, and its activity was not stimulated by calcium phosphodiesterase in high yield in a single step. This general alone. method should be applicable to the purification of other mac- Preparative polyacrylamide gel electrophoresis in an appa- romolecules for which affinity adsorbents are available. Abbreviations: CDR, calcium-dependent regulatory protein; cAMP, The publication costs of this article were defrayed in part by page adenosine 3',5'-cyclic monophosphate; cGMP, guanosine 3',5'-cyclic charge payment. This article must therefore be hereby marked "ad- monophosphate; EGTA, ethylene glycol bis(3-aminoethyl ether)- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate N,N,N',N',-tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; this fact. BES, 2-[bis(2-hydroxyethyl)aminojethanesulfonic acid. 4903 Downloaded by guest on September 25, 2021 4904 Biochemistry: Kincaid and Vaughan Proc. Natl. Acad. Sc.i. USA 76 (1979) ratus designed by Wayne Albers (13) was carried out with a gel volume of 25 mM BES, pH 7.0/1 mM MgCl2/1 mM EGTA/5 buffer of 0.15 M Tris.HCl, pH 8.8/5 mM MgSO4/3 mM NaBr. mM NaN3, and stored at 40C. CDR-Sepharose was reused at Polymerization of the 8% resolving gel (3.5 cm; 25 ml) and 3% least four times for affinity chromatography or sequential ad- stacking gel (0.5 cm; t4 ml) was allowed to take place for 2 hr. sorption-electrophoresis with no apparent change in properties. The acrylamide mixture contained NN'-methylenebisacryl- After each use, it was washed with and stored in 6 M urea/10 amide in a ratio of 1:40 parts of acrylamide, and polymerization mM EGTA/0.8 M NaCl (final pH 4.5). was initiated by the addition of 1/50th vol (of the total gel Phosphodiesterase Assay. cGMP phosphodiesterase activity mixture) of ammonium persulfate (15 mg/ml) followed by was assayed at 30'C in a total volume of 0.3 ml containing 50 1/625th vol of N,N,N',N'-tetramethylethylenediamine. The mM BES (pH 7.0), 0.1 mM EGTA, 8 mM MgCl2, 0.3 mM di- preparative gel was electrophoresed in the gel buffer after thiothreitol, ovalbumin (170 Ag/ml), and 0.5 MAM [3H]cGMP application of 3 ml of 0.005% bromophenol blue in 10% (vol/ (n18,000 cpm). Where indicated, maximally effective amounts vol) glycerol at a current of 50 mA (120-130 V). After elution of CDR (0.2 ,g) and Ca2+ (0.5 mM) were also present. Proce- of the dye, the upper and lower buffers were replaced with dures are described in detail elsewhere (14, 15). Protein was reservoir buffer [25 mM Tris/200 mM glycine, pH 8.3/5 mM estimated by modification of the Coomassie G-250 dye binding MgSO4/3 mM NaBr/1 mM NaN3/0.2 mM EGTA/PMSF (75 assay (16) or as described by Lowry et al. (17) with a gamma ,ug/ml)] and the compartment above the stacking gel was filled globulin protein standard. Values obtained by the two methods with reservoir buffer diluted 1:3. Concentrated enzyme (2-3 were comparable. Analytical gels were prepared as described ml), previously equilibrated with the diluted reservoir buffer, for preparative gels and stained with Coomassie G-250 in 12% was made 10% with glycerol and applied to the gel. Electro- trichloroacetic acid. phoresis was performed at a constant current of 35-50 mA Materials. The sources of reagents and materials and puri- (2100-30 V) and fractions (nt3 ml) were collected every 10 min. fication of CDR are described elsewhere (14). Peak phosphodiesterase fractions were pooled and immediately concentrated by ultrafiltration (Amicon UM-2, 60 psi) to a final RESULTS protein concentration of 1-2 mg/ml. This enzyme preparation Preparative Polyacrylamide Gel Electrophoresis. Table was not activated by 0.5 mM CaCl2. 1 shows the recoveries of phosphodiesterase activity after For chromatography on CDR-Sepharose (Fig. 1), the enzyme electrophoresis in 14 consecutive trials with different amounts (1-2 mg/ml) was made 2 mM in CaCl2 and applied to a column of applied protein and current. With current of _50 mA, re- (0.5 X 2.5 cm) of CbR-Sepharose equilibrated with 25 mM coveries were occasionally low; an applied current of 40 mA BES, pH 7.0/2 mM CaCl2/1 mM EGTA/5 mM MgCl2/1 mM with protein loads of st40 mg appeared to be optimal. The NaN3. The initial eluate was reapplied to the column which was specific activity of the pooled peak phosphodiesterase fractions then washed with 2 ml of the loading buffer followed by 4- ml (representing 85-90% of the recovered activity) was 3-4 times of the same buffer containing 0.4 M NaCl.