ANALYTICAL
BIOCHEMISTRY
204, 296-299 (19%)
A Specific Peroxidase-Coupled for Diamine Oxidases G. Houen and L. Leonardsen Institute of Biochemical Genetics, @.ster Farimagsgade
Received
January
Activity Stain
2A, DK-1353
Copenhagen
21, 1992
MATERIALS
A specific peroxidase-coupled activity staining method for diamine oxidase (DAO) was developed. Diaminobenzidine was found to inhibit DA0 and to give rise to unspecific staining. Among several other reagents 4-Cl-1-naphthol was found to be most suitable. Using specific activity staining DA0 could be visualized in polyacrylamide gels as a high-molecular-weight complex, which could be dissociated by Tween 20 but not by NP-40, Triton X-100, or Chaps. Q 1992 Academic PXSS, Inc.
Amine oxidases are a group of enzymes catalyzing the general reaction (l-3)
R’CH,NR=R”
K., Denmark
+ H,O + 0, --* R’CHO + R2R3NH + H 2 0 2’
Some of these enzymes, especially diamine oxidase (E.C. 1.4.3.6; substituent groups optimally being R’ = H,NCH,CH,CH,CH,, R2 = H, R3 = H) and polyamine oxidase, are involved in regulation of cell growth through the metabolism of histamine and the polyamines putrescine, spermidine, and spermine (4-7). As part of our study of amine oxidases we have developed a specific peroxidase-coupled enzyme activity stain for diamine oxidase. Here we report the visualization of diamine oxidase (DAO)* on nitroceilulose models and in polyacrylamide gels using this activity stain.
’ Abbreviations used: AG, aminoguanidine; BSA, bovine serum albumin; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulfonate; 4-Cl-1-naphthol, 4-chloro-1-naphthol; DAO, diamine oxidase; DAB, diaminobenzidine; DH, dehydrogenase; HRP, horseradish peroxidase; NC, nitrocellulose; PB, phosphate buffer; PUT, putrescine; Tris, tris(hydroxymethyl)aminomethane.
AND
METHODS
Chemicals and Enzymes Tris, diaminobenzidine-4HCl (DAB), 3-amino-g-ethylcarbazole, putrescine-2HC1, acrylamide, N,N-methylene-bis-acrylamide, N,N,N’,N’-tetramethylethylenediamine, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (Chaps), bovineserum albumin fraction V (BSA), DAO, alcohol dehydrogenase, aldehyde dehydrogenase, and horseradish peroxidase (HRP, Type VI) were from Sigma (St. Louis, MO). Ammonium persulfate, bromophenol blue, saccharose, Tween 20, NaH,PO,, and Na,HPO, were from Merck (Darmstadt, Germany). 4-Cl-1-naphthol, o-dianisidine, spermidine-3HC1, and spermine-4HCl were from Fluka (Buchs, Switzerland). Aminoguanidine hemisulfate was from Eastman Kodak (Rochester, NY). [2,3-3H]Putrescine (1 mCi/ml, sp act 16.2 Ci/mmol) was from New England Nuclear (Boston, MA). Fetal calf serum (heat inactivated) was from GIBCO (Paisley, Scotland). Nitrocellulose (NC) paper (0.45 pm) was from Millipore (Bedford, MA). Maxifluor scintillation fluid was from J. T. Baker (Deventer, Holland). Purified bovine serum amine oxidase was a gift from Professor Bruno Mondovi (University of Rome, Italy). DA0 Assay DA0 activity was determined by the method of Okuyama and Kobayashi (8). DA0 (1 mg/ml final concentration) and putrescine (1 mM, 0.5 &i/ml final concentration) were diluted in 0.1 M PB, pH 7.2 (1 ml reaction volume), or PB containing various dilutions of added compound (DAB, Tris, 4-Cl-1-naphthol). Alternatively, DA0 was spotted onto a l-cm2 piece of NC paper; the paper was dried and incubated in 1% BSA in PB for 1 h for the sake of comparison with peroxidasecoupled activity detection, where blocking of the paper is necessary to prevent binding of peroxidase to the
296 All
Copyright 0 1992 rights of reproduction
0003-2697192 $5.00 by Academic Press, Inc. in any form reserved.
PEROXIDASE-COUPLED
ACTIVITY
STAIN
FOR
(gel:reaction (10 pg/ml), (10 mM).
DIAMINE
297
OXIDASE
volume ratio, 4-Cl-1-naphthol
1:20) in (0.1 mM),
PB with HRP and putrescine
RESULTS
L
1
2
3
4
Cont. FIG. 1. Effect of diaminobenzidine trescine by diamine oxidase.
5
6
7
a
9
10
.
of DAB (mM) (DAB)
on the oxidation
of pu-
paper, and, after washing three times for 15 min in PB, the paper was incubated in PB (1 ml) with putrescine (1 mM, 0.5 &Wml). After incubation at 37°C for 1 h, the reaction mixture was extracted with toluene (1 ml) by mixing on a whirlimixer (30 s), samples were frozen after centrifugation (-60”(Z), and the toluene was decanted into scintillation vials. This procedure was found to be reproducible and decantation of the toluene phase was nearly quantitative as the water phase remained frozen during decantation. Four milliliters of Maxifluor was added to the toluene and samples were counted in a Beckman scintillation counter (Model LS 9000). Paper Models DAO-containing samples and control proteins were spotted (2-5 ~1) onto NC paper, which was dried and incubated in 1% BSA in PB for 1 h. After washing in PB strips were incubated overnight (18 h; shorter incubation times produced too little staining) with gentle agitation on a shaking board in PB with HRP (10 yglml) and the indicated combinations of substrate, dye, and inhibitor. Strips were dried and photographed (Kodak Tmax100 film).
Preliminary experiments with small amounts of DA0 spotted onto NC paper followed by incubation with putrescine, HRP, and DAB in 50 mM Tris buffer, pH 8.2, failed to produce a peroxidase-catalyzed dye precipitation. Since this could be due to inactivation of diamine oxidase, inhibition of DA0 by Tris or DAB, or attenuation of the peroxidase-catalyzed reaction with DAB, these possibilities were investigated. Using the radiochemical assay it was shown that the immobilized enzyme was still active, and that Tris in concentrations up to 100 mM had no effect on the formation of A’-pyrroline by DA0 in solution (data not shown), but DAB was found to exert an inhibitory effect (Fig. 1). For this reason the usefulness of several other hydrogen donors was investigated with paper models. From Fig. 2 it can be seen that in phosphate buffer both 4-Cl-1-naphthol, DAB, and 3-amino-9-ethylcarbazole give some staining while o-dianisidine is almost ineffective. Moreover, Tris inhibited the staining reaction of 4-Cl-1-naphthol and further experiments were therefore all performed in phosphate buffer. The specificity and sensitivity of the four hydrogen donors used in Fig. 2 are further illustrated in Table 1. o-Dianisidine produces no staining, while DAB is very unspecific and gives a positive reaction, both in the presence of the diamine oxidase inhibitor aminoguanidine (10) and in the absence of putrescine. 3-Amino-g-ethylcarbazole was more specific but gave some unspecific staining. 4-Cl-1-naphthol was the most specific (although not the most sensitive hydrogen donor, in terms of staining intensity) and was therefore chosen for further studies. Figure 3 shows the effect of varying the 4-Cl-1-naphthol concentration on the sensitivity of the was found to be optimethod; 0.1 mM 4-Cl-1-naphthol
Gel Electrophoresis Nondenaturing gel electrophoresis was done as described (9) in 5% polyacrylamide gels using a Mighty Small electrophoresis apparatus (Hoefer Scientific). Twenty-five microliters of DA0 in PB (10 mg/ml) was mixed with 25 ~1 electrophoresis sample buffer either without detergent or containing 2% Tween 20 (v/v), 2% Triton X-100 (v/v), 2% NP-40 (v/v), or 2% Chaps (w/v). Samples were applied to the gels (0.75 mm X 6 cm X 10 cm) and electrophoresis was carried out for 2 h at 4”C, 15 mA per gel. Gels were stained by incubation overnight (18 h) with gentle agitation on a shaking board
AECDEF
FIG. 2. Paper models showing the usefulness of several hydrogen donors in the peroxidase-coupled activity stain for diamine oxidase. The nitrocellulose strips with DA0 spotted on were incubated in PB with HRP (10 pg/ml) and the following combinations of substrate and hydrogen donor: (Al 4 mM putrescine + 0.1 mM 4-Cl-1-naphthol; (B) 0.1 mM 4-Cl-1-naphthol; (Cl 4 mMputrescine + 0.1 mM DAB; (D) 4 mMputrescine + 0.1 mM 3-amino-9-ethylcarbazole; (E) 4 mMputrestine + 0.1 mM orthodianiside; (F) 4 mM putrescine.
298
HOUEN TABLE
AND
1
Effectiveness of Hydrogen Donors in the PeroxidaseCoupled Spot Test for Diamine Oxidase 3-Amino-gethylcarbazole
0-Dianisidine DA0 +PUT +PUT +AG -PUT Alcohol DH +PUT +PUT +AG -PUT Aldehyde DH +PUT +PUT +AG -PUT
4-Cl-lnaphthol
-
+ -
+ -
+ + +
-
-
-
+ + +
-
+ + -
-
+ + +
mal, allowing detection of as little as 0.1 units of enzyme (1 PcLg,- 10 pmol). The specificity of the assay is further illustrated in Fig. 4. Pig kidney diamine oxidase, which has a high affinity for diamines (ll), produces a spot with putrescine as substrate, but not with spermidine or spermine. Conversely, bovine plasma amine oxidase, which has a high affinity for spermine and spermidine (12) but not for putrescine, produces a spot only with
IJg
DA0
20
-
10
-
-
Ald.dh
-
ESA
-
DAO
-
ESAO
-
DAB
Note. The enzymes were spotted on nitrocellulose and incubated in PB with 0.1 mM of the various hydrogen donors and the indicated combinations of putres’cine (10 mM) and aminoguanidine (1 mM). + indicates a clearly visible spot and - indicates that no spot was visible; 1 unit of DA0 is defined as the amount of enzyme that oxidizes 1 pmol of putrescine per hour at pH 7.2 and 37°C.
Enz.
LEONARDSEN
AECOEFB
A
S
C
D
E
FIG. 4. Substrate specificity of the 4-Cl-l-naphthol-based activity stain for amine oxidases. The enzymes indicated were spotted on nitrocellulose and incubated in PB with HRP (10 pg/ml) 4-Cl-lnaphthol(O.1 mM), and the following substrates: (A) no substrate; (B) putrescine (4 mM); (C) 4 mM spermine; (D) 4 mM spermidine; (E) putrescine (4 mM) + aminoguanidine (0.2 mM).
spermine and spermidine as substrates. When DA0 was subjected to electrophoresis in nondenaturing gels and visualized with the activity stain, DA0 was revealed as a high-molecular-weight complex (Fig. 5) that hardly entered the gel and that Triton X-100, NP-40, and Chaps did not dissociate, but that could be dissociated by Tween 20. DISCUSSION Previous studies have shown that assays for amine oxidase using tetrazolium salts are nonspecific (13) and have serious limitations. An assay for benzylamine oxidase in gels based on the production of benzaldehyde and scanning of the gel at 250 nm has been described (14). Coupled peroxidase reactions have been used for the histochemical localization of monoamine oxidase, benzylamine oxidase (15-17), and p-IVJV-dimethylamine-P-phenylethylamine oxidase (18) and for the localization of various oxidases in gels (19), but no results have previously been presented for diamine oxidase (20). From the results presented here it is clear that great
2l.2
Alc.dh
IO-
Ald.dh
IO-
-
0.1 4-Cl-l
0.2
0.6 -Naphtole
0-E
1-O cone.
1.2
1.2 CmM1
FIG. 3. (A) Effect of varying the concentration of 4-Cl-1-naphthol on the sensitivity of the peroxidase-coupled activity stain for diamine oxidase. The enzymes indicated were spotted on nitrocellulose and incubated in PB containing HRP (10 pg/ml), putrescine (10 mM), and 4-Cl-l-naphthol (concentration as indicated). Lane F also contained 1 mM
aminOgUanidh?.
AECDE
F
FIG. 5. Visualization of DA0 in polyacrylamide gels and effects of detergents. Twenty-five microliters of DA0 (10 mg/ml) in PB was mixed with 25 ~1 electrophoresis sample buffer with (2%) or without detergent: (A) boiled DA0 as control, (B) no detergent, (C) Triton X-100, (D) NP-40, (E) Tween-20, (F) Chaps.
PEROXIDASE-COUPLED
ACTIVITY
care and many precautions must be taken in the study of amine oxidases. Some general guidelines can be made, however: (1) All experiments should be done in phosphate or other nonamine buffers, especially in the case of peroxidase-catalyzed dye reactions. (2) The hydrogen donor should not be an amine, or, for the visualization of DAO, not a diamine. (3) The effects of inhibitors must be interpreted with great care. For the assay of DAO, 4-Cl-1-naphthol was found to be the most suitable staining reagent and for the demonstration of DA0 activity with this dye, omission of substrate or inhibition by aminoguanidine are suitable controls. Preliminary attempts to use the method described here for localization of diamine oxidase on sections from pig kidney or human placenta were not successful. Since model experiments showed that immobilized DA0 was still active after the histochemical fixation, the absence of staining on sections could result from the enzyme being present mainly in an inactive form, which is activated or released only in response to certain stimuli. This hypothesis is compatible with the observation that microinjection of amine oxidases causes growth arrest and has cytotoxic effects in growing cells (21,22). The activity stain described here can be used to localize DA0 in polyacrylamide gels and will be useful for studying the occurrence of DA0 in various tissue extracts and for studying the number of isoenzymes in these extracts. REFERENCES 1. Mondovi, B., and Agro, A. F. (1982) in Advances in Experimental Medicine and Biology (Bossa, F., Chiancone, E., Agro, A. F., and Strom, R., Eds.), Vol. 148, pp. 141-153, Plenum Press, New York. 2. Tipton, K. F. (1986) 3. Blaschko, H. (1952)
Cell Rio&em. Funct. 4, 79-87. Pharmacol. Reu. 4, 415-458.
STAIN
FOR
4. Bachrach,
DIAMINE U. (1970)
299
OXIDASE Ann.
N.Y.
Acad.
Sci. 171,939%959.
5. Andersson, A., Henningson, S., and Rosengren, E. (1980) in Polyamines in Biomedical Research (Gaugas, J. M., Ed.), pp. 273-283, Wiley-Interscience, New York. D. M. L. (1980) in Polyamines in Biomedical Research 6. Morgan, (Gaugas, J. M., Ed.), pp. 285-302, Wiley-Interscience, New York. 7. Gaugas, (Gaugas,
J. M. (1980) in Polyamines in Biomedical J. M., Ed.), pp. 343-362, Wiley-Interscience,
8. Okuyama, 95,242-250. 9. Davis,
T., and Kobayashi,
B. J. (1984)
Ann.
N.Y.
Y. (1961) Acad.
Arch.
Biochem.
Sci. 121,
404-427.
10. Beaven, M. A. (1976) N. Enggl. J. Med. 294, 30. 11. Beardsley, W. G., Hill, G. M., and Lobley, R. W. (1970) J. 117, 1699176.
Research New York. Bioplzys.
Biochem.
12. Mondovi, B., Turini, P., Befani, O., and Sabatini, S. (1983) in Methods in Enzymology (Tabor, H., andTabor, C. W., Eds.), Vol. 94, pp. 314-318, Academic Press, San Diego. 13. Lagnado, J. R., Okamoto, M., and Youdim, M. B. H. (1971) FEBS Lett. 17, 177-120. 14. Lin, A. W. M., and Castell, D. 0. (1975) Anal. Biochem. 69,637642. 15. Ryder, T. A., MacKenzie, M. L., Pryse-Davies, J., Glover, V., Lewinsohn, R., and Sandler, M. (1979) Histochemistry 62,93-100. 16. Ryder, T. A., MacKenzie, M. L., Lewinsohn, R., Pryse-Davies, J., and Sandler, M. (1980) Histochemistry 67, 199-204. 17. Yoshimoto, Y., Sakumoto. T., Arai, R., Miyake, A., Kimura, H., Aono, T., Tanizawa, O., and Maeda, T. (1986) Endocrinology 119, 1800-1804. 18. Shannon, W. A., Wasserkrug, H. L., Plapinger, R. E., and Seligman, A. M. (1976) J. Histochem. Cytochem. 24, 527-539. 19. Feinstein, R. N.. and Lindahl, R. (1973) Anal. Biochem. 56, 353360. 20. Argento-Cerii, M. P., and Autuori, F. (1985) in Structure and Function of Amine Oxidases (Mondovi, B., Ed.), pp. 899104, CRC Press, Boca Raton, FL. 21. Bachrach, U., Ash, I., Abu-Elheiga, L., Hershkovitz, M., and Loyter, A. (1987) J. Cell. Physiol. 131, 92-98. 22. Bachrach, IJ., Ash, I.. and Rahamim, E. (1987) Tissue Cell 19, 39-50.
BIOCHEMISTRY
204, 296-299 (19%)
A Specific Peroxidase-Coupled for Diamine Oxidases G. Houen and L. Leonardsen Institute of Biochemical Genetics, @.ster Farimagsgade
Received
January
Activity Stain
2A, DK-1353
Copenhagen
21, 1992
MATERIALS
A specific peroxidase-coupled activity staining method for diamine oxidase (DAO) was developed. Diaminobenzidine was found to inhibit DA0 and to give rise to unspecific staining. Among several other reagents 4-Cl-1-naphthol was found to be most suitable. Using specific activity staining DA0 could be visualized in polyacrylamide gels as a high-molecular-weight complex, which could be dissociated by Tween 20 but not by NP-40, Triton X-100, or Chaps. Q 1992 Academic PXSS, Inc.
Amine oxidases are a group of enzymes catalyzing the general reaction (l-3)
R’CH,NR=R”
K., Denmark
+ H,O + 0, --* R’CHO + R2R3NH + H 2 0 2’
Some of these enzymes, especially diamine oxidase (E.C. 1.4.3.6; substituent groups optimally being R’ = H,NCH,CH,CH,CH,, R2 = H, R3 = H) and polyamine oxidase, are involved in regulation of cell growth through the metabolism of histamine and the polyamines putrescine, spermidine, and spermine (4-7). As part of our study of amine oxidases we have developed a specific peroxidase-coupled enzyme activity stain for diamine oxidase. Here we report the visualization of diamine oxidase (DAO)* on nitroceilulose models and in polyacrylamide gels using this activity stain.
’ Abbreviations used: AG, aminoguanidine; BSA, bovine serum albumin; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulfonate; 4-Cl-1-naphthol, 4-chloro-1-naphthol; DAO, diamine oxidase; DAB, diaminobenzidine; DH, dehydrogenase; HRP, horseradish peroxidase; NC, nitrocellulose; PB, phosphate buffer; PUT, putrescine; Tris, tris(hydroxymethyl)aminomethane.
AND
METHODS
Chemicals and Enzymes Tris, diaminobenzidine-4HCl (DAB), 3-amino-g-ethylcarbazole, putrescine-2HC1, acrylamide, N,N-methylene-bis-acrylamide, N,N,N’,N’-tetramethylethylenediamine, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (Chaps), bovineserum albumin fraction V (BSA), DAO, alcohol dehydrogenase, aldehyde dehydrogenase, and horseradish peroxidase (HRP, Type VI) were from Sigma (St. Louis, MO). Ammonium persulfate, bromophenol blue, saccharose, Tween 20, NaH,PO,, and Na,HPO, were from Merck (Darmstadt, Germany). 4-Cl-1-naphthol, o-dianisidine, spermidine-3HC1, and spermine-4HCl were from Fluka (Buchs, Switzerland). Aminoguanidine hemisulfate was from Eastman Kodak (Rochester, NY). [2,3-3H]Putrescine (1 mCi/ml, sp act 16.2 Ci/mmol) was from New England Nuclear (Boston, MA). Fetal calf serum (heat inactivated) was from GIBCO (Paisley, Scotland). Nitrocellulose (NC) paper (0.45 pm) was from Millipore (Bedford, MA). Maxifluor scintillation fluid was from J. T. Baker (Deventer, Holland). Purified bovine serum amine oxidase was a gift from Professor Bruno Mondovi (University of Rome, Italy). DA0 Assay DA0 activity was determined by the method of Okuyama and Kobayashi (8). DA0 (1 mg/ml final concentration) and putrescine (1 mM, 0.5 &i/ml final concentration) were diluted in 0.1 M PB, pH 7.2 (1 ml reaction volume), or PB containing various dilutions of added compound (DAB, Tris, 4-Cl-1-naphthol). Alternatively, DA0 was spotted onto a l-cm2 piece of NC paper; the paper was dried and incubated in 1% BSA in PB for 1 h for the sake of comparison with peroxidasecoupled activity detection, where blocking of the paper is necessary to prevent binding of peroxidase to the
296 All
Copyright 0 1992 rights of reproduction
0003-2697192 $5.00 by Academic Press, Inc. in any form reserved.
PEROXIDASE-COUPLED
ACTIVITY
STAIN
FOR
(gel:reaction (10 pg/ml), (10 mM).
DIAMINE
297
OXIDASE
volume ratio, 4-Cl-1-naphthol
1:20) in (0.1 mM),
PB with HRP and putrescine
RESULTS
L
1
2
3
4
Cont. FIG. 1. Effect of diaminobenzidine trescine by diamine oxidase.
5
6
7
a
9
10
.
of DAB (mM) (DAB)
on the oxidation
of pu-
paper, and, after washing three times for 15 min in PB, the paper was incubated in PB (1 ml) with putrescine (1 mM, 0.5 &Wml). After incubation at 37°C for 1 h, the reaction mixture was extracted with toluene (1 ml) by mixing on a whirlimixer (30 s), samples were frozen after centrifugation (-60”(Z), and the toluene was decanted into scintillation vials. This procedure was found to be reproducible and decantation of the toluene phase was nearly quantitative as the water phase remained frozen during decantation. Four milliliters of Maxifluor was added to the toluene and samples were counted in a Beckman scintillation counter (Model LS 9000). Paper Models DAO-containing samples and control proteins were spotted (2-5 ~1) onto NC paper, which was dried and incubated in 1% BSA in PB for 1 h. After washing in PB strips were incubated overnight (18 h; shorter incubation times produced too little staining) with gentle agitation on a shaking board in PB with HRP (10 yglml) and the indicated combinations of substrate, dye, and inhibitor. Strips were dried and photographed (Kodak Tmax100 film).
Preliminary experiments with small amounts of DA0 spotted onto NC paper followed by incubation with putrescine, HRP, and DAB in 50 mM Tris buffer, pH 8.2, failed to produce a peroxidase-catalyzed dye precipitation. Since this could be due to inactivation of diamine oxidase, inhibition of DA0 by Tris or DAB, or attenuation of the peroxidase-catalyzed reaction with DAB, these possibilities were investigated. Using the radiochemical assay it was shown that the immobilized enzyme was still active, and that Tris in concentrations up to 100 mM had no effect on the formation of A’-pyrroline by DA0 in solution (data not shown), but DAB was found to exert an inhibitory effect (Fig. 1). For this reason the usefulness of several other hydrogen donors was investigated with paper models. From Fig. 2 it can be seen that in phosphate buffer both 4-Cl-1-naphthol, DAB, and 3-amino-9-ethylcarbazole give some staining while o-dianisidine is almost ineffective. Moreover, Tris inhibited the staining reaction of 4-Cl-1-naphthol and further experiments were therefore all performed in phosphate buffer. The specificity and sensitivity of the four hydrogen donors used in Fig. 2 are further illustrated in Table 1. o-Dianisidine produces no staining, while DAB is very unspecific and gives a positive reaction, both in the presence of the diamine oxidase inhibitor aminoguanidine (10) and in the absence of putrescine. 3-Amino-g-ethylcarbazole was more specific but gave some unspecific staining. 4-Cl-1-naphthol was the most specific (although not the most sensitive hydrogen donor, in terms of staining intensity) and was therefore chosen for further studies. Figure 3 shows the effect of varying the 4-Cl-1-naphthol concentration on the sensitivity of the was found to be optimethod; 0.1 mM 4-Cl-1-naphthol
Gel Electrophoresis Nondenaturing gel electrophoresis was done as described (9) in 5% polyacrylamide gels using a Mighty Small electrophoresis apparatus (Hoefer Scientific). Twenty-five microliters of DA0 in PB (10 mg/ml) was mixed with 25 ~1 electrophoresis sample buffer either without detergent or containing 2% Tween 20 (v/v), 2% Triton X-100 (v/v), 2% NP-40 (v/v), or 2% Chaps (w/v). Samples were applied to the gels (0.75 mm X 6 cm X 10 cm) and electrophoresis was carried out for 2 h at 4”C, 15 mA per gel. Gels were stained by incubation overnight (18 h) with gentle agitation on a shaking board
AECDEF
FIG. 2. Paper models showing the usefulness of several hydrogen donors in the peroxidase-coupled activity stain for diamine oxidase. The nitrocellulose strips with DA0 spotted on were incubated in PB with HRP (10 pg/ml) and the following combinations of substrate and hydrogen donor: (Al 4 mM putrescine + 0.1 mM 4-Cl-1-naphthol; (B) 0.1 mM 4-Cl-1-naphthol; (Cl 4 mMputrescine + 0.1 mM DAB; (D) 4 mMputrescine + 0.1 mM 3-amino-9-ethylcarbazole; (E) 4 mMputrestine + 0.1 mM orthodianiside; (F) 4 mM putrescine.
298
HOUEN TABLE
AND
1
Effectiveness of Hydrogen Donors in the PeroxidaseCoupled Spot Test for Diamine Oxidase 3-Amino-gethylcarbazole
0-Dianisidine DA0 +PUT +PUT +AG -PUT Alcohol DH +PUT +PUT +AG -PUT Aldehyde DH +PUT +PUT +AG -PUT
4-Cl-lnaphthol
-
+ -
+ -
+ + +
-
-
-
+ + +
-
+ + -
-
+ + +
mal, allowing detection of as little as 0.1 units of enzyme (1 PcLg,- 10 pmol). The specificity of the assay is further illustrated in Fig. 4. Pig kidney diamine oxidase, which has a high affinity for diamines (ll), produces a spot with putrescine as substrate, but not with spermidine or spermine. Conversely, bovine plasma amine oxidase, which has a high affinity for spermine and spermidine (12) but not for putrescine, produces a spot only with
IJg
DA0
20
-
10
-
-
Ald.dh
-
ESA
-
DAO
-
ESAO
-
DAB
Note. The enzymes were spotted on nitrocellulose and incubated in PB with 0.1 mM of the various hydrogen donors and the indicated combinations of putres’cine (10 mM) and aminoguanidine (1 mM). + indicates a clearly visible spot and - indicates that no spot was visible; 1 unit of DA0 is defined as the amount of enzyme that oxidizes 1 pmol of putrescine per hour at pH 7.2 and 37°C.
Enz.
LEONARDSEN
AECOEFB
A
S
C
D
E
FIG. 4. Substrate specificity of the 4-Cl-l-naphthol-based activity stain for amine oxidases. The enzymes indicated were spotted on nitrocellulose and incubated in PB with HRP (10 pg/ml) 4-Cl-lnaphthol(O.1 mM), and the following substrates: (A) no substrate; (B) putrescine (4 mM); (C) 4 mM spermine; (D) 4 mM spermidine; (E) putrescine (4 mM) + aminoguanidine (0.2 mM).
spermine and spermidine as substrates. When DA0 was subjected to electrophoresis in nondenaturing gels and visualized with the activity stain, DA0 was revealed as a high-molecular-weight complex (Fig. 5) that hardly entered the gel and that Triton X-100, NP-40, and Chaps did not dissociate, but that could be dissociated by Tween 20. DISCUSSION Previous studies have shown that assays for amine oxidase using tetrazolium salts are nonspecific (13) and have serious limitations. An assay for benzylamine oxidase in gels based on the production of benzaldehyde and scanning of the gel at 250 nm has been described (14). Coupled peroxidase reactions have been used for the histochemical localization of monoamine oxidase, benzylamine oxidase (15-17), and p-IVJV-dimethylamine-P-phenylethylamine oxidase (18) and for the localization of various oxidases in gels (19), but no results have previously been presented for diamine oxidase (20). From the results presented here it is clear that great
2l.2
Alc.dh
IO-
Ald.dh
IO-
-
0.1 4-Cl-l
0.2
0.6 -Naphtole
0-E
1-O cone.
1.2
1.2 CmM1
FIG. 3. (A) Effect of varying the concentration of 4-Cl-1-naphthol on the sensitivity of the peroxidase-coupled activity stain for diamine oxidase. The enzymes indicated were spotted on nitrocellulose and incubated in PB containing HRP (10 pg/ml), putrescine (10 mM), and 4-Cl-l-naphthol (concentration as indicated). Lane F also contained 1 mM
aminOgUanidh?.
AECDE
F
FIG. 5. Visualization of DA0 in polyacrylamide gels and effects of detergents. Twenty-five microliters of DA0 (10 mg/ml) in PB was mixed with 25 ~1 electrophoresis sample buffer with (2%) or without detergent: (A) boiled DA0 as control, (B) no detergent, (C) Triton X-100, (D) NP-40, (E) Tween-20, (F) Chaps.
PEROXIDASE-COUPLED
ACTIVITY
care and many precautions must be taken in the study of amine oxidases. Some general guidelines can be made, however: (1) All experiments should be done in phosphate or other nonamine buffers, especially in the case of peroxidase-catalyzed dye reactions. (2) The hydrogen donor should not be an amine, or, for the visualization of DAO, not a diamine. (3) The effects of inhibitors must be interpreted with great care. For the assay of DAO, 4-Cl-1-naphthol was found to be the most suitable staining reagent and for the demonstration of DA0 activity with this dye, omission of substrate or inhibition by aminoguanidine are suitable controls. Preliminary attempts to use the method described here for localization of diamine oxidase on sections from pig kidney or human placenta were not successful. Since model experiments showed that immobilized DA0 was still active after the histochemical fixation, the absence of staining on sections could result from the enzyme being present mainly in an inactive form, which is activated or released only in response to certain stimuli. This hypothesis is compatible with the observation that microinjection of amine oxidases causes growth arrest and has cytotoxic effects in growing cells (21,22). The activity stain described here can be used to localize DA0 in polyacrylamide gels and will be useful for studying the occurrence of DA0 in various tissue extracts and for studying the number of isoenzymes in these extracts. REFERENCES 1. Mondovi, B., and Agro, A. F. (1982) in Advances in Experimental Medicine and Biology (Bossa, F., Chiancone, E., Agro, A. F., and Strom, R., Eds.), Vol. 148, pp. 141-153, Plenum Press, New York. 2. Tipton, K. F. (1986) 3. Blaschko, H. (1952)
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