48
5■-NUCLEOTIDASE ASSAY
Paul J. Edelson Robert A. Duncan
I. INTRODUCTION
5'-Nucleotidase (5'-ribonucleotide phosphohydrolase EC 3.1.3.5) hydrolyzes the phosphoester linkage in 5'-mono- nucleotides, liberating a nucleoside and inorganic phosphate.
Because of its presence in a great variety of tissues, in many species, and its localization to the plasma membrane, the enzyme has been widely used as a convenient marker for the plasma membrane in cell fractionation studies. It has been identified as an ecto enzyme (1), oriented with its active site accessible to the cell milieu in guinea pig neutrophils
(2), and subsequently in mouse peritoneal macrophages (3).
Its status in several other cell types is summarized in Table I. Information on its presence in a variety of mouse mono- nuclear phagocyte cell lines has recently been published (4).
The absence of the enzyme has been used as one of several tests for macrophage activation (5).
METHODS FOR STUDYING Copyright © 1981 by Academic Press, Inc.
MONONUCLEAR PHAGOCYTES 4 6 1 All rights of reproduction in any form reserved.
ISBN 0-12-044220-5
Cell type Species 5'-Nucleotidase activity Reference Neutrophil
Monocyte
Alveolar macrophage Peritoneal macrophage
Resident
Thioglycollate-stimulated BCG/PPD-stimulated
Granuloma macrophage Platelet
Erythrocyte Lymphocyte
B lymphocyte T lymphocyte
Guinea pig Human
Mouse Human Mouse Mouse Mouse Mouse Mouse Human Human Human Human
Present Absent Absent Absent Present Present Absent Absent Absent Absent Absent Present Present
(2) (8) (9) (10) (9) (11,12)
(12) (13) (14) (15) (15) (16) (16)
Activity develops over 24-48 Hr in culture.
VI. BIOCHEMICAL CONSTITUENTS 463
Most assays for 5■-nucleotidase depend upon measuring the rate of appearance of one of the two products of hydrolysis of the nucleotide substrate: either the inorganic phosphate or the nucleoside. In the assay described below, the nucleo- tide carries a radioactive label in its nucleoside portion and the labeled nucleoside generated by the enzymatic hydrol- ysis is separated from the substrate and counted in a scin- tillation spectrometer. The method is modified from Avruch and Wallach (6).
II. REAGENTS
(1) Tris-HCl buffer containing 54 mM Tris and 13 mM MgCl^
pH 9.0 (1000 ml) Dissolve 6.51 gm Trizma Base (Sigma Chemi- cal Co., St. Louis, Missouri, Catalog No. T-1503) and 2.44 gm MgCl2-6 Η~0 in suffieient distilled water to make 1 liter of solution. Adjust to pH 9.0 with 2 N HC1 and store indefinite- ly at 4°C.
(2) [3H]-AMP stock (25 yCi/ml in 50% ethanol) (10 ml) Add 9.75 ml ice-cold 50% ethanol to 1 vial Adenosine- [2- H]-53 1-monophosphate, ammonium salt, 250 yCi in 0.250 ml,
>2000 mCi/mmol (Amersham Corp., Arlington Heights, Illinois, Catalog. No. TRK.344). [3H]-AMP stock can be used for approx- mately four months if stored at -20°C.
(3) 0.25 M ZnS04 (100 ml) Add distilled water to 7.19 gm ZnSO^-7 H20 to make 100 ml of solution. This may be stored indefinitely at room temperature.
(4) 0.25 M Ba (OH)2 (100 ml) 7.89 gm Ba(OH)2'8 H20.
Add distilled water to make 100 ml of solution. Ba(0H)2 may be stored indefinitely at room temperature. This is a satura- ted solution and should be filtered through coarse paper
(e.g., Whatman No. 1) immediately before use.
(5) Substrate (25 nCi 51-[3H]-AMP/ml, 0.15 mM 5'-AMP, 6mAf p-NP) (100 ml) 5.8 mg Adenosine-5'-monophosphoric acid, sodium salt, from yeast, crystalline, Type II (Sigma Chemical Corp., St. Louis, Missouri, Catalog No. A-1752) (MW=388.4);
222.6 mg p-Nitrophenyl phosphate, disodium (Sigma Chemical Corp., St. Louis, Missouri, Catalog No. 104.0); 0.1 ml [3H]- AMP stock; 100 ml Tris-HCl buffer. Substrate should be pre- pared immediately prior to assay and kept on ice.
III. PROCEDURE
(1). Place 0.1 ml aliquots of sample (e.g., cell lysate prepared in fresh 0.05% Triton X-100 as described by Edelson,
this volume) or of the appropriate enzyme blank (e.g., 0.05%
Triton X-100 alone) in glass test tubes (culture tubes, dispos- able glass, 12 x 75 mm, Curtin, Matheson Scientific, Inc., Houston, Texas, Catalog No. 339-275) on ice.
(2). Add 0.5 ml substrate, shake well, and incubate in a 37°C water bath for 30 min.
(3). Stop the reaction by returning the tubes to an ice bath and adding 0.2 ml 0.25 M ZnSCK. Vortex briefly.
(4). Add 0.2 ml freshly filtered 0.25 M Ba(0H)2 and vortex thoroughly.
(5). Centrifuge at 1500 g for 20 min at room temperature (or 4°C).
(6). Add 0.5 ml supernatant to 5 ml Aquassure (New England Nuclear, Boston, Massachusetts, Catalog No. NEF-965) in 6 ml plastic minivials (Plastic Sampule liquid scintillation vials, Wheaton Scientific, Millville, New Jersey, Catalog No. 986624) and mix well.
(7). Prepare counting standards (equivalent of 100%
hydrolysis) by diluting 1.0 ml substrate with 1.0 ml water.
Add 0.5 ml to 5 ml Aquassure, as above.
(8). Count for 10 min in a liquid scintillation counter (Packard Instrument Co., Inc., Downers Grove, Illinois).
Typical settings for ^H for a Tricarb counter are A-B channel;
70% gain; 50-4000 gate setting; External standard off.
IV. CALCULATIONS
0.5 ml of 0.15 mM 5f-AMP substrate = 75 nm 5'-AMP per sample. Thus, specific activity can be expressed as mU/mg protein = nm 5'-AMP hydrolyzed/min/ng protein
(CPMEXP - C P MBL) χ 7, x 75 nm (CPM - CPM )
STD BL
(30 min) x (mg protein/0.1 ml)
Protein concentration can be determined as described by Meltzer, this volume.
V. CRITICAL COMMENTS
This is a fairly simple and highly reproducible assay, with a range of sensitivity wide enough to detect activity of between 5 x 104 and 1 x 10^ resident mouse peritoneal macro-
VI. BIOCHEMICAL CONSTITUENTS 465 phages. The activity present in 5 x 10"5 resident macrophages is about 44.2 mU/mg protein.
This method presents a convenient alternative to spectro- photometric assays for 5■-nucleotidase or protocols using
32P-labeled nucleotides. It requires no second enzyme for product conversion as some spectrophotometrie protocols do, and offers the greater sensitivity inherent in assays using radioactive tracers without the hazard of using 32p# j^ ^s
also relatively rapid, a typical assay takes about 3 hr.
It is important to establish that the activity observed is actually due to 5'-nucleotidase, rather than a nonspecific phosphatase. p-Nitrophenyl phosphate is used here as a com- petitive inhibitor of other phosphatases. At pH 9.0 acid phosphatase activity is not of concern and 5 mM ß-glycerophos- phate may be used in place of p-Npp as an effective inhibitor of alkaline phosphatase in situations where confusion with other intracellular phosphatases (e.g., glucose-6-phosphate phosphatase) is not an issue. Tarträte (10 mM) inhibits phos- phatases active at pH 5.O.
5*-Nucleotidase shows peaks in activity at pH 5.5 and 7.0, with an additional peak occurring at about pH 9.2 when MgCl2 is included in the reaction mix. Removal of divalent cations with 20 mM EDTA reduces activity more than 95% at pH 9.O.
Addition of 0.1 mM Zn2 + completely eliminates nucleotidase activity. One should be cautioned, however, that EDTA inter- feres with the BaSO^ precipitation and a different assay meth- od must be used (see reference 7).
51-Nucleotidase is most effective hydrolyzing ribonucleo- side 5'-monophosphates, with little or no cleavage of the corresponding 5■-deoxyribonucleotides or 5'-ribonucleoside di- or triphosphates observed. A nucleotide analog, α,β-meth- ylene adenosine 5■-diphosphate (AOPCP), has been reported to be a very powerful inhibitor of 5'-nucleotidase, though its efficacy may vary among different species.
LITERATURE SOURCES
Much additional useful information may be found in the following:
C. C. Widnell. Purification of rat liver 5'-nucleotidase as a complex with sphingomyelin. Methods Enzymol. 32:
368-374, 1974.
B. L. Reimer, and C. C. Widnell. The demonstration of a specific 5'-nucleotidase activity in rat tissues. Arch.
Biochem. Biophys. 171:343-347, 1975.
Z. A. Werb, and Z. A. Cohn. Plasma membrane synthesis in the macrophage following phagocytosis of polystyrene latex particles. J. Biol Chem. 247:2439-2446, 1972 R. M. Burger, and J. M. Lowenstein. Preparation and properties of 5'-nucleotidase from smooth muscle of small intestine. J. Biol. Chem. 245.-6247-6280, 1970.
REFERENCES
1. P. J. Edelson. Macrophage ecto enzymes: Their identi- fication, metabolism, and control. "Mononuclear Phago- cytes: Functional Aspects" (R. van Furth, ed.), Martinus Nijhoff, The Hague, 1980.
2. J. W. DePierre and M. L. Karnovsky. Ecto enzymes of the guinea pig polymorphonuclear leukocyte. I. Evidence for an ecto adenosine monophosphatase, -adenosine triphos- phatase, and -p-nitrophyl phosphatase. J. Biol. Chem.
249:7111-7120, 1974.
3. P. J. Edelson and Z. A. Cohn. 5'-nucleotidase activity of mouse peritoneal macrophages. II. Cellular distri- bution and effects of endocytosis. J. Exp. Med. 144:
1596-1608, 1976.
4. P. S. Morahan and P. J. Edelson. Mouse mononuclear phago- cyte cell lines. J. Reticuloendothel. Soc. (in press).
5. C. Bianco and P. J. Edelson. Characteristics of the acti- vated macrophage. "Immune Effector Mechanisms in Disease"
(M. E. Weksler et al., eds.), pp. 1-8. Grune and Stratton, New York, 1978.
6. J. Avruch and D. F. H. Wallach. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochem. Biophys. Acta 235:334-347, 1976.
7. B. Glastris and S. E. Pfeiffer. Mammalian membrane marker enzymes: Sensitive assay for 5'-nucleotidase and assay for mammalian 2',3'-cyclic nucleotide-3'-phosphohydrolase.
Methods Enzymol. 32:124-131, 1974.
8. P. S. Shirley, P. Wang, L. R. DeCjatelet, and B. M. Waite.
Absence of the membrane marker enzyme 51-nucleotidase in human polymorphonuclear leukocytes. Anal. Biochem. 64:
624-627, 1975.
9. E. James and P. J. Edelson. Unpublished observations.
VI. BIOCHEMICAL CONSTITUENTS 467 10. W. D. Johnson, B. Mei, and Z. A. Cohn. The separation,
long-term cultivation, and maturation of the human mono- cyte. J. Exp. Med. 146:1613-1626, 1977.
11. R. L. Nachman, B. Ferris, and J. G. Hirsch. Macrophage plasma membranes. I. Isolation and studies on protein components. J. Exp. Med. 133:185, 1971.
12. P. J. Edelson and Z. A. Cohn. 5'-Nucleotidase activity of mouse peritoneal macrophages. I. Synthesis and degradation in resident and inflammatory preparations.
J. Exp. Med. 144:1581-1595, 1976.
13. P. J. Edelson and C. Erbs. Biochemical and functional characteristics of the plasma membrane of macrophages from BCG-infected mice. J. Immunol. 120:1532-1536, 1978.
14. R. J. Bonney, I. Gery, T. Y. Lin, M. F. Meyerhofer, W. Acevedo, and P. Davies. Mononuclear phagocytes from carrageenan-induced granulomas. Isolation, cultivation, and characterization. J. Exp. Med. 148:261-275, 1978.
15. K. Gass and P. J. Edelson. Unpublished observations.
16. J. Schwaber and P. J. Edelson. Unpublished observations.
