Measurement of folylpolyglutamate synthetase in mammalian tissues

ANALYTICAL

BIOCHEMISTRY

Measurement

U&326-342

(1984)

of Folylpolyglutamate

Synthetase

in Mammalian

Tissues’

RICHARD G. MORAN~ AND PAUL D. COLMAN Laboratory of Cellular and Biochemical Pharmacology, Division of Hematology/Oncology, Childrens Hospital of Los Angeles, 4650 Sunset Boulevard, Los Angeles, California 90027 Received November 2, 1983 Previous methods for the measurement of folylpolyghrtamate synthetase have been modified and combined to facilitate assay of this enzyme at the levels found in mammalian tissues. Batch adsorption of product onto charcoal allowed the rapid analysis of multiple samples of partially purified enzyme, e.g., column fractions. This technique, however, was unsuitable for the assay of folylpolyglutamate synthetase in crude cytosols due to the presence of interfering enzyme activities. On the other hand, the sequential use of charcoal adsorption and batch elution from DEAE-cellulose permitted isolation of the folate product from assay mixtures containing crude enzyme fractions. Under these conditions, interference from other enzyme activities and background values were low enough for the quantitation of 10 pmol of oligoglutamyl folate product. Folylpolyghttamate synthetase was measured in a series of mouse tissues and tumors. Enzyme activity was quite low in all cases. Mouse liver and kidney and some of the tumors studied had the highest levels (SO-100 pmol product/h/mg protein); other tumors and spleen had lower levels. Enzyme activity was at the limit of detection in intestine and lung and was below detection in brain, heart, and skeletal muscle.

Although the existence of folate compounds containing several glutamic acid residues per mole of pteridine has been recognized in mammalian tissues for decades, the study of the enzyme system(s) responsible for the synthesis of this class of compounds is still in its infancy. This lag in the investigation of the biosynthesis of these compounds has been due, in part, to the uncertainty that has prevailed concerning the biological function of the folate polyglutamates and, in part, to the intransigence of mammalian folylpolyglutamate syn’ This work was supported by Grant CA-27605 from the National Institutes of Health, DHEW. 2 To whom correspondence should be addressed. 3 Abbreviations used: FPGS, folylpolyglutamate synthetase; NH&, ammonium acetate; CHO, Chinese hamster ovary; PteGlu, folic acid; I-H&Glu, enzymatically prepared tetrahydrofolate; dl-5-CHO-H&Glu, racemic S-formyl tetrahydrofolate; MTX, 4-NH2- IO-CHr-PteGlu, methotrexate, 4-amino-4-deoxy-lo-methylpteroylglutamic acid; Hepes, 4-(2-hydroxyethyl)- I-piperazine ethanesulfonic acid; &ME, 2-mercaptoethanol; Pit A, trade name for tetrabutyl ammonium chloride; ODS, octadecylsilane; BSA, bovine serum albumin.

326

0003-2697184 $3.00 Copyright 0 1984 by Academic Fnx, All rights of reproduction in any fom

thetase (FPGS)3 to study. This enzyme is present at low levels in all mammalian tissues studied to date (1-5) and it has proven to be quite unstable (3,5). These technical difficulties have been amplified by the properties of the assays for FPGS thus far available. Recent experiments have furnished a body of evidence that supports the postulate that the folate polyglutamates are, in fact, the coenzyme forms used intracellularly while the monoglutamates are the transport forms that normally penetrate the plasma membrane. [This topic has been reviewed in Ref. (l).] Furthermore, the fact that mutational deletion of FPGS has been found to be a lethal event under normal growth conditions (6) has suggested that FPGS is a logical target enzyme for cancer chemotherapy (7,8). These observations underscore the importance of this enzyme system and emphasize the need for better methods for the measurement of FPGS. In this work, we report a modification of previously published methods (3,9,10) which allows assay of crude mammalian supematant fractions without interference from folate-in-

Inc. reserved.

MAMMALIAN

FOLYLPOLYGLUTAMATE

dependent reactions and with exceedingly low background values. We also describe an assay procedure for the measurement of FPGS in up to 50 or 100 samples of partially purified enzyme at a time. In both of these assays, FPGS activity is estimated using folic acid as a stable folyl substrate. These techniques have been instrumental in the measurement of FPGS in tissues of low activity and in purification of mammalian FPGS in this laboratory (5). MATERIALS

AND METHODS

Materials. Folic acid (PteGlu), methotrexate, &5-CHO-H$teGlu, soybean trypsin inhibitor, yeast glucose-6-phosphate dehydrogenase (240 IU/mg protein), calf intestine alkaline phosphatase (925 IU/mg protein), and acid-washed activated charcoal (type C4386) were purchased from Sigma Chemical Company (St. Louis, MO.). Pit A reagent (tetrabutyl ammonium chloride) was purchased from Waters Associates (Milford, Mass.). Reagentgrade ammonium acetate (NH&c) was from Baker Chemical Company (Phillipsburg, N. J.), DEAE-cellulose from Eastman Chemical Company (Rochester, N. Y.), and [3H]glutamic acid (lo-16 Ci/mmol) from New England Nuclear (Boston, Mass.). Mice were obtained from Simonsen Laboratories (Gilroy, Calif.) or Jackson Laboratories (Bar Harbor, Maine). Tetrahydrofolate (I-H$teGlu) was prepared enzymatically as previously described (11). Initially, PteGlu3 was obtained from Lederle Laboratories (Pearl River, N. Y.) and a sample of PteGlus was a gift of Dr. Sigmund Zakrzewski of Buffalo, New York. These compounds were purified by DEAE-cellulose chromatography using gradient elution with NH,Ac ( 12). Subsequently, folyl oligoglutamates containing 2-8 glutamate residues per mole of pteridine were obtained from the Southern Alabama Medical Research Foundation. These latter compounds were used as standards in HPLC experiments. The y-[3H]glutamyl conjugates of PteGlu, PteGlu3, I-H,#teGlu, and MTX were prepared by incubation of mouse liver FPGS preparations with [3H]glutamate and the cor-

SYNTHETASE

327

responding folyl compound, as described below. Thymidylate synthase (3.4 IU/mg protein) was purified from Lactobacillus casei/ MTX (originally obtained from Dr. Frank Huennekens) by previously described methods ( 11). A mutant CHO cell line deficient in FPGS, the AUX Bl cell, was kindly provided by Dr. Gordon Whitmore of the Ontario Cancer Center. AUX Bl and CHO cells were grown in alpha medium supplemented with 10% fetal calf serum (Grand Island Biological Co., Grand Island, N. Y.). All cell lines were found to be free of mycoplasm by an agar cloning method. Purljication of [3H]glutamic acid. A microcolumn of charcoal similar to that reported by Ritari et al. (9) was used to purify [3H]glutamic acid. A glass Pasteur pipet was plugged with cotton and a 2-mm layer of cellulose powder (type CC41; Whatman, Inc., Clifton, N. J.). A l- to 2-mm layer of charcoal powder was layered onto this cellulose and then a top layer of cellulose powder was added. This charcoal column was washed with 1 ml of 0.2 M Tris, pH 8.5. [3H]Glutamic acid (0.25-0.75 mCi, 10-l 5 Ci/mmol) was brought to dryness with a stream of N2 and the residue was dissolved in 250 ~1 of 0.2 M Tris, pH 8.5. This solution was passed through the charcoal column under gravity and the column was rinsed with 0.75 ml of Tris (0.2 M, pH 8.5). The eluate was used without further treatment. Several alternate batch or column charcoalpretreatment techniques that we evaluated either did not remove troublesome impurities or adsorbed substantial amounts of radioactivity. Preparation of supernatant fractions of tissues. Sprague-Dawley female rats were sacrificed by a blow to the head. DBA/2 or BDF/ 1 female mice were sacrificed by cervical dislocation and tissues were quickly excised and placed in ice-cold 20 mM Hepes buffer, pH 7.4, containing 0.25 M sucrose. Livers were perfused in situ through the portal vein with 4-10 ml of this solution to remove excess blood prior to further processing. Whole small intestine was excised from stomach to caecum. Lengths of intestine were placed on an inverted

328

MORAN

AND

tray of ice cubes and lumenal contents were removed with gentle pressure from a spatula. Mucosal elements were then extruded with firm pressure and were processed further for enzyme assay. Tissues were weighed quickly, minced in 2 vol of 20 mM Hepes buffer, pH 7.4, containing 0.25 M sucrose and 50 mM b-ME, and then homogenized with a motordriven Teflon pestle. AUX Bl and CHO cells and mouse ascites tumors were harvested by centrifugation, washed once with phosphatebuffered saline, suspended in 2 vol homogenization buffer, and disrupted by three 10-s bursts of sonic oscillation. Cytosol fractions were prepared by centrifuging homogenates at 160,OOOg for 1 h at 4°C and removing the resultant supernatant after aspiration of buoyant lipids. For some studies, a 30% (NH.& SO, precipitate was prepared by treating a crude supernatant fraction with 172 g of (NH&SO4 per liter of supematant fraction. The (NH&S04-precipitated FPGS was desalted on a Sephadex G-25 column immediately before use. Mouse tumors. The L 12 10, P388, and B 16 mouse tumors were obtained from Dr. T. Khwaja of this university and were transplanted in either DBA/2J (L1210, P388) or BDF/ 1 (B 16) female mice. Lewis lung tumors were established in BDF/l mice by the subcutaneous injection of lo6 cells of an established cell culture line. This line was grown in RPM1 1640 medium supplemented with 5% fetal calf serum and was kindly provided by Dr. John Bet-tram of Roswell Park Memorial Institute. Protein determinations. Protein was estimated using the Hartree modification ( 13) of the Lowry assay, scaled down to a total volume of 1.7 ml and with bovine serum albumin used as a standard. For the determination of the protein content of solutions containing compounds that interfere with the Lowry reaction, protein was quantitatively precipitated by trichloroacetic acid in the presence of deoxycholate ( 14). FPGS assay incubation conditions. The activity of FPGS was measured at 37°C in a

COLMAN

reaction mixture containing 500 pM PteGlu, 1 mM [3H]glutamate (4 mCi/mmol), 5 mM ATP, 10 mM MgC12, 30 mM KCl, 20 mM @-ME, and 200 mM Tris, pH 8.5. The total volume of reaction mixture was 0.50 or 1.0 ml for assay of crude supematants while a 0.25-ml system was used for assay of (NH&S04 pellets or column fractions. Duplicate ahquots of assay mixture were prepared for scintillation counting under the same conditions used for quantitation of ‘H-labeled product to allow a direct calculation of the ratio of cpm to picomoles of product. In a typical experiment, there were 3.5 cpm/pmol of product. For some experiments, the radioactivity in samples was determined using a Beckman 7500 liquid scintillation counter with automatic quench corrections utilizing the h-number method (15). Adsorption ofreaction product onto activated charcoal. The conditions for adsorption of reaction products onto charcoal used in this work were essentially those previously used by Masurekar and Brown ( 10). A suspension of charcoal was prepared by the addition of 2.0 g of acid-washed activated charcoal (Sigma Chemical Co.) and 50 mg of Dextran T-70 (Pharmacia, Inc.) to 50 ml of water. Prior to use, the charcoal was washed once with 10 mM glutamate, pH 6.8, containing 10 mM P-ME and was then resuspended in 0.15 M KI-12P04, pH 5.0, at 40 mg/ml. After incubation of crude or partially purified FPGS preparations with reaction mix, aromatic 3H products were adsorbed by the addition of 0.5 ml of this charcoal suspension. The tubes were placed on ice for 10 min, 5 ml of 10 mM glutamate, pH 6.8, containing 10 mM @-ME was added, and the tubes were centrifuged for 10 min at 4000 rpm in a Beckman J-6 centrifuge. The charcoal pellet was washed three additional times with 10 ml of this solution. Ethanolic ammonia (1.5 ml of 3 M NH40H in 60% ethanol) containing 4 mM D-ME was added to the charcoal pellet and the tube was mixed and allowed to stand for 10 min at room temperature; the charcoal was again pelleted. The supematant was either added

MAMMALIAN

FOLYLPOLYGLUTAMATE

SYNTHETASE

329

Quantitation of the isolation of folyl oligodirectly to a scintillation vial with 1.5 ml of glutamates with charcoal. Tritium-labeled water and 14 ml of Budget-Solve scintillation PteGlu2, PteGlu4, 4-NHz- 10-CHJ’teGluz , fluid (Research Products International, Mount and l-H4PteGluz were made by incubating 500 Prospect, Ill.) or was brought to dryness with a gentle stream of dry N2 for analysis using PM PteGlu, PteGlu3, 4-NHz- IO-CH~?teGlu, or 1-H&eGlu for 1 h with desalted, (NH&the combined assay (see below). S04-precipitated mouse liver FPGS under Isolation o~[~H] polyglutamyl folates using DEAE-cellulose minicolumns (combined as- standard reaction conditions. The [3H]folates4 say). Minicolumns of DEAE-cellulose (4.0 formed in these reactions were isolated by adsorption onto charcoal as described above. The f 0.25 cm in height) were prepared in plastic disposable transfer pipets as previously de- eluates were dried under a stream of N2. The that were formed scribed (16), except that the final &ME rinse [3H]folyl-oligoglutamates and purified by this procedure were used to was omitted. The dried residue eluted from charcoal (see above) was dissolved in 2 ml of follow the fate of [3H]folyl reaction product under our assay conditions. In a typical ex50 mM NH4Ac and transferred onto a minicolumn. The tube was rinsed again with 50 periment (see Table l), the dry residue was hydrated with 4.0 ml of a solution containing mM NH4Ac (2 ml) and the rinse was added to the minicolumn. The columns were washed all of the components of the FPGS reaction with an additional 10 ml of 50 mM NH4Ac, mixture except PteGlu (since folates would be and the folate product was eluted with 6 ml contained in the dried residue), protein, and however, unlabeled glutamate of 1 M NH4Ac. This eluate was collected di- [‘Hjglutamate; was added. Duplicate 0.4-ml samples of this rectly into a scintillation vial, and the solution was shell-frozen and lyophilized to dryness. solution were used to determine the total The dried residue was dissolved in water (1 amount of radioactivity in each sample, while ml), 10 ml of scintillation cocktail was added, triplicate l.O-ml samples were transferred to 15-ml centrifuge tubes for charcoal adsorption. and radioactivity was determined by liquid scintillation counting. Charcoal was added, washed, and eluted as Column chromatography. DEAE-cellulose described above. Aliquots of each supematant were prepared for scintillation counting. columns were prepared and eluted as previously described (12). Folate markers were Interference with the activity of mouse liver added to samples prior to their adsorption onto FPGS by intestinal cytosol preparations. For some experiments, the amount of product these columns. High-performance liquid chromatography formed by mouse liver FPGS during a l-h incubation was measured in reaction mixtures (HPLC). Folyl oligoglutamates were analyzed containing increasing amounts of cytosol proby HPLC using a 0.4 X 25-cm Beckman 5-pm Ultrasphere ODS column maintained tein prepared from mouse intestine. Desalted at 40°C. Flow rates were held at 1 ml/min (NH4)2S04-precipitated mouse liver FPGS using a Spectra-Physics SP8000 liquid chro- (0.7 1- 1.2 mg of protein) and intestinal crude matograph. Elution was accomplished with a supematant fraction (O-200 pg of protein) discontinuous methanol& A gradient (initial were incubated under the conditions used for condition, 30% methanol and 70% 5 mM Pit FPGS assays at pH 8.5 in Tris buffer in a total A; linear gradient to 34% methanol over 5 volume of 1 ml. Product was isolated by charmin followed by a linear gradient from 34 to coal adsorption. Folate-independent reactions 40% methanol over the next 35 min). Buffers were continuously degassed with a helium 4 Under these conditions the charcoal-adsorbed products purge system. Markers were detected at 280 formed from PteGlu and PteGlu~ have the chromatonm and 3H-labeled products were localized graphic properties of PteGlu~ and PteGI&, respectively by scintillation counting. [Ref. (5) and Figs. 3 and 61.

330

MORAN

AND TABLE

COLMAN 1

EFFICIENCYOF ADSORPTION AND ELUTION OF OLIGOGLUTAM~

FOLATES FROM CHARCOAL”

Percentage of dpm added Extracted (No. of elution) Compound PteGlu* PteGlu, 4-NH,lO-CHrPteGluz

dpm added

Not adsorbed

Removed inwashes

1

2

3

10,130 f 130

2.8 kO.4

1.6 + 0.4

76.1 + 2.4

6.4 kO.2

1.3 1- 0.1

3.5 + 0.3 1.4 + 0.4

1.4 k 1.1 1.2 + 0.7

74.3 + 2.3 71.5 + 2.0

6.3 rf: 0.4 8.5 + 1.1

1.2 f 0.2

5,850 9,830

k 140 + 140

2.1 + 0.1

Total accounted for 88.2 + 2.5 86.7 + 2.6 84.7 + 2.4

a [3H]PteGluz, [3H]PteGlu,, and [3H]-4-NH,-10-CH,-PteGluZ were added to FPGS reaction mixes containing all components except protein and [3H]glutamate. Charcoal was added and the charcoal was washed four times with 10 mrvtglutamate and then extracted with three successivealiquots of ethanolic ammonia. The charcoal suspension was centrifuged between steps and an aliquot of each supematant fluid was counted. The data presented are the means 5 SD of triplicate samples, For further details, see Materials and Methods.

that incorporate [3H]glutamate into charcoalabsorbable products were not observed at these levels of crude intestinal protein. The effect of intestinal cytosol protein on the activity of other enzymes was examined under identical conditions. For instance, 0.7 1 mg of desalted (NH&S04-precipitated mouse liver FPGS was incubated with O-200 pg of intestinal cytosolic protein and enough pure thymidylate synthase from L. cusei/MTX (3.4 IU/mg protein) (11) to form 0.035 pmol thymidylate/h at pH 8.5. This mixture was incubated with 0.3 ITIM tetrahydrofolate, 6 mM formaldehyde, 115 mM @-mercaptoethanol, and 100 mM deoxyuridylate in a total volume of 1 ml of 0.25 M Tris buffer, pH 8.5. Thus, the incubation conditions were similar to those of the FPGS assay except that ATP was omitted. After 1 h at 37°C the absorbance at 340 nm of each tube was monitored and the relative activity of thymidylate synthase was calculated after subtraction of the blank value seen in the absence of thymidylate synthase. Similarly, glucose-6-phosphate dehydrogenase activity was measured in the presence of mouse liver FPGS (0.7 mg), intestinal protein (O-O.2 mg), NADP (10 IIIM), glucose 6-phosphate (10 mM), and MgClz (20 mM) in 0.25 M Tris, pH 8.5, by measurement of the difference in absorbance at 340 nm from that seen with appropriate controls; total volume

was 1.O ml. Alkaline phosphatase activity was similarly measured using pnitrophenylphosphate as a substrate and the change in absorbance at 4 10 nm as an endpoint. Soybean trypsin inhibitor (100 pg) was added to some of the tubes, as noted. Proteolytic activity measurements.The rate of digestion of bovine serum albumin (BSA) was used as an index to compare the proteolytic activity of intestinal cytosol preparations with that of purified trypsin and protease K. Aliquots of protease or intestinal cytosolic protein were incubated with 10 mg/ml BSA in 50 mM Tris, pH 8.5, at 25°C in a volume of 1 ml. Perchloric acid (100 ~1 of 70% w/v) was added at varying times and the precipitated protein was pelleted by centrifugation for 10 min at 3500 rpm in a Beckman J-6 centrifuge. The absorbance at 280 nm of the supernatant was used to follow the release of acid-soluble peptides. RESULTS

Detection of FPGS activity using columns of DEAE-cellulose. When [‘Hlglutamic acid was incubated with a crude supernatant fmction of mouse liver in the presence of ATP and PteGlu, several radioactive products were formed (Fig. 1). Initially, to detect and measure any folyl oligoglutamates among these prod-

MAMMALIAN

I

I

20

I

I

40

FRACTION

I1

60

I

*

80

FOLYLPGLYGLUTAMATE

1

I

100

I

O

NUMBER

FIG. 1. DEAE-cellulose chromatographic analysis of FPGS incubation mixtures containing crude mouse liver cytosols. High-speed supematant fraction protein (14.2 mg/incubation) was added to standard FPGS reaction mixtures (total volume = 3.0 ml) in the presence (B) and absence (A) of 500 pM PteGlu. After 1 h at 37’C, the mixtures were brought to OT, H&eGlu (1 pmol) and PteGlu ( 1.5 pmol, added only to mixture A) were added, and the mixtures were chromatographed on a 0.62-cm2 X 35cm column of DEAE-cellulose. The columns were. initially eluted with 0.05 M NH,&; at the arrow, a linear gradient of NH&c was initiated (0.05 to 2.5 M; initial volume = 190 ml). All buffers contained 1% &ME. A lml aliquot of each fraction (2.7 ml) was prepared for scintillation counting (0); marker folates were detected by absorbance at 285 nm (- - -). Peaks 1, 2, and 3 had maxima of 6.5 X lo’, 7.2 X lo’, and 4.5 X lo4 cpm/ml, respectively.

ucts, a large amount of mouse liver protein was incubated with complete FPGS reaction mix at 37°C for 1 h and the entire mixture was then chromatographed on a column of DEAE-cellulose. It is usually the case that folyl oligoglutamates elute from DEAE-cellulose in order of increasing glutamyl side-chain length when developed with gradients of most salts, e.g., phosphates or NaCl(2,17,18). However, we have found that this selectivity can be affected by the salt used for gradient elution.

SYNTHETASE

331

As shown in Fig. 2, PteGlu3 and PteGlu5 elute l-3 fractions before marker PteGlu on a column of DEAE-cellulose eluted with a gradient of NH,Ac. Hence, the peak of radioactivity which eluted immediately before PteGlu in Fig. 1 had the chromatographic behavior expected for an oligoglutamyl derivative of folic acid. This product5 was formed in incubations of mouse liver supernatant fractions with [3H]glutamate and ATP in the presence of PteGlu but not in similar incubations that did not contain PteGlu (Fig. 1). It should be noted, however, that this folyl oligoglutamate peak was quite small compared to the magnitude of the other products (peaks 2 and 3 of Fig. 1) formed in the presence or absence of PteGlu. By correcting the radioactivity found in this peak for the specific activity of the glutamic acid used, we calculated that this mouse liver cytosol contained an FPGS activity equivalent to 110 pmol/h/mg protein. Adsorption of folyl oligoglutamates on activated charcoal. Since others have found the adsorption of folates onto activated charcoal useful for the assay of bacterial and fungal FPGS (9,10), we examined the efficiency of batch adsorption of folyl oligoglutamates onto dextran-treated charcoal. When several tritium-labeled di- and tetraglutamyl folate compounds were used as standards, it was found that less than 4% of the radioactivity added to incubation mixtures was not adsorbed onto charcoal under our assay conditions (Table 1). Likewise, only a small proportion (<2%) of these adsorbed folates was removed during the washes. A single treatment of the charcoal with ethanolic ammonia eluted 87-9 1% of the extractable radioactivity. The sum of the radioactivity found in the charcoal supematant, glutamate washes, and ethanolic ammonia extracts approached that originally 5 We have also found that, in the presence of 500 FM PteGlu, partially purified preparations of mouse liver FFGS form a single product that elutes from a DEAE-cellulosechloride column ( 17) midway between the elution positions of PteGlu and PteGlu, (5) and that this product cochromatographs with PteGlu2 on a reverse-phase HPLC column (see Fig. 5).

MORAN

332

AND COLMAN

Use of charcoal adsorption to measure mammalian FPGS activity. We attempted to use activated charcoal adsorption to detect and measure folyl oligoglutamate products formed in FPGS incubations with crude supematant fractions of mammalian tissues. Crude mouse liver cytosol was incubated with [‘HIglutamate and ATP in the presence and absence of PteGlu and the products were adsorbed onto charcoal and eluted as described above. In order to characterize the products that were isolated, the eluted material was chromatographed on a DEAE-cellulose column (Fig. 3). The major product adsorbed by charcoal from the incubation containing PteGlu chromatographed at the position of PteGlu, (Fig.

FRACTION

NUMBER

FIG. 2. &chromatography of EteGlu, PteGluS, and PteGluS on DEAE-cellulose eluted with NH&. [3H]PteGlu was chromatographed with 0.5 mg of either EteGlu (A), PteGlu, (B), or EteGluJ (C) on a 0.9 X 35-cm column of DEAE-cellulose that was eluted with a linear gradient of NH.& (0.05 to 2.5 M; mixer volume was initially 95 ml for each column). The gradient was begun at fraction 1. Each fraction contained 3.5-3.8 ml. Correcting for the differences in molecular weight and taking the AzgJ observed for PteGlu (A) as 100% the absorbancies seen in the EteGlu3 (B) and PteGluS (C) peaks are 96 and 83% of theoretical, respectively.

added to the mock reaction mixtures; that is, 84-88% of the added ‘H could be accounted for. The overall recovery of folyl oligoglutamates in the first ethanolic ammonia wash was acceptably high (72-76%) and was uniform among the unreduced compounds tested (Table 1). A similar recovery experiment was performed using tritium-labeled I-I&PteG1u2. The overall recovery of this labile compound was lower (69%) than that of the unreduced compounds used, as was the proportion (50%) of added radioactivity eluted with a single ethanolic ammonia wash (see Discussion). However, even with this unstable compound, only low levels of label were not adsorbed (5%) or were removed in the washes (3%).

-3

20

Y

40

60

8.’

60

D

100

I

I e--I*+--

0

20

40 FRACTION

60

._-

60

,

0

100

NUMBER

FIG. 3. Chromatographic analysis of the 3H-labeled products of crude FPGS reactions that are adsorbed by charcoal. A crude mouse liver FPGS preparation was incubated with (B) and without (A) 500 pM PteGlu for 1 h under standard conditions (total volume = 2.0 ml). The 3H-labeled products of these reactions were adsorbed onto charcoal and eluted with ethanolic ammonia. This eluate was dried under N2 and the dissolved residue was analyzed by DEAE-cellulose chromatography as described in Fig. 1. The salt gradient was begun at Tube 35. For details, seeMaterials and Methods. A l-ml aliquot of each fraction (3.2 ml) was prepared for scintillation counting (0); marker folates (1 rmol) were detected by absorbance at 285 nm (---).

MAMMALIAN

FOLYLPOLYGLUTAMATE

3B), while a similar peak was absent from the chromatograph of the control reaction (Fig. 3A). A comparison of the data of Fig. 3 with that of Fig. 1 shows that a charcoal adsorption step eliminated the major nonfolate 3H-labeled products but did not allow isolation of exclusively [3H]PteGlu, from incubations containing crude enzyme fractions. For crude supernatant fractions of mouse liver, the rate at which charcoal-adsorbable products formed in the absence of PteGlu was 40- 100% (in different experiments) of that found in the presence of PteGlu (see Table 2). In such experiments, there was often no significant difference between the amount of product formed with and without PteGlu. This was the case even in experiments for which parallel DEAE-cellulose chromatography indicated substantial formation of folyl oligoglutamates (Table 2, experiment 1, and Fig. 1). We concluded from these results that a minus folate blank would not reliably correct for interfering reactions encountered in crude enzyme preparations using charcoal adsorption alone. It should be noted that the major products

SYNTHETASE

333

formed by crude supematant fractions of mouse liver FPGS in the absence of added PteGlu which were isolated by charcoal adsorption were eluted from DEAE-cellulose by 50 mM NH4Ac (Fig. 3A); the earliest eluting folate on this column has previously been shown to be removed only by 0.38 M NH4Ac (12). This indicates that this unidentified product formed in the absence of added folate was not a folyl oligoglutamate synthesized from endogenous folates. This premise is also supported by an experiment in which the protein precipitated by 30% saturation with (NH&SO4 did not catalyze the synthesis of this unidentified product, whereas the protein precipitated by 50% saturation did so to the same degree as an unfractionated supematant (data not shown). The enzyme activities that interfered with the assay of FPGS by charcoal adsorption were easily removed during enzyme purification. For instance, incubation of an (NH4)$04fractionated mouse liver FPGS with complete reaction mix resulted in the incorporation of tritium into charcoal-adsorbable compounds

TABLE 2 UTILITY OF CHARCOAL ADSORPION FOR ASSAY OF FPGS IN CRUDE AND MINIMALLY PURWIED MOUSE LIVER PREPARATIONS 3H-Labeled products eluted from charcoal (cpm) Crude cytosol a

No protein blank No PteGlu 500 /.tM Pt~hI

(NI-I.&S04-precipitated

. cytosol ’

Expt 1

Expt 2

Expt 3

Exnt 2’

191, 198 827, 903 878, 895

177, 168 632, 776 890, 864

90, 103 135, 138 4612.4551

177, 168 201, 219 1784. 1827

* An aliquot of a 160,OOOgsupematant fraction of mouse liver (approximate 2 mg of protein) was incubated with 500 PM PteGlu under FPGS assayconditions (see text) for 1 h at 37°C. Charcoal was added and processed as described in the text. The data represent the radioactivity eluted from charcoal of duplicate incubation mixtures, uncorrected for machine background (20 cpm). Controls (no protein and no PteGlu) contained all other components and were incubated at 37°C for 1 h. The data of Expt 1 were taken from a repeat experiment to that shown in Fig. 1. b The data represent uncorrected cpm eluted from charcoal and directly counted. Each number represents an individual reaction mixture (0.25 ml) containing desalted O-30% (NH&S04-precipitated mouse liver protein (l-3 mg of protein was used per assay) and incubated for 1 h at 37°C. c In Expt 2, a crude mouse liver cytosol and a O-30% (NH&SO4 pellet prepared from it were assayed by the charcoal procedure at the same time.

334

MORAN

AND COLMAN

FRACTION

NUMBER

FIG. 4. Analysis of FPGS incubation mixtures using a (NH&SO,-precipitated mouse liver fraction with (B) and without (A) adsorption of 3H-labeled products onto charcoal prior to chromatography. (NH&S04fractionated mouse liver FPGS was incubated with 500 pM PteGlu for 1 h under standard conditions (total volume = 2.0 ml). An aliquot of this incubation was mixed with marker folates (1 smol) and applied directly onto a 0.62-cm* X 50-cm column of DEAE-cellulose; another aliquot was prepuritied by charcoal adsorption prior to chromatography. Chromatographic conditions were the same as those described for Fig. 1. The peak at tube 50 in (A) had a maximum of 4100 cpm. The symbols used am (0) radioactivity and (0) AzgJ. The arrow indicates the beginning of the gradient.

that chromatographed almost exclusively as folates (Fig. 4B). Radioactivity that adsorbed to charcoal from assays incubated without PteGlu was low (Table 2) and did not seem to be a function of time or protein. On the other hand, when the products of such incubation mixtures were chromatographed on DEAE-cellulose without prior adsorption to charcoal, other nonfolate products were present (compare Figs. 4A and B). For protein fractions without significant folate-independent enzyme activities, a procedure based on simple batch adsorption of product onto charcoal was a reliable measure of FPGS activity. This assay was found to be linear with time for at least one hour and with

protein (Fig. 5). Hence, a l-h incubation was generally used for screening the eluate of chromatographic columns for enzyme activity using this charcoal assay. The various possible blanks (i.e., reaction mixtures either (a) incubated 60 min without protein, (b) containing protein, but kept at 0” for 60 min, or (c) containing protein, but without either ATP or PteGlu, and incubated 60 min at 37“C) gave virtually identical values. Among these blanks, the “minus folate” blank gave slightly higher values on the least-purified enzyme fractions (Table 2). When [3H]glutamate was used without purification, some batches of isotope gave blank values as high as 1100 cpm. However, when prepurified

MAMMALIAN

0

FOLYLPOLYGLUTAMATE

0.2 PROTEIN

0.4 , mg

335

SYIW-HETASE

,

,

(

,

(

0.6

0

30

60

90

TIME

, mln

FIG. 5. Assay of (NH4)$Orpurified mouse liver FF’GS using the charcoal adsorption procedure: Linearity with protein (A) and with time (B). Incubations were performed in a total volume of 250 ~1. Reactions were terminated after 1 h in (A) while the time course of product formation (B) was followed using 0.32 mg of protein. Each point represents the mean of duplicate determinations.

[3H]glutamate was used (see Materials and Methods), blank values would typically be 100-200 cpm immediately after purification and would increase to 200-400 cpm over 2 months. Others have previously reported the removal of troublesome impurities for a similar assay using charcoal pretreatment of isotope (10). Variation between blanks within an assay was minimal; the range observed between duplicate blanks of assays performed over a 6-month period was, on the average, 15% of the blank value. The mean blank value for assays run on sequential days showed low variation between experiments, e.g., the mean blank values of four sequential assays were 278 f 14 cpm. Utilization ofPteGlu as a substrate by FPGS from mammalian tissues. The ability of PteGlu to serve as a substrate for mammalian FPGS was examined using NH4(S0& precipitates of various rodent tissues and the charcoal adsorption assay (Table 3). Reaction rates observed in the absence of folates were negligible for all sources of enzyme used. Enzyme reaction was easily measurable either with l-H$teGlu or with PteGlu or dl-5-CHOH4PteGlu. Of the two stable folate derivatives tested, PteGlu more reliably indicated FPGS activity with I-H4PteGlu. Whereas the reaction

rates observed using high concentrations of PteGlu as a substrate were from 60 to 87% of those measured using I-H&eGlu as a substrate, reaction rates with 5-CHO-HJ’teGlu varied among the tissues studied from 70% less than to 20% more than those seen with l-H$teGlu as a substrate. Identijication of reaction product by HPLC. The product isolated by charcoal adsorption from incubations of mouse liver (NH&SO4 precipitates under the conditions of the FPGS assay was identified by cochromatography with authentic markers of folyl oligoglutamates (Fig. 6). Folyl oligoglutamates were retained on a reverse-phase ODS column that was equilibrated with a tetrabutyl ammonium chloride-methanol mobile phase. A discontinuous gradient of methanol easily resolved the markers with baseline separation and ample room between peaks. However, essentially all (>95%) of the radioactivity isolated by charcoal adsorption from FPGS reactions using PteGlu as substrate chromatographed exactly coincident with PteGlu2 under these conditions. FPGS levels in mouse tissues. The level of FPGS in crude supernatant fractions of various mouse tissues was estimated using a twostep assay procedure (see Materials and Meth-

MORAN

336

AND COLMAN TABLE 3

UTILIZATION

OF pt&h

AS A !&LWRATE

FOR ms

FROM

VARIOUS

TISSUES’

Product formed (nmol/h/mg protein) No folate

H.$PteGlU

PteGlu

Rat Liver

0.007

0.47

0.39 (0.83)b

0.56 (1.20)b

Mouse Liver Kidney L1210 Leukemia

0.016 0.003 0.016

0.84 0.31 1.51

0.73 (0.87) 0.19 (0.60) 0.96 (0.63)

0.51 (0.61) 0.27 (0.88) 0.44 (0.29)

Tissue

5-CHG-H,PteGlu

’ (NH&SO, (30%)-precipitated protein from each tissue was chromatographically desalted, then incubated for 1 h at 37’C with either 50 ELMf-HJYeGlu, 508 @i PteGlu, or 50 PM &5-CHG&PteGlu or in the absence of added folate with otherwise complete FPGS assay mix. Product was isolated by charcoal adsorption as described in the text. Duplicate assayswere run for each condition. The numbers shown in this table are mean reaction rates; duplicates differed by less than 5% for reactions containing folates and by 5-3096 for incubation tubes without folates. ’ The values shown in parentheses indicate the ratio of the rate of reaction for either PteGlu or dl-5-CHO-H$teGlu to that of Z-meGlu.

ods). Using this combined assay, the amount of product formed by crude tissue supematant fractions was a linear function of time for at

0

10

Retention

20

30

40

Time , min

FIG. 6. Chromatography of FPGS reaction product with PteGluz on reverse-phase, ion-paired HPLC. (NH&SOr precipitated mouse liver protein (approx 0.6 mg) was incubated with 500 pM PteGlu for I h under standard conditions. The product of this reaction was isolated by charcoal adsorption, mixed with PteGlu, (n = l-8) markers, and injected onto an Ultrashpere 5-pm ODS column, as described in the text. The elution position of the folyl oligoglutamate markers was detected by Azso (top trace). Fractions (0.5 min) were collected and counted for radioactivity.

least 40 min for some tissues (e.g., mouse liver), but was nonlinear for other tissues (e.g., mouse kidney) (Fig. 7B). Product formation over a fixed time interval increased linearly with the volume of cytosol added up to about 5 mg of protein/l ml reaction mixture for mouse liver (Fig. 7A). The blank values seen using this assay for samples containing all additions but kept at 0°C or for mixtures without PteGlu incubated at 37°C were usually in the range of 15 cpm above scintillation-counter background; however, blanks as high as 50 cpm have been seen. Hence, the limits of detection and quantitation of product (i.e., double and quadruple the blank values) in this system are approximately 5 and 10 pmol of [3H]PteGlu2, respectively. Using the combined assay, FPGS levels in CHO cells (Table 4) were found to be similar to the values previously reported for this cell line (85-150 pmol/h/mg protein (2)) using other methods of assay. FPGS was undetectable in a cytosol preparation of AUX B 1 cells. Among the normal mouse tissues examined (Table 4), liver was found to have the highest FPGS activity while kidney and spleen had lower levels. The level of FPGS was below

MAMMALIAN

FOLYLPOLYGLUTAMATE

protan,mg

SYNTHETASE

TIME

337

, minutes

FIG. 7. Assay of FPGS in crude supematant fractions using the combined assay procedure: Linearity with protein (A) and with time (B). Incubations were performed in a total volume of I ml. Reactions were terminated after incubation with varying amounts of mouse liver protein for 1 h in (A) while the time course of reaction was followed with crude protein from the indicated tissues at 2 to 4.5 mg/assay in (B). The inset shows the formation of product with mouse liver crude supematant protein at a larger ordinate scale. Each point represents the mean of duplicate determinations.

detection in brain, heart, and skeletal muscle and was at the limit of quantitation in lung and intestine. FPGS was easily measurable in all of the mouse tumors studied, and some tumors (e.g., the P388 leukemia) had activities at least as high as liver. It should be noted that the specific activity of mouse liver FPGS determined by this method (Table 4) was not significantly different from that determined by DEAE-cellulose column chromatography (Fig. 1) when correction is made for the efficiency of elution of charcoal by ethanolic ammonia (Table 1). Since (NH4)$04 precipitation was found to remove obstacles to assay of mouse liver FPGS by the charcoal adsorption assay, we simultaneously assayed this protein fraction by both charcoal adsorption and combined assays to directly compare the results obtained with these techniques. The slope of the time course of [3H]PteGlu2 formation was identical for the two assays (data not shown) and individual time points differed, on the average, by 9%. Recovery of FPGS added to cytosols of mouse tissues. The explanation of our inability to detect appreciable levels of FPGS activity

in several mouse tissues was sought in a series of experiments in which (NH&S04-purified mouse liver enzyme was added to crude supematant fractions of various mouse tissues as an internal standard. The amount of FPGS activity found using muscle, spleen, kidney, brain, heart, and lung cytosols approximated the sum of the activity of each tissue plus that of the internal standard (Table 5). However, when mouse liver FPGS was mixed with cytosol protein from mouse intestine prior to assay, the intestinal cytosol preparation suppressed the formation of [3H]PteGlu2 by the mouse liver FPGS (Table 5). In other experiments, it was found that the intestinal factor preventing the activity of liver FPGS was heat labile (Table 5). If intestinal extract was added to reaction mixtures after 1 h of preincubation of mouse liver FPGS with substrates, the preformed [3H]PteGlu2 was not degraded (data not shown). Susceptibility of mouse liver FPGS to proteolysis. The intestinal cytosol preparations used in these experiments had sufficient levels of proteases to cause degradation of mouse liver FPGS added as a standard. A 1-mg amount of intestinal protein had the proteo-

338

MORAN

AND COLMAN

TABLE 4 ACIWITY OF FPGS IN NORMAL AND NEOPLASTIC TISSUESOF THE MOUSER FFGS activity (pmol/h/mg protein)

N

90 cl

1 1

99.8 f 5.9 65 t 30 22 +17 cl 4.1 f 3.4 tl 4.7 + 2.5’ Cl

6 2 2 2 2 2 2 2

Control CHO AUX Bl Tissue Liver Kidney Spleen Brain Lung Heart Intestine Skeletal Muscle Tumor P388 Leukemia L1210 Leukemia B 16 Melanoma Lewis Lung Carcinoma

105 Ik 1 58 t- 7 14 +- 3 44

a4

2 2 2 2

o Crude supematant fractions were prepared from various organs of the mouse and assayed for FPGS activity using the combined assay described in the text. The activities of supematant fractions of CHO cells and AUX BI cells were determined by the same procedure and are included as positive and negative controls (2), respectively. Nrepresents the number of determinations for each value. Each determination was based on the initial slope of a time course defined by duplicate assaysusing at least five time points. Enzyme activities are not corm&xl for the recovery of [3H]PteGluz from charcoal (76%, see Table 1). The mean (*SD) blank value for the experiments in this table was 10 + 6 cpm. For details, see Materials and Methods.

b These assayswere performed under standard conditions. Estimates obtained from assaysperformed in the presence of soybean trypsin inhibitor yield values of 6.9 and 10.2 pmol/h/mg protein using 500 pM PteGlu and 50 f.654I-H.&eGlu as substrate, respectively (see text).

lytic activity equivalent to 15 pg of purified trypsin or 4 pg of protease K. When proteolytically equivalent levels of intestinal protein, trypsin, or protease K were added to mouse liver FPGS, equivalent suppression of FPGS activity was observed (Fig. 8A). In addition, the inhibitory effect of mouse intestinal protein

was eliminated by the addition of soybean trypsin inhibitor (Fig. 8B). However, when the FPGS content of mouse intestinal cytosol preparations was measured in the presence of soybean trypsin inhibitor, FPGS activity was still found to be very low in this tissue (6.9 pmol/mg protein/h when assayed with 500 ELM PteGlu and 10.2 pmol/mg protein/h when assayed with 50 PM I-H&eGlu). The susceptibiiity of FPGS to degradation by intestinal protease was found to be substantially higher than that of several other enzymes under identical conditions (Fig. 8B). Under conditions in which intestinal extract diminished the amount of the product of mouse liver FPGS by >90% during a l-h incubation, the activity of endogenous alkaline phosphatase or glucosed-phosphate dehydrogenase increased with increasing intestinal protein while the activity of exogenously added thymidylate synthase activity was largely unchanged. Whereas the addition of soybean trypsin inhibitor increased the recovery of an internal standard of FPGS added to intestinal extracts back to control levels, it caused little or no change in the level of the three other enzyme activities examined (Fig. 8B). DISCUSSION

Published methods for the estimation of FPGS have been based on the separation of [3H]folyl product from [3H]glutamate after incubation of glutamate with ATP and a folate derivative. This separation has usually been attained by batch elution of small columns of DEAE-cellulose (2,3), although small columns of charcoal (9), batch elution of charcoal (lo), and paper electrophoresis ( 19) have also been used. The assay based on minicolumns of DEAE-cellulose has a variable and substantial blank value (3,4) which contributes to a background value for crude enzyme seen in the absence of added folate (3). Our approach to this specificity problem has been a two-step isolation of [3H]folyl product that relies on both charcoal adsorption and step elution of minicolumns of DEAE-cellulose. As indicated

MAMMALIAN

FOLYLPGLYGLUTAMATE

339

SYNTHETASE

TABLE 5 ACTIVI~

OF

FPGS ASSAYED IN THE PRESENCE OF SUPERNATANT FRACTIONS FROM VARIOUS MOUSE TISSUES” FPGS activity (cpm [“H]PteGlu2 formed/h) mg Tissue protein/assay

Without internal standard

Expt 1 None Brain Heart Lung Spleen Muscle Kidney

1.9 2.4 3.2 2.8 2.9 0.9

37* 7 33+ 4 156+ 5 290 k 20 16+ 3 920 ?I 16

Expt 2 None Intestine Intestine, boiled*

3.2 3.2

25 + 3 -

Tissue

With internal standard 5320 4220 4490 5190 4159 5080 4050

+ + + f + + +

50 150 140 87 390 36 87

6630 + 73 55* 9 4472 + 240

% Recovery 100 79 84 95 75 95 65 100 0.8 68

’ The indicated amounts of high-speed supematant fractions of various mouse tissues were incubated with 500 PM PteGlu, ATP, and [3H]glutamic acid in the presence or absence of desalted, O-30% (NH&SO,-precipitated mouse liver FPGS. Incubations were for 1 h at 37°C. (3H]PteGlu2 product was isolated by the combined assay procedure (see text). b Crude intestinal cytosol was brought to 100°C for 5 min and centrifuged, and the volume of supematant fluid equivalent to 3.2 mg of untreated cytosol was added to each reaction mixture.

by the data of Figs. 1 and 3, the prepurification of reaction product by adsorption onto charcoal prior to DEAE-cellulose chromatography reduces the radioactivity loaded onto the columns from the OS-5 &i used by others (2,3) to about 500-5000 cpm. In addition, the nonfolate products that are adsorbed onto charcoal are more easily separated from [‘H]PteGluz using DEAE-cellulose (Fig. 3) than are the interfering products that are eliminated by charcoal adsorption (Figs. 1,4). Hence, at the expense of the time associated with this charcoal step, blanks are routinely decreased to 550 cpm above machine background, allowing assay of crude enzyme fractions containing low levels of FPGS. Our attempts to isolate ‘H-labeled product from incubation mixtures containing crude enzyme fractions by charcoal adsorption alone have not been successful due to the enzymatic formation of an unidentified nonfolyl product that is charcoal adsorbable. This effect has

been predicted by others (1). Some investigators, however, have successfully used a carefully defined charcoal column (9) adsorption system or batch elution from charcoal ( 10) to study bacterial and fungal FIGS. The utility of charcoal adsorption per se for the assay of crude FPGS from these organisms is most likely due to the much higher enzyme activities in some bacteria and fungi than have been found in mammalian tissues (9,10,20). Elution of several tritium-labeled folyl diglutamates from charcoal under our conditions has proven to be consistent and to ap preach quantitative recovery (Table 1). This is in contrast to the previous experience of our laboratory and of others [see, for instance, Ref. (l)] using different conditions for the adsorption and elution of folates from charcoal. The recovery experiments reported here (see Results and Table 1) rely on the synthesis of labeled folyl diglutamate in a preincubation with FPGS and the isolation of product with

MORAN

340

i0

lntestlnal or

Proteolytic

160

150 Protein

AND COLMAN

260 , pg

Equivalent

FIG. 8. Sensitivity of FPGS to proteolysis. (A) (NH&S04-precipitated mouse liver FIGS was incubated with 500 pM PteGlu under standard assayconditions for 1 h in the presence of the indicated amounts of freshly prepared mouse intestinal crude supematant protein (0) or the amount of trypsin (0) or protease K (0) that had equivalent proteolytic activity. The amount of product formed during this incubation is plotted as a percentage of control. (B) The amount of [sH]folyl oligoglutamate formed during a l-h incubation of (NH&S04-precipitated mouse liver FPGS in the presence of the indicated amounts of intestinal protein is shown for incubations with (0) and without (0) soybean trypsin inhibitor (100 &assay). Likewise, the amount of product formed in 1 h under identical conditions by alkaline phosphatase (inverted triangles), glucose-6-phosphate dehydrogenase (triangles), and thymidylate synthase (squares), is shown as a function of intestinal protein in the presence (solid symbols) and absence (open symbols) of soybean trypsin inhibitor. For details, seeMaterials and Methods. Each symbol represents the mean of two determinations.

charcoal. Any degradation of folyl diglutamate (labeled in the w-glutamate) in the process of the alkaline elution or nitrogen drying during preparation of these standards would cause us

to underestimate recovery. Such degradation was not apparent for unreduced folates (Table l), but would be more significant for tetrahydrofolates, probably the most unstable compounds in the folate series. Hence, the lower proportion of radioactivity from l-I&PteGlu2 in the first alkaline elution (50%) in these recovery experiments represents a minimal figure for the charcoal assay using I-I&PteGlu as a substrate. A difficulty inherent in even the simplest chromatographic assay system is the manip ulation involved in processing large numbers of samples. The limited purification of mammalian FPGS thus far attained required multiple steps, including different types of chromatographic columns (3,5). Localization of FPGS in column fractions by an assay that itself requires chromatography is tedious at best. However, we have found that enzyme activities which interfere with the use of charcoal adsorption as a one-step assay are eliminated in at least some tissues by a simple (NH&S04-precipitation step (Fig. 4 and Table 3). Hence, charcoal adsorption alone can be utilized for assay for all but the crudest fractions during purification of mammalian FPGS. Adaptation of the previously reported (9) charcoal column system to a batch process allows simultaneous assay of up to 100 samples at a time within the space of an 8-h day. It should be noted that blank values are significantly higher with the charcoal as.say than with the combined use of charcoal and DEAEcellulose. However, blanks are constant for a given batch of [3H]glutamate and do not increase appreciably as a function of protein or time of incubation. Our results to date indicate that FPGS reaction rates seen with high concentrations of F’teGlu accurately reflect enzyme activities observed using I-H$teGlu as substrate for FPGS from a number of tissues (Table 3). This was not the case for dl-5-CHO-H$teGlu, the other stable, commercially available folate. In addition, the maximum velocity of mouse liver FPGS using PteGlu as a substrate was not substantially different from that using any

MAMMALIAN

FOLYLPGLYGLUTAMATE

of the substantially more labile reduced folate compounds (5). Hence, the assay procedure for FPGS recommended herein utilizes PteGlu as a standard substrate to avoid potential problems associated with the instability of H,PteGlu and related reduced folates. However, care should be exercised in the use of these conditions to ensure that FPGS activities [e.g., those from bacterial sources (10,20)] which are specific for other folate compounds are not overlooked. FPGS activity was very low in most tissues of the mouse. In brain, intestine, heart, skeletal muscle, and lung, the level of enzyme was at or below the limit of detection with this assay. This was somewhat surprising since the folate pool of at least some of these tissues in adult mammals has been shown to be largely in the form of polyglutamates (2 l-24). The apparent lack of enzyme activity in these tissues could have been due to any one of several factors: (i) the presence of excessive levels of ATPases; (ii) the presence of conjugases, which would digest the folyl oligoglutamate product; (iii) other interfering enzymes, such as glutamateconsuming activities or proteases; and (iv) tissue-specific enzyme inhibitors. We chose to determine whether these artifacts were invalidating our results by adding a partially purified mouse liver FPGS to tissue cytosol preparations as an internal standard. These experiments ruled out all of these possible factors except in the case of the mouse intestine (Table 5). It was clear that the level of protease in intestinal cytosols interfered with our attempts to directly measure FPGS. However, when intestinal proteases were inhibited by soybean trypsin inhibitor, an exogenously added FPGS internal standard was quantitatively recovered (Fig. 8), but endogenous activity was still found to be low. It should also be noted that mouse liver FPGS is much more sensitive to proteolysis than are a number of other enzymes assayed under identical conditions (Fig. 8). It is therefore possible that proteolytic inactivation may be involved in the instability of FPGS observed during purification of this enzyme (3,5,20).

SYNTHETASE

341

We are tempted to speculate that differentiated tissues have a low requirement for FPGS, due perhaps to a minimal rate of tumover of folyl polyglutamates. The low level of activity in intestine may reflect the fact that the majority of the cells of the intestine are terminally differentiated, with significant mitotic activity found only in the dividing crypt cells. In contrast, a report of the activity of FPGS in rat tissues has indicated significant levels of enzyme in brain and in intestine (25). It is not clear why the data on the distribution of activity in the rat do not parallel the levels we find in the corresponding mouse tissues. However, Fry et al. (26) have recently reported that the intact mouse intestine does not form polyglutamates of methotrexate in viva under conditions in which the Ehrlich ascites carcinoma forms substantial antifolyl polyglutamates. It would clearly be disadvantageous to have high levels of FPGS (an enzyme seemingly responsible for impeding efflux of folates from the cell) in the intestinal mucosa, the organ responsible for the absorption of dietary folates. We propose, then, that the FPGS activity of the intestine is largely in the stem cells of the crypts and that the expression of this activity is suppressed as the columnar epithelial cells differentiate. ACKNOWLEDGMENTS We thank Dr. Charles E. Grimshaw and Dr. Shirley Taylor for their critical evaluation of this manuscript, Ms. Valerie Reich for technical assistance,and Ms. Patrice Johnston-Turner for her careful preparation of the manuscript. This work was supported by Grant CA-27605 from the National Institutes of Health.

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342

MORAN

AND COLMAN

7. Moran, R. G., and Colman, P. D. (1980) Proc. Amer. Assoc. Cancer Rex 21, 25. 8. Moran, R. G. (1983) in Folyl and Antifolyl Polyglutamates (Goldman, I. D., Chabner, B. A., and Bertino, J. R., eds.), pp. 327-339, Plenum, New York. 9. Ritari, S. J., Sakami, W., Black, C. W., and Rzepka, J. (1975) Anal. Biochem. 63, 118-129. 10. Masurekar, M., and Brown, G. M. (1975) Biochemistry 14, 2424-2430. 11. Moran, R. G., Spears, C. P., and Heidelberger, C. (1979) Proc. Natl. Acad. Sci. USA 76, 1456-1460. 12. Moran, R. G., We&he&r, W. C., and Zakrzewski, S. F. (1976) J. Biol. Chem. 251, 3569-3575. 13. Hartree, E. F. (1972) Anal. Biochem. 48,422-427. 14. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70,241-250. 15. Horrocks, D. L. Beckman Technical Bulletin 1095NUC-77-IT. 16. Moran, R. G., and Colman, P. D. ( 1982) Anal. Biochem. 122, 70-78. 17. Baugh, C. M., and Krumdieck, C. L.. (1971) Ann. N. Y. Acad. Sci. 186, 7-28.

18. Silverman, M., Law, L. W., and Kaufman, B. ( 196 1) J. Biol. Chem. 236,2530-2533. 19. Rosenberg, I. H., and Neumann, H. ( 1974) J. Biol. Chem. 249, 5126-5130. 20. Shane, B. (1980) J. Biol. Chem. 255, 5655-5662. 2 1. McClain, L. D., and Bridgers, W. F. (1970) J. Neurochem. 17, 763-766. 22. McClain, L. D. (1979) in Folic Acid in Neurology, Psychiatry and Internal Medicine (Botez, M. I., and Reynolds, E. H., eds.), pp. 147-155, Raven Press, New York. 23. Brody, T., Shin, Y. S., and Stokstad, E. L. R. (1976) J. Neurochem. 27, 409-413. 24. Brown, J. P., Davidson, G. E., and Scott, J. M. (1974) B&hem. Biophys. Acta 343, 78-88. 25. McGuire, J. J., Kitamoto, Y., Hsieh, P., Coward, J. K., and Bertino, J. R. (1979) in Chemistry and Biology of Pteridines (Kisliuk, R. L., and Brown, G. M., eds.), pp. 471-476, Elsevier/North-Holland, Amsterdam/New York. 26. Fry, D. W., Goldman, I. D., and White, J. C. (1982) Proc. Amer. Assoc. Cancer Res. 23. 179.