27
Biochimica et Biophysics Acta, 1046 (1990) 27-31 Elsevier
BBALIP
53466
Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes Kazuhide Sumikawa I, Toshikazu Okochi 1 and Kozaburo Ada&i 2 ’ Faculty of Health and Sport Sciences, Osaka University, Toyonaka, Osaka and 2 Department of Food and Nutrition Studies, School of Home Economics, Kobe College, Nishinomiya, Hyogo (Japan) (Received
Key words:
Alkaline
phosphatase
isozyme;
Phosphatidate
23 April 1990)
hydrolysis;
Synthetic
phosphatidate;
Sodium
deoxycholate;
(Human)
Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 34imes higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C,,_,s) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.
Introduction
The measurements of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activities in the serum are routinely used in clinical diagnosis. Alkaline phosphatase is widely distributed in mammalian organs. Its activity is high in intestine, placenta, bone and kidney. Although the hydrolytic functions of alkaline phosphatase have been intensively studied and many roles have been suggested for this enzyme, its physiological function is not yet fully understood [l]. It is well known that there are three main types of alkaline phosphatase isozymes in human tissues, liver/ bone/kidney, intestinal and placental types [2]. Liver, bone and kidney alkaline phosphatases are similar in catalytic and stability properties and are mainly similar in antigenicity [2]. In the previous paper, we reported that human placental ahcaline phosphatase hydrolyzes egg phosphatidate to stoichiometric amounts of diacylglycerol and inorganic phosphate [3]. Calf intestinal alkaline phosphatase has also been reported to dephos-
Correspondence: K. Sumikawa, Faculty of Health Osaka University, Toyonaka, Osaka 560, Japan. 0005-2760/90/$03.50
and Sport Sciences,
0 1990 Elsevier Science Publishers
B.V. (Biomedical
phorylate phosphatidate [4]. In contrast, bacterial alkaline phosphatase has been described not to hydrolyze phosphatidate [5]. The differences in phosphatidate hydrolysis of these reports may come from either the properties of isozymes or the differences in assay conditions. This study was undertaken to elucidate the isozymic difference of alkaline phosphatases in phosphatidate hydrolysis, using the purified human isozymes and various phosphatidates.
Materials and Methods
Materials
Sodium phosphatidate (98% pure) from egg lecithin, synthetic phosphatidates and lipid standards for thinlayer chromatography were purchased from Sigma Chemical (St. Louis, MO). Sephadex G-200 and phenyl-Sepharose were from Pharmacia Fine Chemicals (Uppsala, Sweden). Precoated Silica gel 60 thin-layer chromatography plates were from E. Merck AG (Darmstadt, F.R.G.). Disodium phenyl phosphate, sodium deoxycholate and other chemicals were from Wako Pure Chemical Industries, Osaka, Japan. Division)
28
Purification of human alkaline phosphatase isozymes Placental [3] and liver [6] alkaline phosphatases were purified as described previously. Intestinal alkaline phosphatase was purified by extraction with 30% butanol, precipitation with 30-50s acetone, DEAE-cellulose column chromatography (O-O.2 M NaCl, gradient), TEAE-cellulose column chromatography (O-O.2 M NaCI, gradient), isoelectric focusing and Sephadex G200 column chromatography (unpublished method). The purified human placental, liver and intestinal alkaline phosphatases each gave a single protein band on SDSpolyacrylamide gel electrophoresis, and their specific activities were 680, 1500 and 900 U/mg protein, respectively. Assay of alkaline phosphatase activity Alkaline phosphatase activity was determined with phenyl phosphate as substrate as described previously [7]. One unit of enzyme activity was defined as the amount of enzyme catalyzing the release of 1 pmol of phenol per min. Protein determination Protein was determined by the method of Bradford [8] with bovine serum albumin as a standard. Purification of egg phosphatidate We purified egg phosphatidate by thin-layer chromatography, becuase commercially available egg phosphatidate sometimes contains a small amount of lysophosphatidate. The commercial preparation was applied to a precoated Silica-gel plate with fluorescent indicator (20 cm x 20 cm), developed with chloroform/ methanol/water (65 : 25 : 4, v/v) and located under ultraviolet light. The phosphatidate, co-chromatographed with an authentic standard, was then scraped off the plate and extracted with the same solution. The extract was separated into two layers by addition of water. The chloroform layer was evaporated to dryness and the residue was dissolved in chloroform and stored at - 20 o C until use. The phosphatidate concentration was calculated from Pi determined by a modification of the method of Chen et al. [9]. Assay of phosphatidate hydrolytic activity On the day of use, the phosphatidate solution was dried and dispersed in water by 1-min sonication at 30 W in a Branson sonifier under nitrogen. The standard incubation mixture contained purified alkaline phosphatase (0.02-0.1 U), 1.35 mM sodium phosphatidate, 50 mM Tris-HCl (pH 8.5), 10 g/l sodium deoxycholate and 0.2 mM MgCl, in a total volume of 1.0 ml. As the preliminary experiment showed that a low concentration of Mg2+ stimulated the activity about 2-fold, 0.2 mM MgCl, was added to the assay mixture. The assay was carried out as described previously [3].
Identifications of diacylglycerol and monoacylglvcerol Lipids were extracted as described previously [l]. Diacylglycerol was identified by thin-layer chromatography (TLC) with dipalmitin as a standard as follows: An appropriate amount of the extracted lipid solution and dipalmitin solution in chloroform were applied to a precoated Silica gel plate, developed with hexane/ diethyl ether/acetic acid (50: 50: 1, v/v) and located with iodine vapor. For identifications of lysophosphatidate and monoacylglycerol, lipids were developed with chloroform/methanol/ water (65 : 25 : 4, v/v). Assay of lysophosphatidate hydrolytic activity Sodium lysophosphatidate (oleoyl) was sonicated in the same way as for assay of phosphatidate hydrolytic activity and the assay mixture contained diethanolamine buffer (pH 10.0) and 0.4 mM sodium lysophosphatidate. The assay was carried out at 37” C for 30 min. Released inorganic phosphate was determined by a modification of the method of Chen et al. [91. Results
Effect of sodium deoxycholate on phosphatidate hvdrolytic activity As shown in Fig. 1, in the absence of sodium deoxycholate, none of alkaline phosphatase isozymes hydro-
50
0
1
10 Sodium deoxychdlate
20 (g/l)
Fig. 1. Effects of sodium deoxycholate on phosphatidate hydrolysis by alkaline phosphatase isozymes. The assay was carried out with 0.05 U of enzymes under standard conditions except that the pH was 9.0 and the sodium deoxycholate concentration was varied as indicated. Enzyme source: l, placenta; O. intestine; A, liver. The results are mean values for duplicate determinations.
29
lyzed egg phosphatidate. In the presence of sodium deoxycholate, placental and intestinal alkaline phosphatases hydrolyzed the phosphatidate. The maximal activity was seen in the presence of 10 g/l sodium deoxycholate. The activity of placental alkaline phosphatase was greater than that of intestinal alkaline phosphatase at all the concentrations of sodium deoxycholate examined. Under the optimal conditions, the apparent K, of placental alkaline phosphatase for egg phosphatidate was estimated as 0.44 mM. In contrast, liver alkaline phosphatase did not hydrolyze egg phosphatidate even in the presence of sodium deoxycholate. Of other detergents examined, sodium cholate also stimulated the activities of placental and intestinal alkaline phosphatases, but its stimulation was lower than that of sodium deoxycholate. Triton X-100, Tween 20 and zephiramine (cationic detergent) had no effect on the activity (data not shown). From these reasons we used sodium deoxycholate for the following experiments.
TABLE
I
Hydrolysis of phosphatidates by alkaline phosphatase isozymes Values are shown as nmol Pi released in 30 min. 0.1 U of alkaline phosphatase was used for each assay. The results are mean values for duplicate determinations. Phosphatidate
Placenta
Intestine
Liver
EkX Dioleoyl(18 : 1) Distearoyl(18 : 0) Dipalmitol(16 : 0) Dimyristoyl(14 : 0) Dilauroyl(12 : 0)
93.3 85.5 49.3 54.7 92.6 109.5
37.8 28.2 21.3 20.0 32.3 51.0
0 0 0 0 1.6 6.7
wide pH range, but their maximal activities were at about pH 8.5 and 7.8, respectively. Placental alkaline phosphatase showed several-fold higher activity than intestinal alkaline phosphatase on egg phosphatidate between pH 7.0 and 9.5. Liver alkaline phosphatase did not hydrolyze this phosphatidate at any pH examined.
Effect of pH on phosphatidate hydrolytic activity
For this experiment, Tris-HCl buffer (pH 7.0-9.5) and diethanolamine buffer (pH 8.0-11.0) were used. In the range between pH 8.0 and 9.0, the activity was the same with either buffer. As seen in Fig. 2, placental and intestinal alkaline phosphatases had hydrolytic activities over a relatively
Hydrolysis of synthetic phosphatidates by alkaline phosphatase isozymes
Just before the experiment, phosphatidate was sonicated with sodium deoxycholate, because distearoyl and dipalmitoyl phosphatidates are difficult to disperse without detergent. Table I shows that under standard conditions, all the phosphatidates acted as substrates for placental and intestinal alkaline phosphatases, but that dilauroyl phosphatidate was the best substrate. Placental alkaline phosphatase showed 2- to 3-times higher activity than intestinal enzyme on these phosphatidates. Liver alkaline phosphatase hydrolyzed only dimyristoyl and dilauroyl phosphatidates and its hydrolytic activity on these compounds was very low. Formation of the reaction product, diacylglycerol, was confirmed by TLC. Effect of sodium deoxycholate on phenyl phosphate and lysophosphatidate hydrolytic activities
OL
--A.-.
1.0
8.0
9.0
10.0
11.0
PH
Fig. 2. Effect of pH on phosphatidate hydrolytic activity. The assay was carried out with 0.1 U of enzymes under standard conditions except that the pH was varied as indicated. Enzyme source: 0, placenta; 0, intestine; A, liver. The results are mean values for duplicate determinations.
The above results show that in the presence of sodium deoxycholate, placental and intestinal alkaline phosphatases hydrolyzed phosphatidates but the liver enzyme did not. To determine whether these differences came from those of the effect of sodium deoxycholate on the isozymes, or those of the phosphatidate hydrolytic activities of the isozymes, the effects of sodium deoxycholate on the hydrolytic activities were investigated using phenyl phosphate, which was a representative artificial substrate for alkaline phosphatase and lysophosphatidate, which was the analog of phsphatidate. Sodium deoxycholate activated the phenyl phosphate hydrolytic activity of placental and intestinal alkaline phosphatases, activation of the intestinal enzyme being greater than that of the placental enzyme. In contrast, it
30 TABLE
II
Effect of sodium deoxycholate Activity
is expressed
on phenyl phosphate
as a percentage
and lysophosphatidate
of that without
Addition
Substrate
Sodium deoxycholate
phenyl phosphate
sodium
hydrolytic activities
deoxycholate.
The results
are mean values for duplicate
determinations.
lysophosphatidate
placenta
intestine
liver
placenta
intestine
liver
100 114 116 119 122
100 143 148 152 15.5
100 82 82 83 85
100 54 30 21 15
100 21 2 0 0
100 33 3 1 0
(g/B 0 2 5 10 20
inhibited the activity of liver alkaline phosphatase about 15% (Table II). On the other hand, sodium deoxycholate inhibited the lysophosphatidate hydrolytic activities of all three alkaline phosphatase isozymes. Addition of 10 g/l sodium deoxycholate inhibited the activities of intestinal and liver alkaline phosphatases almost completely, but placental alkaline phosphatase was less sensitive to sodium deoxycholate and 20% of its activity remained in the presence of 10 g/l sodium deoxycholate. Discussion Previously, we reported that human placental alkaline phosphatase can hydrolyze egg phosphatidate [l]. Here, we report that human intestinal alkaline phosphatase also hydrolyzed phosphatidates. Thus, not only calf intestinal alkaline phosphatase [4] but also human intestinal alkaline phosphatase hydrolyzed phosphatidates. In the intestine, bile acids are present in alkaline conditions, and thus our finding that phosphatidates, which may be formed from lecithin. by the action of phospholipase D, can be hydrolyzed by intestinal alkaline phosphatase in these conditions has physiological significance. Of the phosphatidates with saturated fatty acyl chains examined, dilauroyl phosphatidate was the best substrate for placental and intestinal alkaline phosphatases. Dioleoyl phosphatidate was hydrolyzed better than distearoyl phosphatidate. These results may be due to differences in the hydrophobicities of these phosphatidates. Wells et al. [lo] reported similar results for phosphatidate phosphohydrolase. Human placental alkaline phosphatase has been reported to have a lower K, for p-nitrophenyl phosphate of 0.8 mM than for other substrates examined [ll]. Thus, egg phosphatidate seemed to be as a good substrate as p-nitrophenyl phosphate. On the other hand, we found that human liver alkaline phosphatase was unable to hydrolyze phosphatidates with long fatty acyl chains either in the
presence or absence of sodium deoxycholate. This suggests that liver alkaline phosphatase has a different physiological role from placental and intestinal alkaline phosphatases. Placental, intestinal and liver alkaline phosphatases differ in antigenicity [2], electrophoretic mobility [12] and many other properties [6,13-151. All three are regarded as alkaline phosphatase isozymes, because they hydrolyze many substrates of low molecular weight at similar rates at alkaline pH. But with relatively hydrophobic substrates of higher molecular weight, such as phosphatidates as substrates, placental and intestinal alkaline phosphatases may differ from the liver enzyme in hydrolytic activity. At least, as shown here, human liver alkaline phosphatase clearly differed from the other two enzymes with egg phosphatidate as substrate. Thus, this substrate is useful in differentiating the liver enzyme from the other two alkaline phosphatases. As the hydrolytic activity of alkaline phosphatase on phenyl phosphate was affected by sodium deoxycholate (Table II), sodium deoxycholate may act on the enzyme protein. However, sodium deoxycholate also seems to act on the substrate, because its effect on the hydrolytic activity varied with the substrate: it seemed to make phosphatidate a good substrate and lysophosphatidate a poor substrate. References 1 McComb, R.B., Bowers, G.N.. Jr. and Posen, S. (1979) in Alkaline Phosphatase, pp. 865-902, Plenum Press, New York. 2 Moss, D.W. (1982) Clin. Chem. 28, 2007-2016. 3 Sumikawa, K., Saeki, K., Okochi, T., Adachi, K. and Nishimura, H. (1987) Chn. Chim. Acta 167, 321-328. 4 Ehle, H., Mtiller, E. and Horn, A. (1985) FEBS Lett. 183.413-416. 5 Blank, M.L. and Snyder, F. (1970) Biochemistry 9, 5034-5036. 6 Takata, K., Sumikawa, K., Saeki, K., Okochi, T. and Adachi, K. (1988) Clin. Chim. Acta 171, 317-324. 7 Yamamoto, H., Tanaka, M., Nakabayashi, H., Sato, J.. Okochi, T. and Kishimoto, S. (1984) Cancer Res. 44, 339-344. 8 Bradford, M.M. (1976) Anal. B&hem. 72, 248-254. 9 Chen, P.S., Jr., Toribara, T.Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758.
31 10 Wells, G.N., Osborne, L.J. and Jamdar, SC. (1986) B&him. Biophys. Acta 878, 225-237. 11 Harkness, D.R. (1968) Arch. B&hem. Biophys. 126, 513-523. 12 Smith, I., Lightstone, P.J. and Perry, J.D. (1968) Clin. Chim. Acta 19,499-505.
13 Posen, S., Neale, F.C. and Clubb, J.S. (1965) Ann. Int. Med. 62, 1234-1243. 14 Fishman, W.H. and Sie, H.G. (1971) Enzymologia 41,141-167. 15 Okochi, T., Seike, H., Saeki, K., Sumikawa, K., Yamamoto, T. and Higashino, K. (1987) Clin. Chim. Acta 162, 19-27.
Biochimica et Biophysics Acta, 1046 (1990) 27-31 Elsevier
BBALIP
53466
Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes Kazuhide Sumikawa I, Toshikazu Okochi 1 and Kozaburo Ada&i 2 ’ Faculty of Health and Sport Sciences, Osaka University, Toyonaka, Osaka and 2 Department of Food and Nutrition Studies, School of Home Economics, Kobe College, Nishinomiya, Hyogo (Japan) (Received
Key words:
Alkaline
phosphatase
isozyme;
Phosphatidate
23 April 1990)
hydrolysis;
Synthetic
phosphatidate;
Sodium
deoxycholate;
(Human)
Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 34imes higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C,,_,s) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.
Introduction
The measurements of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activities in the serum are routinely used in clinical diagnosis. Alkaline phosphatase is widely distributed in mammalian organs. Its activity is high in intestine, placenta, bone and kidney. Although the hydrolytic functions of alkaline phosphatase have been intensively studied and many roles have been suggested for this enzyme, its physiological function is not yet fully understood [l]. It is well known that there are three main types of alkaline phosphatase isozymes in human tissues, liver/ bone/kidney, intestinal and placental types [2]. Liver, bone and kidney alkaline phosphatases are similar in catalytic and stability properties and are mainly similar in antigenicity [2]. In the previous paper, we reported that human placental ahcaline phosphatase hydrolyzes egg phosphatidate to stoichiometric amounts of diacylglycerol and inorganic phosphate [3]. Calf intestinal alkaline phosphatase has also been reported to dephos-
Correspondence: K. Sumikawa, Faculty of Health Osaka University, Toyonaka, Osaka 560, Japan. 0005-2760/90/$03.50
and Sport Sciences,
0 1990 Elsevier Science Publishers
B.V. (Biomedical
phorylate phosphatidate [4]. In contrast, bacterial alkaline phosphatase has been described not to hydrolyze phosphatidate [5]. The differences in phosphatidate hydrolysis of these reports may come from either the properties of isozymes or the differences in assay conditions. This study was undertaken to elucidate the isozymic difference of alkaline phosphatases in phosphatidate hydrolysis, using the purified human isozymes and various phosphatidates.
Materials and Methods
Materials
Sodium phosphatidate (98% pure) from egg lecithin, synthetic phosphatidates and lipid standards for thinlayer chromatography were purchased from Sigma Chemical (St. Louis, MO). Sephadex G-200 and phenyl-Sepharose were from Pharmacia Fine Chemicals (Uppsala, Sweden). Precoated Silica gel 60 thin-layer chromatography plates were from E. Merck AG (Darmstadt, F.R.G.). Disodium phenyl phosphate, sodium deoxycholate and other chemicals were from Wako Pure Chemical Industries, Osaka, Japan. Division)
28
Purification of human alkaline phosphatase isozymes Placental [3] and liver [6] alkaline phosphatases were purified as described previously. Intestinal alkaline phosphatase was purified by extraction with 30% butanol, precipitation with 30-50s acetone, DEAE-cellulose column chromatography (O-O.2 M NaCl, gradient), TEAE-cellulose column chromatography (O-O.2 M NaCI, gradient), isoelectric focusing and Sephadex G200 column chromatography (unpublished method). The purified human placental, liver and intestinal alkaline phosphatases each gave a single protein band on SDSpolyacrylamide gel electrophoresis, and their specific activities were 680, 1500 and 900 U/mg protein, respectively. Assay of alkaline phosphatase activity Alkaline phosphatase activity was determined with phenyl phosphate as substrate as described previously [7]. One unit of enzyme activity was defined as the amount of enzyme catalyzing the release of 1 pmol of phenol per min. Protein determination Protein was determined by the method of Bradford [8] with bovine serum albumin as a standard. Purification of egg phosphatidate We purified egg phosphatidate by thin-layer chromatography, becuase commercially available egg phosphatidate sometimes contains a small amount of lysophosphatidate. The commercial preparation was applied to a precoated Silica-gel plate with fluorescent indicator (20 cm x 20 cm), developed with chloroform/ methanol/water (65 : 25 : 4, v/v) and located under ultraviolet light. The phosphatidate, co-chromatographed with an authentic standard, was then scraped off the plate and extracted with the same solution. The extract was separated into two layers by addition of water. The chloroform layer was evaporated to dryness and the residue was dissolved in chloroform and stored at - 20 o C until use. The phosphatidate concentration was calculated from Pi determined by a modification of the method of Chen et al. [9]. Assay of phosphatidate hydrolytic activity On the day of use, the phosphatidate solution was dried and dispersed in water by 1-min sonication at 30 W in a Branson sonifier under nitrogen. The standard incubation mixture contained purified alkaline phosphatase (0.02-0.1 U), 1.35 mM sodium phosphatidate, 50 mM Tris-HCl (pH 8.5), 10 g/l sodium deoxycholate and 0.2 mM MgCl, in a total volume of 1.0 ml. As the preliminary experiment showed that a low concentration of Mg2+ stimulated the activity about 2-fold, 0.2 mM MgCl, was added to the assay mixture. The assay was carried out as described previously [3].
Identifications of diacylglycerol and monoacylglvcerol Lipids were extracted as described previously [l]. Diacylglycerol was identified by thin-layer chromatography (TLC) with dipalmitin as a standard as follows: An appropriate amount of the extracted lipid solution and dipalmitin solution in chloroform were applied to a precoated Silica gel plate, developed with hexane/ diethyl ether/acetic acid (50: 50: 1, v/v) and located with iodine vapor. For identifications of lysophosphatidate and monoacylglycerol, lipids were developed with chloroform/methanol/ water (65 : 25 : 4, v/v). Assay of lysophosphatidate hydrolytic activity Sodium lysophosphatidate (oleoyl) was sonicated in the same way as for assay of phosphatidate hydrolytic activity and the assay mixture contained diethanolamine buffer (pH 10.0) and 0.4 mM sodium lysophosphatidate. The assay was carried out at 37” C for 30 min. Released inorganic phosphate was determined by a modification of the method of Chen et al. [91. Results
Effect of sodium deoxycholate on phosphatidate hvdrolytic activity As shown in Fig. 1, in the absence of sodium deoxycholate, none of alkaline phosphatase isozymes hydro-
50
0
1
10 Sodium deoxychdlate
20 (g/l)
Fig. 1. Effects of sodium deoxycholate on phosphatidate hydrolysis by alkaline phosphatase isozymes. The assay was carried out with 0.05 U of enzymes under standard conditions except that the pH was 9.0 and the sodium deoxycholate concentration was varied as indicated. Enzyme source: l, placenta; O. intestine; A, liver. The results are mean values for duplicate determinations.
29
lyzed egg phosphatidate. In the presence of sodium deoxycholate, placental and intestinal alkaline phosphatases hydrolyzed the phosphatidate. The maximal activity was seen in the presence of 10 g/l sodium deoxycholate. The activity of placental alkaline phosphatase was greater than that of intestinal alkaline phosphatase at all the concentrations of sodium deoxycholate examined. Under the optimal conditions, the apparent K, of placental alkaline phosphatase for egg phosphatidate was estimated as 0.44 mM. In contrast, liver alkaline phosphatase did not hydrolyze egg phosphatidate even in the presence of sodium deoxycholate. Of other detergents examined, sodium cholate also stimulated the activities of placental and intestinal alkaline phosphatases, but its stimulation was lower than that of sodium deoxycholate. Triton X-100, Tween 20 and zephiramine (cationic detergent) had no effect on the activity (data not shown). From these reasons we used sodium deoxycholate for the following experiments.
TABLE
I
Hydrolysis of phosphatidates by alkaline phosphatase isozymes Values are shown as nmol Pi released in 30 min. 0.1 U of alkaline phosphatase was used for each assay. The results are mean values for duplicate determinations. Phosphatidate
Placenta
Intestine
Liver
EkX Dioleoyl(18 : 1) Distearoyl(18 : 0) Dipalmitol(16 : 0) Dimyristoyl(14 : 0) Dilauroyl(12 : 0)
93.3 85.5 49.3 54.7 92.6 109.5
37.8 28.2 21.3 20.0 32.3 51.0
0 0 0 0 1.6 6.7
wide pH range, but their maximal activities were at about pH 8.5 and 7.8, respectively. Placental alkaline phosphatase showed several-fold higher activity than intestinal alkaline phosphatase on egg phosphatidate between pH 7.0 and 9.5. Liver alkaline phosphatase did not hydrolyze this phosphatidate at any pH examined.
Effect of pH on phosphatidate hydrolytic activity
For this experiment, Tris-HCl buffer (pH 7.0-9.5) and diethanolamine buffer (pH 8.0-11.0) were used. In the range between pH 8.0 and 9.0, the activity was the same with either buffer. As seen in Fig. 2, placental and intestinal alkaline phosphatases had hydrolytic activities over a relatively
Hydrolysis of synthetic phosphatidates by alkaline phosphatase isozymes
Just before the experiment, phosphatidate was sonicated with sodium deoxycholate, because distearoyl and dipalmitoyl phosphatidates are difficult to disperse without detergent. Table I shows that under standard conditions, all the phosphatidates acted as substrates for placental and intestinal alkaline phosphatases, but that dilauroyl phosphatidate was the best substrate. Placental alkaline phosphatase showed 2- to 3-times higher activity than intestinal enzyme on these phosphatidates. Liver alkaline phosphatase hydrolyzed only dimyristoyl and dilauroyl phosphatidates and its hydrolytic activity on these compounds was very low. Formation of the reaction product, diacylglycerol, was confirmed by TLC. Effect of sodium deoxycholate on phenyl phosphate and lysophosphatidate hydrolytic activities
OL
--A.-.
1.0
8.0
9.0
10.0
11.0
PH
Fig. 2. Effect of pH on phosphatidate hydrolytic activity. The assay was carried out with 0.1 U of enzymes under standard conditions except that the pH was varied as indicated. Enzyme source: 0, placenta; 0, intestine; A, liver. The results are mean values for duplicate determinations.
The above results show that in the presence of sodium deoxycholate, placental and intestinal alkaline phosphatases hydrolyzed phosphatidates but the liver enzyme did not. To determine whether these differences came from those of the effect of sodium deoxycholate on the isozymes, or those of the phosphatidate hydrolytic activities of the isozymes, the effects of sodium deoxycholate on the hydrolytic activities were investigated using phenyl phosphate, which was a representative artificial substrate for alkaline phosphatase and lysophosphatidate, which was the analog of phsphatidate. Sodium deoxycholate activated the phenyl phosphate hydrolytic activity of placental and intestinal alkaline phosphatases, activation of the intestinal enzyme being greater than that of the placental enzyme. In contrast, it
30 TABLE
II
Effect of sodium deoxycholate Activity
is expressed
on phenyl phosphate
as a percentage
and lysophosphatidate
of that without
Addition
Substrate
Sodium deoxycholate
phenyl phosphate
sodium
hydrolytic activities
deoxycholate.
The results
are mean values for duplicate
determinations.
lysophosphatidate
placenta
intestine
liver
placenta
intestine
liver
100 114 116 119 122
100 143 148 152 15.5
100 82 82 83 85
100 54 30 21 15
100 21 2 0 0
100 33 3 1 0
(g/B 0 2 5 10 20
inhibited the activity of liver alkaline phosphatase about 15% (Table II). On the other hand, sodium deoxycholate inhibited the lysophosphatidate hydrolytic activities of all three alkaline phosphatase isozymes. Addition of 10 g/l sodium deoxycholate inhibited the activities of intestinal and liver alkaline phosphatases almost completely, but placental alkaline phosphatase was less sensitive to sodium deoxycholate and 20% of its activity remained in the presence of 10 g/l sodium deoxycholate. Discussion Previously, we reported that human placental alkaline phosphatase can hydrolyze egg phosphatidate [l]. Here, we report that human intestinal alkaline phosphatase also hydrolyzed phosphatidates. Thus, not only calf intestinal alkaline phosphatase [4] but also human intestinal alkaline phosphatase hydrolyzed phosphatidates. In the intestine, bile acids are present in alkaline conditions, and thus our finding that phosphatidates, which may be formed from lecithin. by the action of phospholipase D, can be hydrolyzed by intestinal alkaline phosphatase in these conditions has physiological significance. Of the phosphatidates with saturated fatty acyl chains examined, dilauroyl phosphatidate was the best substrate for placental and intestinal alkaline phosphatases. Dioleoyl phosphatidate was hydrolyzed better than distearoyl phosphatidate. These results may be due to differences in the hydrophobicities of these phosphatidates. Wells et al. [lo] reported similar results for phosphatidate phosphohydrolase. Human placental alkaline phosphatase has been reported to have a lower K, for p-nitrophenyl phosphate of 0.8 mM than for other substrates examined [ll]. Thus, egg phosphatidate seemed to be as a good substrate as p-nitrophenyl phosphate. On the other hand, we found that human liver alkaline phosphatase was unable to hydrolyze phosphatidates with long fatty acyl chains either in the
presence or absence of sodium deoxycholate. This suggests that liver alkaline phosphatase has a different physiological role from placental and intestinal alkaline phosphatases. Placental, intestinal and liver alkaline phosphatases differ in antigenicity [2], electrophoretic mobility [12] and many other properties [6,13-151. All three are regarded as alkaline phosphatase isozymes, because they hydrolyze many substrates of low molecular weight at similar rates at alkaline pH. But with relatively hydrophobic substrates of higher molecular weight, such as phosphatidates as substrates, placental and intestinal alkaline phosphatases may differ from the liver enzyme in hydrolytic activity. At least, as shown here, human liver alkaline phosphatase clearly differed from the other two enzymes with egg phosphatidate as substrate. Thus, this substrate is useful in differentiating the liver enzyme from the other two alkaline phosphatases. As the hydrolytic activity of alkaline phosphatase on phenyl phosphate was affected by sodium deoxycholate (Table II), sodium deoxycholate may act on the enzyme protein. However, sodium deoxycholate also seems to act on the substrate, because its effect on the hydrolytic activity varied with the substrate: it seemed to make phosphatidate a good substrate and lysophosphatidate a poor substrate. References 1 McComb, R.B., Bowers, G.N.. Jr. and Posen, S. (1979) in Alkaline Phosphatase, pp. 865-902, Plenum Press, New York. 2 Moss, D.W. (1982) Clin. Chem. 28, 2007-2016. 3 Sumikawa, K., Saeki, K., Okochi, T., Adachi, K. and Nishimura, H. (1987) Chn. Chim. Acta 167, 321-328. 4 Ehle, H., Mtiller, E. and Horn, A. (1985) FEBS Lett. 183.413-416. 5 Blank, M.L. and Snyder, F. (1970) Biochemistry 9, 5034-5036. 6 Takata, K., Sumikawa, K., Saeki, K., Okochi, T. and Adachi, K. (1988) Clin. Chim. Acta 171, 317-324. 7 Yamamoto, H., Tanaka, M., Nakabayashi, H., Sato, J.. Okochi, T. and Kishimoto, S. (1984) Cancer Res. 44, 339-344. 8 Bradford, M.M. (1976) Anal. B&hem. 72, 248-254. 9 Chen, P.S., Jr., Toribara, T.Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758.
31 10 Wells, G.N., Osborne, L.J. and Jamdar, SC. (1986) B&him. Biophys. Acta 878, 225-237. 11 Harkness, D.R. (1968) Arch. B&hem. Biophys. 126, 513-523. 12 Smith, I., Lightstone, P.J. and Perry, J.D. (1968) Clin. Chim. Acta 19,499-505.
13 Posen, S., Neale, F.C. and Clubb, J.S. (1965) Ann. Int. Med. 62, 1234-1243. 14 Fishman, W.H. and Sie, H.G. (1971) Enzymologia 41,141-167. 15 Okochi, T., Seike, H., Saeki, K., Sumikawa, K., Yamamoto, T. and Higashino, K. (1987) Clin. Chim. Acta 162, 19-27.
