© 1991 Karger AG. Basel 0042-1138/91/0477-0096 $ 2.75/0
Urol Int 1991:47(suppl l):96-99
Characterization of Seminoma-Derived Placental-Like Alkaline Phosphatase K. Koshida, T. Uchibayashi, H. Hisazumi Department of Urology. Kanazawa University. Japan
Key Words. Placental-like alkaline phosphatase • Seminoma • Heterogeneity Abstract. Characteristics of placental-like alkaline phosphatase (PLAP-like enzyme) in seminoma was studied. By use of lectin affinity chromatography, PLAP-like enzyme in seminoma revealed extra sugar chains compared to placen tal alkaline phosphatase (PLAP), indicating heterogeneity of the carbohydrate moiety. However, the glycosylation patterns were found to be essentially similar between seminoma and normal testis. On isoelectric focusing, differences in migration patterns were revealed between seminoma-derived and normal testis-derived PLAP-like enzyme as well as between PLAP-like enzyme and PLAP. The differences in charge were mainly due to differences in sialylation of the molecules. The complex pattern of PLAP-like enzyme from seminoma on isoelectric focusing was not altered by neur aminidase treatment, indicating a considerable charge heterogeneity within the population of the enzyme molecules from the tumor.
Alkaline phosphatase (ALP) consists genetically of three different isoenzymes, namely tissue unspecific or liver ALP, intestinal ALP and placental alkaline phospha tase (PLAP). In addition, tumor-related PLAP activities in sera of cancer patients have been reported as Regan isoenzyme [1] and Nagao isoenzyme [2], The latter is the so-called PLAP-like enzyme since its sensitivity to leucine is different from PLAP. ALP activity in seminoma tissue was shown to be approximately 70 times as high as that of normal testis [3]. The proportions of these three isoen zymes were more than 90% for the unspecific or liver type, 8% for the placental type and less than 1% for the intestinal type. Increase of all three isoenzymes in semi noma tissues suggests that the entire genome coding for ALP appeared to be activated in association with malig nant transformation.
Materials and Methods Tissue Extracts Tissues, stored at -2 0 ° C , were thawed, homogenized with physio logical saline and extracted with n-butanol (1:1) by stirring for 2 h at
room temperature. The homogenate was centrifuged at 10,000 g for 20 min at 4°C. The aqueous layer was collected and dialyz.ed against 10 mM Tris buffer (pi I 7.0) containing 0.5 mAf MgCL. High-Performance Liquid Chromatography After filtration (0.45-um pore size, Millipore, Bedford, Mass.), the extracts were analyzed on a TSK-gcl phenyl-5PW column (Waters, Milford. Mass.) with high-performance liquid chromatography (HPLC) (Waters. Milford. Mass.). The separation condition included a 30-min linear gradient from 1 toO Af of ammonium sulphate in water containing 0.5 mAf MgCL. The fractions collected every 2 min with a flow rate of 1.0 ml/min were dialyzed against 10 m M Tris buffer (pH 7.0) with 0.5 mA/ MgCL, and analyzed by both the catalytic assay and enzyme-linked immunosorbent assay (ELISA) (see below). Lectin Affinity Chromatography The lectin binding properties of PLAP-like enzyme and PLAP were investigated using concanavalin A (Con A)-Sepharose 4B and wheat germ lectin (WGL)-Sepharosc 6MB (Pharmacia, Uppsala. Sweden). The gels were equilibrated with 10 mAf Tris buffer (pH 7.0) containing 0.15 M NaCl and 0.5 mAf MgCL. The heat-stable ALP were applied on small columns containing 1.0 ml Con A- or WGL-Sepharosc. The sam ples were incubated in the column at room temperature overnight, and washed with 12 column vol of the Tris buffer. Elution was performed with two different concentrations of a-methyl-D-mannoside (Sigma, St. Louis, Mo.) (0.01 M followed by 0.2 M) for Con A-Scpharosc and 0.05 M N-acetyl-D-glucosamine (Sigma, St. Louis, Mo.) for WGLSepharose. Fractions of 1.5 ml were collected and the catalytic and immunochemical activities were assayed.
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Introduction
Placental-Like Alkaline Phosphatase of Seminoma
97
Assays fo r Catalytic Activity The catalytic activity in each fraction was measured by a system consisting of 1 M diethanolamine buffer (pH 9.8) containing 0.5 mM MgCLand 8 m M p-nitrophenylphosphate (Sigma, St. Louis, Mo.). 100 pi of each fraction were applied in microtiter plate wells (Nunc, Roskilde, Denmark) and 100 plot'the substrate solution were added to each well. After 30-60 min incubation (depending on activities of samples) at 37°C, the absorbance at 410 nm was measured. Enzyme-Linked Immunosorbent Assay fo r FLAP To determine immunochemical activity of PLAP and PLAP-likc enzyme, an ELISA consisting of protein A-purified rabbit anti-PLAP IgG and horseradish peroxidase-conjugated mouse monoclonal anti body (M Ab) H7 was used [4], Absorbance at 490 nm was recorded in an automated microplate reader. Neuraminidase Digestion One volume of0.2 M acetate buffer pH 6.0, containing 2.3—4.6 units neuraminidase per milliliter (type VI. Sigma. St. Louis, Mo.), was added to 1 vol of ALP solution. The mixture was incubated for 18-72 h at 37 °C. Starch Gel Electrophoresis Butanol extracts of seminoma and normal testis were applied on the gel (pH 5.6) after heat inactivation at 65°C for 10 min. Following elec trophoresis, the enzymatic activity was evidenced with a solution con sisting of 0.25 M Tris/maleicacid pH 9.8.0.8 mAi a-naphthyl phospha tase, 1 mM 4-aminodiphenylamine diazonium sulfate and 5 mM
MgSOj. Isoelectric Focusing Isoelectric focusing (IFF) was performed by an LKB Multiphor apparatus in thin layer polyacrylamide gels (Ampholine PAG plates, LKB. Stockholm. Sweden). pH range 4.0-6.5. The samples were applied by 5 x 10 mm filter papers placed to the cathode. Focusing was performed at 10°C, and with a constant power of 25 W with 0.1 M gluta mic acid in 0.5 M 11^POj as the anodic and 0.1 M b-alanine as the cathodicsolution. After focusing for 2.5 h, the gel was submerged in the same solution as used for starch gel for 3 h at 37°C. After rinsing the gel with destainingsolution (25% v/v ethanol and 16% v/v acetic acid in water), the IE F pattern was photographed.
F ra ctio n No.
Fig. 1. HPLC separation of ALP activity in seminoma. □ = Total enzyme activity; ■ = heat-stable aetivity.
By means of HPLC, we were able to separate the two major isoenzymes of the liver and placental type in semi noma tissue as shown in figure 1. Comparing the separa tion pattern with those of other organs, the first area of the seminoma enzyme, being heat-sensitive, was likely to be the liver type. Enzyme in the second and third areas appeared to be the placental type, since they were heatstable and also reactive with an anti-PLAP MAb (H7). Following the separation of the isoenzymes, we com pared glycosylation patterns of the PLAP activity in semi noma with those in normal testis and in other organs by use of lectin affinity chromatography. A prominent find
ing was that appearance of a Con A unbound fraction (peak I) of placental activity (heat-stable and reactive with H7 MAb) in seminoma while all activities of PLAP from placenta were bound to Con A. The proportion of the unbound fraction of seminoma PLAP varied from 11 to 48% among 9 individuals. Accordingly, proportions of Con A binding fractions (II: weakly bound, III: strongly bound) were variable. Furthermore, an unbound fraction of the enzyme activity for WGL was also disclosed in seminoma but not in placenta (table 1). Thereby, a serial use of these two lectin chromatographies allowed us to differentiate between the following four types of sugar
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Results and Discussion
98
Koshida/Uchibayashi/Hisazumi
Table 1. Affinity of PLAP-likc and PLAP for Con A and WGLSepahrose Tissue
Seminoma Testis Placenta
Lectin
Con A WGL Con A WGL Con A WGL
Catalytic activity, %
(9) (3) (3) (3) (2) (2)
unbound
bound
peak I
peak II
peak III
11-48 52-72 31-42 31-53 0 0
48-84 28-48 49-53 47-69 72-75 100
0-10 5-18 25-28
Figures in parentheses indicate number of samples investigated.
Fig. 2. IEF of heat-stable ALP in seminoma and normal testis (pH range 4.0-6.5). The gel was stained for enzyme activity. Lancs 1-3: nor mal testes; lanes 4-7: seminomas; lane 8: PLAP from placenta.
Table 2- Separation of PLAP-like enzyme by serial lectin chroma tography Tissue
Seminoma 1 2 Testis Placenta
Catalytic activity, %
H7 reactivity, %
I
II
III
IV
I
19 19 17 0
53 43 36 0
7 11 18 0
21 18 27 20 29 18 100 0
II
III
IV
52 41 37 0
7 6 9 0
23 33 36 100
Fig. 3. IEF of heat-stable ALP in seminoma and normal testis (pH range 4.0-6.5). The gel was stained for enzyme activity. Lane 1: semi noma; lane 2; seminoma after digestion with neuraminidase (4.6 U/ml, 72 h at 37 °C); lanes 3 and 4: normal testes after digestion (2.3 U /m l, 18 h at 37 °C).
chains: (1) Con A- and WGL-unbound component, (2) Con A-bound and WGL-unbound component, (3) Con A-unbound and WGL-bound component, and (4) Con Aand WGL-bound component. The last one corresponded to PLAP from placenta. All four types of sugar chains could be demonstrated in seminoma. Thus, heterogeneity of the carbohydrate moiety of seminoma PLAP was revealed. Discrimination of seminoma PLAP from PLAP of placenta was therefore possible in the glycosylation pattern. The modification of the carbohydrate moiety, however, appeared not to be related to the malignant transformation, since normal testis was shown to have essentially the same types of carbohydrate moiety (table 2). Polymorphism of PLAP has been analyzed on starch gel electrophoresis, by which six common phenotypes and
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Type I = Fraction of Con A and WGL unbound; type II = Con A bound, WGL unbound; type III = Con A unbound, WGL bound; type IV = Con a and WGL bound.
Placental-Like Alkaline Phosphatase of Seminoma
References 1 Fishman W II, Inglis NR. Stolbach LL. Krant MJ: A serum alkaline phosphatase isoenzyme of human neoplastic cell origin. Cancer Res 1968;28:150-154. 2 Nakayama T. Yoshida M, Kitamura M: L-Leucine sensitive, heatstable alkaline phosphatase isoenzyme detected in a patient with pleuritis carcinomatosa. Clin Chim Acta 1970;30:546-548. 3 Ilirano K. Matsumoto H, Tanaka T. Hayashi Y, lino S. Domar U. StigbrandT: Specific assay for human alkaline phosphatase isoen zymes. Clin Chim Acta 1987;166:265-273. 4 Wahren B, Hinkula J, Stigbrand T, Jeppsson A , Andersson L, Esposti PL, Edsmyr F. Millan JL: Phenotypes o f placental-type alkaline phosphatase in seminoma sera as defined by monoclonal antibodies. Int J Cancer 1986;37:595-600. 5 Beckman L. Björling G, Christodoulou C: Pregnancy enzymes and placental polymorphism. I. Alkaline phosphatase. Acta Genet 1966;16:59-73. 6 Koshida K, Stigbrand T, Hisazumi H , Wahren B: Electrophoretic heterogeneity of alkaline phosphatase isozymes in seminomas and normal testis. Tumor Biol 1989;10:181-189. 7 Jeppsson A. Wahren B, Brehmer-Andersson E. Silfversward C. Stigbrand T, Millan JL: Eutopic expression of placental-like alka line phosphatase in testicular tumors. Int J Cancer 1984;34: 757-761. 8 Millan JL. Stigbrand T : Antigenic determinants of human placental and testicular placental-like alkaline phosphatases as mapped by monoclonal antibodies. Eur J Biochem 1983;136:1-7.
K. Koshida Department of Urology Kanazawa University 13-1, Takara-machi Kanazawa 920 (Japan)
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several rare variants were classified [5]. We therefore investigated migration patterns of PLAP activity from seminoma and normal testis on starch gel, resulting in dis closing marked heterogeneous migration patterns among individuals [data not shown]. IEF was applied on analysis of the isoenzymes, by which discrimination of PLAP activity between semi noma and normal testis appeared to be possible on the basis of isoelectric points as shown in figure 2. Although isoelectric points of seminoma PLAP and PLAP of pla centa were found to be similar, these two were distin guishable by digestion with neuraminidase, indicating a difference in sialic acid content between these isoenzymes [data not shown]. Microheterogeneity on IEF remained after neuraminidase treatment in the case of seminoma but not in normal testis, implying that factors other than sialylation might contribute to the heterogeneity of semi noma PLAP (fig. 3). Analysis of fractions from lectin affinity chromatography or from HPLC separation sug gested that the structural differences of the carbohydrate moiety and the variation in hydrophobicity of the mole cule possibly contribute to the heterogeneity on IEF [6]. Although similarities between the seminoma- and normal testis-derived enzymes have been shown in immuno chemical and enzyme inhibition studies [7,8], the hetero geneity of the enzyme appears to be considerable in the tumor tissues. In summary, we have demonstrated heterogeneity of the seminoma-derived PLAP with respect to glycosylation and charge of the molecule, by which the enzyme could be discriminated from PLAP of placenta and there fore could be regarded as PLAP-like enzyme. Further more, similar characteristics of the enzyme between semi noma and normal testis imply that a marked elevation of PLAP-like activity in seminoma results from the enhanced eutopic expression of the enzyme normally expressed in the testis.
99
Urol Int 1991:47(suppl l):96-99
Characterization of Seminoma-Derived Placental-Like Alkaline Phosphatase K. Koshida, T. Uchibayashi, H. Hisazumi Department of Urology. Kanazawa University. Japan
Key Words. Placental-like alkaline phosphatase • Seminoma • Heterogeneity Abstract. Characteristics of placental-like alkaline phosphatase (PLAP-like enzyme) in seminoma was studied. By use of lectin affinity chromatography, PLAP-like enzyme in seminoma revealed extra sugar chains compared to placen tal alkaline phosphatase (PLAP), indicating heterogeneity of the carbohydrate moiety. However, the glycosylation patterns were found to be essentially similar between seminoma and normal testis. On isoelectric focusing, differences in migration patterns were revealed between seminoma-derived and normal testis-derived PLAP-like enzyme as well as between PLAP-like enzyme and PLAP. The differences in charge were mainly due to differences in sialylation of the molecules. The complex pattern of PLAP-like enzyme from seminoma on isoelectric focusing was not altered by neur aminidase treatment, indicating a considerable charge heterogeneity within the population of the enzyme molecules from the tumor.
Alkaline phosphatase (ALP) consists genetically of three different isoenzymes, namely tissue unspecific or liver ALP, intestinal ALP and placental alkaline phospha tase (PLAP). In addition, tumor-related PLAP activities in sera of cancer patients have been reported as Regan isoenzyme [1] and Nagao isoenzyme [2], The latter is the so-called PLAP-like enzyme since its sensitivity to leucine is different from PLAP. ALP activity in seminoma tissue was shown to be approximately 70 times as high as that of normal testis [3]. The proportions of these three isoen zymes were more than 90% for the unspecific or liver type, 8% for the placental type and less than 1% for the intestinal type. Increase of all three isoenzymes in semi noma tissues suggests that the entire genome coding for ALP appeared to be activated in association with malig nant transformation.
Materials and Methods Tissue Extracts Tissues, stored at -2 0 ° C , were thawed, homogenized with physio logical saline and extracted with n-butanol (1:1) by stirring for 2 h at
room temperature. The homogenate was centrifuged at 10,000 g for 20 min at 4°C. The aqueous layer was collected and dialyz.ed against 10 mM Tris buffer (pi I 7.0) containing 0.5 mAf MgCL. High-Performance Liquid Chromatography After filtration (0.45-um pore size, Millipore, Bedford, Mass.), the extracts were analyzed on a TSK-gcl phenyl-5PW column (Waters, Milford. Mass.) with high-performance liquid chromatography (HPLC) (Waters. Milford. Mass.). The separation condition included a 30-min linear gradient from 1 toO Af of ammonium sulphate in water containing 0.5 mAf MgCL. The fractions collected every 2 min with a flow rate of 1.0 ml/min were dialyzed against 10 m M Tris buffer (pH 7.0) with 0.5 mA/ MgCL, and analyzed by both the catalytic assay and enzyme-linked immunosorbent assay (ELISA) (see below). Lectin Affinity Chromatography The lectin binding properties of PLAP-like enzyme and PLAP were investigated using concanavalin A (Con A)-Sepharose 4B and wheat germ lectin (WGL)-Sepharosc 6MB (Pharmacia, Uppsala. Sweden). The gels were equilibrated with 10 mAf Tris buffer (pH 7.0) containing 0.15 M NaCl and 0.5 mAf MgCL. The heat-stable ALP were applied on small columns containing 1.0 ml Con A- or WGL-Sepharosc. The sam ples were incubated in the column at room temperature overnight, and washed with 12 column vol of the Tris buffer. Elution was performed with two different concentrations of a-methyl-D-mannoside (Sigma, St. Louis, Mo.) (0.01 M followed by 0.2 M) for Con A-Scpharosc and 0.05 M N-acetyl-D-glucosamine (Sigma, St. Louis, Mo.) for WGLSepharose. Fractions of 1.5 ml were collected and the catalytic and immunochemical activities were assayed.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 12:22:33 PM
Introduction
Placental-Like Alkaline Phosphatase of Seminoma
97
Assays fo r Catalytic Activity The catalytic activity in each fraction was measured by a system consisting of 1 M diethanolamine buffer (pH 9.8) containing 0.5 mM MgCLand 8 m M p-nitrophenylphosphate (Sigma, St. Louis, Mo.). 100 pi of each fraction were applied in microtiter plate wells (Nunc, Roskilde, Denmark) and 100 plot'the substrate solution were added to each well. After 30-60 min incubation (depending on activities of samples) at 37°C, the absorbance at 410 nm was measured. Enzyme-Linked Immunosorbent Assay fo r FLAP To determine immunochemical activity of PLAP and PLAP-likc enzyme, an ELISA consisting of protein A-purified rabbit anti-PLAP IgG and horseradish peroxidase-conjugated mouse monoclonal anti body (M Ab) H7 was used [4], Absorbance at 490 nm was recorded in an automated microplate reader. Neuraminidase Digestion One volume of0.2 M acetate buffer pH 6.0, containing 2.3—4.6 units neuraminidase per milliliter (type VI. Sigma. St. Louis, Mo.), was added to 1 vol of ALP solution. The mixture was incubated for 18-72 h at 37 °C. Starch Gel Electrophoresis Butanol extracts of seminoma and normal testis were applied on the gel (pH 5.6) after heat inactivation at 65°C for 10 min. Following elec trophoresis, the enzymatic activity was evidenced with a solution con sisting of 0.25 M Tris/maleicacid pH 9.8.0.8 mAi a-naphthyl phospha tase, 1 mM 4-aminodiphenylamine diazonium sulfate and 5 mM
MgSOj. Isoelectric Focusing Isoelectric focusing (IFF) was performed by an LKB Multiphor apparatus in thin layer polyacrylamide gels (Ampholine PAG plates, LKB. Stockholm. Sweden). pH range 4.0-6.5. The samples were applied by 5 x 10 mm filter papers placed to the cathode. Focusing was performed at 10°C, and with a constant power of 25 W with 0.1 M gluta mic acid in 0.5 M 11^POj as the anodic and 0.1 M b-alanine as the cathodicsolution. After focusing for 2.5 h, the gel was submerged in the same solution as used for starch gel for 3 h at 37°C. After rinsing the gel with destainingsolution (25% v/v ethanol and 16% v/v acetic acid in water), the IE F pattern was photographed.
F ra ctio n No.
Fig. 1. HPLC separation of ALP activity in seminoma. □ = Total enzyme activity; ■ = heat-stable aetivity.
By means of HPLC, we were able to separate the two major isoenzymes of the liver and placental type in semi noma tissue as shown in figure 1. Comparing the separa tion pattern with those of other organs, the first area of the seminoma enzyme, being heat-sensitive, was likely to be the liver type. Enzyme in the second and third areas appeared to be the placental type, since they were heatstable and also reactive with an anti-PLAP MAb (H7). Following the separation of the isoenzymes, we com pared glycosylation patterns of the PLAP activity in semi noma with those in normal testis and in other organs by use of lectin affinity chromatography. A prominent find
ing was that appearance of a Con A unbound fraction (peak I) of placental activity (heat-stable and reactive with H7 MAb) in seminoma while all activities of PLAP from placenta were bound to Con A. The proportion of the unbound fraction of seminoma PLAP varied from 11 to 48% among 9 individuals. Accordingly, proportions of Con A binding fractions (II: weakly bound, III: strongly bound) were variable. Furthermore, an unbound fraction of the enzyme activity for WGL was also disclosed in seminoma but not in placenta (table 1). Thereby, a serial use of these two lectin chromatographies allowed us to differentiate between the following four types of sugar
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 12:22:33 PM
Results and Discussion
98
Koshida/Uchibayashi/Hisazumi
Table 1. Affinity of PLAP-likc and PLAP for Con A and WGLSepahrose Tissue
Seminoma Testis Placenta
Lectin
Con A WGL Con A WGL Con A WGL
Catalytic activity, %
(9) (3) (3) (3) (2) (2)
unbound
bound
peak I
peak II
peak III
11-48 52-72 31-42 31-53 0 0
48-84 28-48 49-53 47-69 72-75 100
0-10 5-18 25-28
Figures in parentheses indicate number of samples investigated.
Fig. 2. IEF of heat-stable ALP in seminoma and normal testis (pH range 4.0-6.5). The gel was stained for enzyme activity. Lancs 1-3: nor mal testes; lanes 4-7: seminomas; lane 8: PLAP from placenta.
Table 2- Separation of PLAP-like enzyme by serial lectin chroma tography Tissue
Seminoma 1 2 Testis Placenta
Catalytic activity, %
H7 reactivity, %
I
II
III
IV
I
19 19 17 0
53 43 36 0
7 11 18 0
21 18 27 20 29 18 100 0
II
III
IV
52 41 37 0
7 6 9 0
23 33 36 100
Fig. 3. IEF of heat-stable ALP in seminoma and normal testis (pH range 4.0-6.5). The gel was stained for enzyme activity. Lane 1: semi noma; lane 2; seminoma after digestion with neuraminidase (4.6 U/ml, 72 h at 37 °C); lanes 3 and 4: normal testes after digestion (2.3 U /m l, 18 h at 37 °C).
chains: (1) Con A- and WGL-unbound component, (2) Con A-bound and WGL-unbound component, (3) Con A-unbound and WGL-bound component, and (4) Con Aand WGL-bound component. The last one corresponded to PLAP from placenta. All four types of sugar chains could be demonstrated in seminoma. Thus, heterogeneity of the carbohydrate moiety of seminoma PLAP was revealed. Discrimination of seminoma PLAP from PLAP of placenta was therefore possible in the glycosylation pattern. The modification of the carbohydrate moiety, however, appeared not to be related to the malignant transformation, since normal testis was shown to have essentially the same types of carbohydrate moiety (table 2). Polymorphism of PLAP has been analyzed on starch gel electrophoresis, by which six common phenotypes and
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 12:22:33 PM
Type I = Fraction of Con A and WGL unbound; type II = Con A bound, WGL unbound; type III = Con A unbound, WGL bound; type IV = Con a and WGL bound.
Placental-Like Alkaline Phosphatase of Seminoma
References 1 Fishman W II, Inglis NR. Stolbach LL. Krant MJ: A serum alkaline phosphatase isoenzyme of human neoplastic cell origin. Cancer Res 1968;28:150-154. 2 Nakayama T. Yoshida M, Kitamura M: L-Leucine sensitive, heatstable alkaline phosphatase isoenzyme detected in a patient with pleuritis carcinomatosa. Clin Chim Acta 1970;30:546-548. 3 Ilirano K. Matsumoto H, Tanaka T. Hayashi Y, lino S. Domar U. StigbrandT: Specific assay for human alkaline phosphatase isoen zymes. Clin Chim Acta 1987;166:265-273. 4 Wahren B, Hinkula J, Stigbrand T, Jeppsson A , Andersson L, Esposti PL, Edsmyr F. Millan JL: Phenotypes o f placental-type alkaline phosphatase in seminoma sera as defined by monoclonal antibodies. Int J Cancer 1986;37:595-600. 5 Beckman L. Björling G, Christodoulou C: Pregnancy enzymes and placental polymorphism. I. Alkaline phosphatase. Acta Genet 1966;16:59-73. 6 Koshida K, Stigbrand T, Hisazumi H , Wahren B: Electrophoretic heterogeneity of alkaline phosphatase isozymes in seminomas and normal testis. Tumor Biol 1989;10:181-189. 7 Jeppsson A. Wahren B, Brehmer-Andersson E. Silfversward C. Stigbrand T, Millan JL: Eutopic expression of placental-like alka line phosphatase in testicular tumors. Int J Cancer 1984;34: 757-761. 8 Millan JL. Stigbrand T : Antigenic determinants of human placental and testicular placental-like alkaline phosphatases as mapped by monoclonal antibodies. Eur J Biochem 1983;136:1-7.
K. Koshida Department of Urology Kanazawa University 13-1, Takara-machi Kanazawa 920 (Japan)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 12:22:33 PM
several rare variants were classified [5]. We therefore investigated migration patterns of PLAP activity from seminoma and normal testis on starch gel, resulting in dis closing marked heterogeneous migration patterns among individuals [data not shown]. IEF was applied on analysis of the isoenzymes, by which discrimination of PLAP activity between semi noma and normal testis appeared to be possible on the basis of isoelectric points as shown in figure 2. Although isoelectric points of seminoma PLAP and PLAP of pla centa were found to be similar, these two were distin guishable by digestion with neuraminidase, indicating a difference in sialic acid content between these isoenzymes [data not shown]. Microheterogeneity on IEF remained after neuraminidase treatment in the case of seminoma but not in normal testis, implying that factors other than sialylation might contribute to the heterogeneity of semi noma PLAP (fig. 3). Analysis of fractions from lectin affinity chromatography or from HPLC separation sug gested that the structural differences of the carbohydrate moiety and the variation in hydrophobicity of the mole cule possibly contribute to the heterogeneity on IEF [6]. Although similarities between the seminoma- and normal testis-derived enzymes have been shown in immuno chemical and enzyme inhibition studies [7,8], the hetero geneity of the enzyme appears to be considerable in the tumor tissues. In summary, we have demonstrated heterogeneity of the seminoma-derived PLAP with respect to glycosylation and charge of the molecule, by which the enzyme could be discriminated from PLAP of placenta and there fore could be regarded as PLAP-like enzyme. Further more, similar characteristics of the enzyme between semi noma and normal testis imply that a marked elevation of PLAP-like activity in seminoma results from the enhanced eutopic expression of the enzyme normally expressed in the testis.
99
