Proc. Nati. Acad. Sci. USA Vol. 76, No. 10, pp. 4903-4907, October 1979

Sequential adsorption-: Combined procedure for purification of calcium-dependent cyclic nucleotide phosphodiesterase (/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 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. The phosphodies- that of the applied preparation. When several samples were terase was eluted with 3 ml of 25 mM BES, pH 7.0/5 mM pooled and subjected to electrophoresis a second time, the EGTA/5 mM MgCl2/O mM NaN3 and concentrated imme- specific activity was roughly doubled. diately by ultrafiltration (Amicon UM-2, 60 psi). Chromatography with CDR-Sepharose. Initial attempts to For preparative sequential adsorption-electrophoresis, the elute bound phosphodiesterase from CDR-Sepharose yielded enzyme was bound to CDR-Sepharose in the presence of Ca2+ variable, often very low, recoveries. Even after extensive as described above. After extensive washing with the loading washing with EGTA and high-ionic-strength buffers, the buffer containing 0.4 M NaCI, the gel was washed with 5-10 Table 1. Recovery of phosphodiesterase activity after vol of reservoir buffer diluted 1:3 and containing 0.2mM CaCI2. preparative polyacrylamide The washed gel, suspended in 2 vol of the same buffer made electrophoresis 15% with glycerol, was layered on a 6% polyacrylamide gel (1.5 Protein, Conditions, X 2.5 cm). After the Sepharose had sedimented, excess buffer Exp. mg mA/V Recovery,* %

was removed, 2 vol of reservoir buffer diluted 1:3 and con- 1 4 50/290 89 taining 2 mM EGTA and 5% glycerol was carefully added, and 2 8 50/290 103 the upper reservoir was filled with buffer. Electrophoresis was 3 19 50/290 90 performed at constant voltage (120-125 V) in a Savant pre- 4 24 50/300 69 parative gel electophoresis apparatus. Fractions (1.5 ml) were 5 24 50/300 67 collected at a rate of 0.18 ml/min with reservoir buffer as the 6 16 50/280 95 eluant. Peak fractions were pooled and immediately concen- 7 8 50/270 112 trated by ultrafiltration (Amicon PM-10, 25 psi). 8 4 35/210 96 Preparation of CDR-Sepharose. CNBr-activated Sepharose 9 22 40/260 82 4B (20 g dry weight) was washed eight times with 400 ml of 1 10 3 40/250 83 mM HCO on a sintered glass funnel and suspended in O.1 M 11 16 50/280 86 NaHCO3, pH 8.5/0.5 M NaCl. Purified CDR (17.5 mg in 25 12 4 75/330 69 ml of the same buffer) was immediately added, and the sus- 13 59 35/210 97 pension was gently mixed for 18 hr at 4°C. The CDR-Sepharose 14 45 60/310 52 was collected by filtration, washed with 500 ml of the same Samples of phosphodiesterase (protein content indicated) were buffer, and suspended in 150 ml of subjected to electrophoresis; 8% gels were used except in Exp. 1 (5%) 1 M ethanolamine-HCI at and Exp. 12(7%). All enzyme preparations had been precipitated with pH 8.0. After 3 hr at room temperature, the product was col- ammonium sulfate and purified on Ultrogel AcA34. In Exps. 1-12, lected on a sintered glass filter and washed with 200 ml of 0.1 anion-exchange chromatography was carried out before gel filtration; M sodium acetate, pH 4.0/1 M NaCl followed by 200 ml of 0.1 in Exps. 12-14 it was not. The use of anion-exchange chromatography M NaHCOg, pH 8.5/1 M NaCl a total of five times. The gel was did not significantly alter the resolution or recovery of enzyme from then washed with 200 ml of water,* suspended in an equal preparative electrophoresis (data not shown). * Percentage of applied activity (assayed with optimal concentrations * No CDR (protein or activity) was detected in the washes. of Ca2+ plus CDR). Downloaded by guest on September 25, 2021 Biochemistry: Kincaid and Vaughan Proc. Natl. Acad. Sci. USA 76 (1979) 4905 CDR-Sepharose retained a substantial amount of bound enzyme (demonstrated by direct assay) and appeared to have a de- creased capacity for binding of phosphodiesterase. In addition, enzyme eluted with EGTA was sometimes insensitive to stim- ulation by Ca2+ plus CDR. Use of PMSF during homogeniza- tion and routine regeneration of the CDR-Sepharose with 6 M 0~~~~~~~~~~~~~~ urea/EGTA/NaCl increased recovery and reproducibility at this step. V The extent of phosphodiesterase adsorption to the affinity j,~ ~ gel depended on the prior treatment of the enzyme applied. 0 When samples of enzyme at different stages of purification _ were applied to identical affinity columns, there was nearly complete binding in the first three applications of phosphodi- esterase from preparations that had been quantitatively de- pleted of CDR, whereas from fractions not completely depleted of CDR a constant lower percentage of the applied activity, inversely related to Ca2+ responsiveness, was bound (Table 2). cU (The extent of activation by Ca2+ is presumably a function of the amount of CDR present in the enzyme preparation.) The 6.o0L specific activities of preparations from Ultrogel AcA34 before and after QAE-Sephadex treatment (which removed _10% of the protein) were identical, and addition of pure CDR to the (,4.5r latter enzyme markedly decreased the amount bound to CDR-Sepharose (data not shown). Thus, complete removal of E endogenous CDR appears to be critical for maximal adsorption of phosphodiesterase to CDR-Sepharose. With the fourth and fifth applications, the percentage of activity not adsorbed in- creased for all preparations, suggesting that column capacity was limiting binding. 1.50 Under optimal conditions, t90% of the enzyme from pre- < parative electrophoresis bound to CDR-Sepharose; 75-80% of this was recovered in the EGTA eluate and was stimulated 5- to 6-fold by CDR plus Ca2+. These preparations of phospho- 4 8 12 16 20 diesterase, however, contained several protein components. Gel slice Although activity was stable on storage at -60°C in 20% FIG. 1. Sequential adsorption-electrophoresis of CDR-depleted glycerol, only 15% remained after 6 hr at 40C, and attempts at brain supernatant using gels of different acrylamide content. Su- further purification led to large losses. pernatant (20 ml; 60 mg of protein) that had been depleted of CDR Sequential Absorption-Electrophoresis. Conditions for with QAE-Sephadex was made 2 mM in CaCl2 and applied to a col- purification of the enzyme directly from umn (0.7 X 4 cm) of CDR-Sepharose. The column was washed with electrophoretic 20 ml of the loading buffer containing 0.4 M NaCl followed by 1:3 CDR-Sepharose were investigated in experiments like that diluted electrophoresis reservoir buffer containing 0.2 mM CaCl2. The shown in Fig. 1. Samples of brain supernatant depleted of CDR CDR-Sepharose was then suspended in 2 vol of the diluted electro- were applied to CDR-Sepharose, which was then washed and phoresis buffer containing 15% glycerol, and portions were transferred layered over gels of different acrylamide concentration. There to the surface of gels to be stained (0.45 ml) or to be assayed for was only one peak of phosphodiesterase activity that migrated phosphodiesterase activity (0.15 ml). Excess buffer was removed, the CDR-Sepharose was overlayered with 0.3 ml of reservoir buffer con- Table 2. Adsorption of phosphodiesterase to CDR-Sepharose taining 2 mM EGTA and 5% glycerol, and reservoir buffer was added. After completion of electrophoresis at 200 V (constant) in a ther- Stage of Relative Phosphodiesterase mostatted bath (1°C), the CDR-Sepharose was removed and gels were enzyme effect not adsorbed, % either stained or divided into slices (3 mm) for assay. Phosphodies- purification of Ca2+* 1 2 3 4 5 terase activity in 4% (0), 5% (0), 6% (-), and 7% (A) gels is plotted. Supernatant 76 + 10 67 66 69 80 99 Ultrogel AcA34 47 i 3 40 52 55 74 96 QAE-Sephadex 2 i 1 7 7 7 22 23 into the gels. This corresponded to one of the fastest migrating Electrophoresis 4 i 2 7 5 8 22 32 protein components. With higher concentrations of acrylamide, For these experiments, the supernatant was applied directly larger amounts of activity remained at the tops of the gels. [without (NH4)2S04 fractionation] to a column of Ultrogel AcA34. When supernatant was used as the starting material, as in Fig. The pooled active fractions were then treated with QAE-Sephadex in the to remove CDR and subjected to preparative electrophoresis. Five 1, there were significant amounts of other proteins region samples (1 and 2 = 0.5 ml, 3 = 0.8 ml, 4 = 1.0 ml, and 5 = 0.9 ml) of of the phosphodiesterase. With CDR-depleted enzyme from each preparation (containing equivalent amounts of phosphodies- preparative electrophoresis or gel filtration, however, no major terase activity) were successively applied to columns (0.5 X 1 cm) of contaminants were detected in the peak phosphodiesterase CDR-Sepharose. After each sample, the column was washed with 2 fractions (data not shown). ml of the application buffer containing Ca2 , and the phosphodies- The results of sequential adsorption-electrophoresis on a terase activity in the effluent was assayed. The percentage of the ac- tivity applied in each successive sample that was recovered in the preparative scale starting with the CDR-depleted enzyme from eluate (i.e., not adsorbed) is recorded. gel filtration are shown in Fig. 2. Two major peaks of material * Activation by Ca2+ alone as a percentage of that produced by Ca2+ that absorbed light at 280 nm preceded the phosphodiesterase plus CDR (mean ± SEM for three experiments). activity. The first apparently represented unpolymerized Downloaded by guest on September 25, 2021 4906 Biochemistry: Kincaid and Vaughan Proc. Natl. Acad. Sci. USA 76 (1979) that the efficiency of elution of the phosphodiesterase from CDR-Sepharose was somewhat increased by electrophoretic desorption. The major problem, however, was not desorption of the enzyme but its separation from other proteins that were E also bound to CDR and eluted with EGTA. These proteins

o 1 $ ~~ 5,gM cAMP W appeared to be separable by electrophoresis, but the phospho- diesterase at this stage was unstable to concentration and further 0.015 1 5 30 purification. With the use of combined CDR-affinity adsorption

C and electrophoretic fractionation, it was possible to elute, ~~~~~~~~~~~~~Econcentrate, and resolve the phosphodiesterase from major 0.01o -20 interfering proteins in a single step. The procedure takes only 0.5 MiM cGMP a few hours and provides extensive purification of the enzyme in high yield. In four experiments, the average recovery of activity bound to the CDR-Sepharose was ;65%. The specific 0.005 -10 activity of these preparations has not been precisely determined because of their very low protein content. Optimal adsorption of phosphodiesterase to CDR-Sepharose required prior removal of CDR from the enzyme fraction, by 0 10 20 30 40 50 either preparative electrophoresis or anion-exchange chro- Fraction matography. Maximal binding of phosphodiesterase to FIG. 2. Preparative adsorption-electrophoresis of phosphodi- CDR-Sepharose was also dependent on the removal of other esterase. The 100,000 X g supernatant was chromatographed on Ul- CDR-binding proteins from the enzyme fraction. Chroma- trogel AcA34, and the phosphodiesterase was depleted of CDR by use on AcA34 decreased the amount of com- of QAE-Sephadex. The enzyme (12 mg of protein in 5 ml) was diluted tography Ultrogel with 3 ml of the loading buffer (final concentration of NaCl, 0.15 M) peting proteins by t50% and removed certain contaminants and applied to a column (0.15 X 2.5 cm) of CDR-Sepharose. This that migrate on electrophoresis in the region of phosphodies- preparation of CDR-Sepharose contained 3-4 times as much CDR terase activity. Such a preliminary step is therefore important as that described in Experimental Procedures. After electrophoresis, to increase the operational capacity of the CDR-Sepharose for samples of eluted fractions were assayed in the presence of 0.5 mM phosphodiesterase as well as to remove proteins that may be Ca2+ and CDR (0.2 ,ug): 0, 5-,ul samples assayed with 0.5 gM cGMP; more difficult to separate at a later stage. a, 10-,ul samples assayed with 5 ,uM cAMP. The sequential adsorption-electrophoresis procedure de- scribed here differs from that of re- acrylamide, because it was also observed in control (no sample) "affinity electrophoresis" and Kocourek in which the gels. There was very little 280 nm absorption by the fractions ported by Horiejsi (21, 22) affinity containing phosphodiesterase. Most of the protein eluted from ligand is covalently attached to polyacrylamide and polymer- CDR-Sepharose remained in the gel and could be visualized ized in the gel tube, forming a separate region of "affinity gel." after The with A mixture of proteins is electrophoresed through the gel and staining. phosphodiesterase activity assayed the is with that in a without the either 0.5 cGMP or 5 cAMP eluted as a sym- protein pattern compared gel ,gM ,gM single Whereas this method can be useful for metrical peak, and hydrolysis of both substrates was stimulated specific ligand. analysis of protein mixtures in which the components that interact with 5- to 6-fold CDR Ca2+. In this recovery of by plus experiment, the material are in visible it is not phosphodiesterase activity was -75%; in other experiments, affinity present quantity, recoveries from 60 to 75%. readily suited to detection or purification of proteins that exist ranged in small amounts relative to the total Stability of Enzyme Activity. Like the enzyme from applied. Sequential affinity CDR-Sepharose affinity chromatography, that from sequential adsorption and electrophoretic fractionation allows for the treatment of large quantities of crude mixtures, extensive adsorption-electrophoresis was unstable at 40C. However, when with different with buffers diluted with an volume of 40% and washing media, equilibration equal glycerol immediately suitable for and reuse of the matrix after frozen, the enzyme was stable for at least 7 months at -60°C. electrophoresis, affinity was increased 400-600% electrophoresis. The basal activity of these preparations The method described should be useful in the had no and generally by CDR plus Ca2+ (Ca2+ alone significant effect) of macromolecules that interact with was 100 not purification affinity inhibited 50-70% by ,uM spermine (data shown). in cases in which the These responses were quantitatively the same as those of the media, perhaps particularly component of interest is labile. Both the type of affinity interaction (bio- enzyme preparation used for the sequential adsorption-elec- specific ligand, matrix) and the mode of electrophoretic sepa- trophoresis purification. ration can be varied to suit the demands of the particular iso- DISCUSSION lation problem. Preliminary results indicate that both adsorp- tion- and adsorption-isotachophoresis are Partial purification of cGMP phosphodiesterase by polyacryl- effective in separation of phosphodiesterase from other amide gel electrophoresis has been reported, although recovery CDR-binding proteins. Current studies demonstrate that this of enzyme activity was only about 15% (18). In our studies, the method can be used analytically to screen prior fractionation Ca2+_ and CDR-dependent phosphodiesterase was reprodu- steps for those that permit maximal adsorption of the phos- cibly purified by preparative electrophoresis in good yield and phodiesterase to the adsorbent. Such a strategy, coupled with over a wide range of conditions (e.g., protein load, current, judicious selection of electrophoretic conditions, should make enzyme preparation applied). The advantages of electropho- the procedure described directly applicable to the separation retic desorption for elution of antibodies bound to immu- and purification of several other proteins that interact with noadsorbents have been pointed out by Morgan et al. (19). CDR. Electrophoretic desorption also appears to be useful for recovery of ligands that cannot be eluted from dye affinity columns with We thank Dr. Vincent C. Manganiello for helpful discussions, Dr. salt or specific nucleotides (20). We found in early experiments Charles E. Odya for assistance in the preparation of CDR, Mrs. D. Downloaded by guest on September 25, 2021 Biochemistry: Kincaid and Vaughan Proc. Natl. Acad. Sci. USA 76 (1979) 4907 Marie Sherwood for expert secretarial assistance, and an anonymous 12. Miyake, M., Daly, J. W. & Creveling, C. R. (1977) Arch. Biochem. reviewer for bringing to our attention refs. 19 and 20. Biophys. 181,39-45. 13. Jean, D. H., Albers, R. W. & Koval, G. J. (1975) J. Biol. Chem. 1. Cuatrecasas, P., Wilchek, M. & Anfinsen, C. B. (1968) Proc. Nati. 250, 1035-1040. Acad. Sci. USA 61, 636-643. 14. Kincaid, R. L., Manganiello, V. C. & Vaughan, M. (1979) J. Biol. 2. Parikh, I., March, S. & Cuatrecasas, P. (1974) Methods Enzymol. Chem. 245, 4970-4973. 34,77-102. 15. Manganiello, V. C. & Vaughan, M. (1973) J. Biol. Chem. 248, 3. O'Cana, P., Barry, S. & Griffin, T. (1974) Methods Enzymol. 34, 7164-7170. 108-126. 16. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 4. Klee, C. B. & Krinks, M. H. (1978) Biochemistry 17, 120-126. R. 5. Wallace, R. W., Lynch, T. J., Tallant, E. A. & Cheung, W. Y. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, J. (1979) J. Biol. Chem. 254, 377-382. (1951) J. Biol. Chem. 193,265-275. 6. Yazawa, M. & Koichi, Y. (1978) J. Biochem. (Tokyo) 84, 18. Davis, C. W. & Kuo, J. F. (1977) J. Biol. Chem. 252, 4078- 1259-1265. 4084. 7. Dabrowska, R. & Hartshorne, D. J. (1978) Biochem. Biophys. Res. 19. Morgan, M. R. A., Kerr, E. J. & Dean, P. D. G. (1978) J. Steroid Commun. 85,1352-1359. Biochem. 9, 767-770. 8. Adelstein, R. S., Conti, M. A., Hathaway, D. R. & Klee, C. B. 20. Dean, P. D. G. & Watson, D. H. (1978) in Proceedings of First (1978) J. Biol. Chem. 253, 8347-8350. International Symposium on Affinity Chromatography, eds. 9. Westcott, K. R., La Porte, D. C. & Storm, D. R. (1979) J. Biol. Hoffmann-Ostenhof, O., Breitenbach, N., Koller, N. F., Kraft, Chem. 254, 204-208. D. & Scheiner, O. (Pergamon, New York), pp. 25-38. 10. Klee, C. B., Crouch, T. H. & Krinks, M. H. (1979) Biochemistry 21. Horejsi, V. & Kocourek, J. (1974) Biochim. Blophys. Acta 336, 18,722-729. 329-37. 11. Watterson, D. M. & Vanaman, T. C. (1976) Biochem. Biophys. 22. Horeisi, V. & Kocourek, J. (1974) Methods Enzymol. 34, 178- Res. Commun. 73,40-46. 181. Downloaded by guest on September 25, 2021