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Comparison of a tumour-derived form of intestinal alkaline phosphatase with foetal and adult intestinal alkaline phosphatases
CIinica Chimica Acta, 158 (1986) 165-172 Elsevier
165
CCA 03534
Comparison of a tumour-derived form of intestinal alkaline phosphatase with foetal and adult intestinal alkaline phosphatases Donald
W. Moss, Christopher
Royal Postgraduate (Received
R. Parmar and Katrine
Medical School, Hammersmith
B. Whitaker
Hospital, London WI2 OHS (UK)
November 24th 1985; revision received March accepted after revision May 5th, 1986)
12th, 1986;
Key words: Kasahara isoenzyme; Fetal intestinal alkaline phosphatase; Adult intestinal alkaline phosphatase; Electrophoresis; Affinity chromatography; Inhibition
Summary An intestinal alkaline phosphatase-like (Kasahara) isoenzyme has been isolated from the serum of a patient with lung cancer and compared with foetal intestinal alkaline phosphatase from the serum of a premature infant and with adult intestinal phosphatase isolated from serum in the same way. Although the ligand-binding sites of the three enzymes were indistinguishable, the foetal intestinal and Kasahara isoenzymes differed slightly from the adult isoenzyme in heat stability and markedly in electrophoretic mobility and neuraminidase-sensitivity, while themselves being similar in these respects. Neither the Kasahara isoenzyme nor foetal phosphatase reacted with anti-placental phosphatase monoclonal antibodies. These results suggest that the Kasahara isoenzyme corresponds to the reappearance of foetal intestinal alkaline phosphatase, rather than to modification of the adult intestinal isoenzyme.
Introduction Ectopic or inappropriate expression of alkaline phosphatase isoenzymes in tumours, often accompanied by their appearance in the circulation, is now a well-recognized phenomenon (reviewed in [l]). Most examples are of the expression of an isoenzyme essentially identical with term-placental alkaline phosphatase (Regan isoenzymes) and, more recently, of a placental phosphatase-like isoenzyme which is distinguishable from placental alkaline phosphatase in its reaction with certain monoclonal antibodies. The appearance of the placental isoenzyme is attributed to the re-expression of the placental phosphatase gene; an analogous explanation may account for the appearance of the placental-like enzyme, although OC09-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
166
it is as yet by no means certain that this isoenzyme is encoded by a separate gene locus. Ectopic expression by tumours of alkaline phosphatases (Kasahara isoenzymes) which resemble adult intestinal alkaline phosphatase in several of their properties has also been reported, especially in hepatoma [l]. However, the Kasahara isoenzyme is not identical with normal adult intestinal phosphatase, e.g. in the greater anodal electrophoretic mobility of the former, which is partly though not entirely due to the presence of sialic acid residues. Therefore, if the Kasahara isoenzyme originates from the ectopic expression of the adult phosphatase gene, abnormal genetic or post-genetic modifications of the gene product must be postulated. A form of intestinal alkaline phosphatase is present in the foetus up to about 32 wk gestation which, while exhibiting the catalytic properties of the adult isoenzyme, differs from it in being sialylated [2-41. In this respect, therefore, the Kasahara isoenzyme resembles the foetal intestinal alkaline phosphatase more closely than the adult intestinal isoenzyme [5]. Recent structural and immunological evidence suggests that the foetal intestinal phosphatase is encoded by a structural gene different from that of the adult isoenzyme [6-81. Therefore, by analogy with the Regan isoenzyme, the appearance of the Kasahara isoenzyme in cancer may be attributed to re-expression of this postulated foetal intestinal phosphatase gene. We now report a comparison of several properties of foetal intestinal alkaline phosphatase occurring in the serum of a premature baby with those of an intestinal phosphatase-like isoenzyme from the serum of a patient with lung cancer, and with the corresponding properties of adult intestinal alkaline phosphatase isolated from serum in the same way. Materials and methods The source of foetal intestinal alkaline phosphatase was serum obtained from blood samples submitted to the laboratory during the clinical investigation of a 4-wk-old baby, born after 28 wk gestation, who had mild respiratory problems and a history of staphylococcal infection. The source of normal adult intestinal alkaline phosphatase was serum from a patient with no history of malignant disease which was found to contain a high level of the isoenzyme on routine alkaline phosphatase isoenzyme analysis. The patient studied was a retired policeman aged 74 yr who had carcinoma of the left lung. Needle biopsy showed malignant cells of uncertain type. Treatment was by radiotherapy. He had an enlarged liver, and a rising serum alkaline phosphatase level. He had begun to behave irrationally, and died 4 wk later. No post-mortem examination was performed. Electrophoresis was performed on polyacrylamide slab gels and alkaline phosphatase zones were stained using 1-naphthyl phosphate and Fast Blue BB salt [9]. Neuraminidase treatment indicated in the results was carried out for 20 h at 37°C of 1 mg/ml in Tris-HCl buffer (0.02 mol/l, pH 7.6) with a final concentration neuraminidase (Type V, Sigma Chemical Co., St. Louis, MO, USA). Control experiments showed that these conditions were sufficient fully to desialylate liver and bone phosphatases in serum.
167
Blood samples collected into plain glass tubes were allowed to clot at room temperature. Serum was separated by centrifugation at 1000 x g for 10 min. Serum samples were fractionated into intestinal and non-intestinal alkaline phosphatase on mini-columns (2.5 X 0.6 cm) of Reactive Yellow 13 bound to Sepharose 4B [lo]. Twenty to 100 ~1 of serum was applied to the column, which contained 0.7 ml of the matrix in Tris-HCl buffer (0.02 mol/l, pH 7.6) and 4 ml Tris-HCl buffer eluted non-intestinal alkaline phosphatase. Intestinal alkaline phosphatase was eluted with 4 ml of diethanolamine buffer (1 mol/l, pH 10.0 at 25°C with 0.5 mmol/l MgCl,). Alkaline phosphatase was measured by the Scandinavian recommended method at 37°C with 4-nitrophenyl phosphate as substrate [ll]. Inhibition by L-phenylalanine, L-homoarginine and r_-leucine was determined by the addition of these amino-acids to the assay mixture to a final concentration of 5 mmol/l. The percentage inhibition was calculated with reference to the activity obtained in the absence of these amino-acids or, in the case of L-leucine, to the activity in the presence of 5 mmol/l D-leucine. Heat-stability of alkaline phosphatase was determined as the half-inactivation time of the enzyme on incubation at 56.O”C [12] in Tris-HCl buffer (0.02 mol/l, pH 7.6). The sample (usually 250 ~1) was placed into a thin-walled glass tube already pre-heated in the water bath and 30 ~1 volumes were rapidly removed into ice-cold tubes at intervals up to 30 min. The activity at each time was measured and the half-inactivation time was determined from a graph of log (activity) versus time
WI. Placental and placental-like amplified ELISA on microtitre and H17E2 [15], respectively.
alkaline phosphatases were determined by enzymeplates [13] using monoclonal antibodies H317 [14]
Results Both the premature infant’s and the adult cancer patient’s sera contained a fast, anodally-migrating alkaline phosphatase zone on polyacrylamide-gel electrophoresis of the two sera. The mobilities of the zones were similar, although the zone in the cancer serum was slightly faster (Fig. 1 and Table I). The cancer patient’s serum contained also a liver phosphatase zone and the infant’s a zone of bone phosphatase. The alkaline phosphatase in each serum was fractionated into intestinal and non-intestinal components by miniature-column affinity chromatography on Reactive Yellow 13-Sepharose 4B. The intestinal phosphatase-type components, eluted with concentrated diethanolamine buffer, contributed about 22% of the total alkaline phosphatase activities of 2 300 U/l and 1300 U/l, respectively, in the sera of the premature infant and the adult. These components were shown to correspond to the fast-moving zones on electrophoresis (Figs. 2 and 3). The non-intestinal components, eluted with dilute Tris buffer, were characterized as bone-type alkaline phosphatase in the infant’s serum and liver-type phosphatase in that of the adult, by their electrophoretic mobilities before and after neuraminidase digestion (Figs. 2
168
0
B
Du
Wi
Du
Wi
Ad
L
I 1;
t;
Fig. 1. Separation of alkaline phosphatase zones by polyacrylamide gel electrophoresis of serum from the cancer patient (Du) and the premature baby (Wi) together with sera containing predominantly bone (B), adult intestinal (Ad I) and liver (L) alkaline phosphatases; + N indicates neuraminidase treatment prior to electrophoresis.
and 3), and by progressive heat-inactivation at 56°C (half-inactivation time, bone phosphatase 232 s; liver phosphatase 853 s). The intestinal-type phosphatases from the infant’s and adtilt’s sera eluted from the affinity gel by concentrated diethanolamine buffer had similar sensitivities to
0
L
L I;
Du
Du r;
TrisTrisDEA ii
DEA
Ad
Ad
I
I
ii ii
Fig. 2. Alkaline phosphatases separated by polyacrylamide gel electrophoresis of serum from the cancer patient (Du) and its fractions eluted from Reactive Yellow 13 column by Tris buffer (Tris) and diethanolamine buffer (DRA), together with sera containing predominantly liver (L) and adult intestinal (Ad I) alkaline phosphatases; + N indicates neuraminidase treatment prior to electrophoresis.
(%) by:
time at 56OC
75 (I) 8 (1) 48 (1) 134
1.25 + 0.02 (4) 0.93 kO.03 (3)
SD
where availability
24k 1 (3) 853
44_+2 (3) 142
1.00 0.51 + 0.02 (3) 13i2 (3) 68+ 1 (2)
(6)
Liver
of material
26+ 1 (3) 232
0.91 rfI0.03 (7) 0.53 * 0.06 (2) 15&4(3) 69+1 (2)
Bone
analysis
5 * I (2) 38&t(4) Stable
75+_1(4)
Not done
Variable
the
to be
from
Placental
recovered
allowed
with those of the fast-band
(2) (3) (2)
1.29 + 0.02 0.97 * 0.02 7213 10+2
are also shown. Values are shown f
7218 (3) 4rt7(2) 47&l (2) 170
0.80 It 0.02 (5) 0.78 f 0.04 (2)
from serum)
Fast band from patient
(each recovered
Foetal intestinal
phosphatases
Adult intestinal
alkaline
* Corresponding values for liver, bone and placental phosphatases repeated, with numbers of determinations in parentheses.
Half-inactivation
Inhibition
Native
Electrophoretic mobility: (relative to liver isoenzyme) Neuraminidasetreated L-Phenylalanine L-Homoarginine L-Leucine
of adult and foetal intestinal
I
Comparison of properties patient’s serum *
TABLE
170
B
Wi L
L ,-risTrisDEADEAAd r;
IG
/tJ t
Ad 1
N Fig. 3. Alkaline phosphatases separated by polyacrylamide gel electrophoresis of serum from the premature baby (Wi) and its fractions eluted from Reactive Yellow 13 column by Tris buffer (Tris) and diethanolamine buffer (DEA), together with sera containing predominantly bone (B), liver (L) and adult intestinal (Ad I) alkaline phosphatases; + N indicates neuraminidase treatment prior to electrophoresis.
inhibition by amino acids which are recognised as isoenzyme-specific uncompetitive inhibitors of alkaline phosphatase. Their responses were also similar to those of adult intestinal phosphatase recovered from serum of a non-cancer patient by affinity chromatography (Table I). Progressive heat-inactivation of intestinal alkaline phosphatase fractions recovered from sera by affinity chromatography showed the enzymes from the infant’s and the cancer patient’s sera to have essentially similar stabilities (Table I). These values were slightly lower than that found for adult intestinal phosphatase recovered by affinity chromatography from serum. Unlike adult intestinal phosphatase, the electrophoretic mobilities of both the foetal and cancer-serum intestinal phosphatases were reduced by digestion with neuraminidase. The mobilities of the neuraminidase-treated enzymes were similar when compared in several runs, and were distinct from that of adult intestinal phosphatase which is insensitive to neuraminidase (Figs. 1-3; Table I). Neither the infant’s serum nor that of the adult cancer patient contained any alkaline phosphatase activity which bound to the anti-placental phosphatase monoclonal antibodies H317 or H17E2 in an enzyme-amplified ELISA.
The production of foetal intestinal alkaline phosphatase declines to low levels after about 32 wk gestation [4]. It is a sialoprotein, and it is probably for this reason that it persists in the circulation, as in the infant we have studied, unlike the unsialylated adult intestinal phosphatase which is rapidly cleared. The stability of adult intestinal phosphatase is rather variable when tissue-extracts are used as the source of enzyme, and this may also be the case for the foetal form. Furthermore,
171
differing estimates of the heat-stabilities of Kasahara isoenzymes have been reported, ranging from values intermediate between those of placental and liver phosphatases [16,17], to less than liver phosphatase [18] as reported here. Therefore, in order to eliminate possible variability due to different matrix effects between sera, we have based our comparisons on naturally-occurring forms of these enzymes recovered from serum, as being most comparable with the serum-derived Kasahara isoenzyme. This approach has been made possible by the use of the affinity-ligand Reactive Yellow 13, which permits specific and quantitative recoveries of intestinal phosphatases. The variant alkaline phosphatase present in this case of lung cancer was similar to the foetal intestinal alkaline phosphatase in the serum of the premature infant, except for a small difference in electrophoretic mobility before and after digestion with neuraminidase. However, the foetal and cancer-associated alkaline phosphatases are clearly distinct from adult intestinal phosphatase in their electrophoretic mobilities before and after digestion with neuraminidase. Furthermore, there is a small difference between them and the adult isoenzyme in heat-stability. However, all three isoenzymes appear to have identical ligand-binding sites as shown by their response to inhibitors, and in binding to immobilized Reactive Yellow 13, which has previously been shown to be specific for intestinal phosphatase and which does not bind either placental or liver-type phosphatases [19]. Partial structural evidence from comparison of cyanogen-bromide peptides and peptide maps of radioactively-labelled enzymes, as well as reaction with monoclonal antibodies, suggests that foetal and adult intestinal alkaline phosphatases differ in their protein structures [6-S], but that this is not due to the presence of a placental phosphatase subunit in the foetal enzyme, as had earlier been suggested [20]. As reported here, foetal intestinal phosphatase failed to bind to either of two placental monoclonal antibodies specific for placental and placental-like alkaline phosphatases. The foetal intestinal isoenzyme may be encoded by a separate gene which is repressed in postnatal life, or may arise by differential processing of a single mRNA. The possibility that foetal and adult intestinal alkaline phosphatases differ only in their carbohydrate components now seems less likely. The reappearance of intestinal phosphatase-like isoenzymes in cancer may result from both ectopic gene expression and altered post-translational modification. This may account for some discrepancies in the reported properties of intestinal-like phosphatases in cancer patients. In the present case, for example, increased sialylation and perhaps other forms of glycosylation due to the increased activity of sialyl and other glycosyl transferases in cancer tissues may account for the lack of complete identity between the Kasahara isoenzyme and foetal intestinal alkaline phosphatase. However, our results suggest that this Kasahara isoenzyme, at least, is more closely similar to foetal intestinal phosphatase than to the adult isoenzyme, and therefore is unlikely to be a modified adult intestinal phosphatase. Definitive identification of Kasahara isoenzymes with their normal counterparts will come from the use of the monoclonal antibody techniques and partial structural analysis now being applied to the comparison of foetal and adult intestinal phosphatases. However, Kasahara isoenzymes are rarely present in cancer patients, so that the
172
scarcity of the cancer-associated delay more extensive structural
isoenzyme analysis.
in amounts
adequate
for analysis
may
References 1 Stigbrand T, Fishman WH, eds. Human alkaline phosphatases. New York: Alan R. Liss, 1984. 2 Miki K, Suzuki H, Iino S, Oda T, Hirano K, Sigiura M. Human fetal intestinal alkaline phosphatase. Clin Chim Acta 1977; 79: 21-30. 3 Miki K, Oda T, Suzuki H, et al. Human fetal organ alkaline phosphatases. Clin Chim Acta 1978; 85: 115-124. 4 Mulivor RA, Hannig VL, Harris H. Developmental change in human intestinal alkaline phosphatase. Proc Nat1 Acad Sci USA 1978; 75: 3909-3912. 5 Higashino K, Otani R, Kudo S, Yamamura Y. A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue. Clin Chem 1977; 23: 1615-1623. 6 Vockley J, Meyer LJ, Harris H. Differentiation of human adult and fetal intestinal alkaline phosphatases with monoclonal antibodies. Am J Hum Genet 1984; 36: 987-1000. 7 Vockley J, D’Souza MP, Foster CJ, Harris H. Structural analysis of human adult and fetal alkaline phosphatases by cyanogen bromide peptide mapping. Proc Nat1 Acad Sci USA 1984; 81: 6120-6123. 8 Mueller HD, Leung H, Stinson RA. Different genes code for alkaline phosphatases from human fetal and adult intestine. Biochem Biophys Res Commun 1985; 126: 427-433. 9 Moss DW. Alkaline phosphatase isoenzymes. Technical and clinical aspects. Enzyme 1975; 20: 20-34. 10 Williams DG, Byfield PGH, Moss DW. Affinity chromatography of human intestinal alkaline phosphatase. Enzyme 1982; 28: 28-32. 11 Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. Recommended methods for the determination of four enzymes in blood. Stand J Clin Lab Invest 1974; 33: 291-306. 12 Wbitby LG, Moss DW. Analysis of heat inactivation curves of alkaline phosphatase isoenzymes in serum. Clin Chim Acta 1975; 59: 361-367. 13 Self CH. Enzyme-amplification. A general method applied to an immunoassisted assay for placental alkaline phosphatase. J Immunol Methods 1985; 76: 389-393. 14 McLaughlin PJ, Gee H, Johnson PM. Placental-type alkaline phosphatase in pregnancy and malignancy plasma: specific estimation using a monoclonal antibody in a solid phase enzyme immunoassay. Clin Chim Acta 1983; 130: 199-209. 15 Travers P, Bodmer W. Preparation and characterization of monoclonal antibodies against placental alkaline phosphatase and other human trophoblast-associated determinants. Int J Cancer 1984; 33: 633-641. 16 Wamock ML, Reisman R. Variant alkaline phosphatase in human hepatocellular cancers. Clin Chim Acta 1969; 24: 5-11. 17 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. 18 Crofton PM, Smith AF. Regan variant alkaline phosphatase in gastrointestinal carcinoma. Clin Chim Acta 1978; 86: 81-88. 19 Williams DG, Byfield PGH, Moss DW. Inhibition of human alkaline phosphatase isoenzymes by the affinity reagent Reactive Yellow 13. Enzyme 1985; 33: 70-74. 20 Behrens CM, Enns CA, Sussman HH. Characterization of human foetal intestinal alkaline phosphatase. Biochem J 1983; 211: 553-558.
165
CCA 03534
Comparison of a tumour-derived form of intestinal alkaline phosphatase with foetal and adult intestinal alkaline phosphatases Donald
W. Moss, Christopher
Royal Postgraduate (Received
R. Parmar and Katrine
Medical School, Hammersmith
B. Whitaker
Hospital, London WI2 OHS (UK)
November 24th 1985; revision received March accepted after revision May 5th, 1986)
12th, 1986;
Key words: Kasahara isoenzyme; Fetal intestinal alkaline phosphatase; Adult intestinal alkaline phosphatase; Electrophoresis; Affinity chromatography; Inhibition
Summary An intestinal alkaline phosphatase-like (Kasahara) isoenzyme has been isolated from the serum of a patient with lung cancer and compared with foetal intestinal alkaline phosphatase from the serum of a premature infant and with adult intestinal phosphatase isolated from serum in the same way. Although the ligand-binding sites of the three enzymes were indistinguishable, the foetal intestinal and Kasahara isoenzymes differed slightly from the adult isoenzyme in heat stability and markedly in electrophoretic mobility and neuraminidase-sensitivity, while themselves being similar in these respects. Neither the Kasahara isoenzyme nor foetal phosphatase reacted with anti-placental phosphatase monoclonal antibodies. These results suggest that the Kasahara isoenzyme corresponds to the reappearance of foetal intestinal alkaline phosphatase, rather than to modification of the adult intestinal isoenzyme.
Introduction Ectopic or inappropriate expression of alkaline phosphatase isoenzymes in tumours, often accompanied by their appearance in the circulation, is now a well-recognized phenomenon (reviewed in [l]). Most examples are of the expression of an isoenzyme essentially identical with term-placental alkaline phosphatase (Regan isoenzymes) and, more recently, of a placental phosphatase-like isoenzyme which is distinguishable from placental alkaline phosphatase in its reaction with certain monoclonal antibodies. The appearance of the placental isoenzyme is attributed to the re-expression of the placental phosphatase gene; an analogous explanation may account for the appearance of the placental-like enzyme, although OC09-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
166
it is as yet by no means certain that this isoenzyme is encoded by a separate gene locus. Ectopic expression by tumours of alkaline phosphatases (Kasahara isoenzymes) which resemble adult intestinal alkaline phosphatase in several of their properties has also been reported, especially in hepatoma [l]. However, the Kasahara isoenzyme is not identical with normal adult intestinal phosphatase, e.g. in the greater anodal electrophoretic mobility of the former, which is partly though not entirely due to the presence of sialic acid residues. Therefore, if the Kasahara isoenzyme originates from the ectopic expression of the adult phosphatase gene, abnormal genetic or post-genetic modifications of the gene product must be postulated. A form of intestinal alkaline phosphatase is present in the foetus up to about 32 wk gestation which, while exhibiting the catalytic properties of the adult isoenzyme, differs from it in being sialylated [2-41. In this respect, therefore, the Kasahara isoenzyme resembles the foetal intestinal alkaline phosphatase more closely than the adult intestinal isoenzyme [5]. Recent structural and immunological evidence suggests that the foetal intestinal phosphatase is encoded by a structural gene different from that of the adult isoenzyme [6-81. Therefore, by analogy with the Regan isoenzyme, the appearance of the Kasahara isoenzyme in cancer may be attributed to re-expression of this postulated foetal intestinal phosphatase gene. We now report a comparison of several properties of foetal intestinal alkaline phosphatase occurring in the serum of a premature baby with those of an intestinal phosphatase-like isoenzyme from the serum of a patient with lung cancer, and with the corresponding properties of adult intestinal alkaline phosphatase isolated from serum in the same way. Materials and methods The source of foetal intestinal alkaline phosphatase was serum obtained from blood samples submitted to the laboratory during the clinical investigation of a 4-wk-old baby, born after 28 wk gestation, who had mild respiratory problems and a history of staphylococcal infection. The source of normal adult intestinal alkaline phosphatase was serum from a patient with no history of malignant disease which was found to contain a high level of the isoenzyme on routine alkaline phosphatase isoenzyme analysis. The patient studied was a retired policeman aged 74 yr who had carcinoma of the left lung. Needle biopsy showed malignant cells of uncertain type. Treatment was by radiotherapy. He had an enlarged liver, and a rising serum alkaline phosphatase level. He had begun to behave irrationally, and died 4 wk later. No post-mortem examination was performed. Electrophoresis was performed on polyacrylamide slab gels and alkaline phosphatase zones were stained using 1-naphthyl phosphate and Fast Blue BB salt [9]. Neuraminidase treatment indicated in the results was carried out for 20 h at 37°C of 1 mg/ml in Tris-HCl buffer (0.02 mol/l, pH 7.6) with a final concentration neuraminidase (Type V, Sigma Chemical Co., St. Louis, MO, USA). Control experiments showed that these conditions were sufficient fully to desialylate liver and bone phosphatases in serum.
167
Blood samples collected into plain glass tubes were allowed to clot at room temperature. Serum was separated by centrifugation at 1000 x g for 10 min. Serum samples were fractionated into intestinal and non-intestinal alkaline phosphatase on mini-columns (2.5 X 0.6 cm) of Reactive Yellow 13 bound to Sepharose 4B [lo]. Twenty to 100 ~1 of serum was applied to the column, which contained 0.7 ml of the matrix in Tris-HCl buffer (0.02 mol/l, pH 7.6) and 4 ml Tris-HCl buffer eluted non-intestinal alkaline phosphatase. Intestinal alkaline phosphatase was eluted with 4 ml of diethanolamine buffer (1 mol/l, pH 10.0 at 25°C with 0.5 mmol/l MgCl,). Alkaline phosphatase was measured by the Scandinavian recommended method at 37°C with 4-nitrophenyl phosphate as substrate [ll]. Inhibition by L-phenylalanine, L-homoarginine and r_-leucine was determined by the addition of these amino-acids to the assay mixture to a final concentration of 5 mmol/l. The percentage inhibition was calculated with reference to the activity obtained in the absence of these amino-acids or, in the case of L-leucine, to the activity in the presence of 5 mmol/l D-leucine. Heat-stability of alkaline phosphatase was determined as the half-inactivation time of the enzyme on incubation at 56.O”C [12] in Tris-HCl buffer (0.02 mol/l, pH 7.6). The sample (usually 250 ~1) was placed into a thin-walled glass tube already pre-heated in the water bath and 30 ~1 volumes were rapidly removed into ice-cold tubes at intervals up to 30 min. The activity at each time was measured and the half-inactivation time was determined from a graph of log (activity) versus time
WI. Placental and placental-like amplified ELISA on microtitre and H17E2 [15], respectively.
alkaline phosphatases were determined by enzymeplates [13] using monoclonal antibodies H317 [14]
Results Both the premature infant’s and the adult cancer patient’s sera contained a fast, anodally-migrating alkaline phosphatase zone on polyacrylamide-gel electrophoresis of the two sera. The mobilities of the zones were similar, although the zone in the cancer serum was slightly faster (Fig. 1 and Table I). The cancer patient’s serum contained also a liver phosphatase zone and the infant’s a zone of bone phosphatase. The alkaline phosphatase in each serum was fractionated into intestinal and non-intestinal components by miniature-column affinity chromatography on Reactive Yellow 13-Sepharose 4B. The intestinal phosphatase-type components, eluted with concentrated diethanolamine buffer, contributed about 22% of the total alkaline phosphatase activities of 2 300 U/l and 1300 U/l, respectively, in the sera of the premature infant and the adult. These components were shown to correspond to the fast-moving zones on electrophoresis (Figs. 2 and 3). The non-intestinal components, eluted with dilute Tris buffer, were characterized as bone-type alkaline phosphatase in the infant’s serum and liver-type phosphatase in that of the adult, by their electrophoretic mobilities before and after neuraminidase digestion (Figs. 2
168
0
B
Du
Wi
Du
Wi
Ad
L
I 1;
t;
Fig. 1. Separation of alkaline phosphatase zones by polyacrylamide gel electrophoresis of serum from the cancer patient (Du) and the premature baby (Wi) together with sera containing predominantly bone (B), adult intestinal (Ad I) and liver (L) alkaline phosphatases; + N indicates neuraminidase treatment prior to electrophoresis.
and 3), and by progressive heat-inactivation at 56°C (half-inactivation time, bone phosphatase 232 s; liver phosphatase 853 s). The intestinal-type phosphatases from the infant’s and adtilt’s sera eluted from the affinity gel by concentrated diethanolamine buffer had similar sensitivities to
0
L
L I;
Du
Du r;
TrisTrisDEA ii
DEA
Ad
Ad
I
I
ii ii
Fig. 2. Alkaline phosphatases separated by polyacrylamide gel electrophoresis of serum from the cancer patient (Du) and its fractions eluted from Reactive Yellow 13 column by Tris buffer (Tris) and diethanolamine buffer (DRA), together with sera containing predominantly liver (L) and adult intestinal (Ad I) alkaline phosphatases; + N indicates neuraminidase treatment prior to electrophoresis.
(%) by:
time at 56OC
75 (I) 8 (1) 48 (1) 134
1.25 + 0.02 (4) 0.93 kO.03 (3)
SD
where availability
24k 1 (3) 853
44_+2 (3) 142
1.00 0.51 + 0.02 (3) 13i2 (3) 68+ 1 (2)
(6)
Liver
of material
26+ 1 (3) 232
0.91 rfI0.03 (7) 0.53 * 0.06 (2) 15&4(3) 69+1 (2)
Bone
analysis
5 * I (2) 38&t(4) Stable
75+_1(4)
Not done
Variable
the
to be
from
Placental
recovered
allowed
with those of the fast-band
(2) (3) (2)
1.29 + 0.02 0.97 * 0.02 7213 10+2
are also shown. Values are shown f
7218 (3) 4rt7(2) 47&l (2) 170
0.80 It 0.02 (5) 0.78 f 0.04 (2)
from serum)
Fast band from patient
(each recovered
Foetal intestinal
phosphatases
Adult intestinal
alkaline
* Corresponding values for liver, bone and placental phosphatases repeated, with numbers of determinations in parentheses.
Half-inactivation
Inhibition
Native
Electrophoretic mobility: (relative to liver isoenzyme) Neuraminidasetreated L-Phenylalanine L-Homoarginine L-Leucine
of adult and foetal intestinal
I
Comparison of properties patient’s serum *
TABLE
170
B
Wi L
L ,-risTrisDEADEAAd r;
IG
/tJ t
Ad 1
N Fig. 3. Alkaline phosphatases separated by polyacrylamide gel electrophoresis of serum from the premature baby (Wi) and its fractions eluted from Reactive Yellow 13 column by Tris buffer (Tris) and diethanolamine buffer (DEA), together with sera containing predominantly bone (B), liver (L) and adult intestinal (Ad I) alkaline phosphatases; + N indicates neuraminidase treatment prior to electrophoresis.
inhibition by amino acids which are recognised as isoenzyme-specific uncompetitive inhibitors of alkaline phosphatase. Their responses were also similar to those of adult intestinal phosphatase recovered from serum of a non-cancer patient by affinity chromatography (Table I). Progressive heat-inactivation of intestinal alkaline phosphatase fractions recovered from sera by affinity chromatography showed the enzymes from the infant’s and the cancer patient’s sera to have essentially similar stabilities (Table I). These values were slightly lower than that found for adult intestinal phosphatase recovered by affinity chromatography from serum. Unlike adult intestinal phosphatase, the electrophoretic mobilities of both the foetal and cancer-serum intestinal phosphatases were reduced by digestion with neuraminidase. The mobilities of the neuraminidase-treated enzymes were similar when compared in several runs, and were distinct from that of adult intestinal phosphatase which is insensitive to neuraminidase (Figs. 1-3; Table I). Neither the infant’s serum nor that of the adult cancer patient contained any alkaline phosphatase activity which bound to the anti-placental phosphatase monoclonal antibodies H317 or H17E2 in an enzyme-amplified ELISA.
The production of foetal intestinal alkaline phosphatase declines to low levels after about 32 wk gestation [4]. It is a sialoprotein, and it is probably for this reason that it persists in the circulation, as in the infant we have studied, unlike the unsialylated adult intestinal phosphatase which is rapidly cleared. The stability of adult intestinal phosphatase is rather variable when tissue-extracts are used as the source of enzyme, and this may also be the case for the foetal form. Furthermore,
171
differing estimates of the heat-stabilities of Kasahara isoenzymes have been reported, ranging from values intermediate between those of placental and liver phosphatases [16,17], to less than liver phosphatase [18] as reported here. Therefore, in order to eliminate possible variability due to different matrix effects between sera, we have based our comparisons on naturally-occurring forms of these enzymes recovered from serum, as being most comparable with the serum-derived Kasahara isoenzyme. This approach has been made possible by the use of the affinity-ligand Reactive Yellow 13, which permits specific and quantitative recoveries of intestinal phosphatases. The variant alkaline phosphatase present in this case of lung cancer was similar to the foetal intestinal alkaline phosphatase in the serum of the premature infant, except for a small difference in electrophoretic mobility before and after digestion with neuraminidase. However, the foetal and cancer-associated alkaline phosphatases are clearly distinct from adult intestinal phosphatase in their electrophoretic mobilities before and after digestion with neuraminidase. Furthermore, there is a small difference between them and the adult isoenzyme in heat-stability. However, all three isoenzymes appear to have identical ligand-binding sites as shown by their response to inhibitors, and in binding to immobilized Reactive Yellow 13, which has previously been shown to be specific for intestinal phosphatase and which does not bind either placental or liver-type phosphatases [19]. Partial structural evidence from comparison of cyanogen-bromide peptides and peptide maps of radioactively-labelled enzymes, as well as reaction with monoclonal antibodies, suggests that foetal and adult intestinal alkaline phosphatases differ in their protein structures [6-S], but that this is not due to the presence of a placental phosphatase subunit in the foetal enzyme, as had earlier been suggested [20]. As reported here, foetal intestinal phosphatase failed to bind to either of two placental monoclonal antibodies specific for placental and placental-like alkaline phosphatases. The foetal intestinal isoenzyme may be encoded by a separate gene which is repressed in postnatal life, or may arise by differential processing of a single mRNA. The possibility that foetal and adult intestinal alkaline phosphatases differ only in their carbohydrate components now seems less likely. The reappearance of intestinal phosphatase-like isoenzymes in cancer may result from both ectopic gene expression and altered post-translational modification. This may account for some discrepancies in the reported properties of intestinal-like phosphatases in cancer patients. In the present case, for example, increased sialylation and perhaps other forms of glycosylation due to the increased activity of sialyl and other glycosyl transferases in cancer tissues may account for the lack of complete identity between the Kasahara isoenzyme and foetal intestinal alkaline phosphatase. However, our results suggest that this Kasahara isoenzyme, at least, is more closely similar to foetal intestinal phosphatase than to the adult isoenzyme, and therefore is unlikely to be a modified adult intestinal phosphatase. Definitive identification of Kasahara isoenzymes with their normal counterparts will come from the use of the monoclonal antibody techniques and partial structural analysis now being applied to the comparison of foetal and adult intestinal phosphatases. However, Kasahara isoenzymes are rarely present in cancer patients, so that the
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scarcity of the cancer-associated delay more extensive structural
isoenzyme analysis.
in amounts
adequate
for analysis
may
References 1 Stigbrand T, Fishman WH, eds. Human alkaline phosphatases. New York: Alan R. Liss, 1984. 2 Miki K, Suzuki H, Iino S, Oda T, Hirano K, Sigiura M. Human fetal intestinal alkaline phosphatase. Clin Chim Acta 1977; 79: 21-30. 3 Miki K, Oda T, Suzuki H, et al. Human fetal organ alkaline phosphatases. Clin Chim Acta 1978; 85: 115-124. 4 Mulivor RA, Hannig VL, Harris H. Developmental change in human intestinal alkaline phosphatase. Proc Nat1 Acad Sci USA 1978; 75: 3909-3912. 5 Higashino K, Otani R, Kudo S, Yamamura Y. A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue. Clin Chem 1977; 23: 1615-1623. 6 Vockley J, Meyer LJ, Harris H. Differentiation of human adult and fetal intestinal alkaline phosphatases with monoclonal antibodies. Am J Hum Genet 1984; 36: 987-1000. 7 Vockley J, D’Souza MP, Foster CJ, Harris H. Structural analysis of human adult and fetal alkaline phosphatases by cyanogen bromide peptide mapping. Proc Nat1 Acad Sci USA 1984; 81: 6120-6123. 8 Mueller HD, Leung H, Stinson RA. Different genes code for alkaline phosphatases from human fetal and adult intestine. Biochem Biophys Res Commun 1985; 126: 427-433. 9 Moss DW. Alkaline phosphatase isoenzymes. Technical and clinical aspects. Enzyme 1975; 20: 20-34. 10 Williams DG, Byfield PGH, Moss DW. Affinity chromatography of human intestinal alkaline phosphatase. Enzyme 1982; 28: 28-32. 11 Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. Recommended methods for the determination of four enzymes in blood. Stand J Clin Lab Invest 1974; 33: 291-306. 12 Wbitby LG, Moss DW. Analysis of heat inactivation curves of alkaline phosphatase isoenzymes in serum. Clin Chim Acta 1975; 59: 361-367. 13 Self CH. Enzyme-amplification. A general method applied to an immunoassisted assay for placental alkaline phosphatase. J Immunol Methods 1985; 76: 389-393. 14 McLaughlin PJ, Gee H, Johnson PM. Placental-type alkaline phosphatase in pregnancy and malignancy plasma: specific estimation using a monoclonal antibody in a solid phase enzyme immunoassay. Clin Chim Acta 1983; 130: 199-209. 15 Travers P, Bodmer W. Preparation and characterization of monoclonal antibodies against placental alkaline phosphatase and other human trophoblast-associated determinants. Int J Cancer 1984; 33: 633-641. 16 Wamock ML, Reisman R. Variant alkaline phosphatase in human hepatocellular cancers. Clin Chim Acta 1969; 24: 5-11. 17 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. 18 Crofton PM, Smith AF. Regan variant alkaline phosphatase in gastrointestinal carcinoma. Clin Chim Acta 1978; 86: 81-88. 19 Williams DG, Byfield PGH, Moss DW. Inhibition of human alkaline phosphatase isoenzymes by the affinity reagent Reactive Yellow 13. Enzyme 1985; 33: 70-74. 20 Behrens CM, Enns CA, Sussman HH. Characterization of human foetal intestinal alkaline phosphatase. Biochem J 1983; 211: 553-558.