Properties of Alkaline Phosphatase of the Human Dental Pulp M. GOSEKI, S. OIDA, A. NIFUJI1, and S. SASAKI Department of Biochemistry and 11st Department of Oral Surgery, School of Dentistry, Tokyo Medical and Dental University, 5-45, Yushima l-Chome, Bunkyo-ku, Tokyo 113, Japan
Enzymatic and immunological properties of alkaline phosphatase [ALP;
orthophosphoric monoesterphosphohydrolase, alkaline optimum, EC 3.1.3.1.] in the human dental pulp were investigated. In inhibition and thermal inactivation studies, dental pulp ALP showed properties of universal-type ALP (kidney/bone/liver type). Dental pulp ALP crossreacted with polyclonal and monoclonal antibodies against purified swine-kidney ALP, and with monoclonal antibody against ALP of human osteoblast-like cells in the same manner as ALPs of human bone and kidney. The sodium dodecyl sulfate-gel electrophoretic pattern showed a 140, 000-Mr native protein band. These data suggest that dental pulp ALP can be classified as a universal-type ALP having antigenic determinants common to ALP of the kidney and bone. J Dent Res 69(3):909-912, March, 1990
Introduction. Human alkaline phosphatases (ALPs) are classified into three main types: kidney/liver/bone, or "universal" type, intestinal type, and placental type (Badger and Sussman, 1976; Seargeant and Stinson, 1979). They are products of homologous but non-identical genes (Kam et aL, 1985; Weiss et al., 1986; Henthorn et al., 1987) and are distinguished by their enzymatic properties. Although human bone, kidney, liver, intestinal, and placental ALPs are well-characterized, no detailed investigation has yet been made of the ALP of human dental pulp. There were several reports concerning dental tissue ALPs in mammals such as bovines (Harada et al., 1982; Oida et al., 1985), swine (Goseki et al., 1988), and rats (Granstrom and Linde, 1972; Tojyo, 1983), and their biochemical properties were almost similar to those of bone and kidney ALPs, but not to the intestinal or the human placental enzymes (Doellgast and Benirschke, 1979; Goldstein et al., 1982). The cells in the dental pulp are heterogeneous, and include the odontoblast cell layer adjacent to dentin and pulp cells and other cells inside the odontoblast layer. The odontoblasts develop from mesenchymal cells, and it is suggested that the pulp cells have an ability to differentiate into the odontoblasts, even after the completion of tooth development, or in unusual conditions (Kasugai et al., 1988). In the dental pulp, ALP is localized in the odontoblasts and sub-odontoblast layers, which have the highest ALP activity in the pulp (Goggins and Fullmer, 1967; Yoshiki and Kurahashi, 1971). This enzyme might be functional in dentin mineralization. This study was designed to examine enzymatic properties of human dental pulp ALP in detail and to investigate its immunological properties with use of polyclonal antibody (Oida et al., 1984), as well as monoclonal antibodies, against ALP of human osteoblast-like cells (HBC) (Goseki et al., 1989) or purified ALP of swine kidney (Goseki et al., 1988). Received for publication August 10, 1989 Accepted for publication November 2, 1989
Materials and methods. Preparation of tissue ALP.-Human dental pulp was obtained from young normal teeth (third molars) extracted as part of orthodontic treatment. The pulp was homogenized in Trisbuffered saline (TBS; 10 mmol/L Tris-HCl, pH 7.4, containing 0.9% NaCl) with a Polytron homogenizer (Kinematica, Switzerland). Human placenta was from normal labor, and bone was from surgical operations. The other human samples were autopsy materials. Swine and bovine tissues were obtained from a local slaughterhouse. White rabbits (3-4 kg, males) and Wistar-strain rats (250-350 g, males) were from commercial sources. The tissues were homogenized with distilled water (2 mL/g of tissue) and mixed with 1 mL 1-butanol. The aqueous phase was separated, and the precipitates that appeared at pH 4.9 by the addition of acetic acid were removed by centrifugation. The supernatant was adjusted quickly to pH 6.5, and the precipitates that appeared following acetone addition (50% in final concentration) were collected by centrifugation and desiccated to a powder. The human dental pulp, bone tissue, and HBC were homogenized with a Polytron homogenizer in TBS and centrifuged. The supernatant was used for immunological experiments. Enzyme-activity assay. -ALP activity was determined at 370C with 10 mmol/L p-nitro-phenylphosphate as a substrate in 100 mmol/L 2-amino-2-methyl-1,3-propanediol-HCl buffer, pH 10.0, containing 5 mmol/L MgCl2. The enzyme activity was also assayed in the presence of various concentrations of inhibitors, such as levamisole, L-homoarginine, and L-phenylalanine. For thermostability to be assayed, the enzyme preparation was pre-treated at 560C for various time intervals and then reacted with the substrate. The time in minutes required for reduction of ALP activity to 50% of the original activity was determined. Protein determination. -Protein concentration was determined by the method of Lowry et al. (1951). Human osteoblast-like cells. -A primary culture of human osteoblast-like cells (HBC) was used as the source of ALP antigen. Neonatal human long bone was obtained from a stillborn baby who had been dead for several hours. This cell preparation was a gift from Drs. H. Shimokawa and K. Ibaraki, and they will publish the details of its cytological characterization elsewhere. Production of monoclonal antibodies against HBC ALP. Female BALB/c mice were injected intraperitoneally with 107 HBC. Cell fusion was performed between the spleen cells of immunized mice and mouse myeloma cells (Line P3-NS1-Ag41, Flow Lab., Inc.). Hybridoma cells were screened for antiALP antibody production, according to the ELISA method of Wray and Harris (1983). Five stable clones were established; two of them were IgG3, and the others were IgM. One of the most stable clones of IgG3 was designated G3. The hybridoma cells of G3 were inoculated intraperitoneally into BALB/c mice pre-treated with 0.5 mL of pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chemical). When abdominal swelling was detected after one to three weeks, the ascitic fluid was collected, purified by affinity chromatography 909
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the method of Towbin et al. (1979). The transfer medium was 25 mmol/L Tris, 192 mmol/L glycine, pH 8.3, containing 20% methanol. Then, the nitrocellulose sheet was washed with TBS and blocked with 3% skim milk solution in TBS for 40 min and incubated for 24 h with antibody (MCA-G3) solution diluted 500 times and treated as described above. Human-dental-pulp-ALP activity in the sheet was stained with 3-naphthyl phosphate (Kurahashi and Yoshiki, 1972), and molecular-weight standard proteins (ferritin subunit, phosphorylase b, bovine serum albumin, catalase, ovalbumin, lactate dehydrogenase, and carbonic anhydrase) were stained with Amido Black after electro-transfer onto the membrane. (3) The ELISA method was performed as described by Wray and Harris (1983). Northern-blot analysis of mRNA. -RNAs of tissues extracted by the guanidinium/cesium chloride centrifugation method (Maniatis et al., 1982) were electrophoresed on a 1.5% agarose gel with 2.2 mol/L formaldehyde, transferred to a nylon membrane, and hybridized with 32P-labeled cDNA of universal-type ALP, a gift from Dr. M.J. Weiss (1986).
TABLE 1 SPECIFIC ACTIVITIES OF ALKALINE PHOSPHATASES Specific activity Samples (Ua/mg
protein)b (homogenate) 0.341 ± 0.001 Human dental pulp 0.031 ± 0.004 Human bone 0.117 ± 0.009 Human osteoblast-like cells 0.143 ± 0.008 MC3T3-E1 cells (a) U = pumol p-nitrophenol formed/min. (b) Values are the mean ± S.D. of triplicate measurements.
with Protein A (Affi-Gel Protein A MAPS Kit, BioRad Lab.), and stored at - 80°C. The antibody was used at a concentration of 5.4 mg/mL. Production ofpolyclonal and monoclonal antibodies against purified swine-kidney ALP. -Polyclonal antibody against purified swine-kidney ALP was raised in rabbits, as described previously (Oida et al., 1984), and purified by affinity chromatography with Affi-Gel Protein A. Two stable clones of monoclonal antibodies against purified kidney ALP-designated Ml(IgM) and M2(IgM) (Goseki et al., 1988)-were also used. Immunological assay. -(1) Dot-blot assay (Hawkes et al., 1982).-The powdered sample of tissue extract was dissolved in TBS, and 50 ,uL of the solution (0.1 U/ml) was blotted onto a nitrocellulose membrane. After pre-treatment with 3% skim milk solution in TBS, the membrane was incubated with antibody solution diluted 500 times by the buffer for 24 h at 4°C and was soaked in horseradish peroxidase-conjugated goat antimouse IgG (Cappel Lab.) for one h at room temperature. The membrane was washed with TBS and stained with peroxidase substrate solution (a mixture of 30 mg of 4-chloro-1-naphthol dissolved in 10 mL methanol, 50 mL TBS, and 100 1xL of
Results. The ALP specific activity in the human dental pulp homogenate was 0.341 U/mg protein, which was about ten times that of bone homogenate (Table 1). ALP activities of HBC and MC3T3-E1 cells, which are established mouse osteoblast line cells (Sudo et al., 1983), were 0.117 U/mg and 0.143 U/mg,
respectively. From the results of inhibition and thermal inactivation experiments shown in Table 2, it was confirmed that human dental pulp ALP had properties of universal-type ALP, clearly different from human intestinal and placental ALPs. Moreover, the human dental pulp ALP possessed almost the same enzymatic properties as other mammalian dental tissue ALPs. The immuno-cross-reactivity of ALPs from the human dental pulp and other human tissues was investigated by use of various antibodies. The immunological results are summarized in Table 3. By dot-blot assay and ELISA, almost the same results were obtained, and human dental pulp ALP cross-reacted clearly with all of the monoclonal antibodies (MCA-G3, MCA-M1, and M2) and the polyclonal antibody. Human dental pulp ALP was analyzed by 7.5% SDS-PAGE,
30% H202)-
(2) Western-blot assay.-SDS-polyacrylamide gel electrophoresis of human dental pulp ALP (10 pLg protein/lane) was carried out by the method of Weber and Osborn (1969) with 7.5% gel in 50 mmol/L phosphate buffer containing 0.1% SDS at pH 7.0, at 2 mA/lane for two h. Proteins separated in the slab were transferred onto a nitrocellulose membrane (pore size 0.45 ,um; BioRad Lab.) at 5 V/cm for 30 min, according to
TABLE 2 PROPERTIES OF ALKALINE PHOSPHATASES* Concentration or time required for reduction of ALP activity to 50% of original activity Heat stability Lev
L-HA
Tissue (mmol/L) (mmol/L) 0.03 2.08 Human dental pulp 0.03 1.49 Bovine dental pulp Swine dental pulp 0.04 1.96 Rabbit dental pulp 0.02 2.90 Mouse tooth germ 0.03 1.54 Human bone 0.03 1.17 Human kidney 0.03 1.18 Human liver 0.03 1.88 > Human 1 > intestine 10 Human placenta > 10 0.93 *Activity was assayed by the rate of hydrolysis of p-nitrophenyl phosphate. The effects of MgCl2 in the assay mixture. (a) Lev, levamisole; L-HA, L-homoarginine; L-Phe, L-phenylalanine.
Species
L-Phe
(mmol/L)
560C (min)
600C (min) 1.50 1.71 0.99
19.9 2.56 >20 11.33 >20 3.14 >20 9.48 2.73 >20 9.14 1.43 18 2.65 0.72 19.1 5.49 1.51 > 20 3.56 1.85 1.9 21.4 7.22 2.3 >30 > 30 inhibitors were determined in the presence of 5 mmol/L
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ALKALINE PHOSPHATASE OF HUMAN DENTAL PULP
Vol. 69 No. 3
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TABLE 3
IMMUNOLOGICAL EXPERIMENTS ON HUMAN ALKALINE PHOSPHATASES Enzyme Source of ALP Dental Antibody Pulp Kidney Bone Liver Intestine Placenta + + + MCA-G3 + + + MCA-M1 + + + MCA-M2 + + + PCA + MCA-G3, monoclonal antibody against human osteoblast-like cell ALP. MCA-M1,M2, monoclonal antibodies against purified swine-kidney ALP. PCA, polyclonal antibody against purified swine-kidney ALP.
and its molecular weight was estimated to be about 140,000 (Fig., lane 3) when calibrated by the migration positions of standard proteins (lanes 1 and 2). By Western blotting, human dental pulp ALP cross-reacted clearly with MCA-G3 (Fig., lane 5). When dental pulp ALP was treated overnight with 1% 2-mercaptoethanol and 1% SDS, it did not cross-react with MCA-G3 (Fig., lane 6), nor did it have catalytic activity (Fig., lane 4). Northern analysis was performed on cellular RNAs from human and bovine dental pulps, with universal-type ALP cDNA used as a hybridization probe. Universal-type ALP mRNA was detected in both the human and bovine dental pulp tissues (data not shown).
Discussion. In inhibition and thermal inactivation experiments on human dental pulp ALP, it was revealed that the enzyme can be classified as being of the universal (kidney/liver/bone) type. It was rather difficult for us to find substantial difference in enzymatic properties between the human dental ALP and other universaltype ALPs. We have reported that polyclonal antibody against porcinekidney ALP raised in rabbit, cross-reacted with human universal-type ALPs, but not with intestinal or placental ALP (Oida et al., 1984). This antibody could be used so that universal-type ALP could be distinguished clearly from intestinal or placental type; however, no difference in immunological reactivity was observed among universal-type ALPs. Electrophoretic mobility is the only measure routinely used for the distinction of their difference in clinical laboratories (Sundblad et al., 1973). Recently, we found minor immunological differences in crossreactivity among universal-type ALPs by monoclonal antibodies against swine-kidney ALP (Ml and M2; Goseki et al., 1988) or against HBC-cells ALP (G3; Goseki et al., 1989). Human liver ALP scarcely cross-reacted with those monoclonal antibodies. The monoclonal antibody preparations will be useful in distinguishing human kidney and bone ALP from liver ALP. Therefore, the investigation of the cross-reactivity of human dental pulp ALP with those monoclonal antibodies was attractive, and the present results lend support to the concept that human dental pulp, bone, and kidney ALPs have antigenic specificity different from that of liver enzymes. These minor immunological differences among human universal-type ALPs may be caused by a diversity of sugar chain residues. In addition, Northern analysis of the human dental pulp mRNA by a cDNA probe for universal-type ALP confirmed the type of the enzyme. The result in the Fig. shows that enzymatic activity can be detected on a single band corresponding to a native form of
Fig.-SDS-polyacrylamide gel electrophoresis and Western blot immunoassay by MCA-G3. Samples in lanes 4 and 6 were incubated with 1% mercaptoethanol and 1% SDS at 40C overnight, prior to electrophoresis, and those in lanes 3 and 5 were electrophoresed with polyacrylamide gel immediately after the addition of SDS. After electrophoresis, proteins in the gel were transferred onto the nitrocellulose membrane. Standard proteins were stained with Amido Black (lanes 1 and 2); ferritin subunit (220,000), phosphorylase b (94,000), bovine serum albumin (67,000), catalase (60,000), ovalbumin (43,000), lactate dehydrogenase (36,000), and carbonic anhydrase (30,000). Lanes 3-6 are the human dental pulp ALP; enzymatic activity was stained by the P-naphthyl phosphate method (lanes 3 and 4), and immunostaining was carried out with MCA-G3 (lanes 5 and 6).
ALP after electrophoresis and transfer to a nitrocellulose membrane (lanes 3 and 5). Conversion to an inactive monomeric form by prior treatment with mercaptoethanol and SDS was suggested (lanes 4 and 6). These results may indicate that the native ALP of dental pulp is in dimeric form, as are other mammalian ALPs. The kidney/bone/liver-type ALPs are synthesized from a single common gene (Weiss et al., 1986) with minor post-translational heterogeneities. The identity of the molecular scaffold, including catalytic site of universal-type ALPs, may reflect a basically common function of this enzyme in different tissues. Specific localization of ALP activity at the brush-border membrane in kidney tubules or at the sub-odontoblast layer in the dental pulp suggests a role of this enzyme in transport to ions, especially phosphate.
Acknowledgments. We thank Drs. H. Shimokawa and K. Ibaraki in our Department for a gift of cultured human osteoblast-like cells, and Dr. M.J. Weiss for a gift of universal-type ALP cDNA. REFERENCES BADGER, K.S. and SUSSMAN, H.H. (1976): Structural Evidence that Human Liver and Placental Alkaline Phosphatase Isoenzymes are Coded by Different Genes, Proc Natl Acad Sci USA 73:22012205. DOELLGAST, G.J. and BENIRSCHKE, K. (1979): Placental Alkaline Phosphatase in Hominidae, Nature 280:601-602. GOGGINS, J.F. and FULLMER, H.M. (1967): Hydrolytic Enzyme Histochemistry of the Rat Molar Pulp, Arch Oral Biol 12:639644. GOLDSTEIN, D.J.; ROGERS, C.; and HARRIS, H. (1982): Evolution of Alkaline Phosphatases in Primates, Proc Natl Acad Sci USA 79:879-883.
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GOSEKI, M.; OIDA, S.; OGATA, Y.; and SASAKI, S. (1989): Immunological Differentiation of Human Universal Type Alkaline Phosphatases by a Monoclonal Antibody to the Human Bone Cell Enzyme, Clin Chim Acta 180:189-191. GOSEKI, M.; OIDA, S.; and SASAKI, S. (1988): Detection of Minor Immunological Differences Among Human Universal Type Alkaline Phosphatases, J Cellular Biochem 38:155-163. GRANSTROM, G. and LINDE, A. (1972): A Biochemical Study of Alkaline Phosphatase in Isolated Rat Incisor Odontoblasts, Arch Oral Biol 17:1213-1224. HARADA, M.; HIRAOKA, B.Y.; FUKASAWA, K.; and FUKASAWA, K.M. (1982): Purification and Properties of Bovine Dental Pulp Alkaline Phosphatase, Arch Oral Biol 27:69-74. HAWKES, R.; NIDAY, E.; and GORDON, J. (1982): A Dot-immunobinding Assay for Monoclonal and Other Antibodies, Anal Biochem 119:142-147. HENTHORN, P.S.; RADUCHA, M.; EDWARDS, Y.H.; WEISS, M.J.; SLAUGHTER, C.; LAFFERTY, M.A.; and HARRIS, H. (1987): Nucleotide and Amino Acid Sequences of Human Intestinal Alkaline Phosphatase: Close Homology to Placental Alkaline Phosphatase, Proc Natl Acad Sci USA 84:1234-1238. KAM, W.; CLAUSER, E.; KIM, Y.S.; KAN, Y.W.; and RUTTER, W.J. (1985): Cloning, Sequencing, and Chromosomal Localization of Human Term Placental Alkaline Phosphatase cDNA, Proc Natl Acad Sci USA 82:8715-8719. KASUGAI, S.; ADACHI, M.; and OGURA, H. (1988): Establishment and Characterization of a Clonal Cell Line (RPC-C2A) from Dental Pulp of the Rat Incisor, Arch Oral Biol 33:887-891. KURAHASHI, Y. and YOSHIKI, S. (1972): Electron Microscopic Localization of Alkaline Phosphatase in the Enamel Organ of the Young Rat, Arch Oral Biol 17:155-163. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L.; and RANDALL, R.J. (1951): Protein Measurement with the Folin Phenol Reagent, J Biol Chem 193:265-275. MANIATIS, T.; FRITSCH, E.F.; and SAMBROOK, J. (1982): Molecular Cloning: a Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor Lab. OIDA, S.; GOSEKI, M.; and SASAKI, S. (1985): Purification and
J Dent Res March 1990 Subunit Structure of Alkaline Phosphatase from Bovine Enamel Organ, Arch Oral Biol 30:193-196. OIDA, S.; SONE(GOSEKI), M.; and SASAKI, S. (1984): Purification of Swine Kidney Alkaline Phosphatase by Immunoaffinity Chromatography, Anal Biochem 140:117-120. SEARGEANT, L.E. and STINSON, R.A. (1979): Evidence that Three Structural Genes Code for Human Alkaline Phosphatases, Nature 281:152-154. SUDO, H.; KODAMA, H.; AMAGAI, Y.; YAMAMOTO, S.; and KASAI, S. (1983): In vitro Differentiation and Calcification in a New Clonal Osteogenic Cell Line Derived from Newborn Mouse Calvaria, J Cell Biol 96:191-198. SUNDBLAD, L.; WALLIN-NILSSON, M.; and BROHULT, J. (1973): Characterization of Alkaline Phosphatase Isoenzymes in Serum by Agar Gel Electrophoresis, Clin Chim Acta 45:219-223. TOJYO, Y. (1983): A Comparison of the Alkaline Phosphatases of Rat Dental Pulp, Bone, Kidney, Liver and Intestine, Arch Oral Biol 28:103-107. TOWBIN, H.; STAEHELIN, T.; and GORDON, J. (1979): Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications, Proc Natl Acad Sci USA 76:4350-4354. WEBER, K. and OSBORN, M. (1969): The Reliability of Molecular Weight Determinations by Dodecyl Sulfate-polyacrylamide Gel Electrophoresis, J Biol Chem 244:4406-4412. WEISS, M.J.; HENTHORN, P.S.; LAFFERTY, M.A.; SLAUGHTER, C.; RADUCHA, M.; and HARRIS, H. (1986): Isolation and Characterization of a cDNA Encoding a Human Liver/bone/ kidney-type Alkaline Phosphatase, Proc Natl Acad Sci USA 83:7182-7186. WRAY, L.K. and HARRIS, H. (1983): Monoclonal Antibodies to an Ectopically Expressed Alkaline Phosphatase in a Human Malignant Cell Line, Cancer Res 43:758-762. YOSHIKI, S. and KURAHASHI, Y. (1971): A Light and Electron Microscopic Study of Alkaline Phosphatase Activity in the Early Stage of Dentinogenesis in the Young Rat, Arch Oral Biol 16:11431154.
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Enzymatic and immunological properties of alkaline phosphatase [ALP;
orthophosphoric monoesterphosphohydrolase, alkaline optimum, EC 3.1.3.1.] in the human dental pulp were investigated. In inhibition and thermal inactivation studies, dental pulp ALP showed properties of universal-type ALP (kidney/bone/liver type). Dental pulp ALP crossreacted with polyclonal and monoclonal antibodies against purified swine-kidney ALP, and with monoclonal antibody against ALP of human osteoblast-like cells in the same manner as ALPs of human bone and kidney. The sodium dodecyl sulfate-gel electrophoretic pattern showed a 140, 000-Mr native protein band. These data suggest that dental pulp ALP can be classified as a universal-type ALP having antigenic determinants common to ALP of the kidney and bone. J Dent Res 69(3):909-912, March, 1990
Introduction. Human alkaline phosphatases (ALPs) are classified into three main types: kidney/liver/bone, or "universal" type, intestinal type, and placental type (Badger and Sussman, 1976; Seargeant and Stinson, 1979). They are products of homologous but non-identical genes (Kam et aL, 1985; Weiss et al., 1986; Henthorn et al., 1987) and are distinguished by their enzymatic properties. Although human bone, kidney, liver, intestinal, and placental ALPs are well-characterized, no detailed investigation has yet been made of the ALP of human dental pulp. There were several reports concerning dental tissue ALPs in mammals such as bovines (Harada et al., 1982; Oida et al., 1985), swine (Goseki et al., 1988), and rats (Granstrom and Linde, 1972; Tojyo, 1983), and their biochemical properties were almost similar to those of bone and kidney ALPs, but not to the intestinal or the human placental enzymes (Doellgast and Benirschke, 1979; Goldstein et al., 1982). The cells in the dental pulp are heterogeneous, and include the odontoblast cell layer adjacent to dentin and pulp cells and other cells inside the odontoblast layer. The odontoblasts develop from mesenchymal cells, and it is suggested that the pulp cells have an ability to differentiate into the odontoblasts, even after the completion of tooth development, or in unusual conditions (Kasugai et al., 1988). In the dental pulp, ALP is localized in the odontoblasts and sub-odontoblast layers, which have the highest ALP activity in the pulp (Goggins and Fullmer, 1967; Yoshiki and Kurahashi, 1971). This enzyme might be functional in dentin mineralization. This study was designed to examine enzymatic properties of human dental pulp ALP in detail and to investigate its immunological properties with use of polyclonal antibody (Oida et al., 1984), as well as monoclonal antibodies, against ALP of human osteoblast-like cells (HBC) (Goseki et al., 1989) or purified ALP of swine kidney (Goseki et al., 1988). Received for publication August 10, 1989 Accepted for publication November 2, 1989
Materials and methods. Preparation of tissue ALP.-Human dental pulp was obtained from young normal teeth (third molars) extracted as part of orthodontic treatment. The pulp was homogenized in Trisbuffered saline (TBS; 10 mmol/L Tris-HCl, pH 7.4, containing 0.9% NaCl) with a Polytron homogenizer (Kinematica, Switzerland). Human placenta was from normal labor, and bone was from surgical operations. The other human samples were autopsy materials. Swine and bovine tissues were obtained from a local slaughterhouse. White rabbits (3-4 kg, males) and Wistar-strain rats (250-350 g, males) were from commercial sources. The tissues were homogenized with distilled water (2 mL/g of tissue) and mixed with 1 mL 1-butanol. The aqueous phase was separated, and the precipitates that appeared at pH 4.9 by the addition of acetic acid were removed by centrifugation. The supernatant was adjusted quickly to pH 6.5, and the precipitates that appeared following acetone addition (50% in final concentration) were collected by centrifugation and desiccated to a powder. The human dental pulp, bone tissue, and HBC were homogenized with a Polytron homogenizer in TBS and centrifuged. The supernatant was used for immunological experiments. Enzyme-activity assay. -ALP activity was determined at 370C with 10 mmol/L p-nitro-phenylphosphate as a substrate in 100 mmol/L 2-amino-2-methyl-1,3-propanediol-HCl buffer, pH 10.0, containing 5 mmol/L MgCl2. The enzyme activity was also assayed in the presence of various concentrations of inhibitors, such as levamisole, L-homoarginine, and L-phenylalanine. For thermostability to be assayed, the enzyme preparation was pre-treated at 560C for various time intervals and then reacted with the substrate. The time in minutes required for reduction of ALP activity to 50% of the original activity was determined. Protein determination. -Protein concentration was determined by the method of Lowry et al. (1951). Human osteoblast-like cells. -A primary culture of human osteoblast-like cells (HBC) was used as the source of ALP antigen. Neonatal human long bone was obtained from a stillborn baby who had been dead for several hours. This cell preparation was a gift from Drs. H. Shimokawa and K. Ibaraki, and they will publish the details of its cytological characterization elsewhere. Production of monoclonal antibodies against HBC ALP. Female BALB/c mice were injected intraperitoneally with 107 HBC. Cell fusion was performed between the spleen cells of immunized mice and mouse myeloma cells (Line P3-NS1-Ag41, Flow Lab., Inc.). Hybridoma cells were screened for antiALP antibody production, according to the ELISA method of Wray and Harris (1983). Five stable clones were established; two of them were IgG3, and the others were IgM. One of the most stable clones of IgG3 was designated G3. The hybridoma cells of G3 were inoculated intraperitoneally into BALB/c mice pre-treated with 0.5 mL of pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chemical). When abdominal swelling was detected after one to three weeks, the ascitic fluid was collected, purified by affinity chromatography 909
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GOSEKI et al.
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J Dent Res March 1990
the method of Towbin et al. (1979). The transfer medium was 25 mmol/L Tris, 192 mmol/L glycine, pH 8.3, containing 20% methanol. Then, the nitrocellulose sheet was washed with TBS and blocked with 3% skim milk solution in TBS for 40 min and incubated for 24 h with antibody (MCA-G3) solution diluted 500 times and treated as described above. Human-dental-pulp-ALP activity in the sheet was stained with 3-naphthyl phosphate (Kurahashi and Yoshiki, 1972), and molecular-weight standard proteins (ferritin subunit, phosphorylase b, bovine serum albumin, catalase, ovalbumin, lactate dehydrogenase, and carbonic anhydrase) were stained with Amido Black after electro-transfer onto the membrane. (3) The ELISA method was performed as described by Wray and Harris (1983). Northern-blot analysis of mRNA. -RNAs of tissues extracted by the guanidinium/cesium chloride centrifugation method (Maniatis et al., 1982) were electrophoresed on a 1.5% agarose gel with 2.2 mol/L formaldehyde, transferred to a nylon membrane, and hybridized with 32P-labeled cDNA of universal-type ALP, a gift from Dr. M.J. Weiss (1986).
TABLE 1 SPECIFIC ACTIVITIES OF ALKALINE PHOSPHATASES Specific activity Samples (Ua/mg
protein)b (homogenate) 0.341 ± 0.001 Human dental pulp 0.031 ± 0.004 Human bone 0.117 ± 0.009 Human osteoblast-like cells 0.143 ± 0.008 MC3T3-E1 cells (a) U = pumol p-nitrophenol formed/min. (b) Values are the mean ± S.D. of triplicate measurements.
with Protein A (Affi-Gel Protein A MAPS Kit, BioRad Lab.), and stored at - 80°C. The antibody was used at a concentration of 5.4 mg/mL. Production ofpolyclonal and monoclonal antibodies against purified swine-kidney ALP. -Polyclonal antibody against purified swine-kidney ALP was raised in rabbits, as described previously (Oida et al., 1984), and purified by affinity chromatography with Affi-Gel Protein A. Two stable clones of monoclonal antibodies against purified kidney ALP-designated Ml(IgM) and M2(IgM) (Goseki et al., 1988)-were also used. Immunological assay. -(1) Dot-blot assay (Hawkes et al., 1982).-The powdered sample of tissue extract was dissolved in TBS, and 50 ,uL of the solution (0.1 U/ml) was blotted onto a nitrocellulose membrane. After pre-treatment with 3% skim milk solution in TBS, the membrane was incubated with antibody solution diluted 500 times by the buffer for 24 h at 4°C and was soaked in horseradish peroxidase-conjugated goat antimouse IgG (Cappel Lab.) for one h at room temperature. The membrane was washed with TBS and stained with peroxidase substrate solution (a mixture of 30 mg of 4-chloro-1-naphthol dissolved in 10 mL methanol, 50 mL TBS, and 100 1xL of
Results. The ALP specific activity in the human dental pulp homogenate was 0.341 U/mg protein, which was about ten times that of bone homogenate (Table 1). ALP activities of HBC and MC3T3-E1 cells, which are established mouse osteoblast line cells (Sudo et al., 1983), were 0.117 U/mg and 0.143 U/mg,
respectively. From the results of inhibition and thermal inactivation experiments shown in Table 2, it was confirmed that human dental pulp ALP had properties of universal-type ALP, clearly different from human intestinal and placental ALPs. Moreover, the human dental pulp ALP possessed almost the same enzymatic properties as other mammalian dental tissue ALPs. The immuno-cross-reactivity of ALPs from the human dental pulp and other human tissues was investigated by use of various antibodies. The immunological results are summarized in Table 3. By dot-blot assay and ELISA, almost the same results were obtained, and human dental pulp ALP cross-reacted clearly with all of the monoclonal antibodies (MCA-G3, MCA-M1, and M2) and the polyclonal antibody. Human dental pulp ALP was analyzed by 7.5% SDS-PAGE,
30% H202)-
(2) Western-blot assay.-SDS-polyacrylamide gel electrophoresis of human dental pulp ALP (10 pLg protein/lane) was carried out by the method of Weber and Osborn (1969) with 7.5% gel in 50 mmol/L phosphate buffer containing 0.1% SDS at pH 7.0, at 2 mA/lane for two h. Proteins separated in the slab were transferred onto a nitrocellulose membrane (pore size 0.45 ,um; BioRad Lab.) at 5 V/cm for 30 min, according to
TABLE 2 PROPERTIES OF ALKALINE PHOSPHATASES* Concentration or time required for reduction of ALP activity to 50% of original activity Heat stability Lev
L-HA
Tissue (mmol/L) (mmol/L) 0.03 2.08 Human dental pulp 0.03 1.49 Bovine dental pulp Swine dental pulp 0.04 1.96 Rabbit dental pulp 0.02 2.90 Mouse tooth germ 0.03 1.54 Human bone 0.03 1.17 Human kidney 0.03 1.18 Human liver 0.03 1.88 > Human 1 > intestine 10 Human placenta > 10 0.93 *Activity was assayed by the rate of hydrolysis of p-nitrophenyl phosphate. The effects of MgCl2 in the assay mixture. (a) Lev, levamisole; L-HA, L-homoarginine; L-Phe, L-phenylalanine.
Species
L-Phe
(mmol/L)
560C (min)
600C (min) 1.50 1.71 0.99
19.9 2.56 >20 11.33 >20 3.14 >20 9.48 2.73 >20 9.14 1.43 18 2.65 0.72 19.1 5.49 1.51 > 20 3.56 1.85 1.9 21.4 7.22 2.3 >30 > 30 inhibitors were determined in the presence of 5 mmol/L
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ALKALINE PHOSPHATASE OF HUMAN DENTAL PULP
Vol. 69 No. 3
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TABLE 3
IMMUNOLOGICAL EXPERIMENTS ON HUMAN ALKALINE PHOSPHATASES Enzyme Source of ALP Dental Antibody Pulp Kidney Bone Liver Intestine Placenta + + + MCA-G3 + + + MCA-M1 + + + MCA-M2 + + + PCA + MCA-G3, monoclonal antibody against human osteoblast-like cell ALP. MCA-M1,M2, monoclonal antibodies against purified swine-kidney ALP. PCA, polyclonal antibody against purified swine-kidney ALP.
and its molecular weight was estimated to be about 140,000 (Fig., lane 3) when calibrated by the migration positions of standard proteins (lanes 1 and 2). By Western blotting, human dental pulp ALP cross-reacted clearly with MCA-G3 (Fig., lane 5). When dental pulp ALP was treated overnight with 1% 2-mercaptoethanol and 1% SDS, it did not cross-react with MCA-G3 (Fig., lane 6), nor did it have catalytic activity (Fig., lane 4). Northern analysis was performed on cellular RNAs from human and bovine dental pulps, with universal-type ALP cDNA used as a hybridization probe. Universal-type ALP mRNA was detected in both the human and bovine dental pulp tissues (data not shown).
Discussion. In inhibition and thermal inactivation experiments on human dental pulp ALP, it was revealed that the enzyme can be classified as being of the universal (kidney/liver/bone) type. It was rather difficult for us to find substantial difference in enzymatic properties between the human dental ALP and other universaltype ALPs. We have reported that polyclonal antibody against porcinekidney ALP raised in rabbit, cross-reacted with human universal-type ALPs, but not with intestinal or placental ALP (Oida et al., 1984). This antibody could be used so that universal-type ALP could be distinguished clearly from intestinal or placental type; however, no difference in immunological reactivity was observed among universal-type ALPs. Electrophoretic mobility is the only measure routinely used for the distinction of their difference in clinical laboratories (Sundblad et al., 1973). Recently, we found minor immunological differences in crossreactivity among universal-type ALPs by monoclonal antibodies against swine-kidney ALP (Ml and M2; Goseki et al., 1988) or against HBC-cells ALP (G3; Goseki et al., 1989). Human liver ALP scarcely cross-reacted with those monoclonal antibodies. The monoclonal antibody preparations will be useful in distinguishing human kidney and bone ALP from liver ALP. Therefore, the investigation of the cross-reactivity of human dental pulp ALP with those monoclonal antibodies was attractive, and the present results lend support to the concept that human dental pulp, bone, and kidney ALPs have antigenic specificity different from that of liver enzymes. These minor immunological differences among human universal-type ALPs may be caused by a diversity of sugar chain residues. In addition, Northern analysis of the human dental pulp mRNA by a cDNA probe for universal-type ALP confirmed the type of the enzyme. The result in the Fig. shows that enzymatic activity can be detected on a single band corresponding to a native form of
Fig.-SDS-polyacrylamide gel electrophoresis and Western blot immunoassay by MCA-G3. Samples in lanes 4 and 6 were incubated with 1% mercaptoethanol and 1% SDS at 40C overnight, prior to electrophoresis, and those in lanes 3 and 5 were electrophoresed with polyacrylamide gel immediately after the addition of SDS. After electrophoresis, proteins in the gel were transferred onto the nitrocellulose membrane. Standard proteins were stained with Amido Black (lanes 1 and 2); ferritin subunit (220,000), phosphorylase b (94,000), bovine serum albumin (67,000), catalase (60,000), ovalbumin (43,000), lactate dehydrogenase (36,000), and carbonic anhydrase (30,000). Lanes 3-6 are the human dental pulp ALP; enzymatic activity was stained by the P-naphthyl phosphate method (lanes 3 and 4), and immunostaining was carried out with MCA-G3 (lanes 5 and 6).
ALP after electrophoresis and transfer to a nitrocellulose membrane (lanes 3 and 5). Conversion to an inactive monomeric form by prior treatment with mercaptoethanol and SDS was suggested (lanes 4 and 6). These results may indicate that the native ALP of dental pulp is in dimeric form, as are other mammalian ALPs. The kidney/bone/liver-type ALPs are synthesized from a single common gene (Weiss et al., 1986) with minor post-translational heterogeneities. The identity of the molecular scaffold, including catalytic site of universal-type ALPs, may reflect a basically common function of this enzyme in different tissues. Specific localization of ALP activity at the brush-border membrane in kidney tubules or at the sub-odontoblast layer in the dental pulp suggests a role of this enzyme in transport to ions, especially phosphate.
Acknowledgments. We thank Drs. H. Shimokawa and K. Ibaraki in our Department for a gift of cultured human osteoblast-like cells, and Dr. M.J. Weiss for a gift of universal-type ALP cDNA. REFERENCES BADGER, K.S. and SUSSMAN, H.H. (1976): Structural Evidence that Human Liver and Placental Alkaline Phosphatase Isoenzymes are Coded by Different Genes, Proc Natl Acad Sci USA 73:22012205. DOELLGAST, G.J. and BENIRSCHKE, K. (1979): Placental Alkaline Phosphatase in Hominidae, Nature 280:601-602. GOGGINS, J.F. and FULLMER, H.M. (1967): Hydrolytic Enzyme Histochemistry of the Rat Molar Pulp, Arch Oral Biol 12:639644. GOLDSTEIN, D.J.; ROGERS, C.; and HARRIS, H. (1982): Evolution of Alkaline Phosphatases in Primates, Proc Natl Acad Sci USA 79:879-883.
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