309
Clinica Chimica Acra, 186 (1989) 309-314 Elsevier
CCA 04617
Short Communication
An alkaline phosphatase reacting with both monoclonal antibodies to intestinal and placental isoenzymes Hiroyasu Imanishi ‘, Toshikwu Hada ‘, Koji Muratani Kazuyuki Hirano ’ and Kazuya Higashino ’
‘,
’ 3rd Department of Internal Medicine Hyogo College of Medicine, Nishinomrya, Hyogo and 2 Gifu Pharmaceutical University, Gifu (Japan) (Received
14 June 1989; revision received 9 September Key words: Novel alkaline
phosphatase;
1989; accepted
Gastric
11 September
1989)
carcinoma
Introduction (AP), There are at least four genes coding for human alkaline phosphatases germ-cell AP [l], and these APs are namely placental-, intestinal-, liver/bone-, or Among many established cell lines known to consist of homodimeric structure. reported, both placental and intestinal APs are rarely expressed in one cancer cell line simultaneously. Only in Hep2/5 cells (HeLa cell line) [2], FL amnion cells [3] and KB cells [4], a hybrid form of AP consisting of one subunit of placental AP and the other subunit of intestinal AP has been demonstrated. But no one has reported any hybrid form of AP expressed in any tissues of patients with malignant diseases. In the present study, we report a novel AP reacting with both monoclonal antibodies to intestinal and placental APs, suggesting a hybrid form of AP consisting of one subunit of placental AP and the other subunit of intestinal AP, found in metastatic lesion of the liver of a patient with gastric cancer. Materials and methods
Samples Liver with metastatic carcinoma was obtained from a patient with gastric carcinoma. APs from normal intestine, term placenta and liver were prepared as described previously [5]. AP from germ-cell tumor, which was obtained at operation, was prepared as described previously [5].
Correspondence to: Toshikazu Hada, 3rd Department 1-1, Mukogawa-cho, Nishinomiya Hyogo 663, Japan.
0009-8981/89/$03.50
of Internal
0 1989 Elsevier Science Publishers
Medicine
B.V. (Biomedical
Hyogo
Division)
College of Medicine,
310
Assay of alkaline phosphatase AP activity was determined using phenylphosphate as a substrate by the method of Higashino et al. [5]. One unit of the enzyme activity was defined as the amount of enzyme causing the release of 1 pm01 phenol/min. Protein was determined by the method of Lowry et al. [6]. Electrophoresis 5% polyacrylamide gel electrophoresis (PAGE) was carried out by the method of Higashino et al. [5]. Polyacrylamide gradient (4-308) gel electrophoresis was performed according to the method of Higashino et al. [7]. After electrophoresis, the bands were made visible by staining the enzyme activity with 1-naphthylphosphate and Fast blue BB salt as described previously [5]. Purification of a novel alkaline phosphatase from carcinoma tissue AP was extracted from the metastatic lesion of our patient’s liver by the method reported previously [S]. The n-butanol extract from the tissue was treated with acetone. The fraction precipitating between 30 and 60% acetone was dialyzed against 50 mmol/l Tris-HCl buffer, pH 8.0, ~nt~g 0.15 mol/l NaCI and applied onto a Sepharose 4B column coupled with monoclonal antibody to intestinal AP. The enzyme was eluted with 0.2 mol/l Na2C03 containing 0.5 mol/l NaCl. The eluted enzyme was dialyzed, applied onto a Sepharose 4B column coupled with anti-placental AP monoclonal antibody and eluted by the same procedure. Thus, purified AP (named IP-AP) was dialyzed with 20 mmol/l Tris-HCl buffer, pH 7.4, containing 20 pmol/l M&J, and &Cl, and used in the following experiment. Immunological analysis Enzyme-antigen immunoassay was performed using two kinds of monoclonal antibodies (HPMS-1, ZHIMS-1) each of which is specific for human placental or intestinal AP isoenzyme f8]. Briefly, each well of a 96-well plate was incubated with 100 ~1 of goat anti-mouse IgG antibody (20 pg/ml). After the plate had been washed and blocked with 1% bovine serum albumin, 100 ~1 of the monoclonal antibody solution (5 pg/ml) was added to each well, followed by incubation. After the plate had been washed, 100 ~1 of AP (10 mu) was added and incubated at 4°C for 12 h. The plate was washed, and then 200 ~1 of substrate solution (2.7 mmol/l p-nitrophenyl phosphate) was added to each well, followed by incubation at 37°C for 30 min. The absorbance of the resulting solution in each well was measured at 405 nm.
~~i~catio~ of IP-AP 4.2% of total AP activity of metastatic lesion of the patient’s liver bound to ~ti-~t~t~ AP monoclonal antibody column and all the eluted AP from the columr~ was bound to anti-placental AP monoclonal antibody column. The specific activity of the AP was 0.21 U/pg protein. Electrophoretic mobility As shown in Fig. 1, enzyme staining for IP-AP revealed a broad band on 5% PAGE. Neur~d~ treatment of this enzyme decreased its
311
Fig. 1. Electrophoretogram of alkaline phosphatases on 5% polyacrylamide gel. A and B, alkaline phosphatase before and after neuraminidase treatment, respectively; 1, liver AP; 2, intestinal AP; 3, placental AP; 4, partially purified IP-AP.
c
Fig. 2. Electrophoretogram of alkaline phosphatases on polyacrylarnide gradient (4-3046) gel. 1, liver AP; 2, intestinal AP, 3, placental AP; 4 and 5, acetone fraction of AP in the patient’s liver containing cancer tissue before and after heat treatment 65 o C for 5 min, respectively. (4, The faster migrating one is IP-AP, and the slower migrating one is liver AP, 5, x 5 cont.)
312 TABLE
I
Effect of various Inhibitor
inhibitors
’
and heat treatment
Final wnc.
on alkaline
(I of original Intestinal AP
phosphatase
activity
d
activity) Placental AP
Germ-cell AP
IP-AP
L-Phenylahmine
5 mmol/l 10 mmol/l
41.0 31.4
26.2 14.9
13.1 8.9
12.2 7.6
L-Homoarginine
5 mmol/l 10 mmol/l
69.9 61.3
94.8 86.0
71.2 61.2
77.5 68.1
L-Leucine
5 mmol/l 10 mmol/l
46.1 32.9
64.2 42.1
14.8 9.1
19.8 10.2
K,HPO,
5 mmol/l 10 mmol/l
92.5 82.7
37.8 23.6
51.2 35.1
74.9 60.8
102.5 98.8
2.3 0.9
2.1 0.6
38.4 15.6
43.8
85.3
5.05
43.8
SDS
0.5% 5.0%
EDTA
0.1 mmol/l
Heat treatment
65 o C
for 5 min 10 min 15 min
14.6 10.2 3.9
100.2 99.5 98.0
99.9 100.2 105.6
70.2 48.5 35.6
’ The values represent the average of two determinations, ’ Each inhibitor was incorporated in the standard assay mixture so as to give the final concentration indicated. The effect of EDTA on the enzyme activity was studied as described previously [5]. The effect of SDS on the enzyme activity was studied as described previously [6]. Heat stability was examined by incubating the enzyme at 65°C in 20 mmol/l Tris-HCI, pH 7.4, containing 5 mmol/l MgCl, and 5 mmol/l ZnCl, for the time indicated.
electrophoretic mobility, but the position after neuraminidase treatment was different from that of placental, intestinal or liver AP isoenzyme. On polyacrylamide gradient (4-30%) gel electrophoresis, AP extracted from patient’s liver containing me&static lesion contained two bands with AP activity, the faster migrating one in the position between placental and intestinal AP isoenzymes represented IP-AP (Fig. 2). After heat treatment at 65 “C for 5 min, liver AP isoenzyme disappeared but IP-AP retained its enzyme activity (Fig. 2).
Heat stability Activities of IP-AP obtained from our patient’s after incubation at 65°C are shown in Table I. This AP was more heat-labile than placental and germ-cell heat-stable than intestinal AP. IP-AP Effect of amino acids on enzyme activity L-phenylalanine, but resistant to L-homoarginine.
liver remaining APs,
but
more
was sensitive to L-leucine and However, the degree of the
313 TABLE
II
Immunological
phosphatases Monoclonal
Alkaline phosphatase Intestinal Placental Germ-cell IP-AP
study of alkaline
anti-intestinal a
using monoclonal
antibodies
antibodies AP
1.61 b 0 0 0.98
a Enzyme activity of each specimen used was 10 mu. b The value means the actual absorbance at 405 mn of p-nitrophenol two determinations).
anti-placental
AP
0 1.30 1.24 1.28
produced
in each well (average
of
sensitivity of IP-AP to these amino acids was not always compatible with that of placental or intestinal AP, but it resembled germ-cell AP rather than intestinal or placental AP. Effect of inorganic phosphate, sodium dodecyl sulfate (SDS) and EDTA The effect of EDTA on activity of IP-AP were similar to that of intestinal AP isoenzyme, whereas IP-AP showed intermediate properties between those of placental AP (or germ-cell AP) and intestinal AP isoenzymes against inorganic phosphate and sodium dodecyl sulfate (SDS). Immunological analysis Specificities of monoclonal antibodies are shown in Table II. The results of enzyme antigen immunoassay showed that purified IP-AP from metastatic lesion of the patient’s liver reacted with both anti-placental and intestinal AP monoclonal antibodies and had nearly the same affinity for both antibodies. Discussion
An abnormal AP (IP-AP) which was found in metastatic lesion of the liver obtained from a gastric cancer patient had intermediate properties between those of intestinal and placental APs in thermostability, electrophoretic mobility on polyacrylamide gradient gel and sensitivities to some inhibitors such as L-homoarginine, inorganic phosphate and SDS. In addition, this enzyme reacted with both monoclonal antibodies against intestinal and placental APs equally. These data may indicate that IP-AP is a hybrid form of AP, consisting of one subunit of placental AP and the other subunit of intestinal AP. But other enzymic properties of this enzyme were not always consistent with intermediate properties between those of intestinal and placental APs. On the other hand, there is another possibility that IP-AP consists of intestinal AP and germ-cell AP subunits because anti-placental AP monoclonal antibody also reacted with germ-cell AP. But IP-AP did not show intermediate enzymic properties between intestinal and germ-cell APs. These data
314
suggest that the heterodimeric structure of our enzyme may modify the enzyrnic properties of each subunit of intestinal or placental AP (or germ-cell AP). Further investigation will be needed to clarify the subunit structure of our enzyme and the effect of heterodimeric structure of our enzyme on enzymic properties. References 1 Milhm JL, Manes T. Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Nat1 Acad Sci USA 1988;85:3024-3028. 2 Wray L, Harris H. Demonstration using monoclonal antibodies of inter-locus heteromeric isoenzymes of human alkaline phosphatase. J Immunol Meth 1982;55:13-18. 3 Behrens CM, Emrs CA, Sussman HH. Characterization of human foetal intestinal alkaline phosphatase. Biochem J 1983;211:553-558. 4 Luduena MA, Sussman HH. Characterization of KB cell alkaline phosphatase. J Biol Chem 1976;251:2620-2628. 5 Higashino K, Hashinotsume M, Kang K-Y, Takahashi Y, Yamamura Y. Studies on a variant alkaline phosphatase in sera of patients with hepatocellular carcinoma. Clin Chim Acta 1972;40:67-81. 6 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 7 Higashino K, Otani R, Kudo S, Yamamura Y. A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue. Clin Chem 1977;23:1615-1623. 8 Hirano K, Matsumoto H, Tanaka T, et al. Specific assay for human alkaline phosphatase isozymes. Clin Chim Acta 1987;166:265-273.
Clinica Chimica Acra, 186 (1989) 309-314 Elsevier
CCA 04617
Short Communication
An alkaline phosphatase reacting with both monoclonal antibodies to intestinal and placental isoenzymes Hiroyasu Imanishi ‘, Toshikwu Hada ‘, Koji Muratani Kazuyuki Hirano ’ and Kazuya Higashino ’
‘,
’ 3rd Department of Internal Medicine Hyogo College of Medicine, Nishinomrya, Hyogo and 2 Gifu Pharmaceutical University, Gifu (Japan) (Received
14 June 1989; revision received 9 September Key words: Novel alkaline
phosphatase;
1989; accepted
Gastric
11 September
1989)
carcinoma
Introduction (AP), There are at least four genes coding for human alkaline phosphatases germ-cell AP [l], and these APs are namely placental-, intestinal-, liver/bone-, or Among many established cell lines known to consist of homodimeric structure. reported, both placental and intestinal APs are rarely expressed in one cancer cell line simultaneously. Only in Hep2/5 cells (HeLa cell line) [2], FL amnion cells [3] and KB cells [4], a hybrid form of AP consisting of one subunit of placental AP and the other subunit of intestinal AP has been demonstrated. But no one has reported any hybrid form of AP expressed in any tissues of patients with malignant diseases. In the present study, we report a novel AP reacting with both monoclonal antibodies to intestinal and placental APs, suggesting a hybrid form of AP consisting of one subunit of placental AP and the other subunit of intestinal AP, found in metastatic lesion of the liver of a patient with gastric cancer. Materials and methods
Samples Liver with metastatic carcinoma was obtained from a patient with gastric carcinoma. APs from normal intestine, term placenta and liver were prepared as described previously [5]. AP from germ-cell tumor, which was obtained at operation, was prepared as described previously [5].
Correspondence to: Toshikazu Hada, 3rd Department 1-1, Mukogawa-cho, Nishinomiya Hyogo 663, Japan.
0009-8981/89/$03.50
of Internal
0 1989 Elsevier Science Publishers
Medicine
B.V. (Biomedical
Hyogo
Division)
College of Medicine,
310
Assay of alkaline phosphatase AP activity was determined using phenylphosphate as a substrate by the method of Higashino et al. [5]. One unit of the enzyme activity was defined as the amount of enzyme causing the release of 1 pm01 phenol/min. Protein was determined by the method of Lowry et al. [6]. Electrophoresis 5% polyacrylamide gel electrophoresis (PAGE) was carried out by the method of Higashino et al. [5]. Polyacrylamide gradient (4-308) gel electrophoresis was performed according to the method of Higashino et al. [7]. After electrophoresis, the bands were made visible by staining the enzyme activity with 1-naphthylphosphate and Fast blue BB salt as described previously [5]. Purification of a novel alkaline phosphatase from carcinoma tissue AP was extracted from the metastatic lesion of our patient’s liver by the method reported previously [S]. The n-butanol extract from the tissue was treated with acetone. The fraction precipitating between 30 and 60% acetone was dialyzed against 50 mmol/l Tris-HCl buffer, pH 8.0, ~nt~g 0.15 mol/l NaCI and applied onto a Sepharose 4B column coupled with monoclonal antibody to intestinal AP. The enzyme was eluted with 0.2 mol/l Na2C03 containing 0.5 mol/l NaCl. The eluted enzyme was dialyzed, applied onto a Sepharose 4B column coupled with anti-placental AP monoclonal antibody and eluted by the same procedure. Thus, purified AP (named IP-AP) was dialyzed with 20 mmol/l Tris-HCl buffer, pH 7.4, containing 20 pmol/l M&J, and &Cl, and used in the following experiment. Immunological analysis Enzyme-antigen immunoassay was performed using two kinds of monoclonal antibodies (HPMS-1, ZHIMS-1) each of which is specific for human placental or intestinal AP isoenzyme f8]. Briefly, each well of a 96-well plate was incubated with 100 ~1 of goat anti-mouse IgG antibody (20 pg/ml). After the plate had been washed and blocked with 1% bovine serum albumin, 100 ~1 of the monoclonal antibody solution (5 pg/ml) was added to each well, followed by incubation. After the plate had been washed, 100 ~1 of AP (10 mu) was added and incubated at 4°C for 12 h. The plate was washed, and then 200 ~1 of substrate solution (2.7 mmol/l p-nitrophenyl phosphate) was added to each well, followed by incubation at 37°C for 30 min. The absorbance of the resulting solution in each well was measured at 405 nm.
~~i~catio~ of IP-AP 4.2% of total AP activity of metastatic lesion of the patient’s liver bound to ~ti-~t~t~ AP monoclonal antibody column and all the eluted AP from the columr~ was bound to anti-placental AP monoclonal antibody column. The specific activity of the AP was 0.21 U/pg protein. Electrophoretic mobility As shown in Fig. 1, enzyme staining for IP-AP revealed a broad band on 5% PAGE. Neur~d~ treatment of this enzyme decreased its
311
Fig. 1. Electrophoretogram of alkaline phosphatases on 5% polyacrylamide gel. A and B, alkaline phosphatase before and after neuraminidase treatment, respectively; 1, liver AP; 2, intestinal AP; 3, placental AP; 4, partially purified IP-AP.
c
Fig. 2. Electrophoretogram of alkaline phosphatases on polyacrylarnide gradient (4-3046) gel. 1, liver AP; 2, intestinal AP, 3, placental AP; 4 and 5, acetone fraction of AP in the patient’s liver containing cancer tissue before and after heat treatment 65 o C for 5 min, respectively. (4, The faster migrating one is IP-AP, and the slower migrating one is liver AP, 5, x 5 cont.)
312 TABLE
I
Effect of various Inhibitor
inhibitors
’
and heat treatment
Final wnc.
on alkaline
(I of original Intestinal AP
phosphatase
activity
d
activity) Placental AP
Germ-cell AP
IP-AP
L-Phenylahmine
5 mmol/l 10 mmol/l
41.0 31.4
26.2 14.9
13.1 8.9
12.2 7.6
L-Homoarginine
5 mmol/l 10 mmol/l
69.9 61.3
94.8 86.0
71.2 61.2
77.5 68.1
L-Leucine
5 mmol/l 10 mmol/l
46.1 32.9
64.2 42.1
14.8 9.1
19.8 10.2
K,HPO,
5 mmol/l 10 mmol/l
92.5 82.7
37.8 23.6
51.2 35.1
74.9 60.8
102.5 98.8
2.3 0.9
2.1 0.6
38.4 15.6
43.8
85.3
5.05
43.8
SDS
0.5% 5.0%
EDTA
0.1 mmol/l
Heat treatment
65 o C
for 5 min 10 min 15 min
14.6 10.2 3.9
100.2 99.5 98.0
99.9 100.2 105.6
70.2 48.5 35.6
’ The values represent the average of two determinations, ’ Each inhibitor was incorporated in the standard assay mixture so as to give the final concentration indicated. The effect of EDTA on the enzyme activity was studied as described previously [5]. The effect of SDS on the enzyme activity was studied as described previously [6]. Heat stability was examined by incubating the enzyme at 65°C in 20 mmol/l Tris-HCI, pH 7.4, containing 5 mmol/l MgCl, and 5 mmol/l ZnCl, for the time indicated.
electrophoretic mobility, but the position after neuraminidase treatment was different from that of placental, intestinal or liver AP isoenzyme. On polyacrylamide gradient (4-30%) gel electrophoresis, AP extracted from patient’s liver containing me&static lesion contained two bands with AP activity, the faster migrating one in the position between placental and intestinal AP isoenzymes represented IP-AP (Fig. 2). After heat treatment at 65 “C for 5 min, liver AP isoenzyme disappeared but IP-AP retained its enzyme activity (Fig. 2).
Heat stability Activities of IP-AP obtained from our patient’s after incubation at 65°C are shown in Table I. This AP was more heat-labile than placental and germ-cell heat-stable than intestinal AP. IP-AP Effect of amino acids on enzyme activity L-phenylalanine, but resistant to L-homoarginine.
liver remaining APs,
but
more
was sensitive to L-leucine and However, the degree of the
313 TABLE
II
Immunological
phosphatases Monoclonal
Alkaline phosphatase Intestinal Placental Germ-cell IP-AP
study of alkaline
anti-intestinal a
using monoclonal
antibodies
antibodies AP
1.61 b 0 0 0.98
a Enzyme activity of each specimen used was 10 mu. b The value means the actual absorbance at 405 mn of p-nitrophenol two determinations).
anti-placental
AP
0 1.30 1.24 1.28
produced
in each well (average
of
sensitivity of IP-AP to these amino acids was not always compatible with that of placental or intestinal AP, but it resembled germ-cell AP rather than intestinal or placental AP. Effect of inorganic phosphate, sodium dodecyl sulfate (SDS) and EDTA The effect of EDTA on activity of IP-AP were similar to that of intestinal AP isoenzyme, whereas IP-AP showed intermediate properties between those of placental AP (or germ-cell AP) and intestinal AP isoenzymes against inorganic phosphate and sodium dodecyl sulfate (SDS). Immunological analysis Specificities of monoclonal antibodies are shown in Table II. The results of enzyme antigen immunoassay showed that purified IP-AP from metastatic lesion of the patient’s liver reacted with both anti-placental and intestinal AP monoclonal antibodies and had nearly the same affinity for both antibodies. Discussion
An abnormal AP (IP-AP) which was found in metastatic lesion of the liver obtained from a gastric cancer patient had intermediate properties between those of intestinal and placental APs in thermostability, electrophoretic mobility on polyacrylamide gradient gel and sensitivities to some inhibitors such as L-homoarginine, inorganic phosphate and SDS. In addition, this enzyme reacted with both monoclonal antibodies against intestinal and placental APs equally. These data may indicate that IP-AP is a hybrid form of AP, consisting of one subunit of placental AP and the other subunit of intestinal AP. But other enzymic properties of this enzyme were not always consistent with intermediate properties between those of intestinal and placental APs. On the other hand, there is another possibility that IP-AP consists of intestinal AP and germ-cell AP subunits because anti-placental AP monoclonal antibody also reacted with germ-cell AP. But IP-AP did not show intermediate enzymic properties between intestinal and germ-cell APs. These data
314
suggest that the heterodimeric structure of our enzyme may modify the enzyrnic properties of each subunit of intestinal or placental AP (or germ-cell AP). Further investigation will be needed to clarify the subunit structure of our enzyme and the effect of heterodimeric structure of our enzyme on enzymic properties. References 1 Milhm JL, Manes T. Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Nat1 Acad Sci USA 1988;85:3024-3028. 2 Wray L, Harris H. Demonstration using monoclonal antibodies of inter-locus heteromeric isoenzymes of human alkaline phosphatase. J Immunol Meth 1982;55:13-18. 3 Behrens CM, Emrs CA, Sussman HH. Characterization of human foetal intestinal alkaline phosphatase. Biochem J 1983;211:553-558. 4 Luduena MA, Sussman HH. Characterization of KB cell alkaline phosphatase. J Biol Chem 1976;251:2620-2628. 5 Higashino K, Hashinotsume M, Kang K-Y, Takahashi Y, Yamamura Y. Studies on a variant alkaline phosphatase in sera of patients with hepatocellular carcinoma. Clin Chim Acta 1972;40:67-81. 6 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 7 Higashino K, Otani R, Kudo S, Yamamura Y. A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue. Clin Chem 1977;23:1615-1623. 8 Hirano K, Matsumoto H, Tanaka T, et al. Specific assay for human alkaline phosphatase isozymes. Clin Chim Acta 1987;166:265-273.
