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Placental-type alkaline phosphatase from human tumour tissue
CLINICA CHIMICA ACTA
473
CCA 4689
PLACENTAL-TYPE
ALKALINE
PHOSPHATASE
FROM
HUMAN
TUMOUR
TISSUE*
B. JACOBT
AND I<. D. BAGSHAWE
Edgar Laboratory, Britain,) (Received
Char&g
June 14,
Cvoss (Fulham)
Hospital,
St. Dunstan’s
Road, London,
IT’ 6 8RF.
(Great
1971)
SUMMARY
which
An alkaline phosphatase was purified from a primary tumour of the bile duct had metastasised throughout the liver. Its properties were compared with
those of purified placental alkaline phosphatase. Both phosphatases were precipitated by rabbit antiplacental phosphatase antiserum, and behaved similarly on electrophoresis and Sephadex G-zoo chromatography, but the tumour enzyme was more sensitive to L-leucine and ethylene diamine tetra-acetic acid. Although both were stable at 56” at neutral pH, the tumour phosphatase was inactivated with time at pH 11.0. At pH 11.0 and 65” it was the more rapidly inactivated of the two, and appeared to be heterogeneous. Both the tumour phosphatase and a heat-stable phosphatase from the patient’s serum shared the same properties. They were characterised as heatstable and L-phenylalanine-sensitive,
but distinguishable
from placental
phosphatase.
INTRODUCTION
Placental-type alkaline phosphatase has been found in a wide variety of malignant tumours, and in HeLa cells1-4. Fishman et al.5 have suggested that these variants are produced by the tumour tissues themselves. The production of a foetal-type enzyme in adult life may be due to de-repression of the appropriate genes in the cancer celll. With more purified preparations it has been shown that these alkaline phosphatases are similar to, but not identical with, the placental phosphatase2,6. Although these findings are still compatible with a process of genetic de-repression taking place, it is clearly of great interest to further characterise the enzyme variants produced. This report is concerned with the properties of one such tumour phosphatase of the placental type.
* Supported by grants from the Medical Research Council and the Charing Cross Hospital Clinical Research Sub-Committee. Clin. Chim.
Acta,
35 (1971) 473-41
474
JACOBY,
MATERIALS
AND
BAGSHAWE
METHODS
diamine tetra-acetic acid (EDTA) obtained from Sigma (London), DEAE cellulose (DE 52) and Sephadex G-ZOO were obtained from H. Reeve Angel and Co. and Pharmacia (GB) Limited, respectively. and
L-leucine, D- and L-phenylalanine, ethylene z-amino-z-methyl-I-propanol (AMP) were
Tumour sample AAsample of serum was obtained from a patient (J.W.B.) with a poorly differentiated adenocarcinoma of the bile duct with metastases throughout the liver. At autopsy a sample was taken from the liver and stored frozen until used. A sample of the patient’s liver tissue was thawed out and about 5 g(wet weight) was homogenized (Virtis 45) at low speed in 50 ml 0.25 M sucrose, 20% v/v in nbutanol according to Sussman’. The aqueous extract was precipitated at -20' by acetone (6096 v/v) and re-dissolved and dialysed against 0.01 M tris-HCl buffer pH 7.4 containing 0.002 M magnesium chloride. A 2 ml sample was then applied to a DEAE cellulose column (1.6 x 60 cm.) and eluted at 18 ml per hour (9 ml/cm2/h) by pumped downward flow using a Harvard Multispeed Transmission Pump. A salt gradient was provided by linking two beakers of this buffer, with and without 0.3 M potassium chloride, and 5 ml fractions were collected on an LKB 7000 UltroRac fraction collector. After assay for enzyme activity, fractions containing the peak were pooled and stored. The yield at this stage was lower than expected and it is possible that this loss occurred at the acetone precipitation stage. The placental alkaline phosphatase used in this study was prepared from three normal term placentae using butanol extraction followed by ammonium sulphate fractionation, methanol precipitation and DEAE cellulose and Sephadex G-200 chromatography8. The final specific activity of this preparation was 294 pmoles per minute per mg protein at 25’, assayed as below. The placental phenotypes9 were not determined. Crude extracts were made from samples of normal liver, small intestine and bone using the femur of a child, with 20% v/v butanol in 0.25 M sucrose. The aqueous layer was removed
and stored
frozen.
Enzyme assays. Assays were performed in 0.07 M AMP-HCl buffer pH 10.0, 0.004 RI disodium $-nitrophenyl phosphate as substrate and 0.01 M magnesium chloride. It should be noted that the AMP buffers used in this work are quite temperature-sensitive (dpH per “C = 0.03)10 and the indicated pH values were those at room temperature (23”). D- and L-phenylalanine (0.05 ml final concentration 0.005 M) were added, where indicated, and the reaction started by the addition of 0.1 ml of serum or extract making a total volume of 1.5 ml. After IO or 30 min an equal volume of 0.25 N sodium hydroxide was added to stop the reaction, the tubes centrifuged, and the optical density of the supernatants at 410 nm was read on a Unicam SP 500 spectrophotometer. These were compared with controls in which the sodium hydroxide was added before the enzyme. The reaction was linear during the measured time interval. The heat stable activity was defined as that fraction of phosphatase which remained after the sample
TUMOUR
ALKALINE
PHOSPHATASE
had been heated at neutral one hour at 56” & 0.2' EDTA used in this study of activity is defined as a r.o/min, or as the number 25" (specific activity).
475
pH (incubated
with pre-heated
normal human serum) for
(SBz water bath, Grant Instruments, Cambridge). The was adjusted to pH 9.5 with sodium hydroxide. One unit change in the optical density at 410 nm for I cm cuvette of of qoles substrate transformed per min/per mg protein at
Electroplaoresis A vertical flat-bed slab of 5% polyacrylamide was used in tris-borate buffer, pH 9.511. The gel was stained for activity with @-naphthylphosphate and fast blue BB’2. Antiserum preparation Approximately I mg of placental phosphatase, purified as described, was homogenised (Virtis 45) with I ml of Freund’s Complete adjuvant (Difco Limited) and injected at multiple intramuscular sites into three Half-Lop rabbits. Two further injections were given at two weekly intervals, followed by a final injection of 0.3 mg protein in saline, and after seven weeks from the start the rabbits were bled by cardiac puncture and the serum obtained, stored at -13’. The low level of phosphatase present in the antiserum could be destroyed by heating at 56”. Antiserum used for double diffusion was absorbed out with normal human liver tissue. Double diffusion Immunodiffusion
was carried out in 1.5% Ion-Agar
sodium azide as preservative. temperature, or after overnight
(Oxoid Limited)
with 0.02%
The gel was photographed after two days at room washing in saline followed by staining for phosphatase.
Phosphatase precipitation by antiserum. At a final dilution of I :1,000, rabbit antiserum was incubated with enzyme in a volume of 0.1 ml. After I h at 37O and 96 h at 4’ the tubes were centrifuged at 28,000 xg (Griffin-Christ Microhaematocrit with angle head) for 15 min, cold, and samples of 0.04 ml were assayed in duplicate for enzyme activity. These were compared with controls containing
normal rabbit serum at the same dilution.
RESULTS
A placental-type alkaline phosphatase was isolated by Nakayama2 from the serum and the pleural fluid of a patient with “pleuritis carcinomatosa”. Although like placental phosphatase, it was stable when heated at 65”, the tumour phosphatase was more sensitive to the presence of both EDTA and L-leucine. These findings were confirmed with the tumour phosphatase used in this study. When assayed in the presence of L-phenylalanine (5 x IO@ M) the activities of both the tumour and placental phosphatases were only 8.5% and 8.4% respectively of those in the presence of the non-inhibitor D-phenylalanine. However, comparing activities, in the presence and absence of L-leucine (5 x IO-~ M) whilst the placental phosphatase retained 84% of its activity, that of the tumour enzyme was reduced to 38.5%. At this low concentration of L-leucine its activity was thus more strongly CZin. Chim. Acta, 35 (1971) 473-481
476
JACOBI’,
BAGSHAWE
ioa
Percentage actlvlty lo-4M=lOO%
5a
C
1o-4
lo-3 EOTA
10-z
Concentratlon(Molar)
Fig. I. Effect of EDTh on purified placental phosphatase in buffer (-:‘-) and in heated normal serum (-•-). and on purified tumour extract (-A-) and heated “turnour” serum (-A-). EDTA, adjusted to pH 9.5 with sodium hydroxide, was added to the final concentrations shown and phosphatase activity was assayed in the absence of added magnesium chloride. i\ctivities were compared with those at I x IO-~ M EDTA in order to allow for the effects of endogcnous magnesium ion.
inhibited. A similar effect was seen in the greater susceptibility of the tumour phosphatase to the chelating agent, EDTA (Fig. I). The enzyme activity at a final EDTA concentration of I x IO-* M was taken to be IOO~/~in order to allow for low endogenous levels of magnesium ion in the samples. The phosphatase activities of the purified tumour enzyme and of the patient’s heated serum were compared with those of placental phosphatase sampled in the absence and presence of heated normal serum. Samples were heated at 56” for I 11in stoppered tubes on a water bath. Aliquots were removed at intervals and assayed at room temperature for residual activity. Neither the pre-heated extract, nor the purified tumour phosphatase showed any loss of activity. The patient’s serum had a total phosphatase activity of 0.252 pmoles substrate transformed per min per ml and 25.20/o of this (0.064 ~moles/min/ml) was heat stable. These values correspond approximately to 87.0 and 21.9 King-Armstrong Units per IOO ml of serum, but it should be noted that the conditions used were not those of the King-Armstrong procedure. When the stability to heat at 56” was measured at pH 11.0 instead of at neutral pH there was a marked difference of response, the tumour enzyme being inactivated with time (Fig. z) whilst the placental phosphatase activity remained relatively stable. The residual activities were measured at pH 10.0 and at room temperature. In the Clb~.
Chzw.
Acta,
35 (1971)
473-@I
TUMOUR
ALKALINE
PHOSPHATASE
477
A
Percentage activity remaining
50
au Minutes Fig. 2. Heat inactivation at pH I I and 56” of placental phosphatase in buffer (-C-) and heated in normal serum (-•-), and of purified tumour phosphatase (-A-) and heated “tumour” serum (-A-). Samples, 0.02 ml, were heated in 0.1 M AMP buffer in a total volume of 0.15 ml in stoppered tubes. Samples were withdrawn at the time intervals shown, cooled to room temperature, and Methods” section for details). and assayed for residual activity (see “Materials
Time
(minutes)
Fig. 3. Heat inactivation of tumour and placental phosphatases and symbols are as given in the legend to Fig. 2.
at pH I I and 65”.The
Clin. Chzm. Ada,
assay method
35 (1971) 473-481
JACOBT, BAGSHAWE
Enzyme
actwty
Ezeonm
unots per ml.
I.0
Eluted fractions
(ml.)
Fig. 4. Gel filtration of purified tumour phosphatase on Sephadex G-zoo. A 1.0 ml sample was applied to a 1.6 x 60 cm column and eluted by upward flow at 4.6 ml per cm2 per hour. Fractions (4.6 ml) were collected on an LKB UltroRac 7000 collector and assayed for enzyme activity (-O-). Protein (-•-) was estimated by absorption at 280 nm. The main protein peak consisted of bovine serum albumin, added as a marker. In a separate run under the same conditions, 1.0 ml of a 0.27/o solution of Blue Dextran 2000 (Pharmacia) was eluted from the column to mark the
exclusion limit (--o-).
same incubation buffer (at pH 11.0) but heating at 65” both phosphatases showed an irreversible inactivation with time (Fig. 3) but whilst the placental activity decayed at a uniform rate, that of the tumour enzyme showed a biphasic curve; a rapid decay to 20% of the original activity followed by a slower inactivation of the remainder at a rate comparable with that of the placental enzyme. It has been notedI that this type of response may be found where the enzyme involved is heterogeneous, consisting of two or more components. On gel filtration using a 1.6 x 60 cm column of Sephadex G-200 (Fig. 4) the purified tumour enzyme was eluted by upward flow as a single peak of activity, followed by a peak of bovine serum albumin added as a marker. Under the same conditions the placental phosphatase was eluted as an identical peak, together with some higher molecular-weight material which was eluted just under the void volume, that is, before the main peak. When these samples were subjected to electrophoresis on a vertical flat-bed acrylamide gel11 at pH 9.5 and stained for enzyme activity (Fig. 5) the placental phosphatase appeared as three closely-spaced bands9 with the tumour enzyme showing a more diffuse band in the same position. A sample of the higher molecularweight material eluted from the Sephadex column had barely entered the main part of the gel, whilst the phosphatase of a butanol extract of liver tissue migrated the furthest. Clin.
Chum.
Acta,
3j
(1971)
47X-481
TUMOX’R
ALKALINE
479
PHOSPHATASE
The purified tumour and placental phosphatases were both precipitated by rabbit antiserum to human placental alkaline phosphatase at a final dilution in saline of I : I OOO. The supernatant activity was assayed relative to that of controls with
Fig. 5. Vertical slab electrophoresis at pH 9.5 on 5% acrvlamide gel” stained for phosphatase activity-. Phosphatases from liver (I and 5), placenta (a and 3) and the tumour sample (4) were applied at the origin and run for one hour. The placental samples consisted of the main peak (2) and the high molecular-weight phosphatase (3) &ted from a Sephadex G-200 column.
Fig. 6. Immuno-diffusion in agar gel. After two days incubation at room temperature the get was washed overnight in saline and stained for phosphatase activity. In the centre wells were normal rabbit serum (lower left), and antiserum to placental phosphatase (upper right). The peripheral wells wxc loaded with 0.035 ml of phosphatnse extract, from the tnmour sample (I), placenta (2 and 6), bone (3), liver (4) and intestine (j). Cain. Ckim.
Acta,
35
(1971)
~$73-.@1
480
JACOBY,
normal rabbit
serum added. The assays, in duplicate,
BAGSHAWE
showed that in both cases 5.8%
and 6.0% of the activity remained. The phosphatases of bone and liver origin are known not to react with anti-placental antiserum whilst that of intestinal origin is only precipitated at higher antiserum concentrations (final dilution I : 150. Unpublished observation). On double diffusion in agar against the absorbed antiserum, a weak precipitation line developed, common to that of placental phosphatase. Although the most concentrated sample was used, the phosphatase concentration was still well below that of the purified placental enzyme. The immunological activity was better seen when an agar diffusion plate which has been washed overnight in saline to remove nonprecipitated material was then stained for enzyme activity (Fig. 6). Tumour, placental and intestinal phosphatases showed a definite interaction towards rabbit antiserum, whilst bone and liver phosphatases did not. None of the samples showed any reaction when put up against normal rabbit serum.
The purified tumour phosphatase did not differ in any of the properties tested from the heat-stable enzyme in the patient’s serum. It therefore seems unlikely that the purification procedure was itself responsible for the difference of properties between this and the placental phosphatase. Although on agar diffusion the tumour phosphatase could not be distinguished from that of placental or intestinal origin, it was, unlike the intestinal enzyme, stable to heating at 56”. It did not migrate to the intestinal position on electrophoresis, and resembled the placental enzyme on Sephadex gel filtration. Studies of phosphatases from tumour tissue2p6 and from HeLa cells4$15 have shown these to differ from placental phosphatase. From the results of the present study it may be possible to speculate about the extent of these changes. Both phosphatases behaved similarly on Sephadex gel filtration, polyacrylamide electrophoresis and reacted similarly with rabbit antiserum, but the tumour phosphatase activity was more susceptible to the effect of L-leucine and EDTA. However, it was the inactivation of the tumour phosphatase by heating at an alkaline pH which showed the difference most clearly and which suggested a heterogeneity of response (Fig. 3). Thus whilst the overall molecular size and charge of the enzyme are not greatly changed and it is still recognized by rabbit antiserum, it is the stability which is reduced, and even small structural alterations could account for such changes. There remains the question of whether the tumour phosphatases from different patients show the same characteristic properties. The fact that we have been able to confirm two of the findings of the Japanese workers2 (the sensitivity to L-leucine and to EDTA) at least suggests that an alkaline phosphatase with consistent properties may be produced by a number of tumours. It is hoped that the method of measuring heat inactivation at alkaline pH might give meaningful results in this respect. The phosphatase from the patient in this study was originally identified as being of the placental-type through the use of an automated enzyme assay system. An account of this technique will be published elsewhere (R. C. Jennings, personal communication).
Clin. Chim.
Acta,
35 (1971)
473-481
TUMOUR
ALKALINE
PHOSPHATASE
481
ACKNOWLEDGEMENTS
We thank Dr. R. C. Jennings, Mr. D. Brocklehurst and Mr. hf. Hirst of the Department of Pathology, The General Hospital, Altrincham, Cheshire, for the tumour tissue and serum sample and Mr. R. Myers and Dr. A. W. Walker of the Department of Chemical Pathology, Fulham Hospital, London, for the polyacrylamide electrophoresis and the butanol extracts of human bone, intestine and liver. REFERENCES I 2 3 4 5
W.H. FISHMAN,N. I.INGLIS,L.L.STOLBACHANDM. J. KRAST,C~~CW Reseavch, z8(rg68)15o. T. NAKAYAMA, M. YOSHIDA AND M. KITAMURA,CX~. Chim. Acta, 30 (1970) 546. G. hf.mIC AP*'DA. G. RIEGO, Ann. Biol. Chim., 26 (1970) 2x9. N. A. ELSON A~XD R. P. Cox, B&hem. Genet., 3 (1969) 549. W. H. FISHMA~,N.I.INGLIS,S.GREEX,C.L.XNSTISS,N. K.GHOSH, A.E. REIF, KRUSTIGAX,
M.J. KRANTAXD L.L.STOLBACH,N~~W~,ZI~(~~~~)~~~. 6 W. H. FISHMAN, Ann. N.Y. Acad. Sci., 166 (1969) 745. 7 H.H. SUSSMAN, P. A.SMALL JR. ANDE.COTLOVE,~.B~O~. Chem., x+3(1968) 8 D. R. HARKNESS, Arch.Biochem.Biophys., 126 (1968)503. 9 E.B.ROBSON AXD H.HARRIS, Nature, 207(1965) 1257. IO G. N. BOWERS, JR. AR'D R. B.McCOMB,CZ~~. Chem., IZ (1966) 70, I I A. W. WALKER AXD A. C. POLLARD, Clin. Ckim.
12 13 14 1j
Acta,
160.
23 (1969) 270.
I.%ITH, P. J.LICHTSTOXE AND J.D.PERRY,C~~~. Chim. Acta, zj(1969) E.J. KING AND A. R. ARMSTRONG, Canad.Med. Assoc.J., 3~ (1934) 376. J. A. GORMAS ASD A. S. L. Hu,J. Biol.Chem., 244(1g69) 1615. K. I<. TAN ASD S. E. An,, Biochim. Biophys. Acta, 23j (1971) 119. Clin.Chim.
17.
Acta,
35 (1971)
473-481
473
CCA 4689
PLACENTAL-TYPE
ALKALINE
PHOSPHATASE
FROM
HUMAN
TUMOUR
TISSUE*
B. JACOBT
AND I<. D. BAGSHAWE
Edgar Laboratory, Britain,) (Received
Char&g
June 14,
Cvoss (Fulham)
Hospital,
St. Dunstan’s
Road, London,
IT’ 6 8RF.
(Great
1971)
SUMMARY
which
An alkaline phosphatase was purified from a primary tumour of the bile duct had metastasised throughout the liver. Its properties were compared with
those of purified placental alkaline phosphatase. Both phosphatases were precipitated by rabbit antiplacental phosphatase antiserum, and behaved similarly on electrophoresis and Sephadex G-zoo chromatography, but the tumour enzyme was more sensitive to L-leucine and ethylene diamine tetra-acetic acid. Although both were stable at 56” at neutral pH, the tumour phosphatase was inactivated with time at pH 11.0. At pH 11.0 and 65” it was the more rapidly inactivated of the two, and appeared to be heterogeneous. Both the tumour phosphatase and a heat-stable phosphatase from the patient’s serum shared the same properties. They were characterised as heatstable and L-phenylalanine-sensitive,
but distinguishable
from placental
phosphatase.
INTRODUCTION
Placental-type alkaline phosphatase has been found in a wide variety of malignant tumours, and in HeLa cells1-4. Fishman et al.5 have suggested that these variants are produced by the tumour tissues themselves. The production of a foetal-type enzyme in adult life may be due to de-repression of the appropriate genes in the cancer celll. With more purified preparations it has been shown that these alkaline phosphatases are similar to, but not identical with, the placental phosphatase2,6. Although these findings are still compatible with a process of genetic de-repression taking place, it is clearly of great interest to further characterise the enzyme variants produced. This report is concerned with the properties of one such tumour phosphatase of the placental type.
* Supported by grants from the Medical Research Council and the Charing Cross Hospital Clinical Research Sub-Committee. Clin. Chim.
Acta,
35 (1971) 473-41
474
JACOBY,
MATERIALS
AND
BAGSHAWE
METHODS
diamine tetra-acetic acid (EDTA) obtained from Sigma (London), DEAE cellulose (DE 52) and Sephadex G-ZOO were obtained from H. Reeve Angel and Co. and Pharmacia (GB) Limited, respectively. and
L-leucine, D- and L-phenylalanine, ethylene z-amino-z-methyl-I-propanol (AMP) were
Tumour sample AAsample of serum was obtained from a patient (J.W.B.) with a poorly differentiated adenocarcinoma of the bile duct with metastases throughout the liver. At autopsy a sample was taken from the liver and stored frozen until used. A sample of the patient’s liver tissue was thawed out and about 5 g(wet weight) was homogenized (Virtis 45) at low speed in 50 ml 0.25 M sucrose, 20% v/v in nbutanol according to Sussman’. The aqueous extract was precipitated at -20' by acetone (6096 v/v) and re-dissolved and dialysed against 0.01 M tris-HCl buffer pH 7.4 containing 0.002 M magnesium chloride. A 2 ml sample was then applied to a DEAE cellulose column (1.6 x 60 cm.) and eluted at 18 ml per hour (9 ml/cm2/h) by pumped downward flow using a Harvard Multispeed Transmission Pump. A salt gradient was provided by linking two beakers of this buffer, with and without 0.3 M potassium chloride, and 5 ml fractions were collected on an LKB 7000 UltroRac fraction collector. After assay for enzyme activity, fractions containing the peak were pooled and stored. The yield at this stage was lower than expected and it is possible that this loss occurred at the acetone precipitation stage. The placental alkaline phosphatase used in this study was prepared from three normal term placentae using butanol extraction followed by ammonium sulphate fractionation, methanol precipitation and DEAE cellulose and Sephadex G-200 chromatography8. The final specific activity of this preparation was 294 pmoles per minute per mg protein at 25’, assayed as below. The placental phenotypes9 were not determined. Crude extracts were made from samples of normal liver, small intestine and bone using the femur of a child, with 20% v/v butanol in 0.25 M sucrose. The aqueous layer was removed
and stored
frozen.
Enzyme assays. Assays were performed in 0.07 M AMP-HCl buffer pH 10.0, 0.004 RI disodium $-nitrophenyl phosphate as substrate and 0.01 M magnesium chloride. It should be noted that the AMP buffers used in this work are quite temperature-sensitive (dpH per “C = 0.03)10 and the indicated pH values were those at room temperature (23”). D- and L-phenylalanine (0.05 ml final concentration 0.005 M) were added, where indicated, and the reaction started by the addition of 0.1 ml of serum or extract making a total volume of 1.5 ml. After IO or 30 min an equal volume of 0.25 N sodium hydroxide was added to stop the reaction, the tubes centrifuged, and the optical density of the supernatants at 410 nm was read on a Unicam SP 500 spectrophotometer. These were compared with controls in which the sodium hydroxide was added before the enzyme. The reaction was linear during the measured time interval. The heat stable activity was defined as that fraction of phosphatase which remained after the sample
TUMOUR
ALKALINE
PHOSPHATASE
had been heated at neutral one hour at 56” & 0.2' EDTA used in this study of activity is defined as a r.o/min, or as the number 25" (specific activity).
475
pH (incubated
with pre-heated
normal human serum) for
(SBz water bath, Grant Instruments, Cambridge). The was adjusted to pH 9.5 with sodium hydroxide. One unit change in the optical density at 410 nm for I cm cuvette of of qoles substrate transformed per min/per mg protein at
Electroplaoresis A vertical flat-bed slab of 5% polyacrylamide was used in tris-borate buffer, pH 9.511. The gel was stained for activity with @-naphthylphosphate and fast blue BB’2. Antiserum preparation Approximately I mg of placental phosphatase, purified as described, was homogenised (Virtis 45) with I ml of Freund’s Complete adjuvant (Difco Limited) and injected at multiple intramuscular sites into three Half-Lop rabbits. Two further injections were given at two weekly intervals, followed by a final injection of 0.3 mg protein in saline, and after seven weeks from the start the rabbits were bled by cardiac puncture and the serum obtained, stored at -13’. The low level of phosphatase present in the antiserum could be destroyed by heating at 56”. Antiserum used for double diffusion was absorbed out with normal human liver tissue. Double diffusion Immunodiffusion
was carried out in 1.5% Ion-Agar
sodium azide as preservative. temperature, or after overnight
(Oxoid Limited)
with 0.02%
The gel was photographed after two days at room washing in saline followed by staining for phosphatase.
Phosphatase precipitation by antiserum. At a final dilution of I :1,000, rabbit antiserum was incubated with enzyme in a volume of 0.1 ml. After I h at 37O and 96 h at 4’ the tubes were centrifuged at 28,000 xg (Griffin-Christ Microhaematocrit with angle head) for 15 min, cold, and samples of 0.04 ml were assayed in duplicate for enzyme activity. These were compared with controls containing
normal rabbit serum at the same dilution.
RESULTS
A placental-type alkaline phosphatase was isolated by Nakayama2 from the serum and the pleural fluid of a patient with “pleuritis carcinomatosa”. Although like placental phosphatase, it was stable when heated at 65”, the tumour phosphatase was more sensitive to the presence of both EDTA and L-leucine. These findings were confirmed with the tumour phosphatase used in this study. When assayed in the presence of L-phenylalanine (5 x IO@ M) the activities of both the tumour and placental phosphatases were only 8.5% and 8.4% respectively of those in the presence of the non-inhibitor D-phenylalanine. However, comparing activities, in the presence and absence of L-leucine (5 x IO-~ M) whilst the placental phosphatase retained 84% of its activity, that of the tumour enzyme was reduced to 38.5%. At this low concentration of L-leucine its activity was thus more strongly CZin. Chim. Acta, 35 (1971) 473-481
476
JACOBI’,
BAGSHAWE
ioa
Percentage actlvlty lo-4M=lOO%
5a
C
1o-4
lo-3 EOTA
10-z
Concentratlon(Molar)
Fig. I. Effect of EDTh on purified placental phosphatase in buffer (-:‘-) and in heated normal serum (-•-). and on purified tumour extract (-A-) and heated “turnour” serum (-A-). EDTA, adjusted to pH 9.5 with sodium hydroxide, was added to the final concentrations shown and phosphatase activity was assayed in the absence of added magnesium chloride. i\ctivities were compared with those at I x IO-~ M EDTA in order to allow for the effects of endogcnous magnesium ion.
inhibited. A similar effect was seen in the greater susceptibility of the tumour phosphatase to the chelating agent, EDTA (Fig. I). The enzyme activity at a final EDTA concentration of I x IO-* M was taken to be IOO~/~in order to allow for low endogenous levels of magnesium ion in the samples. The phosphatase activities of the purified tumour enzyme and of the patient’s heated serum were compared with those of placental phosphatase sampled in the absence and presence of heated normal serum. Samples were heated at 56” for I 11in stoppered tubes on a water bath. Aliquots were removed at intervals and assayed at room temperature for residual activity. Neither the pre-heated extract, nor the purified tumour phosphatase showed any loss of activity. The patient’s serum had a total phosphatase activity of 0.252 pmoles substrate transformed per min per ml and 25.20/o of this (0.064 ~moles/min/ml) was heat stable. These values correspond approximately to 87.0 and 21.9 King-Armstrong Units per IOO ml of serum, but it should be noted that the conditions used were not those of the King-Armstrong procedure. When the stability to heat at 56” was measured at pH 11.0 instead of at neutral pH there was a marked difference of response, the tumour enzyme being inactivated with time (Fig. z) whilst the placental phosphatase activity remained relatively stable. The residual activities were measured at pH 10.0 and at room temperature. In the Clb~.
Chzw.
Acta,
35 (1971)
473-@I
TUMOUR
ALKALINE
PHOSPHATASE
477
A
Percentage activity remaining
50
au Minutes Fig. 2. Heat inactivation at pH I I and 56” of placental phosphatase in buffer (-C-) and heated in normal serum (-•-), and of purified tumour phosphatase (-A-) and heated “tumour” serum (-A-). Samples, 0.02 ml, were heated in 0.1 M AMP buffer in a total volume of 0.15 ml in stoppered tubes. Samples were withdrawn at the time intervals shown, cooled to room temperature, and Methods” section for details). and assayed for residual activity (see “Materials
Time
(minutes)
Fig. 3. Heat inactivation of tumour and placental phosphatases and symbols are as given in the legend to Fig. 2.
at pH I I and 65”.The
Clin. Chzm. Ada,
assay method
35 (1971) 473-481
JACOBT, BAGSHAWE
Enzyme
actwty
Ezeonm
unots per ml.
I.0
Eluted fractions
(ml.)
Fig. 4. Gel filtration of purified tumour phosphatase on Sephadex G-zoo. A 1.0 ml sample was applied to a 1.6 x 60 cm column and eluted by upward flow at 4.6 ml per cm2 per hour. Fractions (4.6 ml) were collected on an LKB UltroRac 7000 collector and assayed for enzyme activity (-O-). Protein (-•-) was estimated by absorption at 280 nm. The main protein peak consisted of bovine serum albumin, added as a marker. In a separate run under the same conditions, 1.0 ml of a 0.27/o solution of Blue Dextran 2000 (Pharmacia) was eluted from the column to mark the
exclusion limit (--o-).
same incubation buffer (at pH 11.0) but heating at 65” both phosphatases showed an irreversible inactivation with time (Fig. 3) but whilst the placental activity decayed at a uniform rate, that of the tumour enzyme showed a biphasic curve; a rapid decay to 20% of the original activity followed by a slower inactivation of the remainder at a rate comparable with that of the placental enzyme. It has been notedI that this type of response may be found where the enzyme involved is heterogeneous, consisting of two or more components. On gel filtration using a 1.6 x 60 cm column of Sephadex G-200 (Fig. 4) the purified tumour enzyme was eluted by upward flow as a single peak of activity, followed by a peak of bovine serum albumin added as a marker. Under the same conditions the placental phosphatase was eluted as an identical peak, together with some higher molecular-weight material which was eluted just under the void volume, that is, before the main peak. When these samples were subjected to electrophoresis on a vertical flat-bed acrylamide gel11 at pH 9.5 and stained for enzyme activity (Fig. 5) the placental phosphatase appeared as three closely-spaced bands9 with the tumour enzyme showing a more diffuse band in the same position. A sample of the higher molecularweight material eluted from the Sephadex column had barely entered the main part of the gel, whilst the phosphatase of a butanol extract of liver tissue migrated the furthest. Clin.
Chum.
Acta,
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(1971)
47X-481
TUMOX’R
ALKALINE
479
PHOSPHATASE
The purified tumour and placental phosphatases were both precipitated by rabbit antiserum to human placental alkaline phosphatase at a final dilution in saline of I : I OOO. The supernatant activity was assayed relative to that of controls with
Fig. 5. Vertical slab electrophoresis at pH 9.5 on 5% acrvlamide gel” stained for phosphatase activity-. Phosphatases from liver (I and 5), placenta (a and 3) and the tumour sample (4) were applied at the origin and run for one hour. The placental samples consisted of the main peak (2) and the high molecular-weight phosphatase (3) &ted from a Sephadex G-200 column.
Fig. 6. Immuno-diffusion in agar gel. After two days incubation at room temperature the get was washed overnight in saline and stained for phosphatase activity. In the centre wells were normal rabbit serum (lower left), and antiserum to placental phosphatase (upper right). The peripheral wells wxc loaded with 0.035 ml of phosphatnse extract, from the tnmour sample (I), placenta (2 and 6), bone (3), liver (4) and intestine (j). Cain. Ckim.
Acta,
35
(1971)
~$73-.@1
480
JACOBY,
normal rabbit
serum added. The assays, in duplicate,
BAGSHAWE
showed that in both cases 5.8%
and 6.0% of the activity remained. The phosphatases of bone and liver origin are known not to react with anti-placental antiserum whilst that of intestinal origin is only precipitated at higher antiserum concentrations (final dilution I : 150. Unpublished observation). On double diffusion in agar against the absorbed antiserum, a weak precipitation line developed, common to that of placental phosphatase. Although the most concentrated sample was used, the phosphatase concentration was still well below that of the purified placental enzyme. The immunological activity was better seen when an agar diffusion plate which has been washed overnight in saline to remove nonprecipitated material was then stained for enzyme activity (Fig. 6). Tumour, placental and intestinal phosphatases showed a definite interaction towards rabbit antiserum, whilst bone and liver phosphatases did not. None of the samples showed any reaction when put up against normal rabbit serum.
The purified tumour phosphatase did not differ in any of the properties tested from the heat-stable enzyme in the patient’s serum. It therefore seems unlikely that the purification procedure was itself responsible for the difference of properties between this and the placental phosphatase. Although on agar diffusion the tumour phosphatase could not be distinguished from that of placental or intestinal origin, it was, unlike the intestinal enzyme, stable to heating at 56”. It did not migrate to the intestinal position on electrophoresis, and resembled the placental enzyme on Sephadex gel filtration. Studies of phosphatases from tumour tissue2p6 and from HeLa cells4$15 have shown these to differ from placental phosphatase. From the results of the present study it may be possible to speculate about the extent of these changes. Both phosphatases behaved similarly on Sephadex gel filtration, polyacrylamide electrophoresis and reacted similarly with rabbit antiserum, but the tumour phosphatase activity was more susceptible to the effect of L-leucine and EDTA. However, it was the inactivation of the tumour phosphatase by heating at an alkaline pH which showed the difference most clearly and which suggested a heterogeneity of response (Fig. 3). Thus whilst the overall molecular size and charge of the enzyme are not greatly changed and it is still recognized by rabbit antiserum, it is the stability which is reduced, and even small structural alterations could account for such changes. There remains the question of whether the tumour phosphatases from different patients show the same characteristic properties. The fact that we have been able to confirm two of the findings of the Japanese workers2 (the sensitivity to L-leucine and to EDTA) at least suggests that an alkaline phosphatase with consistent properties may be produced by a number of tumours. It is hoped that the method of measuring heat inactivation at alkaline pH might give meaningful results in this respect. The phosphatase from the patient in this study was originally identified as being of the placental-type through the use of an automated enzyme assay system. An account of this technique will be published elsewhere (R. C. Jennings, personal communication).
Clin. Chim.
Acta,
35 (1971)
473-481
TUMOUR
ALKALINE
PHOSPHATASE
481
ACKNOWLEDGEMENTS
We thank Dr. R. C. Jennings, Mr. D. Brocklehurst and Mr. hf. Hirst of the Department of Pathology, The General Hospital, Altrincham, Cheshire, for the tumour tissue and serum sample and Mr. R. Myers and Dr. A. W. Walker of the Department of Chemical Pathology, Fulham Hospital, London, for the polyacrylamide electrophoresis and the butanol extracts of human bone, intestine and liver. REFERENCES I 2 3 4 5
W.H. FISHMAN,N. I.INGLIS,L.L.STOLBACHANDM. J. KRAST,C~~CW Reseavch, z8(rg68)15o. T. NAKAYAMA, M. YOSHIDA AND M. KITAMURA,CX~. Chim. Acta, 30 (1970) 546. G. hf.mIC AP*'DA. G. RIEGO, Ann. Biol. Chim., 26 (1970) 2x9. N. A. ELSON A~XD R. P. Cox, B&hem. Genet., 3 (1969) 549. W. H. FISHMA~,N.I.INGLIS,S.GREEX,C.L.XNSTISS,N. K.GHOSH, A.E. REIF, KRUSTIGAX,
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473-481