Properties and Distribution of Inorganic Pyrophosphatase in Rabbit Dental Pulp SHIUNSUKE FURUYAMA, MASAE MITSUMA, NORIKo Doi, HIROSHI SUGIYA, and SUSUMU OYAMA Department of Physiology, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho-nishi, Matsudo City, Chiba, Japan Some properties and the intracellular distribution of inorganic pyrophosphatase in rabbit dental pulp were determined. This enzyme was sensitive to Mg2+, and not inhibited by imidazol and CN- which are inhibitors of alkaline phosphatase. Inorganic pyrophosphatase was found predominantly in the supernatant fraction. J Dent Res 56(10): 1239-1244 October 1977.
Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, PPase; EC 3.6.1.3) has been found in bacteria' and many mammalian tissues including bone2 and cartilage.3'4 One of the proposed functions of this enzyme is to pull pyrophosphorytic reactions in the direction required for biosynthesis by removing pyrophosphate.5 Alcock and Shils have proposed the other role of this enzyme is calcifying tissues, showing the increase of PPase and decrease of alkaline phosphatase activities in rat costal cartilage before the initiation of calcification. They suggested that PPase functions in calcifying tissues by hydrolysing pyrophosphate, a known inhiitor of calcium phosphate crystal formation.3 In this paper, some characteristics and subcellular distribution of PPase and alkaline phosphatase in rabbit dental pulp have been reported.
Materials and Methods p-Nitrophenyl phosphate (disodium salt)* was converted into tris form by passage through an Amberlite IR-120t H+-form) column and neutralized with tris (hydroxymethyl) aminomethane. Received for publication June 21, 1976. Accepted for publication November 17, 1976. * Wako Pure Chemicals Industries, Ltd., Tokyo, Jap. t Rohm and Haas Co., U.S.A. X Wako Pure Chemicals Industries, Ltd., Tokyo, Jap.
PREPARATION OF ENZYME.-Heads of New Zealand white rabbits were obtained from a local slaughter house. Heads were kept in an ice box below 4 C after the rabbits were killed by a cervical dislocation. Dental pulp was removed from the tooth and chilled in ice cold 0.25 M sucrose. After mincing with scissors, pulp was homogenized with 10 volumes of 0.25 M sucrose in a glass homogenizer with a Teflon pestle at 400 rpm for 90 seconds. The homogenate was passed through an aluminum wire sieve (2 mm mesh) and fractionated by the method of Son et al.6 Briefly, nuclear, mitochondrial and microsomal fractions were precipitated by centrifuging at 750 X g for 10 minutes, at 12,500 X g for 15 minutes and 100,000 X g for 60 minutes, respectively. All the pellets obtained were washed once and suspended in 0.25 M sucrose, and used for enzyme assay. The supernatant fraction was dialysed against 0.25 M sucrose containing 2mM EDTA-Na, (pH 6.5) for 16 hours. All the procedures were carried out below 4 C except where noted. ASSAY OF ENZYME ACTIVITY. PPase activity was determined in a medium containing 50 ytmoles of tris-maleate buffer (pH 7.4), 5 Amoles of MgCl,, 0.5 ismoles of sodium pyrophosphate+ and the appropriate amounts of enzyme solution in a final volume of 0.5 ml. Assays were carried out at 30 C for 15 minutes. Enzyme activity was linear with the incubation time and the amounts of enzyme added. The reaction was stopped by the addition of 1.0 ml of 10% trichloroacetic acid containing 5 mM CuS04. After spinning down at 3,000 X g for 5 minutes, the amount of inorganic phosphate in this deproteinized supernatant fraction was determined by the method of Wolgens and Ahsmann.7
Alkaline phosphatase was determined by the method of Lieberherr et als using a Hitachi 624 recording spectrophotometer. 1239
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1240
FURUYAMA ET AL
J Dent Res October 1977
140 c *0)
1201
0
1 1-
E
00
_
801
4--
E :3 >1)
60 40 .
u
20 5.2
5.6
.
.
6.0
6.4
.
.
a
6.8
7.2
7.6
--
8.0
a
m
8.4
8.8
pH FIG 1. Inorganic pyrophosphatase activity in the supernatant fraction as function of pH. Reaction mixture contained 50 urmoles of tris-
malelate buffer, 5 jumoles of MgCI2 0.5 ,umoles of sodium pyrophosphate and the enzyme solution in a final volume of 0.5 ml.
One enzyme unit was defined as the amount of enzyme which hydrolyzes 1.0 #umoles of pyrophosphate or p-nitrophenyl phosphate in one minute. PROTEIN DETERMINATION.-Protein concentration was determined according to the method of Lowry et a19 with crystalline bovine serum albumin as a standard.
rapidly by increasing pyrophosphate concentration, and was highest between 0.5 to 1.0 mM of pyrophosphate. PPase activity decreased when pyrophosphate concentration was higher than 1.0 mM. The Michaelias constant (Kmi) for pyrophosphate was 80.0 ,uM (Fig 2). EFFECT OF MG2 + CONCENTRATION.-Activation of PPase by Mg2 + was assayed in the
Results The properties of the PPase were determined using the supernatant fraction. PH OPTIMUM. Optimum pH for PPase was determined in 50 ,moles of tris-maleate buffer from pH 5.2 to pH 8.6. High levels of PPase activity were found between pH 6.8 and pH 7.8, but no distinct peak of PPase activity was observed (Fig. 1). EFFECT OF PYROPHOSPHATE CONCENTRATION. -PPase activities at various concentrations of pyrophosphate were determined in the presence of 5 ,imoles of MgCl,. PPase activity increased
High levels of PPase activity were obtained in the presence of 6 to 20 mM of CyCl2, and PPase activity was decreased with the higher concentrations of MgCl,. Michaelis constant for Mg2+ was 1.25 mM (Fig 3).
presence of various concentrations of MgC12.
DIVALENT CATION REQUIREMENT OF PPASE. -The divalent cation requirement of PPase was examined in the presence of one of the following divalent cations: Mg2+, Ca2+, Cd2+, SrI+, Nij2, Co2+, and Cu2+. Each divalent cation was added at 1.0, 5.0, 10.0, and 50.0 mM in chloride form. PPase was activated by Mg2+ alone and other divalent cations tested failed to activate the enzyme. Activation
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Vol. 56 No. 10
INORGANIC PYROPHOSPHATASE IN DENTAL PULP
of PPase was, therefore, highly specific for
Mg2+. SUBSTRATE SPECIFICITY.-Substrate specificity was tested by adding 0.5 dumoles of each substrate to the incubation medium. Negligible activity was detected when substances other than sodium pyrophosphate were used (Table
mM of calcium, inhibition was complete. INTRACELLULAR DISTRIBUTION OF PPASE AND ALKALINE PHOSPHATASE.-PPase activity was found predominantly in the supernatant factors with none detectable in the microsomal fraction. Specific activity of PPase was also highest in the supernatant fraction. Alkaline
1).
EFFECTS OF INHIBITORS.-Effects of various substances on PPase and alkaline phosphatase were tested (Table 2). Alkaline phosphatase was inhibited by theophylline, imidazol, and CN-, which are known inhibitors of alkaline phosphatase in other tissues.10"1° Theophylline gave a moderate inhibition, but imidazol and CN- did not inhibit PPase. Inhibition by tartrate was moderate, but at 0.5 mM of NaF and at 10 mM of ammonium molybdate, PPase activity was less than 10% of that of control. Calcium inhibited PPase in the presence of an optimum concentration of Mg2+, and at 10
1241
TABLE 1 SUBSTRATE SPECIFICITY OF PPASE Relative Substrate
Activity
100.0%
Na4P9O7
Glucose-6-phosphate Adenosine triphosphate Phosphoenolpyruvate Phosphocreatine p-Nitrophenyl phosphate Thiamine pyrophosphate
0
2.28 0.38 0.76 0
0
0.5 ,umoles of each substrate was used. Other conditions were described in the Materials and Methods.
1 50 0,
UL) 0
L-
1 :-'
1 0A
c
.9~ _
E C-
L~~~
-15
-10
2
0
a
0
-5
5
10
[P.] (mM
0
I
0.5
.
1.0'' 2.0
.
.
3.0
4.0
A
5.0
[ PPi](mM) FIG 2. Effect of pyrophosphate concentration on PPase activity in the supernatant fraction. The inset was the Lineweaver-Burk plot of PPase for pyrophosphate. The reaction mixture
contained 50 jmoles of tris-maleate buffer (pH 7.04), 5 pmoles of MgCl2 and the various concentrations of pyrophosphate.
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1242
j Dent Res October 1977
FURUYAMA ET AL
1
a)
0 L Q-
E
60
40 1 30 1
-P
E
I'/
40
20
_
1 0,
_
-
0;
I
.
I
.
1 rMg I
(mM-
KA
*>
I
0
10
II
20
[Mg t FIG 3. Magnesium activation profile of PPase in the supernatant fraction. The inset was the Lineweaver-Burk plot of PPase for Mg2". Reaction mixture contained 50 ,umoles of pyro-
3
2
1
a
a
50
100
(mM)
tris-maleate buffer (pH 7.4), 0.5 ,smoles of phosphate and the various concentrations of MgCl
2.
TABLE 2
EFFECTS OF INHIBITORS ON INORGANIC PYROPHOSPHATASE AND ALKALINE PHOSPHATASE IN MICROSOMAL AND SUPERNATANT FRACTIONS Relative Activity Alkaline Phosphatase
Inhibitor None
Theophylline (lmM) Imidazol (8mM) NaCN (2mM) NaF (0.05mM) Tartrate (10.OmM) Ammonium (10.OmM) Molybdate
Pyrophosphatase
Supernatant
100.0 81.4 123.5 129.1 64.3 82.8 8.0
100.0 26.3 45.0 42.1
101.2 97.4 27.9
Microsome
100.0 3.3 13.3 8.4 122.4 80.2 8.3
Assay conditions were described in the Materials and Methods section. Results the average of three different determinations.
were
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Vol. 56 No. 10
INORGANIC PYROPHOSPHATASE IN DENTAL PULP 5--
uS
(4)
4)4)
0.0
44
phosphatase activity was, however, highest in the microsomal fraction. Specific activity of
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alkaline phosphatase in the supernatant fraction was about 1/40 of that of microsomal fraction (Table 3). Specific activities of PPase in the supernatant fractions from incisors and molars were 104.3 ± 5.5 (N- 4) and 116.5 ± 23.9 (munit/mg protein, N- 4), respectively. In the supernatant fraction, specific activity of alkaline phosphatase in incisors was about two times higher than that of molars. The reason for this difference was unknown. In microsomal fraction, alkaline phosphatase activities in incisors and molars were not significantly different. Discussion Alcock and Shils have reported the direct relationship between PPase activity and calcification in 10,000 X g supernatant fraction of rat costal cartilage.3 Felix and Fleisch reported the separation of PPase from alkaline phosphatase in 25,000 X g supernatant fraction of pig scapula cartilage, but failed to elucidate the direct role of PPase in calcification.4 In rabbit dental pulp, a major part of PPase activity was found in cytosol fraction as reported in other tissues,'2'13 and alkaline phosphatase activity was found predominantly in microsomal fraction. Although purity of mitochondrial fraction was not pure enough to decide the exact distribution of PPase as described below, the enzyme activity was found in the mitochondrial and also in the nuclear fractions. Distributions of PPase in rat liver nuclei and mitochondria have been reported. 14,15 Purities of mitochondrial and microsomal fractions were examined by measuring succinate dehydrogenase and glucose-6- phosphatase in both fractions. Microsomal fraction was contaminated 14.5 + 7.0% (N - 6) by mitochondrial fraction judging from the ratio of specific activities of succinate dehydrogenase in microsomal and mitochondrial fractions. The contamination of microsomal fraction to mitochondrial fraction was 31.0 ± 9.3% (N - 9) by measuring glucose-6-phosphatase activities in both fractions. However, specific activity of alkaline phosphatase in mitochondrial fraction was 19.5% of that of microsomal fraction (Table 3). In this report, purities of nuclear and supernatant fractions were not examined, but the specific activities of PPase and alkaline phosphatase in microsomal fraction were quite different from those in the supernatant
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1244
FURUYAMA ET AL
fraction. Furthermore, inhibitors of alkaline phosphatase did not inhibit PPase from dental pulp. Data presented in this paper may indicate that alkaline phosphatase in microsomal fraction and PPase in the supernatant fraction are separate enzymes. Preliminary experiments indicated that alkaline phosphatase was still detectable in 200,000 X g supernatant fraction, and this enzyme was separated from PPase by DEAE-cellulose column chromatography. PPase activity from incisors was not so different from that from molars; alkaline phosphatase in the supernatant fraction from incisors was about two times higher than that from molars. These results make it less likely that the role of PPase is to promote calcification by destroying pyrophosphate. Kuhlman observed the highest inorganic pyrophosphatase activity in cartilage in the proliferating zone, where cell division but not calcification occurs, whereas alkaline phosphatase activity is highest in the hypertrophic zone and in the calcified zone of the cartilage.16 However, presence of extracellular matrix vesicles was reported in calcifying tissues including dental pulp.17 One possibility of the origin of PPase might, therefore, be the matrix vesicles which are thought to be the initial site of calcification. Further studies are underway in this laboratory to clarify the role of PPase in dental pulp. Conclusions Some properties and the intracellular distribution of PPase in rabbit pulp were described. PPase was sensitive only to Mg+, and inhibited by theophylline, tartrate, NaF, and ammonium molybdate, but not by imidazol and CN- which inhibited alkaline phosphatase in this tissue. Almost all the PPase activity was in the supernatant fraction, but the major parts of alkaline phosphatase were found in the microsemal fraction. References 1. TONO, H., and KORNBERG, A.: Biochemical Studies of Bacterial Sporulation, J Biol Chem 242:2375-2382, 1967. 2. VREVEN, J.; LIEBERHERR, M.; and VAES, G.:
The Acid and Alkaline Phosphatase, Inorganic Pyrophosphatase and Phosphoprotein Phosphatase of Bone. Biochem Biophys Acta 293:170- 177, 1973. 3. ALCOCK, N.W., and SHILS, M.E.: Association of Inorganic Pyrophosphatase Activity with Normal Calcification of Rat Costal Cartilage In Vivo, Biochem J 112:505-513, 1969.
j Dent Res October 1977 4. FELIX, R., and FLEISCH, H.: Properties of Inorganic Pyrophosphatase of Pig Scapula Cartilage, Biochem J 147:11 1-118, 1975. 5. KORNBERG, A.: Horizons in Biochemistry, New York: Academic Press, 1962, p 251. 6. SON, Y.; YOKOYAMA, I.; ASANUMA, T.; ISHIDO, T.; and TAKIGUCHI, H.: Ca2+-stimulated Adenosine Triphosphatase in Dental Pulp of Albino Rabbit, J Dent Res (in
press) .
7. WALTGENS, J., and ASHMANN, W.: Determination of Orthophosphate in the Presence of Inorganic Pyrophosphate in the Assay of Inorganic Pyrophosphatase Activity, A nal Biochemn 35:526-529, 1970. 8. LIEBERHERR, M.; VREVEN, J. and VAES, G.: The Acid and Alkaline Phosphatases, Inorganic Pyrophosphatases and Phosphoprotein Phosphatase of Bone. I. Characterization and Assay, Biochim Biophys Acta 293:160-169, 1973. 9. LOWRY, O.H.; ROSENROUGH, N.J.; FARR, A.L.; and RANDALL, R.J.: Protein Measurement with the Folin Phenol Reagent, J Biol Chem 193:265-275, 1951. 10. BRUNEL, C., and CATHALA, G.: Imidazol; An Inhibitor of L-phenylalanine-Insensitive Alkaline Phosphatases of Tissues other than Intestine and Placenta, Biochim Biophys Acta 268:415-421, 1972. 11. FISHMAN, W.H.; GREEN, S.; and INGLIS, N.I.: Organ-specific Behaviour Exhibited by Rat Intestine and Liver Alkaline Phosphatase, Biochim Biophys Acta 62:363-375, 1962. 12. NORDLE, R C., and LARDY, H.A.: Sub-cellular Distribution of Rat-Liver Inorganic Pyrophosphatase Activity, Biochim Biophyv Acta 50:189-191, 1961. 13. PYNES, G.D., and YOUNATHAN, E.S.: The Subcellular Distribution of Inorganic Pyrophosphatase in Pigeon Pancreas, Biochim Biophys Acta 92:150-151, 1964. 14. KESSELRING, K., and SIEBERT, G.: Eigenschaften einer l6slichen anorganischen Pyrophosphatase aus Rattenleber-Zellkernen, Hoppe-Seyler's Z Physiol Chem 348:585598, 1967. 15. SCHICK, L., and BUTLER, L.G.: Inorganic Pyrophosphatase of Rat Liver Mitochondria. Correlation of Latency with Catalytic Properties and Intramitochondrial Location, J Cell Biol 42:235-240, 1969. 16. KUHILMAN, R.E.: Phosphatases in Epiphyseal Cartilage; Their Possible Role in Tissue Synthesis, J Bone Joint Surg 47A: 545-550, 1965. 17. SLAVKIN, H.C.; CROISSANT, R.; and BRINGAS, P.: Epithelialmesenchymal Interactions During Odontogenesis. III. A Simple Method for the Isolation of Matrix Vesicles. J Cell Biol 53: 841-849, 1972.
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Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, PPase; EC 3.6.1.3) has been found in bacteria' and many mammalian tissues including bone2 and cartilage.3'4 One of the proposed functions of this enzyme is to pull pyrophosphorytic reactions in the direction required for biosynthesis by removing pyrophosphate.5 Alcock and Shils have proposed the other role of this enzyme is calcifying tissues, showing the increase of PPase and decrease of alkaline phosphatase activities in rat costal cartilage before the initiation of calcification. They suggested that PPase functions in calcifying tissues by hydrolysing pyrophosphate, a known inhiitor of calcium phosphate crystal formation.3 In this paper, some characteristics and subcellular distribution of PPase and alkaline phosphatase in rabbit dental pulp have been reported.
Materials and Methods p-Nitrophenyl phosphate (disodium salt)* was converted into tris form by passage through an Amberlite IR-120t H+-form) column and neutralized with tris (hydroxymethyl) aminomethane. Received for publication June 21, 1976. Accepted for publication November 17, 1976. * Wako Pure Chemicals Industries, Ltd., Tokyo, Jap. t Rohm and Haas Co., U.S.A. X Wako Pure Chemicals Industries, Ltd., Tokyo, Jap.
PREPARATION OF ENZYME.-Heads of New Zealand white rabbits were obtained from a local slaughter house. Heads were kept in an ice box below 4 C after the rabbits were killed by a cervical dislocation. Dental pulp was removed from the tooth and chilled in ice cold 0.25 M sucrose. After mincing with scissors, pulp was homogenized with 10 volumes of 0.25 M sucrose in a glass homogenizer with a Teflon pestle at 400 rpm for 90 seconds. The homogenate was passed through an aluminum wire sieve (2 mm mesh) and fractionated by the method of Son et al.6 Briefly, nuclear, mitochondrial and microsomal fractions were precipitated by centrifuging at 750 X g for 10 minutes, at 12,500 X g for 15 minutes and 100,000 X g for 60 minutes, respectively. All the pellets obtained were washed once and suspended in 0.25 M sucrose, and used for enzyme assay. The supernatant fraction was dialysed against 0.25 M sucrose containing 2mM EDTA-Na, (pH 6.5) for 16 hours. All the procedures were carried out below 4 C except where noted. ASSAY OF ENZYME ACTIVITY. PPase activity was determined in a medium containing 50 ytmoles of tris-maleate buffer (pH 7.4), 5 Amoles of MgCl,, 0.5 ismoles of sodium pyrophosphate+ and the appropriate amounts of enzyme solution in a final volume of 0.5 ml. Assays were carried out at 30 C for 15 minutes. Enzyme activity was linear with the incubation time and the amounts of enzyme added. The reaction was stopped by the addition of 1.0 ml of 10% trichloroacetic acid containing 5 mM CuS04. After spinning down at 3,000 X g for 5 minutes, the amount of inorganic phosphate in this deproteinized supernatant fraction was determined by the method of Wolgens and Ahsmann.7
Alkaline phosphatase was determined by the method of Lieberherr et als using a Hitachi 624 recording spectrophotometer. 1239
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1240
FURUYAMA ET AL
J Dent Res October 1977
140 c *0)
1201
0
1 1-
E
00
_
801
4--
E :3 >1)
60 40 .
u
20 5.2
5.6
.
.
6.0
6.4
.
.
a
6.8
7.2
7.6
--
8.0
a
m
8.4
8.8
pH FIG 1. Inorganic pyrophosphatase activity in the supernatant fraction as function of pH. Reaction mixture contained 50 urmoles of tris-
malelate buffer, 5 jumoles of MgCI2 0.5 ,umoles of sodium pyrophosphate and the enzyme solution in a final volume of 0.5 ml.
One enzyme unit was defined as the amount of enzyme which hydrolyzes 1.0 #umoles of pyrophosphate or p-nitrophenyl phosphate in one minute. PROTEIN DETERMINATION.-Protein concentration was determined according to the method of Lowry et a19 with crystalline bovine serum albumin as a standard.
rapidly by increasing pyrophosphate concentration, and was highest between 0.5 to 1.0 mM of pyrophosphate. PPase activity decreased when pyrophosphate concentration was higher than 1.0 mM. The Michaelias constant (Kmi) for pyrophosphate was 80.0 ,uM (Fig 2). EFFECT OF MG2 + CONCENTRATION.-Activation of PPase by Mg2 + was assayed in the
Results The properties of the PPase were determined using the supernatant fraction. PH OPTIMUM. Optimum pH for PPase was determined in 50 ,moles of tris-maleate buffer from pH 5.2 to pH 8.6. High levels of PPase activity were found between pH 6.8 and pH 7.8, but no distinct peak of PPase activity was observed (Fig. 1). EFFECT OF PYROPHOSPHATE CONCENTRATION. -PPase activities at various concentrations of pyrophosphate were determined in the presence of 5 ,imoles of MgCl,. PPase activity increased
High levels of PPase activity were obtained in the presence of 6 to 20 mM of CyCl2, and PPase activity was decreased with the higher concentrations of MgCl,. Michaelis constant for Mg2+ was 1.25 mM (Fig 3).
presence of various concentrations of MgC12.
DIVALENT CATION REQUIREMENT OF PPASE. -The divalent cation requirement of PPase was examined in the presence of one of the following divalent cations: Mg2+, Ca2+, Cd2+, SrI+, Nij2, Co2+, and Cu2+. Each divalent cation was added at 1.0, 5.0, 10.0, and 50.0 mM in chloride form. PPase was activated by Mg2+ alone and other divalent cations tested failed to activate the enzyme. Activation
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Vol. 56 No. 10
INORGANIC PYROPHOSPHATASE IN DENTAL PULP
of PPase was, therefore, highly specific for
Mg2+. SUBSTRATE SPECIFICITY.-Substrate specificity was tested by adding 0.5 dumoles of each substrate to the incubation medium. Negligible activity was detected when substances other than sodium pyrophosphate were used (Table
mM of calcium, inhibition was complete. INTRACELLULAR DISTRIBUTION OF PPASE AND ALKALINE PHOSPHATASE.-PPase activity was found predominantly in the supernatant factors with none detectable in the microsomal fraction. Specific activity of PPase was also highest in the supernatant fraction. Alkaline
1).
EFFECTS OF INHIBITORS.-Effects of various substances on PPase and alkaline phosphatase were tested (Table 2). Alkaline phosphatase was inhibited by theophylline, imidazol, and CN-, which are known inhibitors of alkaline phosphatase in other tissues.10"1° Theophylline gave a moderate inhibition, but imidazol and CN- did not inhibit PPase. Inhibition by tartrate was moderate, but at 0.5 mM of NaF and at 10 mM of ammonium molybdate, PPase activity was less than 10% of that of control. Calcium inhibited PPase in the presence of an optimum concentration of Mg2+, and at 10
1241
TABLE 1 SUBSTRATE SPECIFICITY OF PPASE Relative Substrate
Activity
100.0%
Na4P9O7
Glucose-6-phosphate Adenosine triphosphate Phosphoenolpyruvate Phosphocreatine p-Nitrophenyl phosphate Thiamine pyrophosphate
0
2.28 0.38 0.76 0
0
0.5 ,umoles of each substrate was used. Other conditions were described in the Materials and Methods.
1 50 0,
UL) 0
L-
1 :-'
1 0A
c
.9~ _
E C-
L~~~
-15
-10
2
0
a
0
-5
5
10
[P.] (mM
0
I
0.5
.
1.0'' 2.0
.
.
3.0
4.0
A
5.0
[ PPi](mM) FIG 2. Effect of pyrophosphate concentration on PPase activity in the supernatant fraction. The inset was the Lineweaver-Burk plot of PPase for pyrophosphate. The reaction mixture
contained 50 jmoles of tris-maleate buffer (pH 7.04), 5 pmoles of MgCl2 and the various concentrations of pyrophosphate.
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1242
j Dent Res October 1977
FURUYAMA ET AL
1
a)
0 L Q-
E
60
40 1 30 1
-P
E
I'/
40
20
_
1 0,
_
-
0;
I
.
I
.
1 rMg I
(mM-
KA
*>
I
0
10
II
20
[Mg t FIG 3. Magnesium activation profile of PPase in the supernatant fraction. The inset was the Lineweaver-Burk plot of PPase for Mg2". Reaction mixture contained 50 ,umoles of pyro-
3
2
1
a
a
50
100
(mM)
tris-maleate buffer (pH 7.4), 0.5 ,smoles of phosphate and the various concentrations of MgCl
2.
TABLE 2
EFFECTS OF INHIBITORS ON INORGANIC PYROPHOSPHATASE AND ALKALINE PHOSPHATASE IN MICROSOMAL AND SUPERNATANT FRACTIONS Relative Activity Alkaline Phosphatase
Inhibitor None
Theophylline (lmM) Imidazol (8mM) NaCN (2mM) NaF (0.05mM) Tartrate (10.OmM) Ammonium (10.OmM) Molybdate
Pyrophosphatase
Supernatant
100.0 81.4 123.5 129.1 64.3 82.8 8.0
100.0 26.3 45.0 42.1
101.2 97.4 27.9
Microsome
100.0 3.3 13.3 8.4 122.4 80.2 8.3
Assay conditions were described in the Materials and Methods section. Results the average of three different determinations.
were
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Vol. 56 No. 10
INORGANIC PYROPHOSPHATASE IN DENTAL PULP 5--
uS
(4)
4)4)
0.0
44
phosphatase activity was, however, highest in the microsomal fraction. Specific activity of
co c LO10cc c ,I LLfntcc to(. a r-~ _4 £ ecli crco
cc
tno:
.. -0
CtoLfl
00
Lo LO L.CO.
CDo 0
0 44
Q o0 s 0 > 0 5- 0; o)
.4
4)
4)
4) m 0,4 uo
o)
1243
4 )
5-4:0.3W
alkaline phosphatase in the supernatant fraction was about 1/40 of that of microsomal fraction (Table 3). Specific activities of PPase in the supernatant fractions from incisors and molars were 104.3 ± 5.5 (N- 4) and 116.5 ± 23.9 (munit/mg protein, N- 4), respectively. In the supernatant fraction, specific activity of alkaline phosphatase in incisors was about two times higher than that of molars. The reason for this difference was unknown. In microsomal fraction, alkaline phosphatase activities in incisors and molars were not significantly different. Discussion Alcock and Shils have reported the direct relationship between PPase activity and calcification in 10,000 X g supernatant fraction of rat costal cartilage.3 Felix and Fleisch reported the separation of PPase from alkaline phosphatase in 25,000 X g supernatant fraction of pig scapula cartilage, but failed to elucidate the direct role of PPase in calcification.4 In rabbit dental pulp, a major part of PPase activity was found in cytosol fraction as reported in other tissues,'2'13 and alkaline phosphatase activity was found predominantly in microsomal fraction. Although purity of mitochondrial fraction was not pure enough to decide the exact distribution of PPase as described below, the enzyme activity was found in the mitochondrial and also in the nuclear fractions. Distributions of PPase in rat liver nuclei and mitochondria have been reported. 14,15 Purities of mitochondrial and microsomal fractions were examined by measuring succinate dehydrogenase and glucose-6- phosphatase in both fractions. Microsomal fraction was contaminated 14.5 + 7.0% (N - 6) by mitochondrial fraction judging from the ratio of specific activities of succinate dehydrogenase in microsomal and mitochondrial fractions. The contamination of microsomal fraction to mitochondrial fraction was 31.0 ± 9.3% (N - 9) by measuring glucose-6-phosphatase activities in both fractions. However, specific activity of alkaline phosphatase in mitochondrial fraction was 19.5% of that of microsomal fraction (Table 3). In this report, purities of nuclear and supernatant fractions were not examined, but the specific activities of PPase and alkaline phosphatase in microsomal fraction were quite different from those in the supernatant
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fraction. Furthermore, inhibitors of alkaline phosphatase did not inhibit PPase from dental pulp. Data presented in this paper may indicate that alkaline phosphatase in microsomal fraction and PPase in the supernatant fraction are separate enzymes. Preliminary experiments indicated that alkaline phosphatase was still detectable in 200,000 X g supernatant fraction, and this enzyme was separated from PPase by DEAE-cellulose column chromatography. PPase activity from incisors was not so different from that from molars; alkaline phosphatase in the supernatant fraction from incisors was about two times higher than that from molars. These results make it less likely that the role of PPase is to promote calcification by destroying pyrophosphate. Kuhlman observed the highest inorganic pyrophosphatase activity in cartilage in the proliferating zone, where cell division but not calcification occurs, whereas alkaline phosphatase activity is highest in the hypertrophic zone and in the calcified zone of the cartilage.16 However, presence of extracellular matrix vesicles was reported in calcifying tissues including dental pulp.17 One possibility of the origin of PPase might, therefore, be the matrix vesicles which are thought to be the initial site of calcification. Further studies are underway in this laboratory to clarify the role of PPase in dental pulp. Conclusions Some properties and the intracellular distribution of PPase in rabbit pulp were described. PPase was sensitive only to Mg+, and inhibited by theophylline, tartrate, NaF, and ammonium molybdate, but not by imidazol and CN- which inhibited alkaline phosphatase in this tissue. Almost all the PPase activity was in the supernatant fraction, but the major parts of alkaline phosphatase were found in the microsemal fraction. References 1. TONO, H., and KORNBERG, A.: Biochemical Studies of Bacterial Sporulation, J Biol Chem 242:2375-2382, 1967. 2. VREVEN, J.; LIEBERHERR, M.; and VAES, G.:
The Acid and Alkaline Phosphatase, Inorganic Pyrophosphatase and Phosphoprotein Phosphatase of Bone. Biochem Biophys Acta 293:170- 177, 1973. 3. ALCOCK, N.W., and SHILS, M.E.: Association of Inorganic Pyrophosphatase Activity with Normal Calcification of Rat Costal Cartilage In Vivo, Biochem J 112:505-513, 1969.
j Dent Res October 1977 4. FELIX, R., and FLEISCH, H.: Properties of Inorganic Pyrophosphatase of Pig Scapula Cartilage, Biochem J 147:11 1-118, 1975. 5. KORNBERG, A.: Horizons in Biochemistry, New York: Academic Press, 1962, p 251. 6. SON, Y.; YOKOYAMA, I.; ASANUMA, T.; ISHIDO, T.; and TAKIGUCHI, H.: Ca2+-stimulated Adenosine Triphosphatase in Dental Pulp of Albino Rabbit, J Dent Res (in
press) .
7. WALTGENS, J., and ASHMANN, W.: Determination of Orthophosphate in the Presence of Inorganic Pyrophosphate in the Assay of Inorganic Pyrophosphatase Activity, A nal Biochemn 35:526-529, 1970. 8. LIEBERHERR, M.; VREVEN, J. and VAES, G.: The Acid and Alkaline Phosphatases, Inorganic Pyrophosphatases and Phosphoprotein Phosphatase of Bone. I. Characterization and Assay, Biochim Biophys Acta 293:160-169, 1973. 9. LOWRY, O.H.; ROSENROUGH, N.J.; FARR, A.L.; and RANDALL, R.J.: Protein Measurement with the Folin Phenol Reagent, J Biol Chem 193:265-275, 1951. 10. BRUNEL, C., and CATHALA, G.: Imidazol; An Inhibitor of L-phenylalanine-Insensitive Alkaline Phosphatases of Tissues other than Intestine and Placenta, Biochim Biophys Acta 268:415-421, 1972. 11. FISHMAN, W.H.; GREEN, S.; and INGLIS, N.I.: Organ-specific Behaviour Exhibited by Rat Intestine and Liver Alkaline Phosphatase, Biochim Biophys Acta 62:363-375, 1962. 12. NORDLE, R C., and LARDY, H.A.: Sub-cellular Distribution of Rat-Liver Inorganic Pyrophosphatase Activity, Biochim Biophyv Acta 50:189-191, 1961. 13. PYNES, G.D., and YOUNATHAN, E.S.: The Subcellular Distribution of Inorganic Pyrophosphatase in Pigeon Pancreas, Biochim Biophys Acta 92:150-151, 1964. 14. KESSELRING, K., and SIEBERT, G.: Eigenschaften einer l6slichen anorganischen Pyrophosphatase aus Rattenleber-Zellkernen, Hoppe-Seyler's Z Physiol Chem 348:585598, 1967. 15. SCHICK, L., and BUTLER, L.G.: Inorganic Pyrophosphatase of Rat Liver Mitochondria. Correlation of Latency with Catalytic Properties and Intramitochondrial Location, J Cell Biol 42:235-240, 1969. 16. KUHILMAN, R.E.: Phosphatases in Epiphyseal Cartilage; Their Possible Role in Tissue Synthesis, J Bone Joint Surg 47A: 545-550, 1965. 17. SLAVKIN, H.C.; CROISSANT, R.; and BRINGAS, P.: Epithelialmesenchymal Interactions During Odontogenesis. III. A Simple Method for the Isolation of Matrix Vesicles. J Cell Biol 53: 841-849, 1972.
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