ANALYTICAL

75, 389-401 (1976)

BIOCHEMISTRY

Determination of the Intracellular Concentration 5-Phosphoribosyl-I-pyrophosphate in Cultured Mammalian Fibroblasts

of

S.RANDOLPH MAY AND ROBERT S.KROOTH Department

of Human Genetics and Development, College of Physicians Columbia University, New York. New York 10032

and Surgeons.

Received January 12, 1976; accepted May I I. 1976 The properties of an assay for the 5-phosphoribosyl-1-pyrophosphate (PRPP) content of cultured mammalian fibroblasts are described. The assay is based upon the PRPP-dependent release of “CO, from [carboxy/-“Clorotic acid by a commercially available preparation of yeast orotidine-5’-monophosphate pyrophosphorylase and orotidine-5’-monophosphate decarboxylase. The advantages of the assay include the fact that it is based on the enzymatic recognition of PRPP, employs an irreversible reaction, and does not involve either the chromatographic separation of substrate and product or the purification of a phosphoribosyltransferase. The disadvantage of the assay is that the efficiency of PRPP measurement varies somewhat, in part because the yeast enzyme preparation contains 5’nucleotidase activity. A calibration procedure is described which corrects for variation in efficiency both between and within experiments. This procedure seems to yield highly reliable estimates of PRPP content. The assay will readily detect 0.6 nmol, and the cell strain studied contained 7.76 2 I. 14 nmol of PRPP/I07cells.

cr-5-Phospho-D-ribosyl-1-pyrophosphate (PRPP) plays a central role in the mammalian cell as a substrate for the 5’-phosphoribosyltransferases. These enzymes are involved in the biosynthesis of histidine and tryptophan and the purine, pyrimidine, and pyridine nucleotides (1). The important biosynthetic role of PRPP has prompted the development, during the last decade, of several different assay procedures suitable for use with extracts of mammalian cells. These assay procedures quantitate the PRPP present by reacting PRPP with a purine or pyrimidine base in the presence of a specific phosphoribosyltransferase. The amount of purine or pyrimidine ribonucleotide formed during the course of the reaction is then estimated. One of the earliest demonstrations of an intracellular pool of PRPP was based on pyrimidine nucleotide synthesis. The first published nonradiochemical PRPP assay procedure depended on the decrease in absorbance at 295 nm when orotate was sequentially converted to orotidine 5’-monophosphate (OMP) and uridine 5’-monophosphate (UMP) by orotidine-5’monophosphate pyrophosphorylase (EC 2.4.2. lo), and orotidine-5’monophosphate decarboxylase (EC 4.1.1.23), respectively. In the course 389 Copyright All

rights

0 of

1976 reproduction

by

Academic I”

any

Press,

Inc.

form

reserved.

390

MAY

AND

KROOTH

of these investigations, Kornbergand his co-workers (2) also used the same enzymes to provide evidence that intracellular PRPP is required for the release of 14C0, from [carboXylJ4C]orotic acid. Most of the previously reported radiochemical assays for PRPP depend on purine nucleotide synthesis. One group of assays involves the conversion of radioactive adenine into labeled adenosine 5’-monophosphate by either highly purified (3-5) or partially purified preparations (6- 11,31) of adenine phosphoribosyltransferase (EC 2.4.2.7). Another group of assays employs the conversion of radioactive hypoxanthine into labeled inosine 5’-monophosphate by either crude or partially purified preparations of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) (12-14). Finally, some workers, instead of adding exogenous enzyme, have developed assays which employ the phosphoribosyltransferase activity already present in the cell extract to effect the conversion of radiolabeled adenine or hypoxanthine into the respective purine nucleotides (15,16). Since the initial description by Kornberg and his co-workers (2), there have been very few reports in the literature of investigations of cellular PRPP synthesis which have employed the PRPP-dependent conversion of [carboxylJ4C]orotic acid into 14C0, by the sequential action of orotidine-5’-monophosphate pyrophosphorylase (OMP-ppase) and orotidine-5’-monophosphate decarboxylase (OMP-dcase). Such an approach was used by Green and Martin (30) as an assay of the amount of PRPP produced by the PRPP synthetase of a rat hepatoma cell line. Reem (29) has just reported an assay for the PRPP content of human diploid cell strains in which yeast OMP-ppase and OMP-dcase are employed. In the present paper, we will review our experience with an assay similar to the one described by Reem (29). We shall describe the assay procedure, summarize the properties of the assay, and suggest an experimental design which appears to yield highly reliable estimates of PRPP concentration. MATERIALS

AND METHODS

Materials Tris-HCl, Tris base, 5-phosphoribosyl-I-pyrophosphate (tetrasodium salt, Lot No. 42C-0781-9 and 42C-86301-9), erotic acid, and the crude yeast orotidine-5’-phosphate pyrophosphorylase and orotidine-5’-phosphate decarboxylase (mixed enzymes, Lot No. 124C-8000) were purchased from Sigma Chemical Co., St. Louis, MO. In the presence of excess erotic acid and PRPP, 1 unit of the yeast enzyme preparation is capable of converting 1 pmol of erotic acid (into UMP and COJhr at 25°C and at pH 8.0. Ethylene glycol monoethyl ether, monoethanolamine, perchloric acid, dextrose (o-glucose), sodium acetate, isopropanol,

PRPP

CdNCENTRATION

IN CULTURED

FIBROBLASTS

391

ammonium sulfate, and MgCl, were the products of Fisher Scientific Co., Fairlawn, N.J. [Carboxyl-14C]orotic acid (42.4 mCi/mmol), [6-14C]orotic acid (47.1 mCi/ mmol), and scintillation grade toluene were purchased from New England Nuclear, Boston, Mass. 2,5Diphenyloxazole (PPO) of scintillation grade was supplied by Packard Instrument Co., Inc., Downers Grove, Ill. LSC Complete scintillation fluid was provided by Yorktown Research Corp., South Hackensack, N.J. Plastic center-wells (K-882320-0000) to hold the 14C0, trapping fluid were obtained from Kontes Glass Co., Vineland, N.J. Hanks’ balanced salt solution (Ca2+ and Mg*+ free) was obtained from the Grand Island Biological Co., Grand Island, N.Y. Swim’s 77 medium (20) was prepared from salts (Fisher Scientific Co.), amino acids (Sigma Chemical Co.), and vitamins (Grand Island Biological Co.). Cell Culture The properties of the mouse A9P cells, which are deficient in both adenine and hypoxanthine phosphoribosyltransferase (17,18), and the techniques and materials (other than those described above) employed for the cell culture have been described elsewhere (17). Extraction

of Cellular

PRPP

PRPP was extracted from the cultured mouse cells by the technique of Henderson and Khoo (6). Their procedure was developed for suspension cultures, so we have modified it to accommodate cells growing on the surface of plastic culture flasks. The cells were harvested either by shaking from the surface of the plastic flask or by incubation with a 0.25% solution of trypsin (17). Following harvest, the cells were sedimented by centrifugation at 25g and were then resuspended in a preincubation medium at a concentration of about IO7 cells/ml. The preincubation mixture was composed of Hanks’ balanced salt solution (Ca*+ and Mg*+ free) with 5.5 mM glucose added, Swim’s 77 medium containing 2 mM glutamine (20), or Eagle’s minimal essential medium with Earle’s balanced salt solution for suspension cultures (Ca*+ free) (28). The preincubation was carried out by adding lo- 12 ml of cell suspension to a 25-ml Erlenmeyer flask which was then placed in a shaking water bath at 37°C for 15 min. To extract PRPP, a 1 .O-ml aliquot of the preincubation medium, containing a suspension of about 10’ cells to be assayed, was rapidly transferred to a 15-ml centrifuge tube standing in boiling water. After 30 set, the tube was plunged into an ice-water bath. Denatured protein and~other insoluble materials were then sedimented by centrifugation at 10,OOOg in a Sorvall RC2-B for 15 min at 4°C. The entire clear supernatant was used for each determination of the quantity of cellular PRPP present. The amount of

392

MAYANDKROOTH

PRPP per cell was determined by dividing the estimate of the quantity of PRPP per milliliter of cell suspension by the number of cells per milliliter. The numbers of cells in 1 ml of preincubation medium were determined by hemacytometer counts. The accuracy of the cell counts performed in this way was initially checked by concurrent determinations of cell protein. In these experiments, the cell counts and protein determinations were performed both before and after preincubation to verify the impression that cell loss during preincubation was negligible. Assay for PRPP

The PRPP assay is based upon the PRPP-dependent release of 14C0, from [carboxyl-14C]orotic acid by a commercially available preparation of yeast orotidine-5’-monophosphate pyrophosphorylase (OMP-ppase) (EC 2.4.2.10) and orotidine-5’-monophosphate decarboxylase (OMP-dcase) (EC 4.1.1.23) according to the following scheme: [carboxyl-14C]orotic [carboxyl-

acid + PRPP + Mg2+ i 14C]orotidine 5’-monophosphate

yeast OMP-ppase

J

yeast OMP-dcase

uridine Y-monophosphate

+ 14C0,.

The reaction mixture consisted of 100 mM Tris buffer, pH 8.0; 12.5 mM MgC12; 1.0 mM [carboxyl-14C]orotic acid with a specific radioactivity of 4.24 mCi/mmol (1:9 dilution of radiolabeled to unlabeled); 0.25 unit of crude yeast OMP-ppase and OMP-dcase (capable of converting 0.25 mmol of erotic acid into UMP and CO, in 1 hr); and the solution of PRPP whose concentration was to be determined. When cell extracts were employed, this solution contained the PRPP extracted from IO7 cells, as described earlier. The total reaction volume was 1.4 ml, and the reaction was carried out in 25-ml Erlenmeyer flasks. The reaction was initiated by the addition of the solution containing the unknown concentration of PRPP to the flask which was then immediately closed with a silicone-greased stopper. The center of each stopper held the stem of a plastic centerwell. The centerwell contained 250 ~1 of a 2: 1 solution of ethylene glycol monoethyl ether and monoethanolamine to trap the 14C0, released (21). After 1 hr of incubation at 25°C in a shaking Dubnoff metabolic water bath, 350 ~1 of 60% perchloric acid was injected through the stopper into the reaction vessel to release the dissolved 14C0,. After 40 min of additional incubation with shaking, the centerwells were removed and placed in screw cap vials containing 15 ml of a liquid scintillation fluid consisting of 9.9 g of 2,5-diphenyloxazole in 800 ml of ethylene glycol monoethyl ether mixed with 1000 ml of scintillation grade toluene (21). Efficiency of measurement of radioactivity was approximately 75%.

PRPP CONCENTRATION

IN CULTURED

393

FIBROBLASTS

20 I

‘Ol 5 UNITS

IO OF

20 YEAST

30

OMPppose

and

ASSAY

MIXTURE

OMP-

40 dcose

IDDED

TO

FIG. 1. Decrease in the release of CO, from erotic acid for a reaction mixture containing 10 nmol of PRPP when the amount of yeast OMP-ppase and OMP-dcase in the reaction mixture is increased incrementally from 0.25 to 40.0 units.

The assay was calibrated by adding known quantities of PRPP to the cell suspension immediately prior to boiling and then measuring the amount of 14C02 released. The blank for each such determination contained the suspending medium, without cells, which was treated in the same fashion as the cell suspension. Further calibration data were obtained by adding known quantities of PRPP to the cell-free suspending medium (immediately prior to boiling). Chromatographic and UMP

Assay of the Conversion

of Orotic Acid into OMP

The reaction mixture consisted of 250 mM Tris buffer, pH 8.0; 25 mM MgCl,; 10 mM PRPP; 22.2 nmol of [6-14C]orotic acid (47.1 mCi/mmol); and 5 units of yeast OMP-ppase and OMP-dcase. The total volume was 100 ~1. The reaction was initiated by the addition of enzyme and, after 1 hr of incubation at 25”C, the reaction was terminated by boiling for 3 min. After centrifugation at 5OOOg for I5 min, 20 ~1 of the supernatant were spotted onto Whatman No. 1 paper strips and subjected to 15 hr of ascending chromatography in 80: 18:2 (by volume) of saturated ammonium sulfate: 1 M sodium acetate:isopropanol. Finally, the strips were cut into l-cm sections, and the radioactivity was determined by liquid scintillation counting in a toluene-based scintillator (LSC Complete, Yorktown Research Corp., South Hackensack, N.J.). The Rf values of erotic acid, orotidine, uridine, OMP, and UMP were 0.35, 0.58, 0.74, and 0.74, respectively.

394

MAY

AND

KROOTH

RESULTS Proportionality of the Amount Amount of PRPP Present

of CO, Released from Orotic Acid to the

In the presence of 5 units of yeast enzyme preparation, there is virtual molar equivalence between the quantity of PRPP present, at concentrations of PRPP of more than 100 nmol/ml, and the quantity of CO, released from erotic acid. However, at PRPP concentrations of less than 100 nmol/ ml the equivalence is lost, and the ratio of molecules of CO, released from erotic acid to molecules of PRPP present grows smaller as the PRPP concentration is lowered. The data in Fig. 1 indicate that in the presence of a fixed amount of PRPP t.he quantity of CO, derived from erotic acid decreases as the amount of yeast OMP-ppase and OMP-dcase added to the reaction mixture is increased (above a certain quantity). When the amount of yeast enzyme preparation added becomes large, the curve appears to approach an asymptote corresponding to about 0.2-0.3 mol of CO, released from erotic acid for each mole of PRPP present. The presence of an asymptote may well reflect competition between two simultaneously occurring enzymatic processes: (i) the conversion of PRPP by the yeast OMP-ppase and OMP-dcase into OMP and UMP, and (ii) the hydrolysis of the PRPP by a phosphatase present in the yeast preparation, and/or the degradation of OMP, by a contaminating 5’-nucleotidase (EC 3.1.3 5). A phosphatase which degrades PRPP has recently been characterized in human tissues (12) and may occur in yeast cells as well, in which case it might contaminate the yeast OMP-ppase and OMP-dcase preparation. However, we favor the second explanation listed under (ii) above. We have found that the yeast enzyme preparation tends to degrade the OMP and UMP produced during the course of the reaction. When 5 units of yeast enzyme preparation are incubated for 1 hr with 22.2 nmol of [6J4C]orotic acid and 200 nmol of PRPP, only 33% of the radioactivity in erotic acid can be recovered in chromatographic peaks corresponding to OMP and UMP. The remaining 66% of the label is recovered in peaks whose Rf corresponds to that of orotidine or uridine. This result is prima facie evidence for the contamination of the yeast OMP-ppase and OMP-dcase enzyme preparation with a 5’-nucleotidase activity. In any case, the degradation of PRPP by a phosphatase or of [carboxyl-14C]0MP by 5’nucleotidase could readily explain the decrease in 14C0, recovered from [carboxyl-14C]orotic acid as the concentration of the yeast enzyme preparation in the reaction mixture is increased. It can be shown that for any given range of PRPP concentrations, there is a corresponding range of yeast enzyme concentrations which will yield approximately molar equivalence between the quantity of PRPP in the reaction mixture and the quantity of CO, released from erotic acid. Figure 2 shows that as the quantity of PRPP becomes small, molar equiva-

PRPP CONCENTRATION

IN CULTURED

FIBROBLASTS

395

FIG. 2. (A) Relationship between the amount of PRPP added to the reaction mixtures and the amount of CO, released from erotic acid by the yeast OMP-ppase and OMP-dcase preparation. The quantity of enzyme preparation in units (U) added is indicated above or below the line. (B) Replot of the data presented in (A) to show the near equivalence, over a 250-fold range of PRPP concentrations. between the moles of PRPP present and the moles of CO, released from erotic acid.

lence is obtained with a reduced quantity of enzyme. Molar equivalence is not, however, obtained with the higher quantities of yeast enzyme necessary for equivalence at elevated concentrations of PRPP. In any case, we have found that by controlling the amount of yeast enzyme preparation added to the reaction mixture, we can ensure that there is molar equivalence between the CO, released from erotic acid and the concentration of PRPP. Figure 2 is helpful in determining the quantity of yeast enzyme preparation necessary to assay a particular range of PRPP concentrations. For the determination of the amount of PRPP in about 10’ cultured mouse fibroblasts, an assay is needed which can accurately measure the PRPP content in the range of 0.5 to 50 nmol (vi& infra). Figure 2 suggests that this requirement may be met by the use of 0.25 unit of the yeast enzyme preparation, and, in subsequent experiments, we have shown that this is indeed true. Of course, we have employed but a single preparation of the commercially available yeast pyrimidine enzymes (Sigma Chemical Co., Lot No. 124C-8000). However, similar experiments can determine the concentration of any given preparation of yeast enzymes suitable for a

396

MAY AND KROOTH

IO

20 30 40 50 NANOMOLES OF PRPP ADDED

60

FIG. 3. Determination of the intracellular PRPP content of a mouse cell line, A9P, using various amonts of PRPP as an internal standard to determine the efficiency of measurement of intracellular PRPP. In the same experiment identical concentrations of PRPP were added to a reaction mixture containing no cell extract.

PRPP determination. Moreover, the experimental design we shall describe below provides a method for measuring, in each determination, the molar equivalence between the CO, released from erotic acid and the amount of PRPP in the reaction mixture. Use of the Assay for the Determination Mammalian Cells

of the PRPP

Content of Cultured

In our view, a suitable assay procedure for the PRPP content of cultured mammalian cells involves the simultaneous estimation of two quantities for every assay: the amount of CO, whose release from erotic acid is effected via the PRPP in the cell extract, and the efficiency with which the PRPP concentration is measured by the assay. Within a given experiment, the efficiency of the assay procedure may of course be determined by the addition of several known concentrations of PRPP to individual aliquots of the cell preparation just prior to boiling. Such experiments yield data of the sort shown in Fig. 3. The relationship between CO2 released from erotic acid and the PRPP added to the reaction mixture may be described by the following equation: Ul C = E (Pendo + P,,,L where C equals the number of moles of CO, released from erotic acid, after correction for the efficiency of measurement of radioactivity and for the radioactivity recovered from a blank which contained no cell extract or PRPP; Pendo equals the amount of endogenous PRPP in the cell

PRPP

CONCENTRATION

IN CULTURED

FIBROBLASTS

397

extract; E equals efficiency of the assay within the experiment; and P,,, equals the amount of exogenous PRPP added to the reaction mixture. From the data produced by such an experimental design, regression analysis by the method of least squares yields an estimate of EPendo, which is the amount of CO2 released from erotic acid by the cell preparation in the absence of added PRPP. The slope (E) of the line generated in this way represents the efficiency of the particular assay in which EPendo was determined. The calculated PRPP content of the cell extract (Pendo) then equals the value of ordinate corresponding to the Y-intercept of the regression line divided by the efficiency (E) of the assay, as described in Eq. PI: Calculated concentration of PRPP in extract = EP,,,JE PI The data can in fact be used to obtain three estimates ofPendo. The first of these estimates is generated by a regression relating the quantity of CO, released from erotic acid to the amount of exogenously added PRPP. However. the regression is generated after exclusion of the point corresponding to zero added PRPP. The value of Pendo is then determined by extrapolation of the regression line to the Y-axis and dividing the ordinate of that intercept by the slope of the regression line. We refer to this value of P endoas the “predicted estimate.” An estimate ofPend,, can also be obtained without regression analysis from the amount of CO, released from erotic acid in the absence of added PRPP. This quantity is in fact an estimate of EP endoand can be converted to an estimate of Pendo by dividing it by the slope of the regression line used to obtain the predicted estimate. We refer to the value of Pendo obtained in this way as the “empiric estimate.” The “predicted estimate” and the “empiric estimate” are statistically independent and can be compared. Agreement between these two estimates supports the notion that the relationship, observed in calibrating the assay, between the amount of PRPP present and the amount of CO2 released from erotic acid actually holds in the neighborhood of the PRPP concentration one is attempting to measure. Note that the calibration is accomplished in the presence of extract. The third way of computing Pendo uses all the information and so yields the most accurate estimate, provided of course that the predicted and empiric estimates are found to agree. The third estimate is obtained by computing Pendo from a regression relating the CO, released from erotic acid to the quantity of PRPP added, including in the regression the point or points corresponding to zero added PRPP. We shall refer to the value of P endo computed in this way as the “final estimate.” Table 1 summarizes the regression analysis used to obtain the final estimate for EPendo in four experiments. Note that in three of the four experiments the efficiency of the assays varied from 89 to 100% and that in all four experiments. the final values ofPend,, are very similar. Table 2

398

MAY

AND

KROOTH

TABLE

1

FINALESTIMATEOFINTRACELLULARPRPPCONTENT (Pendo) OFA~PCELLSASINFERRED FROMTHE Y-INTERCEFTOF ALINEARREGRESSION RELATINGTHEAMOUNT OF CO,RELASEDFROMOROTIC ACIDTOTHEQUANTITY OF PRPP ADDEDTO THE REACTIONMIXTURE" Expt.

Estimates

I

Expt.

2

Expt.

3

Expt. A9P

4

A9P

Blank

A9P

Blank

A9P

Blank

Blank

5.464 0. I382 0.8907 0.0018

-0.356 0.0714 I.002 o.coO2

3.904 0.0735 0.5520 0.001 I

-0.038 0.2248 0.9553 0.0007

x.430 0.085I 0.9862 o.OOiI3

-0.119 0.1194 0.9945 0.0003

8.545 0.0441 0.9764 O.OQOl

-0.020 0.0052 I.014 o.om

6.135

-0.355

7.072

-0.040

8.548

-0.120

8.752

-0.020

from regression

EP..,. (in nanomoles

line of of CO2 acid per IO’

released from erotic cells) Variance of EP,.,, in nanomdes Mean efficiency of the assay(E) Variance of E

P,.,, (in nanomoles

of ‘ICO,

released

per IO’ cells) ” The regression

is computed

by the method

of least squares.

The blank

contained

no cell extract.

compares the mean values, for all four experiments, of the three estimates. Note that the empiric, predicted and final estimates agree closely. Level of Sensitivity

of the Assay

The background level of radioactivity, determined by performing the assay on incubation media to which no cells were added, corresponds to 0.18 + 0.21 nmol (mean + SD) of PRPP. Thus, the sensitivity of the assay, two standard deviations above the mean background level, is about 0.6 nmol of PRPP. Reaction mixtures containing neither fibroblast extract nor yeast enzyme did not release detectable quantities of 14C0,. Other Properties

of the Assay

We have confirmed the observation of Henderson and Khoo (6) who noted that a 30-set boiling of a suspension of cells from the preincubation medium does not result in a measurable decrease in the amount of PRPP present. However, there is a significant loss of PRPP if the boiling time is increased to 1 min, and a 2-min boiling lowers the PRPP content by more than one-third. We have also observed that several preincubation mixtures besides Krebs-Ringer with 5.5 mM D-glucose (6) are acceptable for use with the assay, including Hanks’ balanced salt solution (Ca2+ and Mg2+ free) with 5.5 mM D-glucose, Swim’s 77 medium (20) containing 2 mM glutamine (20), and Eagle’s (28) minimal essential medium with Earle’s balanced salt solution.

PRPP CONCENTRATION

IN CULTURED TABLE

399

FIBROBLASTS

2

COMPARISON OF ESTIMATES OF INTRACELLULAR PRPP CONCENTRATION OBTAINED BY THE THREE METHODS”

Estimate

Mean

Predicted estimates Empiric estimates Final estimates

7.93 7.60 7.76

Standard deviation 1.30 I.10 1.14

Standard error of the mean 0.65 0.55 0.57

n The “predicted estimates” were calculated from the Y-intercepts of the linear regressions relating the amount of “C0, released to the quantity of PRPP added to the reaction mixtures. The points corresponding to zero added PRPP were excluded from the data employed to generate these regressions. The Y-intercepts used to estimate the intracellular PRPP concentrations were obtained by extrapolation and were divided by the efficiency (E) of the assay as determined by the slope of the regression. The “empiric estimates” were computed from the quantity of rlCO, recovered in the absence of added PRPP and were divided by the efficiency(E) calculated from the slope of the same regression line used to obtain the predicted estimate in the corresponding experiment. The “final estimates” were calculated in the same way as the predicted estimates. except that the points corresponding to zero added PRPP were included in the data used to generate the regressions. The Y-intercept of the regression of the blanks (containing no cell extract) were subtracted from the Y-intercepts of the regressions in the case of the predicted estimates and the final estimates. In the case of the empiric estimate the r4C0, recovered from the cell-free flasks was simply subtracted from the corresponding figure for flasks containing cells. All standard errors of the mean were calculated empirically, i.e.. by computing the root sum of squares between experiments and dividing by [vN(N - l)]r’*, where N is the number of experiments. namely four. The units are nanomoles of PRPP per IO7 cells.

We have found that the estimate of intracellular PRPP is about one order of magnitude higher when the cells are preincubated in any one of the preincubation media described above. This result confirms the initial observations of Henderson and Khoo (6). The fact that the levels of PRPP are higher in preincubated cells of course means that the PRPP concentration estimated after preincubation need not apply to cells used directly after harvest. Hence, in studies comparing cell strains it is essential that this step in the procedure be uniformly employed (or omitted). DISCUSSION

We have characterized a simple and sensitive assay for the determination of the intracellular concentration of PRPP in cultured mammalian fibroblasts. The assay employs a commercially available preparation of yeast OMP-ppase and OMP-dcase and measures the PRPP-dependent release of 14C0, from carboxyl-labeled erotic acid. The assay procedure is complicated by three factors: the low basal levels of PRPP, the lability

400

MAY AND KROOTH

of the compound, and the contamination of the yeast enzyme preparation by a 5’-nucleotidase activity. However, the basal PRPP levels can be raised IO-fold by preincubation of the cells prior to harvest, and the lability of PRPP and the contamination of the yeast enzyme preparation can each be dealt with by the biochemical and calibration procedures we have just described. We suspect that our experimental design, or an equivalent one, for estimating PRPP concentration should be used, whenever possible, in all quantitative determinations of labile chemicals. The design allows a calibration function to be generated in the presence of cell extract and ensures that variables affecting the efficiency of the assay have no influence on the estimate of the concentration of the substance being measured. Even more importantly, the design permits the investigator, in each determination, to check the validity of a central assumption underlying the assay. This assumption states that the inferred relationship, between the final measurement and the concentration of the substance being measured, holds in the immediate vicinity of the unknown concentration which is being estimated. This same assumption generates a predicted estimate of the unknown concentration which is statistically independent of the actual estimate. The discordance between the two estimates is sensitive to sampling and analytic error, as well as to failure, in a particular experiment, of the molecules to behave in the way predicted. Hence, close agreement between the two estimates provides strong support for the notion that the inferred relationship between measurement and concentration holds in the neighborhood of the unknown concentration being estimated and for the notion that sampling and analytic errors are small. It is seldom realized that nearly all analytic methods in theory permit the estimation of endogenous concentration both from direct measurement and from a calibration function obtained by determining the effect on the final measurement of adding varying quantities of the substance under study. We should perhaps emphasize that another critical assumption underlying every analytic determinations is that, in the absence ofexogenousfy added compound, the final measurement obtained depends on the endogenous quantity of compound present. The experimental design discussed above does not in itself test that assumption. Other methods exist for dealing with this question, such as radiodilution techniques or the use of preparations known, a priori, to be specifically deficient in the substance being measured, e.g., extracts of mutant cells which lack activity for an enzyme necessary for the synthesis of the substance. ACKNOWLEDGMENTS This work was supported by Grants GM 18153 and GM 22103-01 from the United States Public Health Service.

PRPP CONCENTRATION

IN CULTURED

FIBROBLASTS

401

REFERENCES 1. Fox, I. H., and Kelley. W. N. (1971)Ann. Intern. Med 74, 424-433. 2. Komberg, A., Lieberman, I., and Simms, E. S. (1955) J. Biol. Chem. 215, 389-402. 3. Wood, A. W., Becker, M. A., Minna. J. D., and Seegmiller, J. E. (1973) Proc. Nat. Acad.

Sci.

USA

70, 3880-3883.

4. Becker, M. A., Meyer, L. J., Wood, A. W., and Seegmiller, J. E. (1973) Science 179, 1123-1126. 5. Wood, A. W., Becker, M. A., and Seegmiller, J. E. (1973)Biochem. Genet. 9,261-274. 6. Henderson, J. F., and Khoo, M. K. Y. (1965) J. Biol. Chem. 240, 2349-2357. 7. Rosenblum, F. M., Henderson, J. F., Caldwell. I. C.. Kelley. W. N.. and Seegmiller, J. E. (1968) J. Biol. Chetn. 243, I166- 1173. 8. Fox, I. H., Wyngaarden, J. B.. and Kelley. W. N. (1970) N. Eng[. J. Med. 283, 11771182. 9. Bagnara. A. S.. Brox, L. W.. and Henderson, J. F. (1974)Biochim. Biophys. Acta 350, 171- 182. 10. Bagnara, A. S., Letter, A. A., and Henderson, J. F. (1974) Biochim. Biophys. Acta 374, 259-270. 11. Fox, I. H.. and Marchant, P. M. (1974) Cunad. J. Biochem. 52, 1162-I 166. 12. Hershko, A., Razin, A., Shoshani. J.. and Mager. J. (1967) Biochim. Biophys. Acfa 149, 59-73.

13. Hershko, A., Razin. A., and Mager, J. (1969) Biochim. Biophys. Acta 184, 64-76. 14. Meyshkens, F. L., and Williams, H. E. (1971) Mefaholism 20, 737-742. 15. Sperhng, 0.. Eilam. G.. Persky-Brosh, S.. and DeVries, A. (1971)J. Lab. C/in. Med. 79, 1021-1026. 16. Gordon, R. B., Thompson, L., and Emmerson, B. T. (1974) Metabolism 23,921-927. 17. Hashmi, S., May, S. R.. Krooth, R. S., and Miller. 0. J. (1975) J. Cell. Physiol. 86, 191-200. 18. May, S. R., Hashmi, S., Miller, 0. J., and Krooth, R. S. Manuscript in preparation. 19. Greene. M. L., and Seegmiller. J. E. (1969) Arfhrifis Rheum. 12, 666-667. 20. Thompson, E. B., Tomkins, G. M., and Curran, J. F. (1966) Proc. Nar. Acad. Sci. USA 56, 296-303. 21. Jeffay, H. (1963) in Advances in Tracer Methodology, (Rothschild, S., ed.), Vol. I, pp. 113- 114, Plenum, New York. 22. Henderson, J. F. (1972) in Regulation of Purine Biosynthesis, Monogr. 170, pp. 163195. American Chemical Society, Washington, D.C. 23. Wamick, C. T.. and Paterson. A. R. P. (1973) Canad. Res. 33, 1711-1715. 24. Ishii, K., and Green. H. (1973) J. Cell Sci. 13, 429-439. 25. McBumey. M. W., and Whitmore, G. F. (1975) .I. Cell. Physiol. 85, 87- 100. 26. Smith, P. C., Knott. C. E.. and Tremblay, G. C. (1973) Biochem. Biophys. Res. Commun. 55, 1141- 1146. 27. Giilen, S.. Smith, P. C., and Tremblay, G. C. (1974) Biochem. Biophys. Res. Commun. 56, 934-939. 28. Eagle, H. (1959) Science 130, 432-437. 29. Reem, G. H. (1975) Science 190, 1098-1099. 30. Green. C. D., and Martin, D. W., Jr. (1973) Proc. Nat. Acad. Sci. USA 70,3698-3702. 31. Hisata. T. (1975) Anal. Biochem. 68. 448-457.