186,8-13

ANALYTICALBIOCHEMISTRY

(1990)

A Microassay for Mammalian Synthetase’ Bruno Antonsson,2

Julio Barredo,

Folylpolyglutamate

and Richard

G. Moran3

Departments of Biochemistry and Pediatrics, University of Southern California, and Division Hematology/Oncology, Children’s Hospital of Los Angeles, Los Angeles, California 90027

Received

October

l&l989

A new assay for the enzyme folylpoly-y-glutamate synthetase (FPGS) that offers significant advantages over other published procedures has been developed. This assay is based on the addition of high specific activity [3H]glutamic acid to (6-S)-tetrahydrofolate followed by trapping of the labeled tetrahydropteroyldiglutamate product as a covalently bound macromolecular complex by the addition of formaldehyde, fluorodeoxyuridylate, and pure bacterial thymidylate synthase. This complex is then separated from excess labeled glutamic acid by centrifugal elution of a l-ml Sephadex G-50 column. The assay was found to be useful for the measurement of FPGS on small tissue samples and is amenable with the assay of FPGS in cell sonicates. Typically, blank values of 100-200 cpm are seen with a signal normally more than 10 times higher. Analysis of 20-30 samples can be accomplished in less than 90 min. As a result, this assay has proven useful for detection of enzyme in elution fractions from chromatographic columns. 0 1990 Academic P~.SS, h.

The intracellular folate cofactors exist as predominantly or exclusively folylpoly-y-glutamates in mammalian tissues (reviewed in Refs. (1,2)). The enzyme responsible for the synthesis of these compounds catalyzes the addition of several moles of L-glutamic acid to the y-carboxyl group of a wide variety of folates and folate analogs (3-6). This enzyme, folylpoly-y-glutamate syn-

1 Supported in part by Grant CA-27605 from the DHHS. R.G.M. is a Scholar of the Leukemia Society of America; this award was funded by the Scott Helping Hand Fund. ‘Present address: Glaxo Institute for Molecular Biology, S.A., Route des Acacias 46,121l Geneva 24, Switzerland. ‘To whom correspondence should be addressed at University of Southern California Cancer Center, Cancer Research Laboratories, 1303 N Mission Road, Los Angeles, CA 90033. 8

of

thetase (FPGS),4 has proven difficult to study in mammalian tissues for three reasons: it is reasonably unstable, it is present in even relatively rich tissues at very low levels (about one part in 80,000-400,000 of soluble protein), and the assays for this activity are somewhat cumbersome and time-consuming. Polyglutamation of folates circumvents the loss of folates from mammalian cells that is mediated by a low capacity but highly efficient membrane transport system. This function of FPGS is, in fact, essential for the survival of mammalian cells, as evidenced by the observation that somatic cell mutants deficient in FPGS activity cannot maintain intracellular pools of folate cofactors and, as a result, are not viable except in medium supplemented with thymidine and purines (7). This suggests that FPGS is a potential target for cytotoxic chemotherapy of proliferative diseases. In addition, the formation of polyglutamates of the antineoplastic drug methotrexate appears to be centrally involved in its cytotoxicity (8). The folate cofactors (9) and folate antimetabolites (10) bind tighter to the folate-dependent enzymes when they are in polyglutamate forms. Hence, interest in FPGS is substantial because of the apparently critical role that polyglutamation plays both in normal folate metabolism and in the cytotoxicity and, perhaps, in the selective cytotoxicity of a number of folate antimetabolites used in cancer chemotherapy. A major obstacle for the study of mammalian FPGS has been the characteristics of the assays for FPGS presently available. These techniques rely on either column chromatography (3,4) or charcoal adsorption (5,ll) to separate free L-[3H]glutamic acid from [3H]glutamate incorporated into a folyloligoglutamate. These assays are tedious and often not sensitive enough for the very low levels of FPGS present in mammalian tissues. The ’ Abbreviations used: FPGS, folylpolyglutamate FdUMP, 5-Auoro-2’-deoxyuridine-5’-monophosphate; rahydrofolic acid; SDS-PAGE, sodium dodecyl amide gel electrophoresis.

synthetase; H,PteGlu, tetsulfate-polyacryl-

0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

MICROASSAY

FOR

FOLYLPOLYGLUTAMATE

length of time needed to process the number of samples generated by column chromatographic procedures using existing FPGS assays has impaired attempts at purification of this enzyme. Finally, at least one of these procedures, namely, the charcoal adsorption assay, cannot be used for direct measurement of mammalian FPGS on unpurified cell cytosolic protein (11). We have developed a novel assay procedure for FPGS activity that does not have these limitations. This assay has been found useful for the localization of FPGS in column fractions and should have utility for the assay of enzyme in small amounts of tissue, e.g., cytosols of cultured cells or biopsy specimens. MATERIALS

Materials

AND

METHODS

SYNTHETASE

9

mined for each preparation by counting a portion of the buffer under the same conditions as used for counting the samples. Solution C. 1% &mercaptoethanol in water. Solution D. 40 X 1O-6M (6-S)-tetrahydrofolate in solution C. The (6-S)-tetrahydrofolate was stored as a lyophilized powder under dry nitrogen in the dark at -20°C. A fresh solution was prepared every day by dissolving the contents of one ampoule (40 nmol) in 1 ml of solution C. FPGS assay substrate solution. A 1:l mixture of buffer B and solution D. FPGS assay blank solution. A 1:l mixture of buffer B and solution C. Thymidylate synthase solution. 30 mM Na2HP04, pH 7.2,8 mM P-mercaptoethanol, 10 mM formaldehyde, 2 pM FdUMP, 0.1 mg/ml bovine serum albumin, and 0.3 pM thymidylate synthase. When kept on ice, this solution may be used for up to 1 week. As each molecule of thymidylate synthase has two active sites, each of which is capable of forming a complex with 5,10-methylenetetrahydrofolate and FdUMP, the total tetrahydrofolate binding capacity of this solution was 0.6 pM so that each assay mixture had 60 pmol of binding sites. The molarity of thymidylate synthase stock solutions was determined either by titration of active sites with [3H]FdUMP (12) or from an activity measurement assuming 3.4 IU/mg thymidylate synthase at 25°C (12).

Sephadex G-50 (20-80 pm) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden), disposable tuberculin syringes (stock No. 9602) were from BectonDickinson (Rutherford, NJ), 5-fluoro-2’-deoxyuridine5’-monophosphate (FdUMP), formaldehyde, ATP, and L-glutamic acid were from Sigma Chemical Co. Inc. (St. Louis, MO), and miniature polyethylene scintillation vials were from Packard Instrument Co. (Downers Grove, IL). The scintillation cocktail used was BudgetSolve from Research Products International, Corp. (Mount Prospect, IL). [3,4-3H]Glutamic acid was purchased from ICN Radiochemicals (Irvine, CA) and was used without purification. (6-S)-Tetrahydrofolate was prepared from folic acid via dihydrofolate by the enzyMouse Liver FPGS Preparations matic procedure as previously described (12). ThymidylSwiss female mice were sacrificed and livers perfused ate synthase was prepared either from Lactobacillus casei in situ and removed as previously described (11). After resistant to methotrexate (13) (a kind gift of Dr. Bruce Dunlap of the University of South Carolina) or from a centrifugation of a homogenate at 160,OOOgfor 1 h, the strain of Escherichia coli bearing a plasmid (14) which supernatant fraction was brought to 30% saturation allows expression of the L. casei thymidylate synthase (a with (NH&SO., and this precipitate was washed and generous gift of Dr. Daniel Santi of University of Cali- stored under 50% (NH&SO4 at -25°C. Enzyme was used either as a freshly prepared high speed supernatant fornia at San Francisco). Purification of enzyme from either source by (NH&SO, precipitation and step elu- fraction or as a 30% (NH&SO, precipitate dissolved in tion of a phosphocellulose column (15) resulted in an en- buffer A and dialyzed for 24 h against buffer A. zyme preparation that was >90-95% pure as judged by SDS-PAGE. Determination of Protein Solutions

Protein concentration was determined either by the Hartree (16) modification of the Lowry method or spectrophotometrically by measuring the absorption at 228.5 and 234.5 nm. The difference in absorption at these two wavelengths was multiplied by 317.5 to give the protein concentration in micrograms per milliliters (17). These two methods agreed within experimental error when directly compared on the same samples.

Buffer A. 50 mM Tris-HCl, pH 8.5,lO mM MgClz, 2 dithiothreitol, 10 mM phenylmethylsulfonyl fluoride. Buffer B. 800 mM Tris-HCl, pH 8.5 (25”C), 40 mM MgC&, 120 mM KCl, 20 mM ATP, and 4 mM L-[~H]glutamic acid. Sufficient L-[3H]glutamic acid, obtained as a solution from the manufacturer, was transferred to a tube and dried under a stream of N,; the other compo- Preparation of Spun-Columns nents were added to the dry isotope. This buffer was prepared and stored at -25°C. The specific activity was Sephadex G-50 (20-80 pm mesh size) was suspended around 1000 cpm/pmol of glutamic acid and was deterin 5 mM Tris-HCl, pH 8.5, containing 1 mM NaN,. The mM

10

ANTONSSON,

BARREDO,

suspension was heated to 100°C for 5 min, left at room temperature for 24 h, and then stored at 4°C. The pistons were removed from l-ml plastic tuberculin syringes and a small amount of glass wool was compressed in the bottom of each syringe to a total volume of 0.05-0.10 ml. The syringes were placed in 15-ml conical centrifuge tubes so that the syringes were hanging from the top of the tubes. The suspension of Sephadex was added and allowed to sediment to a gel under gravity. The columns were topped off with gel until a cone of gel extended over the top of the syringes to a height of 0.51 cm. The syringes were then centrifuged in these centrifuge tubes in a Beckman J-6 centrifuge for 2.0 min at 2400 rpm (1OOOg). Syringes with less than 0.8 ml of packed gel were discarded.

Basic Assay Procedure The assay consisted of two consecutive reactions (Fig. 1). During the first reaction, any FPGS present in a sample catalyzes the addition of [3H]glutamic acid to the ycarboxylic acid group of tetrahydrofolate. The total volume of this reaction was 5.0 ~1. FPGS substrate solution (2.5 ~1) and sample solution (2.5 ~1) were mixed in 0.5or 1.5-ml Eppendorf tubes on ice. The final concentrations in the reaction mixture were 200 mM Tris-HCl, pH 8.5 (25”C), 10 mM MgCIB, 30 mM KCl, 5 mM ATP, 1 mM L-[3H]glutamic acid, and 10 PM tetrahydrofolate. The incubation was started by transferring the tubes to a 37°C water bath. The standard incubation time was 15 min. At the end of the first incubation, the samples were transferred to ice and 100 ~1 of thymidylate synthase solution was added. During the second incubation, tetrahydrofolate was nonenzymatically converted to 5,10methylenetetrahydrofolate and a covalent complex was formed among 5,10-methylenetetrahydrofolate, FdUMP, and thymidylate synthase. Excess FdUMP and thymidylate synthase over folate were used to ensure that all tetrahydrofolate and [3H]tetrahydropteroyldiglutamate5 in the reaction mixture were bound in a covalently bound macromolecular complex. After 30 min of incubation at 37”C, the second reaction was stopped by transferring the tubes to ice. The high molecular weight complex containing the [3H] tetrahydropteroyldiglutamate was separated from free L-[3H]glutamic acid by passing the reaction mixture through a Sephadex G-50 spun-column. The total reaction mixture (105 ~1) was applied to a prespun Sephadex column. Miniature scintillation vials were placed in the wells of a 14-place 15ml centrifuge tube holder for a Beckman J-6 centrifuge. A plexiglass plate was constructed which fit over the top 5 Under conditions of limited (~10%) near saturating pteroylmonoglutamate have previously determined that product tamate (18).

substrate consumption and substrate concentrations, we was exclusively pteroyldiglu-

AND

H4PteGlu

MORAN

+ [3H]Glu

HqPteGlu([3H]Glu), ----->

FPGS, ATP, Mg2+ -------------------a HqPteGlu([3H]Glu),

+ H2CO

+ TS + FdUMP

------>

TS.FdUMP.5,10-CH2-HqPteGlu([3H]Glu)n FIG.

1.

Basis

of microassay

for FPGS.

of each adapter and had holes drilled that corresponded to the centers of each position in the adapter. The syringes were hung into the holes in the plexiglass plate so that the tips of the syringes were inside of the scintillation vials and the wings on the syringes supported the syringes on the plexiglass. The columns were centrifuged for 5 min at 400 rpm followed by 2.0 min at 2400 rpm (1OOOg). Scintillation liquid (5 ml) was added to the eluate and the tritium in the samples was determined by liquid scintillation counting. Enzyme activity was calculated and expressed as international units (IU), i.e., micromoles of glutamic acid incorporated into tetrahydropteroyldiglutamate per minute. Modification of Basic Assay for Use on Crude Protein Fractions The total volume of the first incubation was increased to 10 ~1. Sample protein (2.5 ~1) was added to FPGS substrate solution (5 ~1) and 2.5 ~1 of buffer A or a similar buffer. Prior to addition of cytosolic protein fractions for assay, it is recommended that protein be desalted by Sephadex chromatography or passage of small samples through a centrifugally eluted Sephadex G-50 column (see Results). RESULTS The principle of this assay is illustrated in Fig. 1. FPGS present in protein samples catalyzes the reaction of high specific activity [3H]glutamic acid with ATP and tetrahydrofolate to form [3H] tetrahydropteroyldiglutamate in an initial reaction carried out at pH 8.5 in a small volume (5-10 ~1). Thereafter, product and excess unlabeled tetrahydrofolate are both trapped as a covalently bound, macromolecular complex in a second reaction run in 100 ~1 at neutral pH in the presence of bacterial thymidylate synthase, fluorodeoxyuridylate, and formaldehyde. (Because of the dilution of the reactants and the very low activity of mammalian FPGS at neutral pH, the FPGS reaction was effectively quenched by the addition of thymidylate synthase solution.) The thymidylate synthase-bound [3H]tetrahydropteroyldiglutamate is then quickly separated from excess unreacted [3H]glutamic acid using a centrifugally eluted l-ml column of Sephadex. The sensitivity of this assay is a result of the efficiency of such a “spun-column” separa-

MICROASSAY

psr:

““i; :ti;*

FOR

FOLYLPOLYGLUTAMATE

v Blank,

- H4PteGlu

Sample

>3,000,000

>3,000,000

638.060

I

584,990

24,540

45,735

5,105

19,025

1,070

8,540

360

3,285

116

780

5.z

Eluate

142

99

3255

FIG. 2. Separation of excess [“Hlglutamic acid substrate from thymidylate synthase-bound [3H]tetrahydropteroyldiglutamate on spun-columns of Sephadex. Ammonium sulfate-precipitated mouse liver protein was incubated with the components of the FPGS reaction with and without the addition of tetrahydrofolate for 15 min and the [3H]tetrahydropteroyldiglutamate formed was trapped as a macromolecular complex with thymidylate synthase. Reaction mixes (105 ~1) were applied to l-ml Sephadex columns and the columns were centrifugally eluted. Each column bed was cut into 0.1.ml segments following centrifugation, and the radioactivity in each fraction was determined.

tion of protein-bound radioactivity and excess substrate, which is illustrated by the data of Fig. 2. Two reaction mixtures containing mouse liver FPGS and either a complete reaction mixture or a mixture lacking tetrahydrofolate were incubated and processed as described under Materials and Methods. After centrifugal elution of these samples through columns of Sephadex packed in l-ml syringes, the bed of resin was physically cut into O-l-ml fractions and the amount of radioactivity in each fraction was determined. Of a total of 6.25 &i of tritium in each reaction, almost all was present in the top two O.l-ml fractions. However, a full 0.9-1.0 ml of resin was required to reduce the background to the point needed for the low levels of FPGS commonly found in mammalian tissues. The background shown in Fig. 2 was typical; for example, in an experiment in which five determinations of FPGS activity were made on a single sample and on the same sample in the absence of tetrahydrofolate, raw data on the complete mixture gave 1801 k 38 cpm, while the blanks were 155 + 12 cpm. The specific activity used was 1100 cpm/pmol of [3H]glutamic acid. Despite this low background, recovery of the preformed ternary complex was found to be 92-96% in a series of experiments under the conditions used for this assay procedure. However, minor changes in the method of packing of the Sephadex spun-columns was found to result either in incomplete recovery or in higher background values. Alternative methods of separation of free and proteinbound [3H]glutamic acid (11,19) resulted in a substantially increased blank value in this assay.

11

SYNTHETASE

Some characteristics of this assay when used for FPGS partially purified from mouse liver by (NH&SO, precipitation are illustrated by the data of Fig. 3. The level of FPGS activity was found to be 27 pIU/mg protein from the slope of a plot of product vs protein/assay (Fig. 3A); this value agreed with simultaneous assays on this same sample using the charcoal adsorption procedure (11). With a 15min incubation, FPGS activity was four times background using 1 yg of this protein source in this new procedure, while this level of signal would require 75 times more enzyme and a l-h incubation using the charcoal adsorption assay due to the difference in specific activity that was compatible with a reasonable blank value, The reaction catalyzed by this source of FPGS was linear for 30-40 min at a protein content on this range (Fig. 3B). As a compromise between speed of assay and sensitivity, an incubation time of 15 min is recommended for assay of the FPGS content of column fractions. Figure 4 shows the elution of caIf thymus FPGS from a column of Blue-Sepharose as detected by this assay. Enzyme activity was quantitatively retained

pg protein/assay

o0

20

40 Time,

60

mln

FIG. 3. Linearity of this microassay with protein content (A) and with time (B). Increasing amounts of ammonium sulfate-precipitated mouse liver protein were added to assays that were incubated for 15 min in A. In B, the first reaction shown in Fig. 1 was allowed to proceed for different times in the presence of either 10 or 22 /*g of this same protein sample. Each symbol indicates the mean of two determinations. Error bars indicate SD.

12

ANTONSSON.

BARREDO.

0.5

1000

0

10

20

Fraction

30

40

50

60

Number

FIG. 4. Fractionation of calf thymus FPGS on Blue-Sepharose. A solution (60 ml) of partially purified calf thymus FPGS was applied to a 2.8-ml column of Cibricon Blue-Sepharose equilibrated with buffer A and packed in a 3-ml syringe. At fraction 13, the column was rinsed with buffer A. At fraction 16, a linear gradient of O-l.5 M KC1 in buffer A was applied to the column. The gradient was complete at fraction 46. At fraction 4’7, the column was eluted with buffer A containing 1 M KC1 and 20 mM ATP. Protein was detected by A, (0); absorption of light at this wavelength indicated the breakthrough of ATP in fractions > 47. Aliquots (2.5 ~1) were assayed for FPGS activity; fractions that contained high salt or ATP were desalted using a Sephadex spuncolumn prior to assay. FPGS data (0) are not corrected for assay background, which is indicated by the horizontal arrow. Fractions 1-15 contained 6.2 ml, fractions 16-57 were 1.25 ml.

by Blue-Sepharose and was released from the column in a sharp peak after addition of ATP to the elution buffer. The relative rapidity of this assay allowed the peak of enzyme activity to be detected shortly after elution and, hence, allowed the next step of purification to be begun the same afternoon. Because of the low levels of FPGS in mammalian tissues, the sensitivity of this assay has been useful for minimizing lossesof enzyme during purification simply due to consumption during detection. The utility and limitations of this procedure for the determination of FPGS activity in cytosol fractions of mammalian tissues are illustrated in Fig. 5. The activity of FPGS in mouse liver has previously been shown to be relatively high (5), but still only amounts to l-2 pIU/ mg protein. FPGS activity was easily measurable on this source and the reaction was well behaved, unless high levels of protein were added to assays (Fig. 5A). Thus, product formation was linear for up to 1 h at 22 and 44 pg/6 ~1 assay, but linearity was limited to about 30 min at 85 pg/assay. Addition of fresh ATP to these reactions at 15 and 30 min allowed the reaction to continue, indicating that consumption of ATP by competing reactions was limiting the assay. Conventional ATP regenerating systems are not effective at the alkaline pH of this assay. At higher protein input (170 pg/assay), product formation reached a clear maximum, indicating the destruction of product. These limitations will probably hold true for any tissue at some point, so that an initial velocity should be determined from time curves for an appropriate assay. We found that the range of protein con-

AND

MORAN

tents compatible with acceptable linearity with time could be extended simply by increasing the volume of the standard assay to 10 ~1. Tissue folylpolyglutamates, which have been shown to be inhibitors of FPGS activity (3,20), are a potential source of error for assays on crude cytosol preparations. In agreement with this concern, when crude cytosolic protein was assayed, control reactions for which tetrahydrofolate was not added were often found to allow the formation of the macromolecular tritium-labeled product from [3H]glutamic acid, whereas controls missing ATP or thymidylate synthase were not different from a no protein blank. This is probably the result of the pres-

3ow1A

0

1'5

30

Time,

4.5

60

min

Assay of crude mouse liver cytosolic protein for FPGS activFIG. 5. ity. (A) Increasing amounts of mouse liver cytosolic protein were incubated for the indicated periods of time with the components of the FPGS reaction in a total volume of 5 ~1 and then with thymidylate synthase solution for 20 min. Protein contents were 22 pg (0); 44 pg (D); 88 pg (0); and 176 pg (0). Controls for which 66 pg of protein was incubated for 60 min in the absence of tetrahydrofolate are indicated by A. (B) Either crude (0) or desalted (0) cytosolic protein from mouse liver was incubated for the indicated periods in a 10-J reaction volume prior to the addition of thymidylate synthase solution. Controls were no tetrahydrofolate (A), no thymidylate synthase during second incubation (m), and no FdUMP during second incubation (Cl). Each point is the mean of two determinations; error bars indicate SDS, but are no included for the data of A.

MICROASSAY

FOR

FOLYLPOLYGLUTAMATE

ence of endogenous tetrahydrofolate in the protein samples. The desalting of mouse liver cytosol using a Sephadex spun-column prior to assay was found to remove some endogenous inhibitor of the reaction (or tissue glutamic acid which diluted the specific activity in the assay) and also to virtually eliminate reaction in assays for which tetrahydrofolate was not added (Fig. 5B). DISCUSSION

FPGS is present in mammalian cells at low levels and has proven to be fairly unstable during purification (3,4,18). A sensitive and rapid assay method would be an advantage for the purification and study of this enzyme. We have found the technique described in this report to be about 75 times more sensitive and about 4 times faster than the charcoal adsorption assay previously in use in this laboratory. With preparation of materials beforehand, the location of a peak of FPGS activity in the effluent of a chromatographic column (which involves 20-50 enzyme assays) required about 75 min. Before developing this assay, we found that a substantial proportion of total enzyme was consumed during purification of FPGS with a multistep procedure just determining where it eluted from each column, In addition, the instability of this enzyme often meant that it was inactivated while standing in dilute protein fractions during the time required to locate enzyme activity using other assays. This assay has rectified these problems. In this laboratory, we have previously used a combination of a charcoal adsorption assay with a mini-column to isolate the product formed during the FPGS assay of tissue samples. This procedure has very low blanks, but requires 3-4 days and is cumbersome. Others have used a mini-DEAE-cellulose system for such assays, which is substantially more convenient but also is somewhat incompatible with a large number of replicates or samples. The assay described herein will allow the simultaneous manipulation of a number of samples, being limited only by the tedium of prepacking spun-columns and by the number of samples accommodated by a laboratory centrifuge. Using a Beckman J-6B, we have processed up to 84 samples at a time. This assay has some disadvantages. As presently designed, the only folyl substrates that can be used are tetrahydrofolate and 5,10-methylenetetrahydrofolate. However, aminopterin, which is an excellent substrate for all species of FPGS so far examined (3,4,6), could also be used if a plentiful dihydrofolate reductase were used as a trapping protein in place of thymidylate synthase. Another disadvantage is that the method requires the availability of substrate amounts of thymidylate synthase as a trapping protein. However, L. casei strains resistant to methotrexate have been developed (13) which overproduce thymidylate synthase up to a level of 2-3% of their soluble protein. More recently, the gene for the L. casei enzyme has been cloned and expressed in E. coli at levels up to 30% of the total soluble protein

13

SYNTHETASE

(14). Using this strain, we have obtained about 250 mg of pure enzyme from 4 liters of bacterial growth. Other advantages of this assay should be mentioned that are not immediately evident. A major practical advantage is that [3H]glutamic acid can be used without prepurification and without prescreening labeled glutamic acid for background in this assay. This has not been our experience with the other available assays. A second advantage for some experiments is that the chain length of the polyglutamate tail added in the course of assays can be analyzed directly on the ternary complex that is formed as the endpoint of this assay using the gel system developed by Priest and Diog (21). ACKNOWLEDGMENT We thank Dr. John McGuire velopment of this assay.

for helpful

discussions

during

the de-

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G. F. (1974) Cell 2,173-182. Goldman, I. D. (1982) J. Biol.

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K., Davisson,

N. G. L., and Huennekens,

V. J., and Santi,

D. V. (1988)

R. K., and Kisliuk, R. L. (1975) Biochem. 64,648-655. E. F. (1972) Anal. Biochem. 48,422-427.

F. M. (1971) DNA Biophys.

7, 235Res.

17. Ehresman, B., Imhault, P., and Weil, J. H. (1971) Anal. Biochem. 54,454-463. 18. Moran, R. G., and Colman, P. D. (1984) Biochemistry 23,45804589. 19. Moore, M. A., Ahmed, F., and Dunlap, R. B. (1984) Biochem. Biophys. Res. Commun. 124,37-43. 20. Baiinska, M., Nimec, Z., and Galivan, J. (1982) Arch, Biochem. Biophys. 216,466-476. 21. Priest, D. G., and Diog, M. T. (1986) in Methods in Enzymology (Chytil, F., and McCormick, D. G., Eds.), Vol. 122, pp. 313-319, Academic Press, San Diego, CA.