- Email: [email protected]
An in vitro production of bone specific alkaline phosphatase
Printed in Sweden CopyriRhr @ 1975 by Academic Press, Inc. All riehrs of reproducrion in uny form reserved
Experimental
AN
IN
VITRO
Cell Research
PRODUCTION
ALKALINE
95 (1975) 347-358
OF
BONE
SPECIFIC
PHOSPHATASE
I. SINGH and K. Y. TSANG Orthopaedic
Research
Laboratories,
Medical
College
of Ohio,
Toiedo.
OH 43614,
USA
SUMMARY Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadavetic bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.
Mammalian cell alkaline phosphatases are a heterogenous group of enzymes whose physiologic functions have not been fully defined. The ability of the enzyme (extracted from various tissues) to hydrolyze p-nitrophenylphosphate at an alkaline pH has been associated with numerous attempts to isolate organ specific alkaline phosphatases. Schlamowitz & Bodansky [13] investigated the tissue sources of alkaline phosphatases found in human serum. Their data, using immunochemical technics, indicated that the serum with “antibone” activity contained antibodies not only to phosphatase of bone but other tissues as well. Sussman and co-workers [ 193, using a two-step method of antigen-antibody reaction, demonstrated that human hepatic and placental isozymes could be distinguished from each other and from the isoenzymes of bone, kidney, and intestine
by immunochemical means. Cox and associates [4] have established that the enzyme extracted from HeLa cells behaves as the placental isozyme. Eaton & Moss [5] partially purified alkaline phosphatase from human bone, but the yield of the enzyme was relatively low. Although not able to purify the enzyme from bone to the same degree as the hepatic and intestinal isozyme, they were able to confirm the pyrophosphatase activity and the effect of neuramidase on electrophoretic mobility of the enzyme from bone. Furthermore, it has not been established that a specific alkaline phosphatase from bone can be separated for study of its role in osseous metabolism. Boyer [2] indicated that many phosphatases can be distinguished electrophoretically and by chromatography. However, his data indicated that alkaline phosphatase of liver, bone, Exprl
Cell
Res 95 (1975)
348
Singh und Tsmng
Fig. I. Abscissa: time (set); ordinate: % activity remaining. Cm, Normal human bone enzyme; O-O, induced cell enzyme; O-O, normal liver enzyme. A composite of the thermolability of the enzyme from osteosarcoma cells in tissue culture, normal human bone and normal human liver, indicates that all three forms of alkaline phosphatase are rapidly inactivated at 56°C. 100% spec. enzyme act. (Mlmgl min) was: Induced cell enzyme, 106x IO-ia; bone, 360~ IO-‘“* . liver 7224x10-lo.
spleen and kidney were a closely related class of proteins with similar antigenicity and thus there was inter-reaction between these various isozymes. The present investigation was undertaken to provide a model for in vitro production, isolation and purification of a specific alkaline phosphatase from bone. METHODS
AND MATERIALS
Tissue culture methods
pellet was washed and centrifuged in cold O.Y’,c NaCI 3 times at 600 g for 10 min. The final pellet was 5~15. pended in ice cold sucrose (0.2.5 M) and homogenized in a Potter-Elvehjem teflon homogenizer. The cell homogenate was centrifuged in the cold room at 600 g for 10 min and the supernatant stored at -20°C until ready for use. To rule out possible immunologic differences related to blood type, bone, liver, intestine and kidney were obtained at a fresh autopsy on a young man who died immediately after an accident. Placental tissue was obtained following a normal delivery. Liver. A piece of normal human liver was obtained, cut into small pieces and rinsed in many rinses of cold 0.9% NaCl solution. The specimen was then homogenized in a Vertis homogenizer, and the mixture was centrifuged 3 times in cold 0.9% NaCl at 600 g for 10 min to remove contaminating blood and bile. The pellet obtained was then homogenized in a Potter-Elvehjem homogenizer in ice cold sucrose (0.25 M). The liver homogenate was centrifuged in the cold at 600 g for 10 min and the supernatant stored at -20°C until ready for extraction. Similar procedures for extraction were used for kidney, intestine and placenta.
Alkaline
phosphatase
assay
The substrate used for alkaline phosphatase activity assay was 0.016 M p-nitrophenylphosphate (p-Npp) (Sigma 104) with an equal volume of I M 2-amino-2methyl-I-propanol (Ammenol buffer) at pH 10.3 with lO-3 M MgC& added. The absorbance was read at 410 nm in a Coleman spectrophotometer. The resultant activity was expressed as PM or nM/min/mg protein. The enzymes extracted from normal bone, liver, kidney, intestine, placenta and from osteosarcoma cells grown in tissue culture were investigated.
Purification
ofthe enzyme
extraction. Five ml of the supernatant were thawed and magnetically stirred at 4°C with n-butanol being added in a dropwise fashion over a period of I5 min to give a 20% solution. The stirring was continued for 30 more minutes. The mixture was centrifuged for 30 min at 38 000 g at 4°C and the aqueous layer collected and dialyzed for 20 h against 100 vol of distilled water at 4°C.
Bumto/
Osteosarcoma cells were cultured as a monolayer [ 141. Alkaline uhosohatase activity was inducible in these . . cells by the addition of 0.5 -pg/ml of hydrocortisone by gel filtration phosphate to the tissue culture medium and the enzyme Purification extracted 12 to 15 days after incubation as reported The partially purified butanol extract of the enzyme earlier [14]. The extracted enzyme was stored at -20°C preparation from osteosarcoma cells in tissue culture until ready for use. was layered on a column (2.6~40 cm) packed with Sephadex G-200 (Pharmacia Fine Chemicals) and eluted with 0.05 M Tris-HCI buffer, pH 8.6. The elutant Tissues was collected in a Buchler fraction collector at 4°C. The Bone. A piece of normal human bone excised in the eluted alkaline phosphatase from the column was concentrated tenfold by ultrafiltration in an Amicon course of an arthroplasty procedure in a young patient micro-ultrafiltration system using an XM-50 filter at 20 was washed in multiple changes of ice cold Ringer’s atm of pressure. This concentrated enzyme (from tissue lactate to remove as much of the blood as possible. The bone was then cut into small pieces and pulverized in a cultures) was used as antigen for antibody production cold pestle and mortar. The pulverate was suspended in (see antibody production). Gel filtration was also ice cold sucrose (0.25 M) and centrifuged at 600 g for 10 performed on the enzyme from normal human bone and min. The blood-tinged supernatant was discarded. The liver. Exptl
Cell
Res 95 (1973-J
Specific alkaline LOO 90
of osseous
+
80 I lo60SO403020IO-
Gel electrophoresis
of Smith, Lightstone 100 90 80 I
+
-
70 60
\ *
b
II
50 40 30 20 10 l!h 012
3
4
5
LL 6 7
Figs 2, 3. Abscissa: penetration into the gel (cm); ordinate: optical density. Fig. 2. A tracing of the densitometric scan of gel electrophoresis of the enzyme extracted from osteosarcoma cells grown in tissue culture (a) and the enzyme extracted from bone (b) removed during an operation (see Methods). The three peaks of activity in each enzyme are marked by arrows (J).
Pyrophosphatase
tissue
349
the enzyme was determined at pH 7.4 and pH 10.0. The released inorganic phosphate was determined by a modified Fiske & Subbarow technic [6]. The enzyme extracts from normal cadaveric bone, liver, kidney, intestine, fresh placenta and from osteosarcoma cells grown in tissue culture were investigated. The protein content of each of the enzyme extracts was determined and the results expressed as nM/min/mg of protein.
a -
phosphatase
assay
The substrate used for pyrophosphatase assay was 3.3 mM pyrophosphate (tetra sodium salt), with an equal volume of 1 .O M Tris HCI buffer to which 2x 10m3 M MgCI, was added. One ml of the buffer substrate mixture was incubated with 0.2 ml of the enzyme at 37°C for 1 h. The reaction was stopped by adding 4 ml of TCA to the mixture. The pyrophosphatase activity of
(modification & Perry [I83
Electrophoresis was carried out in a Hoefer disc electrophoresis apparatus. Only running 5 % polyacrylamide gels were used, as adequate results were obtained without the need of spacer or sample gels. In all experiments, control serum to which a trace of bromphenol blue (albumin-BPB) had been added to the dye was run as a tracer. Upon completion, the gels were rimmed directly from their running tube into a slightly larger tube containing the location buffer, and 2 mg/ml of substrate (alpha Naphthylphosphate), with 1 mglml Fast Blue RR (Harleco, Philadelphia) added to the location buffer just prior to use. The tubes were incubated in a dark room at room temperature (25°C) for 1 h and were scanned with a Canalco Model .I densitometer. The penetration of the enzyme and its peaks was expressed in cm on a standard scale.
Heat stability The enzyme extracted from normal human bone, from cells in tissue culture and from liver were assayed for specific activity and were then placed in a fixed temperature shaking water bath (56°C). The tubes were removed at intervals of 15,30,45,60,90, 120 and 300 sec. The tubes were immediately placed in crushed ice and assayed for the remaining enzymatic activity.
Inhibition studies To the substrate for alkaline freshly prepared L-homoarginine mixture, 0. I ml of the enzyme or osteosarcoma cells in tissue incubated at 37°C for 30 min.
phosphatase, 8 mM of was added. To this preparation (from bone culture) was added and The remaining activity
Table 1. Inhibition study Normal
Spec Initial activity L-Homoarginine L-Phenylalanine L-Tryptophan
’
Spec.
bone
act.”
34.4 x10-9 13.26~10-~ +2.5 x10-9 23.46x 1O-9 +2.2 x10-9 18.02x lo+ k2.8 x10-9
Induced Percentage inhibition
6;+7.5 31k6.5 4758.3
Spec
enzyme
act.‘”
32.76x lO-9 15.23~10-~ +1.8 x10+’ 26.04x IO+ k2.7 x1022.11 x 10-S +2.1 x10-9
Percentage inhibition
G.5U.7 20.5k8.3 32.5
t6.5
act.-moles/mg/min.
Exp~lCel~Res
95 (1975)
b
loo90. so-
+
lo60sow
QI
2
I
4
5
6
7
13 I4
Fig. 3. A composite of tracings of densitometric scans of the enzyme extracted from osteosarcoma cells grown in tissue culture; intestine; liver; liver+Ha;
placenta; bone; bone+Ha; kidney. Ha represents the effect of 8 mM homoarginine on the enzymes extracted from hone and liver.
was determined and expressed as percentage of inhibition of normal activity. The same experiment was repeated with L-tryptophan and L-phenylalanine. Following L-homoarginine inhibition experiment, gel electrophoresis was performed on the enzyme from bone and liver with 8 mM of homoarginine added to the enzyme.
antigen for antibody production. This avoided the possibility of contamination with human serum which may have remained in the bone in spite of repeated washings of cadaveric or fresh human bone.
Immunologic studies Only the enzyme extracted and purified from osteosarcoma cells grown in tissue culture was used as Exprl
Cell Res 95 (1975)
Antibody production Adjuvant mixtures were prepared by emulsifying purified alkaline phosphatase from cells in tissue culture with an equal volume of Freund’s complete adjuvant (1.5 parts Artacel, 8.5 parts mineral oil, 5 mg/ml mycobacterium). The initial injections were into
Specific alkaline phosphatase Table 2. Enzyme penetration Enzyme peaks First Second Third End of activity
Bone
Bone +Ha
1.80 3.00
1.70 3.00
6.25
i.70
of osseous tissue
35 1
into the gel (cm) Cells
Liver
Liver +Ha
1.70 3.00 4.15 6.50
1.60 4.40 5.70 7.30
1.55 3.80 5.30 6.80
the foot pads of all four feet and in both thighs of rabbits. In total, approx. 125 pg of protein was given to each animal throughout the six sites. The injections were repeated 2 weeks later and rabbits bled 7 days later. Booster injections were given 30 days later and the rabbits bled 7-9 days after that. The titre of the antibody was measured by immunofluorescence technics. When titres of one to 250 or higher had been obtained, the rabbits were bled for preparation of the antibody.
Pur$cation of the antibody and separation of 7s gamma globulin fraction The blood from the rabbit was allowed to clot and the serum separated by centrifugation at 600 g for 10 min. The serum was diluted with an equal volume of 0.85 % NaCI. To each 10 ml of the diluted serum, 2.05 g of anhydrous Na*SO, was slowly added, while the mixture was being continuously stirred to ensure even distribution. The mixture was allowed to stand at room temperature for 2 h and then centrifuged at 2 000 g for 30 min. The supernantant fluid was discarded and the precipitate dissolved in a volume of distilled water equal to two thirds of the initial volume of the diluted serum. The proteins were reprecipitated by adding 1.93 g of Na$O, for each 10 ml of distilled water used in the previous step. The mixture was allowed to stand at room temperature for 2 h and then centrifuged at 2 000 g for 30 min. The supernatant was discarded. This precipitation was carried out four times. The final precipitate was dissolved in distilled water and was dialyzed against 0.01 M phosphate buffered saline pH 7.2 at 4°C overnight. This mixture was dialyzed against distilled water overnight at 4°C. The final dialysate was centrifuged at 2000 g for 15 min. The precipitate was discarded and the supernatant was concentrated in an Amicon ultrafiltration cell using a XM-50 filter at 20 atm pressure.
Determination of the antibody concentration necessary for precipitation of alkaline phosphatase The rabbit antiserum was heated at 56°C for 30 min to inactivate the endogenous heat labile enzyme. Antibody precipitation of the enzyme was carried out by adding a constant amount of alkaline phosphatase extract from normal human bone, liver, kidney, and from cells in tissue culture (antigen 0.2 ml) to serial dilutions of rabbit antisera (0.2 ml) and incubating at 37°C for ) h.
Kidney
Placenta
Intestine
2.15 2.65
2.85 4.4
6.55
i.30
2.00 3.40 5.25 6.30
To this mixture, 0.2 ml solution of antirabbit globulin was added. The reaction mixture was kept at 4°C for 24 h, following which it was centrifuged at 900 g for 10 min and the supernatant decanted. The precipitate was washed in ice cold 0.9 % NaCl and resuspended in 0.05 M Tris HCI pH 7.4, equal to the volume of the decanted supernatant. The supernatant and the resuspended precipitate were assayed for alkaline phosphatase activity. The amount of endogenous alkaline phosphatase activity in the rabbit antiserum was determined and appropriate allowances were made in calculating the inhibition of activity by the antisera.
Zmmunodiffusion One per cent Noble Agar was used for immunodiffusion. The alkaline phosphatase extracted from normal human bone, liver, kidney, intestine, placenta and that obtained from osteosarcoma cells were placed in individual wells and the rabbit antiserum against the alkaline phosphatase from cell culture was placed in the center well.
RESULTS Heat stability Heat stability tests showed that the enzyme extracted from normal human bone and that from tissue culture cells as well as liver was heat labile at 56°C. There was rapid inactivation of the enzymes at this temperature (fig. 1). Znhibition studies The enzyme extracted from the cells as well as from bone showed maximal induction with L-homoarginine. There was some inhibition with L-phenylalanine and L-tryptophan, but not to the extent seen with homoarginine (table 1). Electrophoresis Gel electrophoresis on bone removed surgically showed three peaks, as did the enzyme Exprl
Cell Res 95 (1975)
352
Singh and Tsung a .>'
50-
>"\ II
)- '\ '\
4 o-
\: "
30-
/ c
-80
-40
0 50
52
54
56
56
60
62
64
66
68
70
72
74
76
76
80
b 20
Fig. 4. Abscissa: eluate (ml); ordinate: (lefr) nM/min; (right) @g/ml protein. Sephadex column chromatography of alkaline phosphatase extracts. (a) In the eluate from normal human bone the enzymatic activity appeared, directly after the void volume and nearly all of the enzyme passed through the column in 28 ml of the eluate. No activity was demon-
strable in the tube at 29 ml; (b) in the eluate obtained from the liver enzyme the activity appeared directly after the void volume and most of the enzyme activity had passed through the column in 37 ml. O-O, nmoles of enzyme activity as measured by its ability to hydrolyze p-nitrophenylphosphatelmin at 3PC; O-O, *g protein/ml of eluate).
extracted from tissue culture cells (fig. 2a, b). The electrophoretic migration of enzyme from liver indicated a fast moving peak and two other peaks that were in the same area of migration as the peak found in the study of enzyme from bone (fig. 3~). A composite of the gel electrophoresis of the enzyme from cadaveric tissue extracts of bone, liver, kidney, intestine and from a fresh placenta is shown in fig. 3a and b. The densitometric scans showed (table 2) that there were three distinct peaks of activ-
ity in the enzyme extracted from cells in tissue culture. The second and third peaks appeared to be confluent in the enzyme extracted from cadaveric human bone. The effect of 8 mM of homoarginine on the bone enzyme indicated no difference in the peaks, but the total penetration was decreased, indicating that the homoarginine effect was mostly on the third isozyme or peak. The enzyme from liver showed three distinct peaks of activity and the third was a fast penetrating isozyme which penetrated the
ExptlCeNRes
95 (1975)
Specific
5. Indirect fluorescent antibody test. Rabbit antisera against enzyme extract from osteosarcoma cells was reacted with fluorescent conjugated sheep antirabbit serum. (a) Strongly positive reaction against osteosarcoma cells grown in tissue culture. x1040;
Fig.
alkaline phosphatase
of osseous
tissue
353
(&) a positive reaction against normal bone cell smear; occasional bone tibres (arrow) showing reaction are also seen. x500; (c) a negative reaction to cells from normal human liver. x 1040. Exptl
Cell Res 95 (1975)
354
Singh und Tsung
Table 3 Pyrophosphatase nmoles/min/mg
Enzyme origin
Protein content mglml
pH
Cells Bone Intestine Kidnev Liver. Placenta
0.971 0.142 0.625 0.770 0.791 0.225
219.0 168.0 157.0 400.0 307.0 105.0
7.4
act. protein pH
10.0
97.0 79.0 62.0 290.0 295.0 88.0
farthest into the gel. The homoarginine not only inhibited the enzyme in regard to total penetration into the gel, but in addition, there was a distinct difference in electrophoretic mobility of the enzyme. All three peaks of the enzyme from liver appeared at an earlier penetration of the enzyme into the gel with homoarginine (table 2). The placental enzyme had only two peaks. However, a distinct shoulder was noticeable between the first and second peaks at approx. 3.35 cm (1) penetration of the gel. The intestinal enzyme had three distinct peaks and the first isozyme had a completely different penetration rate than the enzyme from liver, bone, or cells in tissue culture. The enzyme from kidney showed two peaks of penetration which were closely located. Sephadex column chromatography
When the results of Sephadex G-200 column chromatography were compared, the data indicated that the enzyme activity in bone, liver, and cells in tissue culture appeared immediately after the void volume. It was observed that all of the butanol extracted alkaline phosphatase from bone had passed through the Sephadex G-200 column in 28 ml of the eluate (fig. 4a). No enzyme activity could be demonstrated in the last tube, even Exptl
Cell
Res 95 (1975)
though traces of protein were detectable. However, the enzyme from liver was present in 37 ml of the eluate (fig. 4b). The concentration of protein in the fractions of the eluate from the liver and osseous enzyme were as shown in fig. 4a and h. The enzymatic activity and protein concentration in the eluate showed a much higher concentration of protein in peak 1 and lower concentration of the protein in peaks 2 and 3 of the enzyme from bone (fig. 4a). These data indicated that the specific activity of alkaline phosphatase expressed as nmoles/mg of protein/min are the greatest in peaks 2 and 3, rather than in peak 1. Pyrophosphatase
activity
All of the enzyme extracts showed definite pyrophosphatase activity in that there was liberation of inorganic phosphate from the tetra sodium pyrophosphate. Table 3 shows the pyrophosphatase activity of the enzyme at pH 7.4 and pH 10.0 in the presence of MgCl,. It was noted that the pyrophosphatase activity of all of the enzymes indicated that the release of inorganic phosphatase was greater at pH 7.4 (normal) than at the highly alkaline pH 10.0. Immunojluorescence
The results of immunofluorescence showed that the antibody titre was one to 512. The antisera reacted positively against cells from tissue culture and bone (fig. 5a, b) but results against hepatic cells were negative (fig. 5c). Zmmunodiffusion
The results of immunodiffusion were as shown in fig. 6a, b and c. There was a strong positive precipitate against the enzyme extracted from bone and the enzyme extracted from cells in tissue culture. No precipitate line appeared against alkaline phosphatase
Specific
alkaline phosphatase
b
of osseous
tissue
355
c
Fig. 6. Results of immunodiffusion. In (a) and (c) rabbit antiserum (Ab) to alkaline nhosnhatase derived from osteosarcoma cells grown in tissue culture was used as the antibodv in the center well. In (b) nuritied 7s gamma globulin (Ab) was used as the antibody. B, enzyme from bone; C, enzyme from osteosarcoma
cells in tissue culture; I, intestinal enzyme; K, kidney enzyme; L, liver enzyme; P, placental enzyme; S, saline. A positive reaction seen as a precipitate line occurred only against the enzyme extracted from cells grown in tissue culture (c) and against bone (b).
extracted from liver, kidney, placenta.
physiologic functions and substrates of alkaline phosphatases are still not totally known. The estimation of the enzymatic activity in clinical practice has been based upon the enzyme’s ability to hydrolyze p-nitrophenylphosphate at an alkaline pH of 8 or greater. p-Nitrophenylphosphate is an artificial compound and it is highly unlikely that the high pH’s at which this substrate is hydrolyzed exist under normal conditions in man. Recent work indicates that alkaline phosphatases do hydrolyze pyrophosphate at a neutral pH [3, 51. The concentration of this enzyme in plasma has been used as a diagnostic aid in metabolic bone disease. In general, the amount of the enzyme in plasma has been roughly correlated with the rate of bone formation, being high in Paget’s disease, osteomalacia and rickets, although no correlation has been found between the level of this enzyme and rate of bone resorption. Patients with a deficiency of cytoplasmic alkaline phosphatase in certain cells (hypophosphatasia, osteogenesis imperfecta) have pyrophosphaturia, suggesting that an impaired metabolism of inorganic pyro-
intestine
and
Precipitation test
The precipitation of the enzyme extracts from cells in tissue culture, normal cadaveric human bone, liver and kidney were as shown in fig. 7a, b and c. Allowances were made in the calculations for the endogenous enzyme in antiserum from the rabbit. No enzymatic activity was detected in the supernatant with normal bone and cells in tissue culture and antisera at dilutions of one in two and one in one. The entire enzymatic activity was recoverable in the precipitate. However, all of the enzymatic activity remained in the supernatant from the preparation from liver and kidney (fig. 7c). DISCUSSION Robison [12] postulated that the alkaline phosphatase of chondroblasts and osteoblasts was responsible for precipitation of calcium salts in bone by hydrolytic liberation of inorganic phosphate. However, the
Exptl
Cell Res 95 (1975)
5432I-
Ii28
I64
1’32
I‘,8
1’8
1’4
I’2
;I
Fig. 7. Abscissa: anti-serum dilutions; ordinate: alkaline phosphatase activityx 10e4 urn. Precipitate test. As the concentration of the antiserum was increased, the enzyme activity recoverable in the supernatant of the enzyme extracts from osteosarcoma cells grown in tissue culture (a) and normal human bone (b) decreased. At dilutions of 1 : 1, no activity was demonstrable in the supernatant and all of the activity was recovered in the precipitate (a. b). There was no decrease in the activity in the supernatant with the enzymes of liver and kidney (c) extraction. (Appropriate allowances were made for endogenous alkaline phosphatase in rabbit antiserum.) Exptl
Cell
Res 95 (1975)
phosphate is associated with this inborn error of metabolism. Another problem in correlating the levels of alkaline phosphatase with disease processes has been a general assumption that changes in the plasma levels are a direct reflection of changes at sites of bone formation. In the present investigation, the authors have isolated an alkaline phosphatase in vitro by growing osteosarcoma cells in tissue culture medium to which hydrocortisone (0.5 kg/ml) had been added. In the present investigation, the characteristics of this in vitro produced enzyme were compared with those of enzymes extracted from cadaveric normal human bone, liver, kidney, intestine, and placenta. It was observed that heat lability of this enzyme was about the same as for the enzyme extracted from liver and bone. The electrophoretic mobility of this in vitro produced enzyme closely resembled that of the enzyme extracted from normal human bone but not that of liver, kidney, intestine, or placenta. The results of Sephadex column chromatography indicated that the molecular size of the variants (bands, peaks) in the in vitro produced enzyme were approximately the same as in alkaline phosphatase extracted from human bone but were different from those of liver. By using the in vitro enzyme as the antigen for antibody production, possible sources of contamination with human serum were avoided. This was important since the serum contains all of the various forms of alkaline phosphatases except placental in cadaver extracts used in this investigation. Immunofluorescence studies (fig. 5~7, 6, c) conclusively indicated that the antibody was specific against the enzyme from cells in tissue culture and bone but not from liver. Immunodiffusion (fig. 6) also proved that the antibody induced by injection of the in vitro
Specific
produced enzyme was specific against the enzyme extracted from normal human bone and cells in tissue culture, but not against the enzyme extracted from human placenta, kidney, liver or intestine. Fleisch and co-workers [8, 9, lo] have suggested that alkaline phosphatase from bone is a pyrophosphatase and is responsible for regulating mineral ions, an extracellular process. In their view, the dissolution of both mineral of bone and matrix of bone is under enzymatic control, and parathyroid and thyrocalcitonin could affect the deposition of minerals or resorption of bone minerals either by controlling the rate of release of alkaline phosphatase into extracellular osseous fluid or by altering the activity of the released enzyme. Firschein & Urist [7] studied the alkaline phosphatase activity in mesenchymal cells invading active and inactive implants of matrix of bone. Their data indicated that induction of alkaline phosphatase activity occurred in the pre-osseous stage of morphogenesis and preceded the deposition of calcium by 5-7 days. No direct relationship was observed between the alkaline phosphatase and calcification in their investigation. These and other in vivo investigations have established a correlation between levels of enzymatic activity and osteogenesis. However, these data have not established whether enzymatic activity is concerned with cell modulation or with mineral deposition or both. The work of Morrill, Kestow & Murphy [I l] and Borle [l] suggests that pyrophosphatase and alkaline phosphatase may be synonymous and that there may be multiple forms in each tissue; one form being membrane bound and involved with the transport of ions and others within the cell involved in the hydrolysis of pyrophosphate. Three variants or peaks have been
alkaline phosphatase
of osseous
tissue
357
observed in the enzyme from bone by gel electrophoresis. It has been observed that the first variant of the enzyme extracted from bone had low specific activity per mg of protein in the present investigation. These findings were also observed in the enzyme extracted from osteosarcoma cells in tissue culture. The findings further confirm the data of Cox and associates [3] and Eaton & Moss [5] that alkaline phosphatases hydrolyze pyrophosphates at a neutral pH (7.4). In conclusion, a model for in vitro production, isolation, and purification of a specific alkaline phosphatase (pyrophosphatase) of osseous origin has been described. This work is dedicated to the memory of George D. Ludwig, M.D., late Professor and Chairman, Department of Medicine, Medical College of Ohio, Toledo, Ohio. During his lifetime he was a source of continuing inspiration and encouragement to the investigators. This research was in part supported by ACS Institutional Grant no. IN-98A and the Ohio Chapter of the ACS.
REFERENCES I. Borle, A B, Phosphate et mttabolisme phosphocalcique (ed D Hioco) pp. 295. Sandoz, Paris (1971). 2. Boyer, S H, Ann NY acad sci 103 (1963) 938. 3. Cox, R P, Gilbert, P & Griffin, M J, Biochem j I05 (1967) 155. 4. Cox, R P. Personal communication (1974). 5. Eaton, H R & Moss, D W, Enzymologia 35 (1968) 31. 6. Fiske, C H & Subbarow, Y, Inorganic phosphorus in clinical chemistry, principles agd procedures (ed J S Annino) pp __ 201-204, 3rd edn. Little. Brown & Co, Boston, MA (1964). 7. Firschein, H E & Urist, M R, Calicum tissue res 7 (1971) 108. 8. Fleisch, H, Maecki, J & Russell, R G G, Proc sot exp biol med 122 (1966) 317. 9. Fleisch, H & Russell, R G G, Int encyclopaedia of pharmacology and therapeutics, sect. 51. Pharmacology of the endocrine system and related drugs. Parathvroid hormone. thvrocalcitonin and related drugs section (ed H Rasmussen) vol. I, pp 61-100. Pergamon Press, Oxford (1970). IO. Fleisch, H,kussell, R G G & Straumann, F, Science I65 (1969) 1262. 11. Merrill, G A, Kostellow, A D & Murphy, J B, Exp cell res 66 (197 I) 289. Expr/
Cd
Res 95 (1975)
12.
Robison, R & Soames, K M, Biochem j 18 (1924) 740. 13. Schlamowitz, M & Bodansky, 0, J biol them 234 (1959) 1433. 14. Singh, I, Bartolomeo, L & Ludwig, G D. J biothem. Submitted for publication. 15. -J biol them. Submitted for oublication. 16. Singh, I, Tsang, K Y & Ludwig, G D, Cancer res 34 (1974) 2946.
Expd
Cell
Res 95 (1975)
17. - Em surg res 6 (1974) 247. 18. Smith, I, Lightstone, P J & Perry. J 0. acta 19 (1968) 499. 19. Sussman, H H, Small, P A & Cotlove. them 243 (1968) 160.
Received
March
25,
1975
Clin
chim
E, J biol
Experimental
AN
IN
VITRO
Cell Research
PRODUCTION
ALKALINE
95 (1975) 347-358
OF
BONE
SPECIFIC
PHOSPHATASE
I. SINGH and K. Y. TSANG Orthopaedic
Research
Laboratories,
Medical
College
of Ohio,
Toiedo.
OH 43614,
USA
SUMMARY Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadavetic bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.
Mammalian cell alkaline phosphatases are a heterogenous group of enzymes whose physiologic functions have not been fully defined. The ability of the enzyme (extracted from various tissues) to hydrolyze p-nitrophenylphosphate at an alkaline pH has been associated with numerous attempts to isolate organ specific alkaline phosphatases. Schlamowitz & Bodansky [13] investigated the tissue sources of alkaline phosphatases found in human serum. Their data, using immunochemical technics, indicated that the serum with “antibone” activity contained antibodies not only to phosphatase of bone but other tissues as well. Sussman and co-workers [ 193, using a two-step method of antigen-antibody reaction, demonstrated that human hepatic and placental isozymes could be distinguished from each other and from the isoenzymes of bone, kidney, and intestine
by immunochemical means. Cox and associates [4] have established that the enzyme extracted from HeLa cells behaves as the placental isozyme. Eaton & Moss [5] partially purified alkaline phosphatase from human bone, but the yield of the enzyme was relatively low. Although not able to purify the enzyme from bone to the same degree as the hepatic and intestinal isozyme, they were able to confirm the pyrophosphatase activity and the effect of neuramidase on electrophoretic mobility of the enzyme from bone. Furthermore, it has not been established that a specific alkaline phosphatase from bone can be separated for study of its role in osseous metabolism. Boyer [2] indicated that many phosphatases can be distinguished electrophoretically and by chromatography. However, his data indicated that alkaline phosphatase of liver, bone, Exprl
Cell
Res 95 (1975)
348
Singh und Tsmng
Fig. I. Abscissa: time (set); ordinate: % activity remaining. Cm, Normal human bone enzyme; O-O, induced cell enzyme; O-O, normal liver enzyme. A composite of the thermolability of the enzyme from osteosarcoma cells in tissue culture, normal human bone and normal human liver, indicates that all three forms of alkaline phosphatase are rapidly inactivated at 56°C. 100% spec. enzyme act. (Mlmgl min) was: Induced cell enzyme, 106x IO-ia; bone, 360~ IO-‘“* . liver 7224x10-lo.
spleen and kidney were a closely related class of proteins with similar antigenicity and thus there was inter-reaction between these various isozymes. The present investigation was undertaken to provide a model for in vitro production, isolation and purification of a specific alkaline phosphatase from bone. METHODS
AND MATERIALS
Tissue culture methods
pellet was washed and centrifuged in cold O.Y’,c NaCI 3 times at 600 g for 10 min. The final pellet was 5~15. pended in ice cold sucrose (0.2.5 M) and homogenized in a Potter-Elvehjem teflon homogenizer. The cell homogenate was centrifuged in the cold room at 600 g for 10 min and the supernatant stored at -20°C until ready for use. To rule out possible immunologic differences related to blood type, bone, liver, intestine and kidney were obtained at a fresh autopsy on a young man who died immediately after an accident. Placental tissue was obtained following a normal delivery. Liver. A piece of normal human liver was obtained, cut into small pieces and rinsed in many rinses of cold 0.9% NaCl solution. The specimen was then homogenized in a Vertis homogenizer, and the mixture was centrifuged 3 times in cold 0.9% NaCl at 600 g for 10 min to remove contaminating blood and bile. The pellet obtained was then homogenized in a Potter-Elvehjem homogenizer in ice cold sucrose (0.25 M). The liver homogenate was centrifuged in the cold at 600 g for 10 min and the supernatant stored at -20°C until ready for extraction. Similar procedures for extraction were used for kidney, intestine and placenta.
Alkaline
phosphatase
assay
The substrate used for alkaline phosphatase activity assay was 0.016 M p-nitrophenylphosphate (p-Npp) (Sigma 104) with an equal volume of I M 2-amino-2methyl-I-propanol (Ammenol buffer) at pH 10.3 with lO-3 M MgC& added. The absorbance was read at 410 nm in a Coleman spectrophotometer. The resultant activity was expressed as PM or nM/min/mg protein. The enzymes extracted from normal bone, liver, kidney, intestine, placenta and from osteosarcoma cells grown in tissue culture were investigated.
Purification
ofthe enzyme
extraction. Five ml of the supernatant were thawed and magnetically stirred at 4°C with n-butanol being added in a dropwise fashion over a period of I5 min to give a 20% solution. The stirring was continued for 30 more minutes. The mixture was centrifuged for 30 min at 38 000 g at 4°C and the aqueous layer collected and dialyzed for 20 h against 100 vol of distilled water at 4°C.
Bumto/
Osteosarcoma cells were cultured as a monolayer [ 141. Alkaline uhosohatase activity was inducible in these . . cells by the addition of 0.5 -pg/ml of hydrocortisone by gel filtration phosphate to the tissue culture medium and the enzyme Purification extracted 12 to 15 days after incubation as reported The partially purified butanol extract of the enzyme earlier [14]. The extracted enzyme was stored at -20°C preparation from osteosarcoma cells in tissue culture until ready for use. was layered on a column (2.6~40 cm) packed with Sephadex G-200 (Pharmacia Fine Chemicals) and eluted with 0.05 M Tris-HCI buffer, pH 8.6. The elutant Tissues was collected in a Buchler fraction collector at 4°C. The Bone. A piece of normal human bone excised in the eluted alkaline phosphatase from the column was concentrated tenfold by ultrafiltration in an Amicon course of an arthroplasty procedure in a young patient micro-ultrafiltration system using an XM-50 filter at 20 was washed in multiple changes of ice cold Ringer’s atm of pressure. This concentrated enzyme (from tissue lactate to remove as much of the blood as possible. The bone was then cut into small pieces and pulverized in a cultures) was used as antigen for antibody production cold pestle and mortar. The pulverate was suspended in (see antibody production). Gel filtration was also ice cold sucrose (0.25 M) and centrifuged at 600 g for 10 performed on the enzyme from normal human bone and min. The blood-tinged supernatant was discarded. The liver. Exptl
Cell
Res 95 (1973-J
Specific alkaline LOO 90
of osseous
+
80 I lo60SO403020IO-
Gel electrophoresis
of Smith, Lightstone 100 90 80 I
+
-
70 60
\ *
b
II
50 40 30 20 10 l!h 012
3
4
5
LL 6 7
Figs 2, 3. Abscissa: penetration into the gel (cm); ordinate: optical density. Fig. 2. A tracing of the densitometric scan of gel electrophoresis of the enzyme extracted from osteosarcoma cells grown in tissue culture (a) and the enzyme extracted from bone (b) removed during an operation (see Methods). The three peaks of activity in each enzyme are marked by arrows (J).
Pyrophosphatase
tissue
349
the enzyme was determined at pH 7.4 and pH 10.0. The released inorganic phosphate was determined by a modified Fiske & Subbarow technic [6]. The enzyme extracts from normal cadaveric bone, liver, kidney, intestine, fresh placenta and from osteosarcoma cells grown in tissue culture were investigated. The protein content of each of the enzyme extracts was determined and the results expressed as nM/min/mg of protein.
a -
phosphatase
assay
The substrate used for pyrophosphatase assay was 3.3 mM pyrophosphate (tetra sodium salt), with an equal volume of 1 .O M Tris HCI buffer to which 2x 10m3 M MgCI, was added. One ml of the buffer substrate mixture was incubated with 0.2 ml of the enzyme at 37°C for 1 h. The reaction was stopped by adding 4 ml of TCA to the mixture. The pyrophosphatase activity of
(modification & Perry [I83
Electrophoresis was carried out in a Hoefer disc electrophoresis apparatus. Only running 5 % polyacrylamide gels were used, as adequate results were obtained without the need of spacer or sample gels. In all experiments, control serum to which a trace of bromphenol blue (albumin-BPB) had been added to the dye was run as a tracer. Upon completion, the gels were rimmed directly from their running tube into a slightly larger tube containing the location buffer, and 2 mg/ml of substrate (alpha Naphthylphosphate), with 1 mglml Fast Blue RR (Harleco, Philadelphia) added to the location buffer just prior to use. The tubes were incubated in a dark room at room temperature (25°C) for 1 h and were scanned with a Canalco Model .I densitometer. The penetration of the enzyme and its peaks was expressed in cm on a standard scale.
Heat stability The enzyme extracted from normal human bone, from cells in tissue culture and from liver were assayed for specific activity and were then placed in a fixed temperature shaking water bath (56°C). The tubes were removed at intervals of 15,30,45,60,90, 120 and 300 sec. The tubes were immediately placed in crushed ice and assayed for the remaining enzymatic activity.
Inhibition studies To the substrate for alkaline freshly prepared L-homoarginine mixture, 0. I ml of the enzyme or osteosarcoma cells in tissue incubated at 37°C for 30 min.
phosphatase, 8 mM of was added. To this preparation (from bone culture) was added and The remaining activity
Table 1. Inhibition study Normal
Spec Initial activity L-Homoarginine L-Phenylalanine L-Tryptophan
’
Spec.
bone
act.”
34.4 x10-9 13.26~10-~ +2.5 x10-9 23.46x 1O-9 +2.2 x10-9 18.02x lo+ k2.8 x10-9
Induced Percentage inhibition
6;+7.5 31k6.5 4758.3
Spec
enzyme
act.‘”
32.76x lO-9 15.23~10-~ +1.8 x10+’ 26.04x IO+ k2.7 x1022.11 x 10-S +2.1 x10-9
Percentage inhibition
G.5U.7 20.5k8.3 32.5
t6.5
act.-moles/mg/min.
Exp~lCel~Res
95 (1975)
b
loo90. so-
+
lo60sow
QI
2
I
4
5
6
7
13 I4
Fig. 3. A composite of tracings of densitometric scans of the enzyme extracted from osteosarcoma cells grown in tissue culture; intestine; liver; liver+Ha;
placenta; bone; bone+Ha; kidney. Ha represents the effect of 8 mM homoarginine on the enzymes extracted from hone and liver.
was determined and expressed as percentage of inhibition of normal activity. The same experiment was repeated with L-tryptophan and L-phenylalanine. Following L-homoarginine inhibition experiment, gel electrophoresis was performed on the enzyme from bone and liver with 8 mM of homoarginine added to the enzyme.
antigen for antibody production. This avoided the possibility of contamination with human serum which may have remained in the bone in spite of repeated washings of cadaveric or fresh human bone.
Immunologic studies Only the enzyme extracted and purified from osteosarcoma cells grown in tissue culture was used as Exprl
Cell Res 95 (1975)
Antibody production Adjuvant mixtures were prepared by emulsifying purified alkaline phosphatase from cells in tissue culture with an equal volume of Freund’s complete adjuvant (1.5 parts Artacel, 8.5 parts mineral oil, 5 mg/ml mycobacterium). The initial injections were into
Specific alkaline phosphatase Table 2. Enzyme penetration Enzyme peaks First Second Third End of activity
Bone
Bone +Ha
1.80 3.00
1.70 3.00
6.25
i.70
of osseous tissue
35 1
into the gel (cm) Cells
Liver
Liver +Ha
1.70 3.00 4.15 6.50
1.60 4.40 5.70 7.30
1.55 3.80 5.30 6.80
the foot pads of all four feet and in both thighs of rabbits. In total, approx. 125 pg of protein was given to each animal throughout the six sites. The injections were repeated 2 weeks later and rabbits bled 7 days later. Booster injections were given 30 days later and the rabbits bled 7-9 days after that. The titre of the antibody was measured by immunofluorescence technics. When titres of one to 250 or higher had been obtained, the rabbits were bled for preparation of the antibody.
Pur$cation of the antibody and separation of 7s gamma globulin fraction The blood from the rabbit was allowed to clot and the serum separated by centrifugation at 600 g for 10 min. The serum was diluted with an equal volume of 0.85 % NaCI. To each 10 ml of the diluted serum, 2.05 g of anhydrous Na*SO, was slowly added, while the mixture was being continuously stirred to ensure even distribution. The mixture was allowed to stand at room temperature for 2 h and then centrifuged at 2 000 g for 30 min. The supernantant fluid was discarded and the precipitate dissolved in a volume of distilled water equal to two thirds of the initial volume of the diluted serum. The proteins were reprecipitated by adding 1.93 g of Na$O, for each 10 ml of distilled water used in the previous step. The mixture was allowed to stand at room temperature for 2 h and then centrifuged at 2 000 g for 30 min. The supernatant was discarded. This precipitation was carried out four times. The final precipitate was dissolved in distilled water and was dialyzed against 0.01 M phosphate buffered saline pH 7.2 at 4°C overnight. This mixture was dialyzed against distilled water overnight at 4°C. The final dialysate was centrifuged at 2000 g for 15 min. The precipitate was discarded and the supernatant was concentrated in an Amicon ultrafiltration cell using a XM-50 filter at 20 atm pressure.
Determination of the antibody concentration necessary for precipitation of alkaline phosphatase The rabbit antiserum was heated at 56°C for 30 min to inactivate the endogenous heat labile enzyme. Antibody precipitation of the enzyme was carried out by adding a constant amount of alkaline phosphatase extract from normal human bone, liver, kidney, and from cells in tissue culture (antigen 0.2 ml) to serial dilutions of rabbit antisera (0.2 ml) and incubating at 37°C for ) h.
Kidney
Placenta
Intestine
2.15 2.65
2.85 4.4
6.55
i.30
2.00 3.40 5.25 6.30
To this mixture, 0.2 ml solution of antirabbit globulin was added. The reaction mixture was kept at 4°C for 24 h, following which it was centrifuged at 900 g for 10 min and the supernatant decanted. The precipitate was washed in ice cold 0.9 % NaCl and resuspended in 0.05 M Tris HCI pH 7.4, equal to the volume of the decanted supernatant. The supernatant and the resuspended precipitate were assayed for alkaline phosphatase activity. The amount of endogenous alkaline phosphatase activity in the rabbit antiserum was determined and appropriate allowances were made in calculating the inhibition of activity by the antisera.
Zmmunodiffusion One per cent Noble Agar was used for immunodiffusion. The alkaline phosphatase extracted from normal human bone, liver, kidney, intestine, placenta and that obtained from osteosarcoma cells were placed in individual wells and the rabbit antiserum against the alkaline phosphatase from cell culture was placed in the center well.
RESULTS Heat stability Heat stability tests showed that the enzyme extracted from normal human bone and that from tissue culture cells as well as liver was heat labile at 56°C. There was rapid inactivation of the enzymes at this temperature (fig. 1). Znhibition studies The enzyme extracted from the cells as well as from bone showed maximal induction with L-homoarginine. There was some inhibition with L-phenylalanine and L-tryptophan, but not to the extent seen with homoarginine (table 1). Electrophoresis Gel electrophoresis on bone removed surgically showed three peaks, as did the enzyme Exprl
Cell Res 95 (1975)
352
Singh and Tsung a .>'
50-
>"\ II
)- '\ '\
4 o-
\: "
30-
/ c
-80
-40
0 50
52
54
56
56
60
62
64
66
68
70
72
74
76
76
80
b 20
Fig. 4. Abscissa: eluate (ml); ordinate: (lefr) nM/min; (right) @g/ml protein. Sephadex column chromatography of alkaline phosphatase extracts. (a) In the eluate from normal human bone the enzymatic activity appeared, directly after the void volume and nearly all of the enzyme passed through the column in 28 ml of the eluate. No activity was demon-
strable in the tube at 29 ml; (b) in the eluate obtained from the liver enzyme the activity appeared directly after the void volume and most of the enzyme activity had passed through the column in 37 ml. O-O, nmoles of enzyme activity as measured by its ability to hydrolyze p-nitrophenylphosphatelmin at 3PC; O-O, *g protein/ml of eluate).
extracted from tissue culture cells (fig. 2a, b). The electrophoretic migration of enzyme from liver indicated a fast moving peak and two other peaks that were in the same area of migration as the peak found in the study of enzyme from bone (fig. 3~). A composite of the gel electrophoresis of the enzyme from cadaveric tissue extracts of bone, liver, kidney, intestine and from a fresh placenta is shown in fig. 3a and b. The densitometric scans showed (table 2) that there were three distinct peaks of activ-
ity in the enzyme extracted from cells in tissue culture. The second and third peaks appeared to be confluent in the enzyme extracted from cadaveric human bone. The effect of 8 mM of homoarginine on the bone enzyme indicated no difference in the peaks, but the total penetration was decreased, indicating that the homoarginine effect was mostly on the third isozyme or peak. The enzyme from liver showed three distinct peaks of activity and the third was a fast penetrating isozyme which penetrated the
ExptlCeNRes
95 (1975)
Specific
5. Indirect fluorescent antibody test. Rabbit antisera against enzyme extract from osteosarcoma cells was reacted with fluorescent conjugated sheep antirabbit serum. (a) Strongly positive reaction against osteosarcoma cells grown in tissue culture. x1040;
Fig.
alkaline phosphatase
of osseous
tissue
353
(&) a positive reaction against normal bone cell smear; occasional bone tibres (arrow) showing reaction are also seen. x500; (c) a negative reaction to cells from normal human liver. x 1040. Exptl
Cell Res 95 (1975)
354
Singh und Tsung
Table 3 Pyrophosphatase nmoles/min/mg
Enzyme origin
Protein content mglml
pH
Cells Bone Intestine Kidnev Liver. Placenta
0.971 0.142 0.625 0.770 0.791 0.225
219.0 168.0 157.0 400.0 307.0 105.0
7.4
act. protein pH
10.0
97.0 79.0 62.0 290.0 295.0 88.0
farthest into the gel. The homoarginine not only inhibited the enzyme in regard to total penetration into the gel, but in addition, there was a distinct difference in electrophoretic mobility of the enzyme. All three peaks of the enzyme from liver appeared at an earlier penetration of the enzyme into the gel with homoarginine (table 2). The placental enzyme had only two peaks. However, a distinct shoulder was noticeable between the first and second peaks at approx. 3.35 cm (1) penetration of the gel. The intestinal enzyme had three distinct peaks and the first isozyme had a completely different penetration rate than the enzyme from liver, bone, or cells in tissue culture. The enzyme from kidney showed two peaks of penetration which were closely located. Sephadex column chromatography
When the results of Sephadex G-200 column chromatography were compared, the data indicated that the enzyme activity in bone, liver, and cells in tissue culture appeared immediately after the void volume. It was observed that all of the butanol extracted alkaline phosphatase from bone had passed through the Sephadex G-200 column in 28 ml of the eluate (fig. 4a). No enzyme activity could be demonstrated in the last tube, even Exptl
Cell
Res 95 (1975)
though traces of protein were detectable. However, the enzyme from liver was present in 37 ml of the eluate (fig. 4b). The concentration of protein in the fractions of the eluate from the liver and osseous enzyme were as shown in fig. 4a and h. The enzymatic activity and protein concentration in the eluate showed a much higher concentration of protein in peak 1 and lower concentration of the protein in peaks 2 and 3 of the enzyme from bone (fig. 4a). These data indicated that the specific activity of alkaline phosphatase expressed as nmoles/mg of protein/min are the greatest in peaks 2 and 3, rather than in peak 1. Pyrophosphatase
activity
All of the enzyme extracts showed definite pyrophosphatase activity in that there was liberation of inorganic phosphate from the tetra sodium pyrophosphate. Table 3 shows the pyrophosphatase activity of the enzyme at pH 7.4 and pH 10.0 in the presence of MgCl,. It was noted that the pyrophosphatase activity of all of the enzymes indicated that the release of inorganic phosphatase was greater at pH 7.4 (normal) than at the highly alkaline pH 10.0. Immunojluorescence
The results of immunofluorescence showed that the antibody titre was one to 512. The antisera reacted positively against cells from tissue culture and bone (fig. 5a, b) but results against hepatic cells were negative (fig. 5c). Zmmunodiffusion
The results of immunodiffusion were as shown in fig. 6a, b and c. There was a strong positive precipitate against the enzyme extracted from bone and the enzyme extracted from cells in tissue culture. No precipitate line appeared against alkaline phosphatase
Specific
alkaline phosphatase
b
of osseous
tissue
355
c
Fig. 6. Results of immunodiffusion. In (a) and (c) rabbit antiserum (Ab) to alkaline nhosnhatase derived from osteosarcoma cells grown in tissue culture was used as the antibodv in the center well. In (b) nuritied 7s gamma globulin (Ab) was used as the antibody. B, enzyme from bone; C, enzyme from osteosarcoma
cells in tissue culture; I, intestinal enzyme; K, kidney enzyme; L, liver enzyme; P, placental enzyme; S, saline. A positive reaction seen as a precipitate line occurred only against the enzyme extracted from cells grown in tissue culture (c) and against bone (b).
extracted from liver, kidney, placenta.
physiologic functions and substrates of alkaline phosphatases are still not totally known. The estimation of the enzymatic activity in clinical practice has been based upon the enzyme’s ability to hydrolyze p-nitrophenylphosphate at an alkaline pH of 8 or greater. p-Nitrophenylphosphate is an artificial compound and it is highly unlikely that the high pH’s at which this substrate is hydrolyzed exist under normal conditions in man. Recent work indicates that alkaline phosphatases do hydrolyze pyrophosphate at a neutral pH [3, 51. The concentration of this enzyme in plasma has been used as a diagnostic aid in metabolic bone disease. In general, the amount of the enzyme in plasma has been roughly correlated with the rate of bone formation, being high in Paget’s disease, osteomalacia and rickets, although no correlation has been found between the level of this enzyme and rate of bone resorption. Patients with a deficiency of cytoplasmic alkaline phosphatase in certain cells (hypophosphatasia, osteogenesis imperfecta) have pyrophosphaturia, suggesting that an impaired metabolism of inorganic pyro-
intestine
and
Precipitation test
The precipitation of the enzyme extracts from cells in tissue culture, normal cadaveric human bone, liver and kidney were as shown in fig. 7a, b and c. Allowances were made in the calculations for the endogenous enzyme in antiserum from the rabbit. No enzymatic activity was detected in the supernatant with normal bone and cells in tissue culture and antisera at dilutions of one in two and one in one. The entire enzymatic activity was recoverable in the precipitate. However, all of the enzymatic activity remained in the supernatant from the preparation from liver and kidney (fig. 7c). DISCUSSION Robison [12] postulated that the alkaline phosphatase of chondroblasts and osteoblasts was responsible for precipitation of calcium salts in bone by hydrolytic liberation of inorganic phosphate. However, the
Exptl
Cell Res 95 (1975)
5432I-
Ii28
I64
1’32
I‘,8
1’8
1’4
I’2
;I
Fig. 7. Abscissa: anti-serum dilutions; ordinate: alkaline phosphatase activityx 10e4 urn. Precipitate test. As the concentration of the antiserum was increased, the enzyme activity recoverable in the supernatant of the enzyme extracts from osteosarcoma cells grown in tissue culture (a) and normal human bone (b) decreased. At dilutions of 1 : 1, no activity was demonstrable in the supernatant and all of the activity was recovered in the precipitate (a. b). There was no decrease in the activity in the supernatant with the enzymes of liver and kidney (c) extraction. (Appropriate allowances were made for endogenous alkaline phosphatase in rabbit antiserum.) Exptl
Cell
Res 95 (1975)
phosphate is associated with this inborn error of metabolism. Another problem in correlating the levels of alkaline phosphatase with disease processes has been a general assumption that changes in the plasma levels are a direct reflection of changes at sites of bone formation. In the present investigation, the authors have isolated an alkaline phosphatase in vitro by growing osteosarcoma cells in tissue culture medium to which hydrocortisone (0.5 kg/ml) had been added. In the present investigation, the characteristics of this in vitro produced enzyme were compared with those of enzymes extracted from cadaveric normal human bone, liver, kidney, intestine, and placenta. It was observed that heat lability of this enzyme was about the same as for the enzyme extracted from liver and bone. The electrophoretic mobility of this in vitro produced enzyme closely resembled that of the enzyme extracted from normal human bone but not that of liver, kidney, intestine, or placenta. The results of Sephadex column chromatography indicated that the molecular size of the variants (bands, peaks) in the in vitro produced enzyme were approximately the same as in alkaline phosphatase extracted from human bone but were different from those of liver. By using the in vitro enzyme as the antigen for antibody production, possible sources of contamination with human serum were avoided. This was important since the serum contains all of the various forms of alkaline phosphatases except placental in cadaver extracts used in this investigation. Immunofluorescence studies (fig. 5~7, 6, c) conclusively indicated that the antibody was specific against the enzyme from cells in tissue culture and bone but not from liver. Immunodiffusion (fig. 6) also proved that the antibody induced by injection of the in vitro
Specific
produced enzyme was specific against the enzyme extracted from normal human bone and cells in tissue culture, but not against the enzyme extracted from human placenta, kidney, liver or intestine. Fleisch and co-workers [8, 9, lo] have suggested that alkaline phosphatase from bone is a pyrophosphatase and is responsible for regulating mineral ions, an extracellular process. In their view, the dissolution of both mineral of bone and matrix of bone is under enzymatic control, and parathyroid and thyrocalcitonin could affect the deposition of minerals or resorption of bone minerals either by controlling the rate of release of alkaline phosphatase into extracellular osseous fluid or by altering the activity of the released enzyme. Firschein & Urist [7] studied the alkaline phosphatase activity in mesenchymal cells invading active and inactive implants of matrix of bone. Their data indicated that induction of alkaline phosphatase activity occurred in the pre-osseous stage of morphogenesis and preceded the deposition of calcium by 5-7 days. No direct relationship was observed between the alkaline phosphatase and calcification in their investigation. These and other in vivo investigations have established a correlation between levels of enzymatic activity and osteogenesis. However, these data have not established whether enzymatic activity is concerned with cell modulation or with mineral deposition or both. The work of Morrill, Kestow & Murphy [I l] and Borle [l] suggests that pyrophosphatase and alkaline phosphatase may be synonymous and that there may be multiple forms in each tissue; one form being membrane bound and involved with the transport of ions and others within the cell involved in the hydrolysis of pyrophosphate. Three variants or peaks have been
alkaline phosphatase
of osseous
tissue
357
observed in the enzyme from bone by gel electrophoresis. It has been observed that the first variant of the enzyme extracted from bone had low specific activity per mg of protein in the present investigation. These findings were also observed in the enzyme extracted from osteosarcoma cells in tissue culture. The findings further confirm the data of Cox and associates [3] and Eaton & Moss [5] that alkaline phosphatases hydrolyze pyrophosphates at a neutral pH (7.4). In conclusion, a model for in vitro production, isolation, and purification of a specific alkaline phosphatase (pyrophosphatase) of osseous origin has been described. This work is dedicated to the memory of George D. Ludwig, M.D., late Professor and Chairman, Department of Medicine, Medical College of Ohio, Toledo, Ohio. During his lifetime he was a source of continuing inspiration and encouragement to the investigators. This research was in part supported by ACS Institutional Grant no. IN-98A and the Ohio Chapter of the ACS.
REFERENCES I. Borle, A B, Phosphate et mttabolisme phosphocalcique (ed D Hioco) pp. 295. Sandoz, Paris (1971). 2. Boyer, S H, Ann NY acad sci 103 (1963) 938. 3. Cox, R P, Gilbert, P & Griffin, M J, Biochem j I05 (1967) 155. 4. Cox, R P. Personal communication (1974). 5. Eaton, H R & Moss, D W, Enzymologia 35 (1968) 31. 6. Fiske, C H & Subbarow, Y, Inorganic phosphorus in clinical chemistry, principles agd procedures (ed J S Annino) pp __ 201-204, 3rd edn. Little. Brown & Co, Boston, MA (1964). 7. Firschein, H E & Urist, M R, Calicum tissue res 7 (1971) 108. 8. Fleisch, H, Maecki, J & Russell, R G G, Proc sot exp biol med 122 (1966) 317. 9. Fleisch, H & Russell, R G G, Int encyclopaedia of pharmacology and therapeutics, sect. 51. Pharmacology of the endocrine system and related drugs. Parathvroid hormone. thvrocalcitonin and related drugs section (ed H Rasmussen) vol. I, pp 61-100. Pergamon Press, Oxford (1970). IO. Fleisch, H,kussell, R G G & Straumann, F, Science I65 (1969) 1262. 11. Merrill, G A, Kostellow, A D & Murphy, J B, Exp cell res 66 (197 I) 289. Expr/
Cd
Res 95 (1975)
12.
Robison, R & Soames, K M, Biochem j 18 (1924) 740. 13. Schlamowitz, M & Bodansky, 0, J biol them 234 (1959) 1433. 14. Singh, I, Bartolomeo, L & Ludwig, G D. J biothem. Submitted for publication. 15. -J biol them. Submitted for oublication. 16. Singh, I, Tsang, K Y & Ludwig, G D, Cancer res 34 (1974) 2946.
Expd
Cell
Res 95 (1975)
17. - Em surg res 6 (1974) 247. 18. Smith, I, Lightstone, P J & Perry. J 0. acta 19 (1968) 499. 19. Sussman, H H, Small, P A & Cotlove. them 243 (1968) 160.
Received
March
25,
1975
Clin
chim
E, J biol