ANALYTICAL
BIOCHEMISTRY
A Simple, SUNIL
95, 497-506 (1979)
Specific
K. CHATTERIEE, Department
Radiometric
Assay for 5’-Nucleotidase
MALAYA BHATTACHARYA,ANDJOSEPH of Gynecology, 666 Elm Street,
Roswell Park Memorial Buffalo, New York 14263
J. BARLOW
Institute.
Received November 1, 1978 A radiometric assay for 5’-nucleotidase (EC 3.1.3.5) has been developed. which is applicable for all 5’-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were found to be the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 M TrisHCI, pH 7.4; subsequent elution of the 5’-nucleotide was then accomplished with 0.2 M sodium phosphate, pH 7.4. Differential measurement of 5’nucleotidase can be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavahn A into the reaction mixture. It completely inhibits 5’nucleotidase without effecting the phosphatases. The applicability of this assay has been demonstrated by studying the properties of 5’-nucleotidase present in a purified plasma membrane preparation from a rat tumor which is enriched with both S’nucleotidase and alkaline phosphatase.
5’-Nucleotidase (EC 3.1.3.5; ribonucleotide phosphohydrolase) is widely used as a plasma membrane marker enzyme (1). Elevation of the levels of this enzyme in human sera has been observed in a number of clinical conditions (2,3). Commonly used calorimetric (4) and spectrophotometric (5) methods for the assay of 5’-nucleotidase are laborious and not suitable for low levels of activity in crude extracts. This prompted the development of various radiometric procedures (6,7) which are sensitive, but are applicable only when 5’-AMP or CMP are used as substrates. For systems which prefer IMP or GMP as substrates (8,9), or in crude extracts where other enzymes capable of metabolizing the nucleotides or nucleoside products are present, these methods cannot be used. These drawbacks can be overcome by the use of 32P-labeled mononucleotide substrates (10,ll). However, the short half-life (14 days) of the radioisotope is a serious limitation of this technique. Another problem encountered in the
assay of 5’-nucleotidase is the nonspecific hydrolysis of the 5’-monophosphates by acid or alkaline phosphatases. Plant iectin concanavalin A is a powerful inhibitor of 5’-nucleotidase, but has no effect on acid or alkaline phosphatases. Described in this communication is a specific method for measuring 5’-nucleotidase activity with any ribonucleotide substrate in the presence of interfering phosphatases. This has been accomplished by the inclusion of a lectin into the reaction mixture and the use of neutral alumina columns for the separation of products. MATERIALS
[ U-14C]AMP, diammonium salt (specific activity 508 Ci/mol) and Triton X-100 have been obtained from New England Nuclear (Boston, Mass.). All 5’-nucleotides, 3’AMP, nucleosides, purine and pyrimidine bases, deoxycholate, P-phenylphosphate, P-glycerophosphate, glucose &phosphate, p-nitrophenyl phosphate, neutral alumina,
497
0003-2697/79/080497-10$02.00/O Copyrtght All rights
0 1979 by Academic Press, Inc. of reproduction m any form rewrvcd
498
CHATTERJEE,
BHATTACHARYA,
a-methyl mannoside and Crotalus atrox venom 5’-nucleotidase, grade IV, were purchased from Sigma Chemical Company (St. Louis, MO.). Resin AG l-X2, in the chloride form, 200-400 mesh, was obtained from Bio-Rad Laboratories (Richmond, Calif.). QAE-Sephadex was bought from Pharmacia Fine Chemicals (Piscataway, N. J.). Concanavalin A (3 x crystallized) was from Miles Laboratories, Inc. (Elkhart, Ind.). EDTA, disodium salt was obtained from Eastman Kodak Company (Rochester, N. Y.). All other chemicals were the purest available from commercial sources. METHODS Preparation of columns. Columns were prepared in disposable Pasteur pipets loosely plugged with glass wool. AG l-X2 was successively washed with 0.5 N NaOH, HzO, 0.5 N HCl, and H,O until neutral. QAE-Sephadex was equilibrated and washed with HzO. Resin slurry in HZ0 (1:3) was pipetted (1.5 ml) in columns to obtain a bed volume of 0.5 ml. For alumina columns, 0.6 g of the material was poured into the columns. Assay
procedure
for
5’-nucleotidase.
AND
BARLOW
alumina columns. Adenosine and other products were washed out with 0.1 M Tris-HCl, pH 7.4 and 10 ml was collected directly on scintillation vials, while adsorbed 5’-AMP was eluted with 10 ml of 0.2 M sodium phosphate, pH 7.4 in another set of scintillation vials. Radioactivity in the samples was determined after mixing with 10 ml of Aquasol II in a Packard Tri-Carb scintillation counter using an external standard method of quench correction. Counts were normalized with respect to total added, and background counts were subtracted from each value. For comparison, the procedure of Gentry and Olsson (6) was employed. Other procedures. Procedures for the preparation of plasma membranes and assay of alkaline phosphatase have been described (12). Descending paper chromatography was performed in S&S orange 589C papers (Schleicher & Schuell, Inc., Keene, N. H.) developing with 86% normal butanol in HZ0 for 25-30 h at room temperature. RESULTS El&ion of Possible Metabolites Ribonucleotides and 5’-AMP Ion-Exchange Columns
of from
The reaction mixture (100 ~1) contained 0.5 Three types of column materials were ~1 of [U-14C]AMP (0.01 &i), 0.1-0.5 pmol 5’-AMP, 50 mM Tris-HCl, pH 7.5, evaluated for their effectiveness in the separation of 5’-nucleotides 10 mM MgClz, 0.5% Triton X-100, and an quantitative appropriate amount of enzyme. One set of from their possible metabolites. Data shown tubes contained 100 pg concanavalin A and in Table 1 suggest that neutral alumina all incubations were conducted at 37°C with columns were the most effective. More than continuous shaking in a Dubnoff metabolic 90% of the nucleosides and their bases, shaker incubator for 15-30 min. The reac- but none of the AMP, was eluted in the pH 7.4 wash. tion was first terminated by placing the first 10 ml of 0.1 M Tris-HCl, A method for the assay of 5’-nucleotidase tubes in dry-ice-ethanol mixture and later boiling all of them together at 100°C for 5 using AG l-X2 columns and 0.1 M glycinemin. In a second procedure, 100 ~1 of 0.2 M NaOH, pH 8.5 as eluant has been described EDTA (pH adjusted to 7.4) was used to ter- (7). Such a procedure will not work when minate the reaction, which also eliminated IMP or GMP is used as substrate, or even the problem of clogging of the columns by with AMP in crude extracts where high the precipitate formed during boiling. Prod- levels of adenosine deaminase are present, ucts of the reaction were separated on pre- since neither inosine nor guanosine was washed (10 ml, 0.1 M Tris-HCl, pH 7.4) eluted by the glycine buffer. With AG
499
ASSAY OF 5’-NUCLEOTIDASE TABLE ELUTION
OF POWBLE
METABOLITES
1
OF RIBONUCLEOTIDES
AND
S-AMP
FROM
ION-EXCHANGE
COLUMNS”
AC 1-X2 Nucleoside derivatives
Hz0 eluant
0.1 M NaHCO, eluant
0.1 M GlycineNaOH, pH 8.5, eluant
Adenosine Inosine Guanosine* Uridine Cytidine Hypoxanthine Adenine Guanine Uracil Cytosine 5’-AMP
95 68 75 81 97 88 58 85 98 1
97 90 90 98 94 97 88 95 97 0.6
91 0 0 96 97 2
QAE-Sephadex J&O eluant 68 0
Neutral alumina Tris-HCI, pH 7.4, eluant 91 90 93 97 97 95 94 94 loo 98 0
a Unlabeled S-AMP, nucleotides, their bases (5 mM) were mixed with 50 mM Tris-HCl, pH 7.5, 10 mM MgCI,, 0.5% Triton X-100 in a total volume of 100 ~1 and transferred to columns without any incubation. A,,, of the fractions (0.5 ml) were read in a Gilford Spectrophotometer, Model 250, against H,O blank. Percentage of total A,,, applied to the column in 10 ml of the eluant is presented. b Precipitates when pH lowered to pH 7.4.
l-X2 columns, 0.1 M NaHCO, is very effective for the elution of nucleosides and their bases, but some AMP (0.6%) also emerges with this eluant. Adsorbed AMP can be quantitatively eluted from the alumina columns by 10 ml of 0.2 M sodium phosphate buffer, pH 7.4. One can, therefore, normalize the values for unequal recoveries and examine the stoichiometry of the disappearance of 5’-AMP and appearance of its metabolites. IMP, GMP, CMP, and UMP behave similarly as AMP in these columns. Chromatographic Pattern of the 5’-Nucleotidase Reaction Products in Alumina Columns
The elution patterns of a complete incubation mixture performed with venom 5’nucleotidase and its zero time control are presented in Fig. 1. Unadsorbed radioactivity was all collected in 10 ml of Tris buffer; further washings with an additional 15 ml of buffer did not elute any more counts. After washing with Tris buffer,
residual radioactivity can be eluted with 10 ml of 0.2 M phosphate buffer. Recovery of the added radioactivity from the columns was more than 98%, and a 1: 1 stoichiometry between the appearance of counts in the Tris wash and disappearance from phosphate an eluate was observed in every incubation throughout these studies. About 2-3% of the counts appear in the Tris washings of the zero-min control. It was felt necessary to check the purity of [C14]AMP and to reduce this background value to increase the sensitivity of the assay, Descending paper chromatography of a mixture of 5 ~1 [C14]AMP, carrier unlabeled AMP, adenosine, and adenine was performed as described under Methods. In this system, AMP remained in the origin well separated from adenosine (R, 0.19) and adenine (R, 0.35). An unidentified compound with an Rf of 0.11 was also present in the [C14]AMP preparation and contributed 1.1% of the total radioactivity. Adenosine and adenine spots had 0.9 and
500
CHATTERJEE,
BHATTACHARYA,
Elurnt
AND BARLOW
(ml )
FIG. 1. Chromatographic pattern of the S’nucleotidase reaction products in alumina columns. Incubation mixture (100 ~1) contained 50 mM Tris-HCl, pH 7.5, 10 mM MgClz, 5 mM AMP, 0.5 ~1 [UJ4C]AMP (0.01 &i) and 2 pg of venom 5’-nucleotidase. Reaction was terminated at 0 min (0) or at 30 min (0) and products fractionated as described under Methods. One-milliliter fractions were collected for radioactivity determination. Position of the eluants is indicated by downward arrows.
0.4% of the total counts, respectively. Purification of [C14]AMP by adsorption in AG l-X2 column followed by elution with
0.003-0.05 N HCl reduced this background to 1.4- 1.8% of total counts. After purification by paper chromatography the assay background can be further reduced to below 0.5%. Comparison with Other Assay Procedure of 5’-Nucleotidase
Enzyme (pl)
FIG. 2. Comparison of values obtained by Ba(OH),&SO, precipitation vs alumina column chromatography. Duplicate tubes were incubated under conditions described in Fig. 1 with indicated amounts of venom 5’-nucleotidase (40 @ml). In one set of tubes reactions were terminated at 0 and 30 min by the successive addition of 100 pl each of Ba(OH), and Z&O, (6), and 200 pl of the supernatant obtained by centrifugation at 4°C was counted after addition of 10 ml of Aquasol 11. In another set of tubes the reaction was stopped and analyzed by alumina columns as described under Methods. Ba(OH)z-ZnSO, method (0); alumina chromatography (0). Zero-minute values are connected by discontinuous lines.
Validity of this procedure was established by parallel assay of serial dilutions of Snucleotidase by the method of Gentry and Olsson (6) and by the alumina chromatographic procedure. Results presented in Fig. 2 show that similar values are obtained by the two procedures, both for the control (zero time) and complete incubation. In a separate experiment, 50 ~1 of the venom 5’-nucleotidase was incubated with a purified [C14]AMP and the products were analyzed by paper chromatography. Only spots for AMP and adenosine were detected on the paper, suggesting the absence of enzymes capable of further metabolizing adenosine or deaminate AMP. This preparation from snake venom did not contain any alkaline phosphatase activity determined by both &glycerophosphate and p-nitro-
501
ASSAY OF 5’-NUCLEOTIDASE
FIG. 3. Effect of 5’-AMP on alkaline phosphatase activity. Alkaline phosphatase in plasma membrane prepared from rat mammary tumor MT-W9B was assayed as described in (12). Inset is the double-reciprocal plot.
phenylphosphate substrates. Since the method of Gentry and Olsson (6) is accurate for pure enzyme with S-AMP as the substrate, the validity of our separation procedure is established. Effect of 5’-AMP Activity
on Alkaline
Phosphatase
In systems containing alkaline or acid phosphatases 5’-nucleotidase can be demonstrated only under experimental conditions designed to minimize the background of these phosphatases. One approach for correcting the interference of alkaline phosphatase has been to add an excess of a preferential substrate for alkaline phosphatase (substrate diverter) to the reaction mixture for 5’-nucleotidase assay (11,13,14). The effect of AMP on the alkaline phosphatase activity of a rat tumor preparation (12) was studied and the results are summarized in Fig. 3. Even in the presence of 5 mM of the preferred substrate p-nitrophenyl phosphate the alkaline phosphatase activity (K,, 35-70 PM) was inhibited competitively by low concentrations of AMP in this system, suggesting that the enzyme is in fact a nonspecific monophospho-esterase.
Effect of Different Monophosphate Esters and Concanavalin A on 5’-Nucleotidase Activity The effect of a number of monophosphate esters on the activity of venom 5’nucleotidase is shown in Fig. 4. All of these
25P---b
2’5
6 Concanavolin
4 (mg/mll
is
lb
FIG. 4. Effect of different monophosphate esters and concanavalin A on 5’-nucleotidase. Incubation conditions are described in legend to Fig. I. Monophosphate esters were neutralized immediately before addition to the incubation mixture. Concanavalin A was dissolved and diluted in saturated solution of NaCl. /I-glycerophosphate (0): glucose 6-phosphate (0); &phenylphosphate (A): 3’-AMP (A): concanavalin A (Cl).
502
CHA’ITERJEE,
0
t Co”co”o”Dlln
BHATTACHARYA,
10 A ,pg/mll
100
AND BARLOW
0
I 5
t 10
I 15
o-M.thyl-D-Monno$ide~rnMl
FIG. 5. (A) Effect of concanavalin A on 5’-nucleotidase, acid, and alkaline phosphatases present in the plasma membrane fractions from rat tumor. Phosphatases were assayed using /3-glycerophosphate at pH 4.5 (A) and 9.5 (A) using plasma membrane from the tumor MT-W9B (12). 5’Nucleotidase was assayed using plasma membranes from the tumors MT-W9B (0) and SMT-2A (0). Concentration of concanavalin A is plotted in logarithmic scale. (B) Reversal of concanavalin A inhibition by a-methyl mannoside. Data with MT-W9B is shown.
compounds inhibited the 5’-nucleotidase. Their inclusion at a 20-fold concentration of substrate would underestimate 5’-nucleotidase activity by 30-60%. Concanavalin
A has been reported to be a rather specific inhibitor for 5’-nucleotidase (15- 17). With venom 5’-nucleotidase, concanavalin A at a concentration of 0.5 mg/ml inhibited the enzyme by 80%. Therefore, differential assay in the presence and absence of lectin could determine the 5’-nucleotidase activity with at least 80% accuracy. Effect of Concanavalin A on 5’-Nucleotidase, Acid, and Alkaline Phosphatases Present in Plasma Membranes from Rat Tumors
Time of incubation
(min)
FIG. 6. Linearity of the assay with increasing incubation time and concentration of enzyme protein. Details of the assay procedure are described under Methods.
Plasma membrane preparation isolated from two rat mammary tumors, one nonmetastasizing (MT-W9B) and one metastasizing (SMT-2A), are enriched in alkaline phosphatase and 5’-nucleotidase compared to homogenate by 15- to 20-fold and also have some acid phosphatase activity (12). The effect of concanavalin A on these plasma membrane bound enzymes was studied. 5’-Nucleotidase was inhibited by 90-100% with a lectin concentration of lo-25 pg/rnl of the reaction mixture. Neither acid nor alkaline phosphatase was af-
503
ASSAY OF S’-NUCLEOTIDASE
fected by concanavalin A up to 50 &ml (Fig. 5A). The inhibition of 5’-nucleotidase can be completely reversed by preincubation with a-methyl mannoside (Fig. 5B). This suggests that concanavalin A inhibition is a specific effect which involves the binding of this macromolecule to the saccharide containing active center of the enzyme. Validity of our separation technique and the procedure for the assay of 5’-nucleotidase in the presence of high activities of nonspecific phosphatases by using concanavalin A were then investigated using the plasma membrane preparation from the tumor MT-W9B (12).
I’-. /“” L B el
4
6
mM)
FIG. 8. Effect of divalent cation on S’nucleotidase from plasma membrane. Activities in the presence of MgC1, (O), with EDTA (O), in the absence of M&I,, and in the presence of 4 IIIM EDTA plus varying concentrations of MgCI, (A) have been determined as described under Methods. EDTA was always added before MgCI, and the reaction was preincubated with EDTA for 10 min at 37°C.
Linearity of Assay and Optimum pH of 5’-Nucleotidase from Plasma Membrane Linearity of the assay with increasing incubation time and concentration of enzyme protein is shown in Fig. 6. 5’-Nucleotidase showed two pH optima, one around pH 7-8 and a second around 9.7. This pattern is maintained both in the presence or absence of MgCI, (Fig. 7), or replacing bicarbonate buffer by a glycine-NaOH system. Venom 5’-nucleotidase also showed two pH optima (data not shown). /
6
EDTA/MgCi21
/
IO
I
,
12
PH FIG. 7. Optimum pH of the 5’nucleotidase from plasma membrane in the presence and absence of magnesium. All the buffers were 50 mM, and in the pH range 3.8-5.3 sodium acetate-acetic acid; at 5.98.3 Tris-maleate at 9.2-10.6 sodium carbonate bicarbonate buffer systems were used. The difference in the activity in the presence (0) and absence (0) of 1 mgiml of concanavalin A is indicated by discontinuous line. The assay was conducted as described under Methods without MgCI, (A) or in the presence of 10 mM MgCI, (B), except the addition of 100 ~1 of 1 M Tris-HCI, pH 7.5 before boiling for the termination of reaction.
Effect of Divalent Cation on 5’-Nucleotidase from Plasma
Membrane
Addition of MgCl, to the incubation mixture did not stimulate the enzyme activity; rather slight inhibition was observed. Preincubation with EDTA caused inhibition of 5’-nucleotidase activity, being total at 20 mM EDTA. The EDTA inhibition was completely reversed with MgCI, (Fig. 8) suggesting the presence of an endogenous divalent cation which is either Mg2+ or some other ion replaceable by Mg2+. The EDTA effect was used as an alterna-
504
CHATTERJEE,
BHATTACHARYA,
AND BARLOW
plasma membranes from MT-W9B tumor. Optimum stimulation with Triton X-100 is obtained at 0.2%, any higher concentration being inhibitory. With deoxycholate the plateau was obtained at 1%. Maximum stimulation with both the detergents was 50% over the control activity in the absence of any detergent (Fig. 9). I 0
I 1
I 2
Percent
1 3
detergent
I 4
I 5
Effect of Substrate Concentration on 5’-Nucleotidase from Plasma Membrane
(weight/volume1
FIG. 9. Effect of detergents on S-nucleotidase from plasma membrane. In the standard incubation media (see Methods) either Triton X-108 (0) or deoxycholate (0) was added.
tive procedure for the termination of Snucleotidase reaction. Incubations terminated by 100 ~1 of 0.2 M EDTA (pH 7.4) gave results identical to boiling, with one additional advantage; the flocculent protein precipitate resulting from boiling, which slows down the columns, can be avoided.
5’-Nucleotidase was determined with a dilute plasma membrane suspension using a range of substrate concentration from 0.04 to 5 mM. Under these conditions, in 15 min, less than 25% of the substrate was utilized, and reaction rate was linear. The plateau of enzyme activity was reached at 1 mM of substrate concentration (Fig. 10). K, and V determined by double-reciprocal plot (Fig. 10, inset) were 35 PM and 1.85 nmoY 15 min, respectively.
Effect of Detergents on 5’-Nucleotidase from Plasma Membrane
Spectrophotometric Adaptation of the 5’-Nucleotidase Assay
Both Triton X-100 and deoxycholate stimulated 5’-nucleotidase activity from
Eluates from the alumina columns can be read at 260 nm instead of being counted
.*
a
-
.E d
.
1.0
i
.
s 1.5d s WI
ci
1
0.6
1 p7j
.
0.6
’
0.4
3 0.2
Is e *
0.5
(Ir 5
to
I5
20
2s
ii h
SUBSTRATE
CONC. (mM)
FIG. 10. Effect of substrate concentration on 5’-nucleotidase from plasma membrane. Concentration of AMP in the incubation mixture varied from 0.04 to 5 mM (800-100,000 cpm). The plasma membrane suspension was diluted 2S-fold compared to standard assay and incubation time was reduced to 15 min. Inset, double-reciprocal plot. The line was fitted to the points by the method of least squares.
ASSAY
505
OF 5’-NUCLEOTIDASE
in a liquid scintillation counter. By this spectrophotometric procedure, 5’-nucleotidase activity in the plasma membrane with AMP, GMP, UMP, CMP, and IMP as substrates was determined and their relative specific activities were 100:63:81:63:59. DISCUSSION
A radiometric method of 5’-nucleotidase assay using ZnSO,-Ba(OH), (6) is based on the original observation of Krishna et al. (18) that AMP is quantitatively precipitated by the reagent, while adenosine remains in solution. However, adenine, guanine, guanosine, hypoxanthine, and inosine are also precipitated by ZnSO,-Ba(OH),. In crude systems where AMP-deaminase, adenosine deaminase, or adenosine nucleosidase are present, this procedure will underestimate the level of 5’-nucleotidase. Besides, in systems which prefer IMP or GMP as substrates (8,9), this method cannot be used. Separation using AG 1-X2 columns (7) has the same drawback, since inosine and guanosine are not eluted by the glycine buffer (Table 1). Our procedure is based on the observation of Ramchandran (19) that nucleotides are multivalent anions at neutral pH and are adsorbed strongly on alumina, while nucleosides and their metabolic products pass through such columns. Advantages of this separation technique are twofold, One can use a crude enzyme source and use any ribonucleoside monophosphate as substrate. The method can also be adopted for spectrophotometric determination of 5’-nucleotidase. Suran (20) has applied this separation technique to the assay of 5’-nucleotidase in particulate preparations of brain and spinal cord, but did not investigate the possible use of substrates other than AMP, or the effect of nonspecific phosphatases on the assay. Our study suggests that a number of monophosphate esters inhibit the 5’-nucleotidase assay and their use as substrate
diverters for alkaline phosphatase (11,13,14) may underestimate the enzyme activity by 30-60%. By differential assay, in the presence and absence of concanavalin A and using alumina columns, it is possible to determine the activity of 5’-nucleotidase with more than 80% accuracy, even in a crude system. While our study was in progress, a calorimetric technique using concanavalin A inhibition as the basis for a specific assay of serum 5’-nucleotidase, was described (21). The validity of our assay procedure is confirmed by studying the properties of 5’-nucleotidase in plasma membrane preparations containing high levels of alkaline phosphatase and considerable activity of acid phosphatase. These properties are similar to those from other systems (22), which suggests that salts, detergents, EDTA or different concentrations of AMP do not effect the separation technique to any significant extent. We used this technique to assay serum 5’-nucleotidase in a number of normal individuals. Using purified AMP and 50 ~1 of serum, we obtained 1000-2500 cpm in 10 ml of Tris eluate after 20 min of incubation and control (zero min) values of 30-40 cpm. In contrast, by the calorimetric procedure (4), we had to use 0.25 ml of serum and at least 2 h of incubation time to obtain reproducible optical density for the same set of serum samples. Even then the value (A.& of the experimental tube was only twofold that of control. At present we are assaying 5’-nucleotidase activity in the sera of a large number of cancer patients by this technique in order to correlate the alterations of the levels of this enzyme with the clinical status of these patients. ACKNOWLEDGMENT We thank assistance.
Koong-Shian
S. Ou
for
her
technical
506
CHATTERJEE,
BHATTACHARYA,
REFERENCES
4.
5. 6. 7.
8.
9. 10.
DePierre, J. W., and Kamovsky, M. L. (1973) J. Cell Biol. 56, 275-303. Schwartz, M. K., and Bodansky, 0. (1965) Cancer 18, 886-892. Korsten, J. B., Persijn, J. P., and Van der Slik, W. (1974)Z. Klin. Chem. Klin. Biochem. 12, 116120. Heppel, L., and Hilmoe, R. S. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 547-550, Academic Press, New York. Ipata, P. L. (1%7) Anal. Biochem. 20, 30-36. Gentry, M. K., and Olsson, R. A. (1975) Anal. Biochem. 64,624-627. Glastris, B., and Pfeiffer, S. E. (1974) in Methods in Enzymology (Fleischer, S., ed.), Vol. 32, pp. 124-131, Academic Press, New York. Naito, Y., and Tsushima, K. (1976) Biochim. Biophys. Acta 438, 159-168. Olson, A. C., and Fraser, M. J. (1974) Biochim. Biophys. Acta 334, 156-176. DePierre, J. W., and Kamovsky, M. L. (1974) J. Biol. Chem. 249, 7121-7129.
AND BARLOW
11. Quagliata, F., Faig, D., Conklyn, M., and Silber, R. (1974) Cancer Res. 34, 3197-3202. 12. Chattejee, S. K., Kim, U., and Bielat, K. (1976) Brit. 1. Cancer 33, 15-26. 13. Belfield, A., and Goldberg, D. M. (1968) Nature (London) 219, 73-75. 14. Persijn, J. P., Van der Slik, W., and Bon, A. W. M. (1%9) Z. Klin. Chem. Klin. Biochem 7, 493497.
15. Riorden, J. R., and Slavik, M. (1974) Biochim. Biophys. Acta 373, 356-360. 16. Stefanovic, V., Mandel, P., and Rosenberg, A. (1975) J. Biol. Chem. 250,7081-7083. 17. Carraway, K. L., Fogle, D. D., Chestnut, R. W., Huggins, J. W., and Carraway, C. A. C. (1976) J. Biol. Chem. 251, 6173-6178. 18. Krishna, G., Weiss, B., and Brodie, B. B. (1%8) J. Pharmocol. Exp. Ther. 163, 379-385. 19. Ramchandran, J. (1971) Anal. Biochem. 43, 227239.
Suran, A. A. (1973) Anal. Biochem. 55, 593-600. 21. Zygowicz, E. R., Sunderman, F. W., Jr., Horak, E., and Dooley, J. F. (1977) Clin. Chem. 23, 2311-2323. 22. Bodansky, O., and Schwartz, M. K. (1968)Advan. Clin. Chem. 11, 277-328. 20.
BIOCHEMISTRY
A Simple, SUNIL
95, 497-506 (1979)
Specific
K. CHATTERIEE, Department
Radiometric
Assay for 5’-Nucleotidase
MALAYA BHATTACHARYA,ANDJOSEPH of Gynecology, 666 Elm Street,
Roswell Park Memorial Buffalo, New York 14263
J. BARLOW
Institute.
Received November 1, 1978 A radiometric assay for 5’-nucleotidase (EC 3.1.3.5) has been developed. which is applicable for all 5’-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were found to be the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 M TrisHCI, pH 7.4; subsequent elution of the 5’-nucleotide was then accomplished with 0.2 M sodium phosphate, pH 7.4. Differential measurement of 5’nucleotidase can be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavahn A into the reaction mixture. It completely inhibits 5’nucleotidase without effecting the phosphatases. The applicability of this assay has been demonstrated by studying the properties of 5’-nucleotidase present in a purified plasma membrane preparation from a rat tumor which is enriched with both S’nucleotidase and alkaline phosphatase.
5’-Nucleotidase (EC 3.1.3.5; ribonucleotide phosphohydrolase) is widely used as a plasma membrane marker enzyme (1). Elevation of the levels of this enzyme in human sera has been observed in a number of clinical conditions (2,3). Commonly used calorimetric (4) and spectrophotometric (5) methods for the assay of 5’-nucleotidase are laborious and not suitable for low levels of activity in crude extracts. This prompted the development of various radiometric procedures (6,7) which are sensitive, but are applicable only when 5’-AMP or CMP are used as substrates. For systems which prefer IMP or GMP as substrates (8,9), or in crude extracts where other enzymes capable of metabolizing the nucleotides or nucleoside products are present, these methods cannot be used. These drawbacks can be overcome by the use of 32P-labeled mononucleotide substrates (10,ll). However, the short half-life (14 days) of the radioisotope is a serious limitation of this technique. Another problem encountered in the
assay of 5’-nucleotidase is the nonspecific hydrolysis of the 5’-monophosphates by acid or alkaline phosphatases. Plant iectin concanavalin A is a powerful inhibitor of 5’-nucleotidase, but has no effect on acid or alkaline phosphatases. Described in this communication is a specific method for measuring 5’-nucleotidase activity with any ribonucleotide substrate in the presence of interfering phosphatases. This has been accomplished by the inclusion of a lectin into the reaction mixture and the use of neutral alumina columns for the separation of products. MATERIALS
[ U-14C]AMP, diammonium salt (specific activity 508 Ci/mol) and Triton X-100 have been obtained from New England Nuclear (Boston, Mass.). All 5’-nucleotides, 3’AMP, nucleosides, purine and pyrimidine bases, deoxycholate, P-phenylphosphate, P-glycerophosphate, glucose &phosphate, p-nitrophenyl phosphate, neutral alumina,
497
0003-2697/79/080497-10$02.00/O Copyrtght All rights
0 1979 by Academic Press, Inc. of reproduction m any form rewrvcd
498
CHATTERJEE,
BHATTACHARYA,
a-methyl mannoside and Crotalus atrox venom 5’-nucleotidase, grade IV, were purchased from Sigma Chemical Company (St. Louis, MO.). Resin AG l-X2, in the chloride form, 200-400 mesh, was obtained from Bio-Rad Laboratories (Richmond, Calif.). QAE-Sephadex was bought from Pharmacia Fine Chemicals (Piscataway, N. J.). Concanavalin A (3 x crystallized) was from Miles Laboratories, Inc. (Elkhart, Ind.). EDTA, disodium salt was obtained from Eastman Kodak Company (Rochester, N. Y.). All other chemicals were the purest available from commercial sources. METHODS Preparation of columns. Columns were prepared in disposable Pasteur pipets loosely plugged with glass wool. AG l-X2 was successively washed with 0.5 N NaOH, HzO, 0.5 N HCl, and H,O until neutral. QAE-Sephadex was equilibrated and washed with HzO. Resin slurry in HZ0 (1:3) was pipetted (1.5 ml) in columns to obtain a bed volume of 0.5 ml. For alumina columns, 0.6 g of the material was poured into the columns. Assay
procedure
for
5’-nucleotidase.
AND
BARLOW
alumina columns. Adenosine and other products were washed out with 0.1 M Tris-HCl, pH 7.4 and 10 ml was collected directly on scintillation vials, while adsorbed 5’-AMP was eluted with 10 ml of 0.2 M sodium phosphate, pH 7.4 in another set of scintillation vials. Radioactivity in the samples was determined after mixing with 10 ml of Aquasol II in a Packard Tri-Carb scintillation counter using an external standard method of quench correction. Counts were normalized with respect to total added, and background counts were subtracted from each value. For comparison, the procedure of Gentry and Olsson (6) was employed. Other procedures. Procedures for the preparation of plasma membranes and assay of alkaline phosphatase have been described (12). Descending paper chromatography was performed in S&S orange 589C papers (Schleicher & Schuell, Inc., Keene, N. H.) developing with 86% normal butanol in HZ0 for 25-30 h at room temperature. RESULTS El&ion of Possible Metabolites Ribonucleotides and 5’-AMP Ion-Exchange Columns
of from
The reaction mixture (100 ~1) contained 0.5 Three types of column materials were ~1 of [U-14C]AMP (0.01 &i), 0.1-0.5 pmol 5’-AMP, 50 mM Tris-HCl, pH 7.5, evaluated for their effectiveness in the separation of 5’-nucleotides 10 mM MgClz, 0.5% Triton X-100, and an quantitative appropriate amount of enzyme. One set of from their possible metabolites. Data shown tubes contained 100 pg concanavalin A and in Table 1 suggest that neutral alumina all incubations were conducted at 37°C with columns were the most effective. More than continuous shaking in a Dubnoff metabolic 90% of the nucleosides and their bases, shaker incubator for 15-30 min. The reac- but none of the AMP, was eluted in the pH 7.4 wash. tion was first terminated by placing the first 10 ml of 0.1 M Tris-HCl, A method for the assay of 5’-nucleotidase tubes in dry-ice-ethanol mixture and later boiling all of them together at 100°C for 5 using AG l-X2 columns and 0.1 M glycinemin. In a second procedure, 100 ~1 of 0.2 M NaOH, pH 8.5 as eluant has been described EDTA (pH adjusted to 7.4) was used to ter- (7). Such a procedure will not work when minate the reaction, which also eliminated IMP or GMP is used as substrate, or even the problem of clogging of the columns by with AMP in crude extracts where high the precipitate formed during boiling. Prod- levels of adenosine deaminase are present, ucts of the reaction were separated on pre- since neither inosine nor guanosine was washed (10 ml, 0.1 M Tris-HCl, pH 7.4) eluted by the glycine buffer. With AG
499
ASSAY OF 5’-NUCLEOTIDASE TABLE ELUTION
OF POWBLE
METABOLITES
1
OF RIBONUCLEOTIDES
AND
S-AMP
FROM
ION-EXCHANGE
COLUMNS”
AC 1-X2 Nucleoside derivatives
Hz0 eluant
0.1 M NaHCO, eluant
0.1 M GlycineNaOH, pH 8.5, eluant
Adenosine Inosine Guanosine* Uridine Cytidine Hypoxanthine Adenine Guanine Uracil Cytosine 5’-AMP
95 68 75 81 97 88 58 85 98 1
97 90 90 98 94 97 88 95 97 0.6
91 0 0 96 97 2
QAE-Sephadex J&O eluant 68 0
Neutral alumina Tris-HCI, pH 7.4, eluant 91 90 93 97 97 95 94 94 loo 98 0
a Unlabeled S-AMP, nucleotides, their bases (5 mM) were mixed with 50 mM Tris-HCl, pH 7.5, 10 mM MgCI,, 0.5% Triton X-100 in a total volume of 100 ~1 and transferred to columns without any incubation. A,,, of the fractions (0.5 ml) were read in a Gilford Spectrophotometer, Model 250, against H,O blank. Percentage of total A,,, applied to the column in 10 ml of the eluant is presented. b Precipitates when pH lowered to pH 7.4.
l-X2 columns, 0.1 M NaHCO, is very effective for the elution of nucleosides and their bases, but some AMP (0.6%) also emerges with this eluant. Adsorbed AMP can be quantitatively eluted from the alumina columns by 10 ml of 0.2 M sodium phosphate buffer, pH 7.4. One can, therefore, normalize the values for unequal recoveries and examine the stoichiometry of the disappearance of 5’-AMP and appearance of its metabolites. IMP, GMP, CMP, and UMP behave similarly as AMP in these columns. Chromatographic Pattern of the 5’-Nucleotidase Reaction Products in Alumina Columns
The elution patterns of a complete incubation mixture performed with venom 5’nucleotidase and its zero time control are presented in Fig. 1. Unadsorbed radioactivity was all collected in 10 ml of Tris buffer; further washings with an additional 15 ml of buffer did not elute any more counts. After washing with Tris buffer,
residual radioactivity can be eluted with 10 ml of 0.2 M phosphate buffer. Recovery of the added radioactivity from the columns was more than 98%, and a 1: 1 stoichiometry between the appearance of counts in the Tris wash and disappearance from phosphate an eluate was observed in every incubation throughout these studies. About 2-3% of the counts appear in the Tris washings of the zero-min control. It was felt necessary to check the purity of [C14]AMP and to reduce this background value to increase the sensitivity of the assay, Descending paper chromatography of a mixture of 5 ~1 [C14]AMP, carrier unlabeled AMP, adenosine, and adenine was performed as described under Methods. In this system, AMP remained in the origin well separated from adenosine (R, 0.19) and adenine (R, 0.35). An unidentified compound with an Rf of 0.11 was also present in the [C14]AMP preparation and contributed 1.1% of the total radioactivity. Adenosine and adenine spots had 0.9 and
500
CHATTERJEE,
BHATTACHARYA,
Elurnt
AND BARLOW
(ml )
FIG. 1. Chromatographic pattern of the S’nucleotidase reaction products in alumina columns. Incubation mixture (100 ~1) contained 50 mM Tris-HCl, pH 7.5, 10 mM MgClz, 5 mM AMP, 0.5 ~1 [UJ4C]AMP (0.01 &i) and 2 pg of venom 5’-nucleotidase. Reaction was terminated at 0 min (0) or at 30 min (0) and products fractionated as described under Methods. One-milliliter fractions were collected for radioactivity determination. Position of the eluants is indicated by downward arrows.
0.4% of the total counts, respectively. Purification of [C14]AMP by adsorption in AG l-X2 column followed by elution with
0.003-0.05 N HCl reduced this background to 1.4- 1.8% of total counts. After purification by paper chromatography the assay background can be further reduced to below 0.5%. Comparison with Other Assay Procedure of 5’-Nucleotidase
Enzyme (pl)
FIG. 2. Comparison of values obtained by Ba(OH),&SO, precipitation vs alumina column chromatography. Duplicate tubes were incubated under conditions described in Fig. 1 with indicated amounts of venom 5’-nucleotidase (40 @ml). In one set of tubes reactions were terminated at 0 and 30 min by the successive addition of 100 pl each of Ba(OH), and Z&O, (6), and 200 pl of the supernatant obtained by centrifugation at 4°C was counted after addition of 10 ml of Aquasol 11. In another set of tubes the reaction was stopped and analyzed by alumina columns as described under Methods. Ba(OH)z-ZnSO, method (0); alumina chromatography (0). Zero-minute values are connected by discontinuous lines.
Validity of this procedure was established by parallel assay of serial dilutions of Snucleotidase by the method of Gentry and Olsson (6) and by the alumina chromatographic procedure. Results presented in Fig. 2 show that similar values are obtained by the two procedures, both for the control (zero time) and complete incubation. In a separate experiment, 50 ~1 of the venom 5’-nucleotidase was incubated with a purified [C14]AMP and the products were analyzed by paper chromatography. Only spots for AMP and adenosine were detected on the paper, suggesting the absence of enzymes capable of further metabolizing adenosine or deaminate AMP. This preparation from snake venom did not contain any alkaline phosphatase activity determined by both &glycerophosphate and p-nitro-
501
ASSAY OF 5’-NUCLEOTIDASE
FIG. 3. Effect of 5’-AMP on alkaline phosphatase activity. Alkaline phosphatase in plasma membrane prepared from rat mammary tumor MT-W9B was assayed as described in (12). Inset is the double-reciprocal plot.
phenylphosphate substrates. Since the method of Gentry and Olsson (6) is accurate for pure enzyme with S-AMP as the substrate, the validity of our separation procedure is established. Effect of 5’-AMP Activity
on Alkaline
Phosphatase
In systems containing alkaline or acid phosphatases 5’-nucleotidase can be demonstrated only under experimental conditions designed to minimize the background of these phosphatases. One approach for correcting the interference of alkaline phosphatase has been to add an excess of a preferential substrate for alkaline phosphatase (substrate diverter) to the reaction mixture for 5’-nucleotidase assay (11,13,14). The effect of AMP on the alkaline phosphatase activity of a rat tumor preparation (12) was studied and the results are summarized in Fig. 3. Even in the presence of 5 mM of the preferred substrate p-nitrophenyl phosphate the alkaline phosphatase activity (K,, 35-70 PM) was inhibited competitively by low concentrations of AMP in this system, suggesting that the enzyme is in fact a nonspecific monophospho-esterase.
Effect of Different Monophosphate Esters and Concanavalin A on 5’-Nucleotidase Activity The effect of a number of monophosphate esters on the activity of venom 5’nucleotidase is shown in Fig. 4. All of these
25P---b
2’5
6 Concanavolin
4 (mg/mll
is
lb
FIG. 4. Effect of different monophosphate esters and concanavalin A on 5’-nucleotidase. Incubation conditions are described in legend to Fig. I. Monophosphate esters were neutralized immediately before addition to the incubation mixture. Concanavalin A was dissolved and diluted in saturated solution of NaCl. /I-glycerophosphate (0): glucose 6-phosphate (0); &phenylphosphate (A): 3’-AMP (A): concanavalin A (Cl).
502
CHA’ITERJEE,
0
t Co”co”o”Dlln
BHATTACHARYA,
10 A ,pg/mll
100
AND BARLOW
0
I 5
t 10
I 15
o-M.thyl-D-Monno$ide~rnMl
FIG. 5. (A) Effect of concanavalin A on 5’-nucleotidase, acid, and alkaline phosphatases present in the plasma membrane fractions from rat tumor. Phosphatases were assayed using /3-glycerophosphate at pH 4.5 (A) and 9.5 (A) using plasma membrane from the tumor MT-W9B (12). 5’Nucleotidase was assayed using plasma membranes from the tumors MT-W9B (0) and SMT-2A (0). Concentration of concanavalin A is plotted in logarithmic scale. (B) Reversal of concanavalin A inhibition by a-methyl mannoside. Data with MT-W9B is shown.
compounds inhibited the 5’-nucleotidase. Their inclusion at a 20-fold concentration of substrate would underestimate 5’-nucleotidase activity by 30-60%. Concanavalin
A has been reported to be a rather specific inhibitor for 5’-nucleotidase (15- 17). With venom 5’-nucleotidase, concanavalin A at a concentration of 0.5 mg/ml inhibited the enzyme by 80%. Therefore, differential assay in the presence and absence of lectin could determine the 5’-nucleotidase activity with at least 80% accuracy. Effect of Concanavalin A on 5’-Nucleotidase, Acid, and Alkaline Phosphatases Present in Plasma Membranes from Rat Tumors
Time of incubation
(min)
FIG. 6. Linearity of the assay with increasing incubation time and concentration of enzyme protein. Details of the assay procedure are described under Methods.
Plasma membrane preparation isolated from two rat mammary tumors, one nonmetastasizing (MT-W9B) and one metastasizing (SMT-2A), are enriched in alkaline phosphatase and 5’-nucleotidase compared to homogenate by 15- to 20-fold and also have some acid phosphatase activity (12). The effect of concanavalin A on these plasma membrane bound enzymes was studied. 5’-Nucleotidase was inhibited by 90-100% with a lectin concentration of lo-25 pg/rnl of the reaction mixture. Neither acid nor alkaline phosphatase was af-
503
ASSAY OF S’-NUCLEOTIDASE
fected by concanavalin A up to 50 &ml (Fig. 5A). The inhibition of 5’-nucleotidase can be completely reversed by preincubation with a-methyl mannoside (Fig. 5B). This suggests that concanavalin A inhibition is a specific effect which involves the binding of this macromolecule to the saccharide containing active center of the enzyme. Validity of our separation technique and the procedure for the assay of 5’-nucleotidase in the presence of high activities of nonspecific phosphatases by using concanavalin A were then investigated using the plasma membrane preparation from the tumor MT-W9B (12).
I’-. /“” L B el
4
6
mM)
FIG. 8. Effect of divalent cation on S’nucleotidase from plasma membrane. Activities in the presence of MgC1, (O), with EDTA (O), in the absence of M&I,, and in the presence of 4 IIIM EDTA plus varying concentrations of MgCI, (A) have been determined as described under Methods. EDTA was always added before MgCI, and the reaction was preincubated with EDTA for 10 min at 37°C.
Linearity of Assay and Optimum pH of 5’-Nucleotidase from Plasma Membrane Linearity of the assay with increasing incubation time and concentration of enzyme protein is shown in Fig. 6. 5’-Nucleotidase showed two pH optima, one around pH 7-8 and a second around 9.7. This pattern is maintained both in the presence or absence of MgCI, (Fig. 7), or replacing bicarbonate buffer by a glycine-NaOH system. Venom 5’-nucleotidase also showed two pH optima (data not shown). /
6
EDTA/MgCi21
/
IO
I
,
12
PH FIG. 7. Optimum pH of the 5’nucleotidase from plasma membrane in the presence and absence of magnesium. All the buffers were 50 mM, and in the pH range 3.8-5.3 sodium acetate-acetic acid; at 5.98.3 Tris-maleate at 9.2-10.6 sodium carbonate bicarbonate buffer systems were used. The difference in the activity in the presence (0) and absence (0) of 1 mgiml of concanavalin A is indicated by discontinuous line. The assay was conducted as described under Methods without MgCI, (A) or in the presence of 10 mM MgCI, (B), except the addition of 100 ~1 of 1 M Tris-HCI, pH 7.5 before boiling for the termination of reaction.
Effect of Divalent Cation on 5’-Nucleotidase from Plasma
Membrane
Addition of MgCl, to the incubation mixture did not stimulate the enzyme activity; rather slight inhibition was observed. Preincubation with EDTA caused inhibition of 5’-nucleotidase activity, being total at 20 mM EDTA. The EDTA inhibition was completely reversed with MgCI, (Fig. 8) suggesting the presence of an endogenous divalent cation which is either Mg2+ or some other ion replaceable by Mg2+. The EDTA effect was used as an alterna-
504
CHATTERJEE,
BHATTACHARYA,
AND BARLOW
plasma membranes from MT-W9B tumor. Optimum stimulation with Triton X-100 is obtained at 0.2%, any higher concentration being inhibitory. With deoxycholate the plateau was obtained at 1%. Maximum stimulation with both the detergents was 50% over the control activity in the absence of any detergent (Fig. 9). I 0
I 1
I 2
Percent
1 3
detergent
I 4
I 5
Effect of Substrate Concentration on 5’-Nucleotidase from Plasma Membrane
(weight/volume1
FIG. 9. Effect of detergents on S-nucleotidase from plasma membrane. In the standard incubation media (see Methods) either Triton X-108 (0) or deoxycholate (0) was added.
tive procedure for the termination of Snucleotidase reaction. Incubations terminated by 100 ~1 of 0.2 M EDTA (pH 7.4) gave results identical to boiling, with one additional advantage; the flocculent protein precipitate resulting from boiling, which slows down the columns, can be avoided.
5’-Nucleotidase was determined with a dilute plasma membrane suspension using a range of substrate concentration from 0.04 to 5 mM. Under these conditions, in 15 min, less than 25% of the substrate was utilized, and reaction rate was linear. The plateau of enzyme activity was reached at 1 mM of substrate concentration (Fig. 10). K, and V determined by double-reciprocal plot (Fig. 10, inset) were 35 PM and 1.85 nmoY 15 min, respectively.
Effect of Detergents on 5’-Nucleotidase from Plasma Membrane
Spectrophotometric Adaptation of the 5’-Nucleotidase Assay
Both Triton X-100 and deoxycholate stimulated 5’-nucleotidase activity from
Eluates from the alumina columns can be read at 260 nm instead of being counted
.*
a
-
.E d
.
1.0
i
.
s 1.5d s WI
ci
1
0.6
1 p7j
.
0.6
’
0.4
3 0.2
Is e *
0.5
(Ir 5
to
I5
20
2s
ii h
SUBSTRATE
CONC. (mM)
FIG. 10. Effect of substrate concentration on 5’-nucleotidase from plasma membrane. Concentration of AMP in the incubation mixture varied from 0.04 to 5 mM (800-100,000 cpm). The plasma membrane suspension was diluted 2S-fold compared to standard assay and incubation time was reduced to 15 min. Inset, double-reciprocal plot. The line was fitted to the points by the method of least squares.
ASSAY
505
OF 5’-NUCLEOTIDASE
in a liquid scintillation counter. By this spectrophotometric procedure, 5’-nucleotidase activity in the plasma membrane with AMP, GMP, UMP, CMP, and IMP as substrates was determined and their relative specific activities were 100:63:81:63:59. DISCUSSION
A radiometric method of 5’-nucleotidase assay using ZnSO,-Ba(OH), (6) is based on the original observation of Krishna et al. (18) that AMP is quantitatively precipitated by the reagent, while adenosine remains in solution. However, adenine, guanine, guanosine, hypoxanthine, and inosine are also precipitated by ZnSO,-Ba(OH),. In crude systems where AMP-deaminase, adenosine deaminase, or adenosine nucleosidase are present, this procedure will underestimate the level of 5’-nucleotidase. Besides, in systems which prefer IMP or GMP as substrates (8,9), this method cannot be used. Separation using AG 1-X2 columns (7) has the same drawback, since inosine and guanosine are not eluted by the glycine buffer (Table 1). Our procedure is based on the observation of Ramchandran (19) that nucleotides are multivalent anions at neutral pH and are adsorbed strongly on alumina, while nucleosides and their metabolic products pass through such columns. Advantages of this separation technique are twofold, One can use a crude enzyme source and use any ribonucleoside monophosphate as substrate. The method can also be adopted for spectrophotometric determination of 5’-nucleotidase. Suran (20) has applied this separation technique to the assay of 5’-nucleotidase in particulate preparations of brain and spinal cord, but did not investigate the possible use of substrates other than AMP, or the effect of nonspecific phosphatases on the assay. Our study suggests that a number of monophosphate esters inhibit the 5’-nucleotidase assay and their use as substrate
diverters for alkaline phosphatase (11,13,14) may underestimate the enzyme activity by 30-60%. By differential assay, in the presence and absence of concanavalin A and using alumina columns, it is possible to determine the activity of 5’-nucleotidase with more than 80% accuracy, even in a crude system. While our study was in progress, a calorimetric technique using concanavalin A inhibition as the basis for a specific assay of serum 5’-nucleotidase, was described (21). The validity of our assay procedure is confirmed by studying the properties of 5’-nucleotidase in plasma membrane preparations containing high levels of alkaline phosphatase and considerable activity of acid phosphatase. These properties are similar to those from other systems (22), which suggests that salts, detergents, EDTA or different concentrations of AMP do not effect the separation technique to any significant extent. We used this technique to assay serum 5’-nucleotidase in a number of normal individuals. Using purified AMP and 50 ~1 of serum, we obtained 1000-2500 cpm in 10 ml of Tris eluate after 20 min of incubation and control (zero min) values of 30-40 cpm. In contrast, by the calorimetric procedure (4), we had to use 0.25 ml of serum and at least 2 h of incubation time to obtain reproducible optical density for the same set of serum samples. Even then the value (A.& of the experimental tube was only twofold that of control. At present we are assaying 5’-nucleotidase activity in the sera of a large number of cancer patients by this technique in order to correlate the alterations of the levels of this enzyme with the clinical status of these patients. ACKNOWLEDGMENT We thank assistance.
Koong-Shian
S. Ou
for
her
technical
506
CHATTERJEE,
BHATTACHARYA,
REFERENCES
4.
5. 6. 7.
8.
9. 10.
DePierre, J. W., and Kamovsky, M. L. (1973) J. Cell Biol. 56, 275-303. Schwartz, M. K., and Bodansky, 0. (1965) Cancer 18, 886-892. Korsten, J. B., Persijn, J. P., and Van der Slik, W. (1974)Z. Klin. Chem. Klin. Biochem. 12, 116120. Heppel, L., and Hilmoe, R. S. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 547-550, Academic Press, New York. Ipata, P. L. (1%7) Anal. Biochem. 20, 30-36. Gentry, M. K., and Olsson, R. A. (1975) Anal. Biochem. 64,624-627. Glastris, B., and Pfeiffer, S. E. (1974) in Methods in Enzymology (Fleischer, S., ed.), Vol. 32, pp. 124-131, Academic Press, New York. Naito, Y., and Tsushima, K. (1976) Biochim. Biophys. Acta 438, 159-168. Olson, A. C., and Fraser, M. J. (1974) Biochim. Biophys. Acta 334, 156-176. DePierre, J. W., and Kamovsky, M. L. (1974) J. Biol. Chem. 249, 7121-7129.
AND BARLOW
11. Quagliata, F., Faig, D., Conklyn, M., and Silber, R. (1974) Cancer Res. 34, 3197-3202. 12. Chattejee, S. K., Kim, U., and Bielat, K. (1976) Brit. 1. Cancer 33, 15-26. 13. Belfield, A., and Goldberg, D. M. (1968) Nature (London) 219, 73-75. 14. Persijn, J. P., Van der Slik, W., and Bon, A. W. M. (1%9) Z. Klin. Chem. Klin. Biochem 7, 493497.
15. Riorden, J. R., and Slavik, M. (1974) Biochim. Biophys. Acta 373, 356-360. 16. Stefanovic, V., Mandel, P., and Rosenberg, A. (1975) J. Biol. Chem. 250,7081-7083. 17. Carraway, K. L., Fogle, D. D., Chestnut, R. W., Huggins, J. W., and Carraway, C. A. C. (1976) J. Biol. Chem. 251, 6173-6178. 18. Krishna, G., Weiss, B., and Brodie, B. B. (1%8) J. Pharmocol. Exp. Ther. 163, 379-385. 19. Ramchandran, J. (1971) Anal. Biochem. 43, 227239.
Suran, A. A. (1973) Anal. Biochem. 55, 593-600. 21. Zygowicz, E. R., Sunderman, F. W., Jr., Horak, E., and Dooley, J. F. (1977) Clin. Chem. 23, 2311-2323. 22. Bodansky, O., and Schwartz, M. K. (1968)Advan. Clin. Chem. 11, 277-328. 20.
