- Email: [email protected]
Further characterization of a heat-stable alkaline phosphatase with low sensitivity to L-phenylalanine
193
Clinica Chimicu Aria, 194 (1990) 193-202 Elsevier
CCA 04884
Further characterization of a heat-stable alkaline phosphatase with low sensitivity to L-phenylalanine Doina Department (Received
Onica,
Kerstin
of Clinical Chemistry,
17 November
Key words: Alkaline
Rosendahl Karolinska
1989: revision received phosphatase;
and Lennart
Institutet
Waldenlind
at Sijdersjukhuset
10 September
Siockholm
1990; accepted
Heat stability; pH optimum: Monoclonal antibody
K,,
(Sweden)
11 September
1990)
value: L-Phenylalanine:
Summary A heat-stable alkaline phosphatase, hitherto found in two famiIies with inherited hyperphosphatasemia, was further characterized. The enzyme was similar to serum placental alkaline phosphatase from pregnant women concerning its apparent affinity constant (K,,,) for 4-nitrophenyl phosphate and its reactivity with H7 mon~lonal ~ti-placental alkaline phosphatase (PLAP) antibodies, but different in the following respects: it exhibited greater heat stability. a higher pH optimum, lower sensitivity to inhibition by L-phenylalanine, and no reactivity with C2 monoclonal anti-PLAP antibodies. The low sensitivity to L-phenylalanine suggests that the enzyme might correspond to a rare phenotype of placental alkaline phosphatase found in human term placenta.
Introduction We previously reported on the occurrence in a family of an alkaline phosphatase (AP; EC 3.1.3.1) that was similar to the PLAP found in serum during pregnancy in that it had high heat stability (65”C, 5 min) and was reactive with polyclonal anti-PLAP antibodies. However, the enzyme showed a distinct pattern of inhibition by certain amino acids and tripeptides and it also differed from PLAP in its electrophoretic mobility, isoelectric heterogeneity and apparent molecular mass [l]. The purpose of the present investigation was to further characterize the thermal stability, the catalytic and the i~unologic properties of this enzyme and to compare it with a heat stable AP found in another unrelated patient.
Correspondence to: Doina Onica. S-100 64 Stockholm, Sweden.
~09-~981/~/$03.50
Ph.D..
Department
of Clinical
if) 1990 Elsevier Science Publishers
Chemistry.
B.V. (Biomedical
Siidersjukhuset,
Division)
Box 38100.
194
Materials and methods Putients Patient R.S., a 4%year-old, nonpregnant woman, was investigated in the period 1972-1986 for joint pains of unclear origin, coagulation anomalies, and a persistent increase in serum AP (3.6 times higher than the upper limit of the reference range for adults which was 0.8-4.6 pkat/l). Liver function tests (serum y-glutamyltransferase, alanine aminotransferase, aspartate arninotransferase, lactate dehydrogenase, bilirubin), serum electrophoresis, and concentrations of acute phase reactants (orosomucoid, haptoglobin) and of immunoglobulins (IgG, IgA, IgM) were normal. In this patient we identified a heat stable alkaline phosphatase (65”C, 5 min) which represented 88% of the total AP activity. It was repeatedly demonstrated in the patient’s serum and was further characterized in 1986 when the patient was 48-year-old. The enzyme was also found in three other members of the patient’s family. Data concerning the patient and her family have been published previously [ 11. Patient M.K., a 53-year-old, nonpregnant woman, unrelated to the above patient, was investigated in 1987 for an increase in serum AP (2.7 times higher than the upper limit of the reference range for adults) and for diffuse pains. Other laboratory values were within the reference ranges. The elevated AP activity was due to a heat stable alkaline phosphatase (65”C, 5 mm), which represented 85% of the total AP activity.
Serum samples were stored at -70°C. trimester served as PLAP controls.
Sera from pregnant
women
in the third
Chemicals 4-Nitrophenyl phosphate (CNPP), diethanolarnine (DEA), magnesium chloride. magnesium acetate, zinc sulphate, and 2-amino-2-methyl-l-propanol (AMP) were obtained from Merck, Darmstadt, FRG, and N-hydroxyethylethylenediaminetriacetic acid, trisodium salt (HEDTA) from Sigma Chemical Co, St Louis, MO, USA. Enzyme
assa)
Alkaline phosphatase activity was measured by the method recommended by the Scandinavian Society for Clinical Chemistry (SSCC) [2] with some modifications. The assay medium contained 16 mmol/l 4-NPP and 0.5 mmol/l Mg2+ in 1 mol/l DEA buffer, pH 10.0. Enzyme activity was also determined by the reference method of the American Association for Clinical Chemistry (AACC) and the International Federation of Clinical Chemistry (IFCC) [3,4], using 16 mmol/l 4-NPP, 2 mmol/l Mg 2+. 1 mmol/l Zn2+ and 2 mmol/l HEDTA in 0.35 mol/l AMP buffer, pH 10.4.
195
In the assays, we continuously measured the production of 4-nitrophenol at 405 nm and 37°C with an LKB 8600 reaction rate analyzer (LKB, Bromma, Sweden). The reaction was initiated by adding substrate, and the volume fraction of the sample was kept the same in all methods as 0.0116 (1 : 86) according to the instructions of the manufacturer for the kinetic AP test, using the LKB 8600 analyzer.
Electrophoresis
and isoelectric focusing
Electrophoresis was performed in 1.5-mm layers of 0.9% (w/v) agar gel in 0.082 mol/l sodium barbital-HCl buffer, pH 8.4, as previously described [l]. Isoelectric focusing was performed as previously described [l] in ready-made Ampholine PAG plates (245 x 110 X 1 mm) (LKB. Bromma, Sweden), pH range 4.0-6.5 containing 5% polyacrylamide and 2.2% ampholine (w/v).
pH optimum pH optimum was determined and at a substrate concentration
in 1 mol/l DEA buffer of 16 mmol/l 4-NPP.
in the pH range 9.0-12.0
Thermostability Heat inactivation curves were established for whole sera diluted fourfold with 0.154 mol/l NaCl. Samples of 1.5 ml in stoppered 10 X 55-mm glass tubes were incubated in a water bath at 70°C and 79“C. These temperatures were chosen according to Holmgren and Stigbrand [5]. Portions of 100 ~1 were removed every 2.5 min during a 30-min period and cooled in an ice bath. Residual AP activity was then determined by the SSCC method in 1 mol/l DEA buffer, pH 10.0. Plots of the logarithm of percentage residual AP activity versus time were biphasic for all sera examined. The first part of the curve was assumed to correspond to the inactivation of labile nonplacental isoenzymes and the latter part to the inactivation of the stable AP enzymes. The half-life of the stable AP was calculated from the latter, linear part of the inactivation curve, the equation of which was determined by regression analysis. In the calculation only the O-20 min time interval was included, since after 20 min of incubation the thermal inactivation curve seemed to deviate from linearity at least for the AP activity in patients’ samples.
Determination
of K,
values
In the enzyme assay using either 1 mol/l DEA buffer, pH 10.0, or 0.35 mol/l AMP buffer, pH 10.4, the concentration of 4-NPP was varied from 0.4 to 8 mmol/l. Linear regression analysis was used for the Lineweaver-Burk double reciprocal plotting of reaction velocities versus substrate concentrations.
196
Inhibition
studies
Inhibition constants ( Ki) were determined as described by Dixon [6], in both 1 mol/l DEA buffer, pH 10.0, and 0.35 mol/l AMP buffer, pH 10.4. Two substrate (CNPP) concentrations (0.6 and 16 mmol/l) and six inhibitor (L-phenylalanine) concentrations (0.5510 mmol/l) were used. Enzyme
immunoassay
(EIA) for PLAP
The activities of PLAP in serum were determined with the Prolifigena PLAP-EIA kit (Sangtec Medical, Bromma, Sweden). Serum samples (50 ~1) were added to a microtiter plate precoated with the H7 or C2 monoclonal antibodies reacting with PLAP. After overnight incubation (17 h) at room temperature, the plate was washed three times with 10 mmol/l Tris buffer, pH 7.4, containing 1 mmol/l Mg2+, 0.154 mol/l NaCl, 15 mmol/l sodium azide and 5 g/l bovine serum albumin. The enzymatic activity of the bound PLAP was determined by adding 100 ~1 of substrate solution containing 10 mmol/l 4-NPP, 0.5 mmol/l Mg2+ and 15 mmol/l sodium azide in 1 mol/l DEA buffer, pH 9.8, according to the instructions of the manufacturer. After 1 h incubation at 37°C the absorbance at 405 nm was measured. Results The electrophoretic and isoelectric properties of the heat stable AP found in sera from patients R.S. and M.K. were apparently the same (Figs. 1 and 2). The pH optimum was found to be identical (10.8) for the patients’ enzymes, while for PLAP from pregnant women (n = 7) it was 10.4-10.6, in the presence of 1 mol/l DEA buffer and 16 mmol/l 4-NPP at 37°C. (Fig. 3). The sera were heat treated at 65°C for 5 min before the assays in order to eliminate the nonplacental isoenzymes.
L B/PLAP I abcdef
9
h
Fig. 1. Electrophoretic separation in agar gel of AP isoenzymes in sera from patients and in control sera. The gel was stained for enzyme activity. a, Liver and intestinal AP; b, bone AP; c, d, pregnant woman; e, f, patient R.S.; g, h, patient M.K. Sera in d, f and h were heat treated at 65°C for 5 min. The approximate positions of various isoenzymes are given: L, liver AP; B, bone AP; I, intestinal AP; PLAP, placental AP. The arrow indicates the start position.
197
PH m-3.65 *- 3.95 a-4.25 -4.75 -4.85 -4.95
*- 5.25
f
e--5.35
abcdef Fig. 2. Isoelectric focusing stained for enzyme activity.
in polyacrylamide gel of serum AP isoenzymes at pH 4.0-6.5. The gel was a, b, patient M.K.; c, d, pregnant woman; e, f, patient R.S. Sera in b, d and f were heat treated
at 65°C for 15 min.
Fig. 3. Effect of pH on the heat stable AP from patient R.S. (Ol) and patient M.K. (A. . . .A), and on PLAP from a pregnant woman (O0). Serum samples were heat treated (65”C, 5 min) before the measurements. The assay was performed in 1 mol/l DEA buffer and at a substrate concentration of 16 mmol/l4-NPP. The AP activity at pH 10.0 was considered to be 100%.
198
70’ c 70’ c
70‘ c
79’ c 79. c
5’ 0
5
10
15
20
25
30
Time (min) Fig. 4. Effect of heat treatment on AP activity in serum from patient R.S. (0 -0) and patient M.K. (r------r), and from a pregnant woman (a -•). The sera were diluted fourfold with 0.154 moi/l NaCl before heat treatment. For patient M.K. the measuring interval was 5 min instead of 2.5 min.
The enzymes from the two patients were more heat stable than PLAP from pregnant women at 79OC (Fig. 4). The half-lives for the enzymes from patient R.S. and patient M.K. were similar, 20.5 f 1.3 min (mean i SE, 7 determinations) and 23.0 i 2.0 mm (6 deter~nations), respectively, while PLAP from pregnant women exhibited half-lives of 8-11 min (n = 12). At 70°C both the patients’ enzymes and PLAP from pregnant women were stable. The apparent affinity constants (K, ) of the patients’ enzymes were similar both in the DEA buffer, pH 10.0 and in the AMP buffer, pH 10.4. No distinct differences in K, could be seen between the patients’ enzymes and PLAP (Table I).
TABLE
I
K, values (mmol/l) for 4-nitrophenyl from pregnant women a
Patient R.S. h Patient M.K. PLAP =
phosphate
of heat stable AP enzymes
in sera from patients and
DEA buffer, pH 10.0
AMP buffer, pH 10.4
0.37 0.40 0.41 f 0.05
0.37 0.37 0.51+ 0.07
’ All sera were heat treated at 65°C for 5 mm before the kinetic measurements. b Mean value for sera from the patients (3-5 dete~ations). ’ Mean valuef SD for sera from pregnant women (a = 12 in DEA buffer and n = 6 in AMP buffer).
199
TABLE
II
Inhibition women a
by L-phenylalanine
of heat
stable
AP enzymes
in sera from
patients
and
from
pregnant
Ki (mmol/l)
Patient R.S. a Patient M.K. PLAP ’
DEA buffer, pH 10.0
AMP buffer,
6.7 7.0 2.1kO.2
8.9 10.8 2.8 + 0.3
pH 10.4
-
a All sera were heat treated at 65°C for 5 min before the inhibition experiments. b Mean value for sera from the patients (2 determinations). ’ Mean value+ SD for sera from pregnant women (n = 6 in DEA buffer and n = 3 in AMP buffer).
TABLE Reactivity women
III of H7 and C2 monoclonal
anti-PLAP
Alkaline
Patient R.S. Patient M.K. Pregnant women
antibodies
phosphatase
with sera from patients
activity
and from pregnant
(pkat/l)
Reactive with H7 antibodies
Reactive with C2 antibodies
11.1 8.8 3.8 0.7 1.2 2.5 1.1 2.6 2.7 1.1 1.9 1.1 1.4
0.008 0.026 3.7 0.7 1.2 2.3 1.1 2.2 2.5 1.0 1.8 1.0 1.3
The inhibition constants (Ki) for L-phenylalanine were also similar for the patients’ enzymes (Table II). However, the enzymes differed from PLAP since they were less inhibited by L-phenylalanine both in the DEA and in the AMP buffer. The patients’ enzymes displayed practically no reaction with the C2 monoclonal anti-PLAP antibodies, while PLAP from pregnant women showed a strong reaction. The H7 monoclonal anti-PLAP antibodies reacted with both the enzymes and PLAP (Table III). Discussion The enzymes from the two patients, who belong to different families, had similar biochemical (apparent affinity constant for 4-NPP, pH optimum, sensitivity to
TABLE
IV
Comparison of the properties reviewed by Fishman [II] Properties
of the patients’
enzymes
alkaline
phosphatases
as
lsoenzymes PLAP (term placenta/Regan)
Molecular mass pH optimum Heat stability 65’C, 5 min 70°C 79°C. t l/2 (min) Inhibition by L-Phenylalanine (5 mmol/l) L-Homoarginine (5 mmol/l) L-Leucine (Smmol/l) Neuraminidase sensitivity Reaction with antibodies Polyclonal anti-PLAP H7 Monoclonal anti-PLAP C2 Monoclonal anti-PLAP
with those of placental
PLAP-like AP (testis, thymus/Nagao)
Patients’
128000 10.6
130000 10.6
140000 10.X
stable stable U-11
stable n.d. n.d.
stable stable 20.5-23.0
+++
tit
t
+
*
*
+ sensitive
it+
(+)
sensitive
sensitive
reactive reactive some variants reactive
reactive reactive non-reactive
reactive reactive reactive
’ The origin of the enzymes is indicated in parentheses: sociated enzyme. ’ The properties of patients’ enzymes are summarized
a
normal
are
tissue/name
enzymes
of the respective
on the basis of the data presented
h
tumor-as-
in this and a
previous paper [l]. n.d.. not determined.
L-phenylalanine), electrophoretic, isoelectric, thermal and immunologic properties. These results suggest that the enzymes are the same. In Table IV the properties of the patients’ enzymes are compared with those of PLAP and PLAP-like AP of normal and tumor tissue origin. The reactivity of the enzymes from patient R.S. [l] and patient M.K. (unpubl. data) with polyclonal anti-PLAP antibodies, and with H7 monoclonal anti-PLAP antibodies, which have been shown by Millan and Stigbrand [7] to react both with PLAP and PLAP-like AP, indicates that the enzymes from the two patients are alkaline phosphatases of the placental type. However, they differed from PLAP by the lack of reactivity with C2 monoclonal anti-PLAP antibodies, which are known to react with the common phenotypes of PLAP [7]. This result suggests that there are differences in the antigenic properties of the patients’ enzymes and common PLAP. The patients’ enzymes and PLAP from pregnant women have quite similar active the same. sites, as shown by their K, values for 4-NPP, which were essentially
201
However, the patients’ enzymes were different from PLAP in the following respects: they exhibited greater heat stability, a higher pH optimum and lower sensitivity to inhibition by L-phenylalanine. It seems that the patients’ enzymes and PLAP, although related, may be structurally different. In our previous study we also showed that the enzyme from patient R.S. has a lower susceptibility than PLAP to inhibition by the tripeptides L-leucyl-glycyl-glycine and L-phenylalanyl-glycylglycine. The great heat stability and the inhibition profile suggest that the patients’ enzymes are a variant of PLAP, with a relatively low sensitivity to L-phenylalanine, which might correspond to a rare phenotype of PLAP found in human term placenta [8]. An L-phenylalanine-insensitive, heat stable alkaline phosphatase has also been found in human milk [9]. The relatively low sensitivity of the enzyme from patient R.S. [l] and patient M.K. (unpubl. data) to L-leucine, indicates that the enzymes are not of the PLAP-like AP (Nagao) type [lo]. Hitherto we have found the heat stable alkaline phosphatase in four members of the family of patient R.S., as previously reported [l], and in patient M.K. (this study), who is not related to the first family. Preliminary results indicate that the occurrence of the enzyme in patient M.K. is also inherited, since a small amount of it has been found in the serum of her daughter (unpubl. data). All these persons were apparently healthy, except patients R.S. and M.K., who were suffering from joint pains of unclear origin. There was no history of cancer in any of the persons concerned. Since an elevated level of PLAP or PLAP-like AP will alert the clinician to consider malignancy in a nonpregnant patient, it is clinically important that among the causes of increased AP activity, the possibility of a familial increase in AP of the placental type, not associated with malignant disease, should be kept in mind. The occurrence of the enzyme by inheritance in patients without malignant disease suggests that the regulation of its biosynthesis is different from that of placental phosphatases (Regan and Nagao isoenzymes), which are tumor-associated and thought to be products of derepressed oncotrophoblast genes [ll]. Acknowledgement The Prolifigen@ PLAP-EIA Sweden.
kit was kindly provided
by Sangtec Medical,
Bromma,
References 1 Onica D, Rosendahl K, Waldenlind L. Inherited occurrence of a heat stable alkaline phosphatase in the absence of malignant disease. Clin Chim Acta 1989;180:23-34. 2 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. 3 Enzyme working group of the Subcommittee on Standards, American Association for Clinical Chemistry, Study group on alkaline phosphatase. A reference method for measurement of alkaline phosphatase activity in human serum. Clin Chem 1983;29:751-761. 4 International Federation of Clinical Chemistry. Expert panel on enzymes. Part 5. IFCC method for alkaline phosphatase (orthophosphoric - monoester phosphohydrolase. alkaline optimum, EC 3.1.3.1). Clin Chim Acta 1983;135:339F_67F.
202 5 Holmgren PA, Stigbrand T. Catalytic properties and stability of three common variants of placental alkaline phosphatase. Biochem Gen 1978;16:433-442. 6 Dixon M. The determination of enzyme inhibitor constants. Biochem J 1953;55:170-171. 7 Millan JL, Stigbrand T. Antigenic determinants of human placental and testicular placental-like alkaline phosphatases as mapped by monocional antibodies. Eur J Biochem 1983;136:1-7. 8 Mulivor RA, Plotkin LJ, Harris H. Differential inhibition of the products of the human alkaline phosphatase loci. Ann Hum Genet 1978;42:1-13. 9 Chuang NN. Alkaline phosphatase in human milk: a new heat stable enzyme. Clin Chim Acta 1987:169:165-174. 10 Nakayama T, Yoshida M, Kitamura M. L-leucine sensitive. heat-stable alkaline phosphatase isoenzyme detected in a patient with pleuritis carcinomatosa. Clin Chim Acta 1970;30:5466548. 11 Fishman WH. Oncotrophoblast gene expression: placental alkaline phosphatase. Adv Cancer Res 1987;48:1-35.
Clinica Chimicu Aria, 194 (1990) 193-202 Elsevier
CCA 04884
Further characterization of a heat-stable alkaline phosphatase with low sensitivity to L-phenylalanine Doina Department (Received
Onica,
Kerstin
of Clinical Chemistry,
17 November
Key words: Alkaline
Rosendahl Karolinska
1989: revision received phosphatase;
and Lennart
Institutet
Waldenlind
at Sijdersjukhuset
10 September
Siockholm
1990; accepted
Heat stability; pH optimum: Monoclonal antibody
K,,
(Sweden)
11 September
1990)
value: L-Phenylalanine:
Summary A heat-stable alkaline phosphatase, hitherto found in two famiIies with inherited hyperphosphatasemia, was further characterized. The enzyme was similar to serum placental alkaline phosphatase from pregnant women concerning its apparent affinity constant (K,,,) for 4-nitrophenyl phosphate and its reactivity with H7 mon~lonal ~ti-placental alkaline phosphatase (PLAP) antibodies, but different in the following respects: it exhibited greater heat stability. a higher pH optimum, lower sensitivity to inhibition by L-phenylalanine, and no reactivity with C2 monoclonal anti-PLAP antibodies. The low sensitivity to L-phenylalanine suggests that the enzyme might correspond to a rare phenotype of placental alkaline phosphatase found in human term placenta.
Introduction We previously reported on the occurrence in a family of an alkaline phosphatase (AP; EC 3.1.3.1) that was similar to the PLAP found in serum during pregnancy in that it had high heat stability (65”C, 5 min) and was reactive with polyclonal anti-PLAP antibodies. However, the enzyme showed a distinct pattern of inhibition by certain amino acids and tripeptides and it also differed from PLAP in its electrophoretic mobility, isoelectric heterogeneity and apparent molecular mass [l]. The purpose of the present investigation was to further characterize the thermal stability, the catalytic and the i~unologic properties of this enzyme and to compare it with a heat stable AP found in another unrelated patient.
Correspondence to: Doina Onica. S-100 64 Stockholm, Sweden.
~09-~981/~/$03.50
Ph.D..
Department
of Clinical
if) 1990 Elsevier Science Publishers
Chemistry.
B.V. (Biomedical
Siidersjukhuset,
Division)
Box 38100.
194
Materials and methods Putients Patient R.S., a 4%year-old, nonpregnant woman, was investigated in the period 1972-1986 for joint pains of unclear origin, coagulation anomalies, and a persistent increase in serum AP (3.6 times higher than the upper limit of the reference range for adults which was 0.8-4.6 pkat/l). Liver function tests (serum y-glutamyltransferase, alanine aminotransferase, aspartate arninotransferase, lactate dehydrogenase, bilirubin), serum electrophoresis, and concentrations of acute phase reactants (orosomucoid, haptoglobin) and of immunoglobulins (IgG, IgA, IgM) were normal. In this patient we identified a heat stable alkaline phosphatase (65”C, 5 min) which represented 88% of the total AP activity. It was repeatedly demonstrated in the patient’s serum and was further characterized in 1986 when the patient was 48-year-old. The enzyme was also found in three other members of the patient’s family. Data concerning the patient and her family have been published previously [ 11. Patient M.K., a 53-year-old, nonpregnant woman, unrelated to the above patient, was investigated in 1987 for an increase in serum AP (2.7 times higher than the upper limit of the reference range for adults) and for diffuse pains. Other laboratory values were within the reference ranges. The elevated AP activity was due to a heat stable alkaline phosphatase (65”C, 5 mm), which represented 85% of the total AP activity.
Serum samples were stored at -70°C. trimester served as PLAP controls.
Sera from pregnant
women
in the third
Chemicals 4-Nitrophenyl phosphate (CNPP), diethanolarnine (DEA), magnesium chloride. magnesium acetate, zinc sulphate, and 2-amino-2-methyl-l-propanol (AMP) were obtained from Merck, Darmstadt, FRG, and N-hydroxyethylethylenediaminetriacetic acid, trisodium salt (HEDTA) from Sigma Chemical Co, St Louis, MO, USA. Enzyme
assa)
Alkaline phosphatase activity was measured by the method recommended by the Scandinavian Society for Clinical Chemistry (SSCC) [2] with some modifications. The assay medium contained 16 mmol/l 4-NPP and 0.5 mmol/l Mg2+ in 1 mol/l DEA buffer, pH 10.0. Enzyme activity was also determined by the reference method of the American Association for Clinical Chemistry (AACC) and the International Federation of Clinical Chemistry (IFCC) [3,4], using 16 mmol/l 4-NPP, 2 mmol/l Mg 2+. 1 mmol/l Zn2+ and 2 mmol/l HEDTA in 0.35 mol/l AMP buffer, pH 10.4.
195
In the assays, we continuously measured the production of 4-nitrophenol at 405 nm and 37°C with an LKB 8600 reaction rate analyzer (LKB, Bromma, Sweden). The reaction was initiated by adding substrate, and the volume fraction of the sample was kept the same in all methods as 0.0116 (1 : 86) according to the instructions of the manufacturer for the kinetic AP test, using the LKB 8600 analyzer.
Electrophoresis
and isoelectric focusing
Electrophoresis was performed in 1.5-mm layers of 0.9% (w/v) agar gel in 0.082 mol/l sodium barbital-HCl buffer, pH 8.4, as previously described [l]. Isoelectric focusing was performed as previously described [l] in ready-made Ampholine PAG plates (245 x 110 X 1 mm) (LKB. Bromma, Sweden), pH range 4.0-6.5 containing 5% polyacrylamide and 2.2% ampholine (w/v).
pH optimum pH optimum was determined and at a substrate concentration
in 1 mol/l DEA buffer of 16 mmol/l 4-NPP.
in the pH range 9.0-12.0
Thermostability Heat inactivation curves were established for whole sera diluted fourfold with 0.154 mol/l NaCl. Samples of 1.5 ml in stoppered 10 X 55-mm glass tubes were incubated in a water bath at 70°C and 79“C. These temperatures were chosen according to Holmgren and Stigbrand [5]. Portions of 100 ~1 were removed every 2.5 min during a 30-min period and cooled in an ice bath. Residual AP activity was then determined by the SSCC method in 1 mol/l DEA buffer, pH 10.0. Plots of the logarithm of percentage residual AP activity versus time were biphasic for all sera examined. The first part of the curve was assumed to correspond to the inactivation of labile nonplacental isoenzymes and the latter part to the inactivation of the stable AP enzymes. The half-life of the stable AP was calculated from the latter, linear part of the inactivation curve, the equation of which was determined by regression analysis. In the calculation only the O-20 min time interval was included, since after 20 min of incubation the thermal inactivation curve seemed to deviate from linearity at least for the AP activity in patients’ samples.
Determination
of K,
values
In the enzyme assay using either 1 mol/l DEA buffer, pH 10.0, or 0.35 mol/l AMP buffer, pH 10.4, the concentration of 4-NPP was varied from 0.4 to 8 mmol/l. Linear regression analysis was used for the Lineweaver-Burk double reciprocal plotting of reaction velocities versus substrate concentrations.
196
Inhibition
studies
Inhibition constants ( Ki) were determined as described by Dixon [6], in both 1 mol/l DEA buffer, pH 10.0, and 0.35 mol/l AMP buffer, pH 10.4. Two substrate (CNPP) concentrations (0.6 and 16 mmol/l) and six inhibitor (L-phenylalanine) concentrations (0.5510 mmol/l) were used. Enzyme
immunoassay
(EIA) for PLAP
The activities of PLAP in serum were determined with the Prolifigena PLAP-EIA kit (Sangtec Medical, Bromma, Sweden). Serum samples (50 ~1) were added to a microtiter plate precoated with the H7 or C2 monoclonal antibodies reacting with PLAP. After overnight incubation (17 h) at room temperature, the plate was washed three times with 10 mmol/l Tris buffer, pH 7.4, containing 1 mmol/l Mg2+, 0.154 mol/l NaCl, 15 mmol/l sodium azide and 5 g/l bovine serum albumin. The enzymatic activity of the bound PLAP was determined by adding 100 ~1 of substrate solution containing 10 mmol/l 4-NPP, 0.5 mmol/l Mg2+ and 15 mmol/l sodium azide in 1 mol/l DEA buffer, pH 9.8, according to the instructions of the manufacturer. After 1 h incubation at 37°C the absorbance at 405 nm was measured. Results The electrophoretic and isoelectric properties of the heat stable AP found in sera from patients R.S. and M.K. were apparently the same (Figs. 1 and 2). The pH optimum was found to be identical (10.8) for the patients’ enzymes, while for PLAP from pregnant women (n = 7) it was 10.4-10.6, in the presence of 1 mol/l DEA buffer and 16 mmol/l 4-NPP at 37°C. (Fig. 3). The sera were heat treated at 65°C for 5 min before the assays in order to eliminate the nonplacental isoenzymes.
L B/PLAP I abcdef
9
h
Fig. 1. Electrophoretic separation in agar gel of AP isoenzymes in sera from patients and in control sera. The gel was stained for enzyme activity. a, Liver and intestinal AP; b, bone AP; c, d, pregnant woman; e, f, patient R.S.; g, h, patient M.K. Sera in d, f and h were heat treated at 65°C for 5 min. The approximate positions of various isoenzymes are given: L, liver AP; B, bone AP; I, intestinal AP; PLAP, placental AP. The arrow indicates the start position.
197
PH m-3.65 *- 3.95 a-4.25 -4.75 -4.85 -4.95
*- 5.25
f
e--5.35
abcdef Fig. 2. Isoelectric focusing stained for enzyme activity.
in polyacrylamide gel of serum AP isoenzymes at pH 4.0-6.5. The gel was a, b, patient M.K.; c, d, pregnant woman; e, f, patient R.S. Sera in b, d and f were heat treated
at 65°C for 15 min.
Fig. 3. Effect of pH on the heat stable AP from patient R.S. (Ol) and patient M.K. (A. . . .A), and on PLAP from a pregnant woman (O0). Serum samples were heat treated (65”C, 5 min) before the measurements. The assay was performed in 1 mol/l DEA buffer and at a substrate concentration of 16 mmol/l4-NPP. The AP activity at pH 10.0 was considered to be 100%.
198
70’ c 70’ c
70‘ c
79’ c 79. c
5’ 0
5
10
15
20
25
30
Time (min) Fig. 4. Effect of heat treatment on AP activity in serum from patient R.S. (0 -0) and patient M.K. (r------r), and from a pregnant woman (a -•). The sera were diluted fourfold with 0.154 moi/l NaCl before heat treatment. For patient M.K. the measuring interval was 5 min instead of 2.5 min.
The enzymes from the two patients were more heat stable than PLAP from pregnant women at 79OC (Fig. 4). The half-lives for the enzymes from patient R.S. and patient M.K. were similar, 20.5 f 1.3 min (mean i SE, 7 determinations) and 23.0 i 2.0 mm (6 deter~nations), respectively, while PLAP from pregnant women exhibited half-lives of 8-11 min (n = 12). At 70°C both the patients’ enzymes and PLAP from pregnant women were stable. The apparent affinity constants (K, ) of the patients’ enzymes were similar both in the DEA buffer, pH 10.0 and in the AMP buffer, pH 10.4. No distinct differences in K, could be seen between the patients’ enzymes and PLAP (Table I).
TABLE
I
K, values (mmol/l) for 4-nitrophenyl from pregnant women a
Patient R.S. h Patient M.K. PLAP =
phosphate
of heat stable AP enzymes
in sera from patients and
DEA buffer, pH 10.0
AMP buffer, pH 10.4
0.37 0.40 0.41 f 0.05
0.37 0.37 0.51+ 0.07
’ All sera were heat treated at 65°C for 5 mm before the kinetic measurements. b Mean value for sera from the patients (3-5 dete~ations). ’ Mean valuef SD for sera from pregnant women (a = 12 in DEA buffer and n = 6 in AMP buffer).
199
TABLE
II
Inhibition women a
by L-phenylalanine
of heat
stable
AP enzymes
in sera from
patients
and
from
pregnant
Ki (mmol/l)
Patient R.S. a Patient M.K. PLAP ’
DEA buffer, pH 10.0
AMP buffer,
6.7 7.0 2.1kO.2
8.9 10.8 2.8 + 0.3
pH 10.4
-
a All sera were heat treated at 65°C for 5 min before the inhibition experiments. b Mean value for sera from the patients (2 determinations). ’ Mean value+ SD for sera from pregnant women (n = 6 in DEA buffer and n = 3 in AMP buffer).
TABLE Reactivity women
III of H7 and C2 monoclonal
anti-PLAP
Alkaline
Patient R.S. Patient M.K. Pregnant women
antibodies
phosphatase
with sera from patients
activity
and from pregnant
(pkat/l)
Reactive with H7 antibodies
Reactive with C2 antibodies
11.1 8.8 3.8 0.7 1.2 2.5 1.1 2.6 2.7 1.1 1.9 1.1 1.4
0.008 0.026 3.7 0.7 1.2 2.3 1.1 2.2 2.5 1.0 1.8 1.0 1.3
The inhibition constants (Ki) for L-phenylalanine were also similar for the patients’ enzymes (Table II). However, the enzymes differed from PLAP since they were less inhibited by L-phenylalanine both in the DEA and in the AMP buffer. The patients’ enzymes displayed practically no reaction with the C2 monoclonal anti-PLAP antibodies, while PLAP from pregnant women showed a strong reaction. The H7 monoclonal anti-PLAP antibodies reacted with both the enzymes and PLAP (Table III). Discussion The enzymes from the two patients, who belong to different families, had similar biochemical (apparent affinity constant for 4-NPP, pH optimum, sensitivity to
TABLE
IV
Comparison of the properties reviewed by Fishman [II] Properties
of the patients’
enzymes
alkaline
phosphatases
as
lsoenzymes PLAP (term placenta/Regan)
Molecular mass pH optimum Heat stability 65’C, 5 min 70°C 79°C. t l/2 (min) Inhibition by L-Phenylalanine (5 mmol/l) L-Homoarginine (5 mmol/l) L-Leucine (Smmol/l) Neuraminidase sensitivity Reaction with antibodies Polyclonal anti-PLAP H7 Monoclonal anti-PLAP C2 Monoclonal anti-PLAP
with those of placental
PLAP-like AP (testis, thymus/Nagao)
Patients’
128000 10.6
130000 10.6
140000 10.X
stable stable U-11
stable n.d. n.d.
stable stable 20.5-23.0
+++
tit
t
+
*
*
+ sensitive
it+
(+)
sensitive
sensitive
reactive reactive some variants reactive
reactive reactive non-reactive
reactive reactive reactive
’ The origin of the enzymes is indicated in parentheses: sociated enzyme. ’ The properties of patients’ enzymes are summarized
a
normal
are
tissue/name
enzymes
of the respective
on the basis of the data presented
h
tumor-as-
in this and a
previous paper [l]. n.d.. not determined.
L-phenylalanine), electrophoretic, isoelectric, thermal and immunologic properties. These results suggest that the enzymes are the same. In Table IV the properties of the patients’ enzymes are compared with those of PLAP and PLAP-like AP of normal and tumor tissue origin. The reactivity of the enzymes from patient R.S. [l] and patient M.K. (unpubl. data) with polyclonal anti-PLAP antibodies, and with H7 monoclonal anti-PLAP antibodies, which have been shown by Millan and Stigbrand [7] to react both with PLAP and PLAP-like AP, indicates that the enzymes from the two patients are alkaline phosphatases of the placental type. However, they differed from PLAP by the lack of reactivity with C2 monoclonal anti-PLAP antibodies, which are known to react with the common phenotypes of PLAP [7]. This result suggests that there are differences in the antigenic properties of the patients’ enzymes and common PLAP. The patients’ enzymes and PLAP from pregnant women have quite similar active the same. sites, as shown by their K, values for 4-NPP, which were essentially
201
However, the patients’ enzymes were different from PLAP in the following respects: they exhibited greater heat stability, a higher pH optimum and lower sensitivity to inhibition by L-phenylalanine. It seems that the patients’ enzymes and PLAP, although related, may be structurally different. In our previous study we also showed that the enzyme from patient R.S. has a lower susceptibility than PLAP to inhibition by the tripeptides L-leucyl-glycyl-glycine and L-phenylalanyl-glycylglycine. The great heat stability and the inhibition profile suggest that the patients’ enzymes are a variant of PLAP, with a relatively low sensitivity to L-phenylalanine, which might correspond to a rare phenotype of PLAP found in human term placenta [8]. An L-phenylalanine-insensitive, heat stable alkaline phosphatase has also been found in human milk [9]. The relatively low sensitivity of the enzyme from patient R.S. [l] and patient M.K. (unpubl. data) to L-leucine, indicates that the enzymes are not of the PLAP-like AP (Nagao) type [lo]. Hitherto we have found the heat stable alkaline phosphatase in four members of the family of patient R.S., as previously reported [l], and in patient M.K. (this study), who is not related to the first family. Preliminary results indicate that the occurrence of the enzyme in patient M.K. is also inherited, since a small amount of it has been found in the serum of her daughter (unpubl. data). All these persons were apparently healthy, except patients R.S. and M.K., who were suffering from joint pains of unclear origin. There was no history of cancer in any of the persons concerned. Since an elevated level of PLAP or PLAP-like AP will alert the clinician to consider malignancy in a nonpregnant patient, it is clinically important that among the causes of increased AP activity, the possibility of a familial increase in AP of the placental type, not associated with malignant disease, should be kept in mind. The occurrence of the enzyme by inheritance in patients without malignant disease suggests that the regulation of its biosynthesis is different from that of placental phosphatases (Regan and Nagao isoenzymes), which are tumor-associated and thought to be products of derepressed oncotrophoblast genes [ll]. Acknowledgement The Prolifigen@ PLAP-EIA Sweden.
kit was kindly provided
by Sangtec Medical,
Bromma,
References 1 Onica D, Rosendahl K, Waldenlind L. Inherited occurrence of a heat stable alkaline phosphatase in the absence of malignant disease. Clin Chim Acta 1989;180:23-34. 2 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. 3 Enzyme working group of the Subcommittee on Standards, American Association for Clinical Chemistry, Study group on alkaline phosphatase. A reference method for measurement of alkaline phosphatase activity in human serum. Clin Chem 1983;29:751-761. 4 International Federation of Clinical Chemistry. Expert panel on enzymes. Part 5. IFCC method for alkaline phosphatase (orthophosphoric - monoester phosphohydrolase. alkaline optimum, EC 3.1.3.1). Clin Chim Acta 1983;135:339F_67F.
202 5 Holmgren PA, Stigbrand T. Catalytic properties and stability of three common variants of placental alkaline phosphatase. Biochem Gen 1978;16:433-442. 6 Dixon M. The determination of enzyme inhibitor constants. Biochem J 1953;55:170-171. 7 Millan JL, Stigbrand T. Antigenic determinants of human placental and testicular placental-like alkaline phosphatases as mapped by monocional antibodies. Eur J Biochem 1983;136:1-7. 8 Mulivor RA, Plotkin LJ, Harris H. Differential inhibition of the products of the human alkaline phosphatase loci. Ann Hum Genet 1978;42:1-13. 9 Chuang NN. Alkaline phosphatase in human milk: a new heat stable enzyme. Clin Chim Acta 1987:169:165-174. 10 Nakayama T, Yoshida M, Kitamura M. L-leucine sensitive. heat-stable alkaline phosphatase isoenzyme detected in a patient with pleuritis carcinomatosa. Clin Chim Acta 1970;30:5466548. 11 Fishman WH. Oncotrophoblast gene expression: placental alkaline phosphatase. Adv Cancer Res 1987;48:1-35.