49
ALKALINE PHOSPHODIESTERASE I
Paul J. Edel son Katherine D. Gass
INTRODUCTION
Phosphodiesterase I (EC 3.1.4.1) liberates 5'-nucleotides from polyribonucleotides or oligodeoxyribonucleotides in a step-wise manner starting from a nucleotide with a free 3'-OH group at the tail of the chain. In this assay, we use an arti- ficial substrate in which a single nucleotide is coupled at its 5*-end to a phosphate group that is then bound to a p- nitrophenyl group instead of to a length of coupled nucleo- tides . When the 5'-mononucleotide is liberated from the p- nitrophenyl group, the group can be detected by its strong yellow color in alkaline solutions.
The assay may conveniently be used on cell lysates, cell fractions, or extracellular fluid. Sufficient activity is present in 10^ resident mouse peritoneal macrophages for con- venient detection.
METHODS FOR STUDYING Copyright © 1981 by Academic Press, Inc.
MONONUCLEAR PHAGOCYTES 4 6 9 All rights of reproduction in any form reserved.
ISBN 0-12-044220-5
470 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES I I . REAGENTS
Sorensen's Glycine II Buffer (with 2 mM Zinc acetate) Solution A (0.1 M glycine + 0.1 M NaCl)
7.5 gm glycine 5.85 gm NaCl
Disolve in sufficient distilled water to make 1 liter of solution.
Solution B (0.1 N NaOH): 4 gm NaOH
Dissolve in sufficient distilled water to make 1 liter of solution.
Combine 732 ml solution A + 238 ml solution B. Add 440 mg zinc acetate to 1 liter of buffer. Adjust pH to 9.6.
Store refrigerated. Buffer can be indefinitely stored.
Substrate (1.5 mAf Thymidine-5'-phosphate-p-nitrophenolJ Dissolve 8.11 mg TMP-p-NP (Calbiochem-Behring Corp., La Jolla, California Catalog No. 48786), MW 537.5, in 10 ml Sorensen's glycine II-Zn acetate buffer (pH 9.6).
Substrate should be stored dessicated, refrigerated, and protected from light. Substrate solution should be pre- pared on the day of assay, and may be held on ice for several hours if protected from light.
III. PROCEDURE
(1). Place 50 yl aliquots of cell lysates (prepared in 0.05% Triton X-100 as described by Edelson, this volume.) or Triton X-100 alone (for enzyme blank) in small disposable glass test tubes (culture tubes,, disposable glass, 12 x 75 mm, Curtin, Matheson Scientific, Inc., Houston, Texas, Catalog No. 339-275.)
(2). Add 0.5 ml substrate, prewarmed to 37°C.
(3). Shake briskly, cover rack with aluminum foil, and incubate tubes in 37°C water bath for 30 min.
(4). At end of incubation, immediately place tubes on ice, and stop reaction with 1.0 ml 0.1 N NaOH.
(5). Vortex thoroughly each tube.
(6). Read absorbance within 30 min at 400 nm.
VI. BIOCHEMICAL CONSTITUENTS 471 IV. CALCULATION
One unit equals that amount of enzyme which hydrolyzes 1 ym of substrate per minute under above assay conditions.
mU/mg protein =
absorbance (sample) - absorbance (blank) 1
1.55 ml protein cone
(mg/ml) The factor 6.7 is derived as follows:
Molar extinction coefficient of p-nitrophenol = 12 X 103 OD U/l M (at 400 nm).
Then, specific activity in mU/mg = nmol/min/mg protein=
(absorbance) 3
^ ΧΪ 2 ~ Χ 301ΪΪΓ X 1.5 X 1.55 = (protein cone)
(mg/ml) (absorbance)
ml protein cone
(mg/ml)
X 6.7
V. CRITICAL COMMENTS
This is a simple, straightforward, and highly reliable assay. Because it is based on a colorimetric measurement, it is about an order of magnitude less sensitive than assays us- ing radioactive substrates, but even so it readily detects the activity present in 106 resident mouse peritoneal macrophages that is about 1.43 mU/mg.
REFERENCES
1. The artificial substrate used in this assay was originally described in
W. E. Razzell and H. G. Khorana. Studies on polynucleo- tides. III. Enzymic degradation. Substrate specificity and properties of snake venom phosphodiesterase. J. Biol.
Chem. 234:2105, 1959.
472 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES 2. The various phosphodiesterases are reviewed in
H. G. Khorana. In "The Enzymes" Vol. 5 (P. D. Boyer, H. Lardy, and K. Myrback, eds.), PP· 79. Academic Press, New York, 1961. 2nd edition.
3. The assay was adapted from the protocol presented by H. Beaufay, A. Amar-Costesec, E. Feytmans, D. Thines- Simpoux, M. Wibo, M. Robbi, and J. Berthet. Analytical study of microsomes and isolated subcellar membranes from rat liver. I. Biochemical methods. J. Cell Biol. 61:188, 1974.
4. An example of the application of this assay to macrophages is
P. J. Edelson and C. Erbs. Plasma membrane localization and metabolism of alkaline phosphodiesterase I in mouse peritoneal macrophages. J. Exp. Med. 147:17, 1978.
