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Improved purification of human liver alkaline phosphatase by phenyl-Sepharose column chromatography
317
Clinica Chimica Aeta, 171 (1988) 317-324
Elsevier CCA 04070
Short communication
improves purification of human liver alkaline phosphatase by phenyl-Sepharose column chromatography Ken-iti Takata a, Kazuhide Sumikawa a, Koh Saeki a, Toshikazu Okochi a and Kozaburo Ada&i ’ a Faculty of Heailh and Sport Sciences, Osaka Universiiy, Tqonaka, Osaka. ’ Department of Food and Nutrition Studies, School of Home Economics, K&e CollegeP ~jsh~nvrn~ya, Hyogo {Japan)
(Received 23 June 1987; revision received 31 August 1987; accepted after revision 3 September 1987) && words: Alkaline phosphat~e;
Human liver; ~henyl-~pharose;
hydrophobic
chromato~apby
Introduction Isozymes of human alkaline pbosphatase (AP, orthophospho~c mon~ster phosphohydrolase (alkaline optimum), EC 3.1.3.1) have been purified from the placenta, intestine and liver, respectively. Their physiochemical and immunological properties suggest that they are coded by three different genes [l-3]. Liver AP, which is widely used in clinical diagnosis, has recently been purified by various procedures, such as chromatography on concanavalin A-Sepharose [4-61, TEAE-cellulose [7], p-aminobenzyl phosphonic acid-Sepharose [8,9] and phenylSepharose [lO,llJ. However, human liver AP is the most difficult of the three AP isozymes to purify because of its low activity in liver tissue and its loss of activity during purification. This paper reports that a procedure using NaCl-mediated hydrophobic chromatography on phenyl-~pharose is effective for purification of human liver AP. By this procedure, liver AP could be separated from placental and intestinal APs due to their differing hydrophobicities. Materials A specimen of normal human liver was obtained 8 h after death at the Department of Legal Medicine, Osaka University Medical School. Intestinal and placental APs were purified in our laboratory as reported previously [12]. DEAE-cellulose was obtained from Brown Co., Berlin, NH, USA. Phenyl-Sepharose and Sephadex G-200 were obtained from Pharmacia Fine Chemicals, Upp-
Correspondence to: Dr. K. Takata, Faculty of Health and Sport Sciences, Osaka Urtiversity. Toyonaka, Osaka 560, Japan. 0009-8981/88/$03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
318
sala, Sweden. Bovine serum albumin was obtained from Sigma Chemical Co., St. Louis, MO, USA. Disodium phenyl phosphate and other chemicals were obtained from Wako Pure Chemical Industries, Osaka, Japan. Methods Protein determination Protein concentration was measured serum albumin as a standard.
by the method
of Bradford
[13] with bovine
Alkaline phosphatase assay AP activity was determined with phenyl phosphate as substrate by the method of Higashino et al [14]. One unit of enzyme activity was defined as the activity catalyzing the release of 1 pmol of phenol/mm at 37’ C. Purification procedure Extraction Normal human liver was cut into small pieces, and homogenized in a Polytron homogenizer in 3 vol of 20 mmol/l Tris-HCl buffer, pH 7.5, containing 2 mmol/l MgCl, and 0.025 mmol/l ZnCl, (buffer A). n-Butanol was added to the homogenate to a final concentration to 300 ml/l, and the mixture was stirred for 2 h at room temperature. After centrifugation at 6000 X g for 20 min at 4OC, the water phase containing the enzyme was collected. Acetone precipitation Cold acetone ( - 20 o C) was added’ to the extracted enzyme solution to give a final concentration of 500 ml/l. The precipitate, which contained most of the enzyme, was immediately collected by centrifugation at 15 000 X g for 20 min at - 10 o C, and dissolved in buffer A. DEAE-cellulose chromatography The enzyme solution was applied to a column (3.0 x 28 cm) of DEAE-cellulose previously equilibrated with buffer A. The column was washed with 30 mmol/l NaCl in buffer A, and developed with 2000 ml of a linear gradient of 30 to 120 mmol/l of NaCl in buffer A. The active fractions were pooled and concentrated. The enzyme sample was adjusted to a Phenyl-Sepharose column chromatography final concentration of 2.7 mol/l NaCl in buffer A with 5 mol/l NaCl in buffer A. Then, the sample was applied to a phenyl-Sepharose column (2.0 x 25 cm) which had been equilibrated with buffer A containing 2.7 mol/l NaCl. Stepwise elution was carried out with 240 ml of NaCl at 2.7 mol/l in buffer A and 460 ml of NaCl at 2.5 mol/l in buffer A. Fractions of 10 ml were collected at a flow rate of 25 ml/h. The fractions of eluate with activity were concentrated by ultrafiltration, and dialyzed against 50 mmol/l Tris-HCl, pH 7.5, containing 2 mmol/l MgCl, and 0.025 mmol/l ZnCl,.
319
TABLE I Purification of human liver alkaline phosphatase Procedure
Total protein (mg)
Total act (U)
Spec act (KU/g protein)
Purification factor
Yield (W
Crude homogenate 30% (v/v) butanol extract 50% (v/v) acetone precipitate DEAE-cellulose Phenyl-Sepharose Sephadex G-200
163000
3260
0.020
1
100
17800
2310
0.130
6.50
71
2290 1160 814 620
0.295 5.80 502 1510
14.8 290 25 100 75 500
70 36 25 19
7750 200 1.62 0.411
Fig 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 10 (*g of enzyme stained for protein. Electrophoresis was carried out as described in the ‘Methods’.
320
Sephadex G-200 chromatography Chromatography was performed in 50 mmol/l Tris-HCl buffer, pH 7.5, containing 2 mmol/l MgCl, and 0.025 mmol/l ZnCl, on Sephadex G-200 (2.5 X 90 cm) at a flow rate of 12 ml/h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out by the method of Weber et al [15]. Protein was stained with Coomassie Brilliant Blue R. Results Table I summarizes the purification of human liver AP. The specific activity of the final preparation was 1510 KU/g protein, which represents an increase of about 75 500-fold over that of the crude homogenate. The yield was 19%. The purified enzyme gave a single protein band on electrophoresis on sodium dodecyl sulfatepolyacrylamide gel (Fig. 1). Figure 2 shows the elution profile of the enzyme from the phenyl-Sepharose column. In this step, the active enzyme was eluted from the column with 2.5 mol/l NaCl in buffer A, and the peak of AP activity was obtained with 2 column volumes of elution buffer. Higher concentrations of NaCl removed inactive protein. This salt-mediated hydrophobic chromatography achieved 87-fold purification of the enzyme (increase from 5.80 to 502 KU/g protein). This step, in which the yield was 70%, was the most effective of the purification steps. Neither intestinal nor placental AP was eluted with 2.5 mol/l NaCl in buffer A. The peak of intestinal AP activity was eluted at 3.9 column volumes in 1.75 mol/l NaCl in buffer A, and the peak of placental AP activity was eluted at 3.6 column
Fraction
number
Fig. 2. Elution profile of human liver alkaline phosphatase from a phenyl-Sepharose column (2.0~25 cm). Fractions of 10 ml were collected at a flow rate of 25 ml/h. Enzyme activity (o), protein (0). Conditions of elution buffers are presented as concentrations of NaCl in buffer A at the top of the figure.
321
(mol/l)
C”,,ccntrati<,n
_ 4
0.3-
$
0.2-
2
0.1
ci e
NnCI
,,I
,,ut*rr
n
--1.25-
(a)
.i > .rl -I :
of
w-2.0--1.75--1.5
-
0
--I (b)
0.10
-
zo.05 2
-
Oo
IO
20
30
40
Fraction
50
60
70
00
90
number
Fig. 3. Elution profile of human intestinal (a) and placental (b) alkaline phosphatases from a phenyl-sepharose column (1.4 x 10 cm). Fractions of 5 ml were collected at a flow rate of 20 ml/h. Enzyme activity (0). Conditions of elution buffers are presented as concentrations of NaCl in buffer A at the top of the figure.
volumes in 1.25 mol/l NaCl in buffer A. Therefore, the human AP isozymes, placental, intestinal and liver APs, were eluted in order of their hydrophobicities. Figure 3 shows the elution profiles of human intestinal and placental APs on hydrophobic chromatography on phenyl-Sepharose. Discussion of human In the present purification procedure, based on the hydrophobicity liver AP as a glycoprotein [16-181, we developed a simple, efficient method for its purification. This principle was at first applied in purification of human placental AP on L-phenylalanine-Sepharose [19]. Since then, several other methods for purification of AP based on its hydrophobicity have been reported. For example, the APs from Dictyostelium discoideum [20] and Sinclair swine melanoma [21] were purified by phenyl-Sepharose column chromatography, and AP from thermophilic Actinomycetes was purified by chromatographies on octyl-Sepharose and pentyl-Sepharose columns [22]. But phenyl-Sepharose chromatography has not previously been used to efficient purification of human liver AP. Seiffer et al [lo] used this material for repeated chromatography just after the n-butanol extraction step to isolate two forms of human liver AP [23]. They were the first to use a phenyl-Sepharose column for purification of human liver AP, but in their method recovery was sacrificed for purity. In contrast, our NaCl-mediated phenyl-Sepharose chromatography method was effective and increased the specific activity 87-fold, with 70% recovery of AP activity. In NaCl at 2.5 mol/l in buffer A, human liver AP is in weak adsorption
322
equilibrium with the column, the peak of activity is eluted after two column volumes of eluent and is separated from various other proteins. Our procedure for purification of liver AP consists of only 5 steps including n-butanol extraction and acetone precipitation. In contrast, conventional procedures for purification of human liver AP require 6 or more steps. In these procedures, affinity chromatography on concanavalin A-Sepharose [5] or phosphonic acid-Sepharose [8] is most effective in increasing the specific activity (17.4-fold and 51-fold, respectively), and the final specific activities are 650 KU/g protein and 1300 KU/g protein, respectively, with p-nitrophenyl phosphate as substrate. The specific activity of the enzyme purified by our procedure was 1510 KU/g protein with phenyl phosphate as substrate. This specific activity is comparable with reported values of 1360 KU/g protein after a 6-step purification procedure [7] and 1450 KU/g protein after a 9-step procedure [4]. Our method using phenyl-Sepharose is very effective not only for purification of human AP but also for its separation from intestinal and placental APs. References 1 Moss DW. Alkaline phosphatase isoenzymes. Clin Chim 1982;28:2007-2016. 2 Seargeant LE, Stinson RA. Evidence that three structural genes code for human alkaline phosphatase. Nature (London) 1979;281:152-154. MJ, Hamilton A, Sussman H. Comparison of human alkaline phosphatase isoenzymes. 3 McKenna Biochem J 1979;181:67-73. purified by affinity chromatography, 4 Latner AL, Hodson AW. Human liver alkaline phosphatase ultracentrifugation and polyacrylamide gel electrophoresis. Biochem J 1976; 159:697-705. LE, Stinson RA. Affinity purification and some molecular-properties of 5 Trepanier JM, Seargeant human liver alkaline phosphatase. Biochem J 1976;155:653-660. and some properties of human liver alkaline phos6 Komoda T, Sakagishi Y. Partial purification phatase. Biochim Biophys Acta 1976;438:138-152. OH. Human alkaline phosphatases. I. Purification and some 7 Gerbitz KD, Kolb HJ, Wieland structural properties of the enzyme from human liver. Hoppe-Seyler’s Z Physiol Chem 1977;358:435-446. LE, Stinson RA. Affinity elution from a phosphonic acid-Sepharose derivative in the 8 Seargeant purification of human liver alkaline phosphatase. J Chromatogr 1979;173:101-108. LE. Comparative studies of pure alkaline phosphatases from five human 9 Stinson RA, Seargeant tissues. Clin Chim Acta 1981;110:261-272. in human liver 10 Seiffer UB, Sied WH, Welsch GJ, Oremek G. Multiple forms of alkaline phosphatases tissue. Clin Chim Acta 1984;144:17-27. MIE, Leijnse B. Hydrophobic properties of alkaline phosphatases. Int J 11 Wulkan RW, Huijskes-Heins Biochem 1986;18:1045-1051. 12 Okochi T, Seike H, Saeki K, Sumikawa K, Yamamoto T, Higashino K. A novel alkaline phosphatase isozyme in human adipose tissue. Clin Chim Acta 1987;162:19-27. of microgram quantities of protein 13 Bradford MM. A rapid and sensitive method for the quantitation utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-254. M, Kang K, Takahashi Y, Yamamura Y. Studies on a variant alkaline 14 Higashino K, Hashinotsume phosphatase in sera of patients with hepatocellular carcinoma. Clin Chim Acta 1972;40:67-81. of molecular weights by electrophoresis on SDS15 Weber K, Pringle JR, Osborn M. Measurement acrylamide gel. Methods Enzymol 1972;26c:3-27. 16 Robinson JC, Pierce JE. Differential action of neuraminidase on human serum alkaline phosphatase. Nature (London) 1964;204:473-473.
323 17 Moss DW, Eaton RH, Smith JK, Whitby LG. Alteration in the electrophoretic mobility of alkaline phosphatase after treatment with neuraminidase. Biochem J 1966;98:32c-33~. 18 Ghosh NK. Purification and molecular properties of placental and intestinal alkaline phosphatase. Ann NY Acad Sci 1969;166:604-640. 19 Doellgast GJ, Fishman WH. ~~fi~tion of human placental alkaline phosphatase. B&hem J 1974;141:103-112. 20 Armant DR, Rutherford CL. Copurification of alkaline phosphatase and 5’-AMP specific nucleotidase in Dictyosfelium discoideum. J Biol Chem 1981;256:12710-12718. 21 Williams MV, Hook RR. Properties of an alkaline phosphatase from Sinclair swine melanoma. J Invest Dermatol 1984;82:526-531. 21 Iny D, Sofer J, Pinsky A. isolation of a thermopbili~ alkaline phosphatase by either hydrophobic or Pro&n red Sepharose c~omato~aphy. J Chromatogr 1986;3~:437-~2. 23 Simon FR, Sutherland E. Hepatic alkaline phosphatase isoenzymes: isolation, characterization and differential alteration. Enzyme 1977;22:80-90.
Clinica Chimica Aeta, 171 (1988) 317-324
Elsevier CCA 04070
Short communication
improves purification of human liver alkaline phosphatase by phenyl-Sepharose column chromatography Ken-iti Takata a, Kazuhide Sumikawa a, Koh Saeki a, Toshikazu Okochi a and Kozaburo Ada&i ’ a Faculty of Heailh and Sport Sciences, Osaka Universiiy, Tqonaka, Osaka. ’ Department of Food and Nutrition Studies, School of Home Economics, K&e CollegeP ~jsh~nvrn~ya, Hyogo {Japan)
(Received 23 June 1987; revision received 31 August 1987; accepted after revision 3 September 1987) && words: Alkaline phosphat~e;
Human liver; ~henyl-~pharose;
hydrophobic
chromato~apby
Introduction Isozymes of human alkaline pbosphatase (AP, orthophospho~c mon~ster phosphohydrolase (alkaline optimum), EC 3.1.3.1) have been purified from the placenta, intestine and liver, respectively. Their physiochemical and immunological properties suggest that they are coded by three different genes [l-3]. Liver AP, which is widely used in clinical diagnosis, has recently been purified by various procedures, such as chromatography on concanavalin A-Sepharose [4-61, TEAE-cellulose [7], p-aminobenzyl phosphonic acid-Sepharose [8,9] and phenylSepharose [lO,llJ. However, human liver AP is the most difficult of the three AP isozymes to purify because of its low activity in liver tissue and its loss of activity during purification. This paper reports that a procedure using NaCl-mediated hydrophobic chromatography on phenyl-~pharose is effective for purification of human liver AP. By this procedure, liver AP could be separated from placental and intestinal APs due to their differing hydrophobicities. Materials A specimen of normal human liver was obtained 8 h after death at the Department of Legal Medicine, Osaka University Medical School. Intestinal and placental APs were purified in our laboratory as reported previously [12]. DEAE-cellulose was obtained from Brown Co., Berlin, NH, USA. Phenyl-Sepharose and Sephadex G-200 were obtained from Pharmacia Fine Chemicals, Upp-
Correspondence to: Dr. K. Takata, Faculty of Health and Sport Sciences, Osaka Urtiversity. Toyonaka, Osaka 560, Japan. 0009-8981/88/$03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
318
sala, Sweden. Bovine serum albumin was obtained from Sigma Chemical Co., St. Louis, MO, USA. Disodium phenyl phosphate and other chemicals were obtained from Wako Pure Chemical Industries, Osaka, Japan. Methods Protein determination Protein concentration was measured serum albumin as a standard.
by the method
of Bradford
[13] with bovine
Alkaline phosphatase assay AP activity was determined with phenyl phosphate as substrate by the method of Higashino et al [14]. One unit of enzyme activity was defined as the activity catalyzing the release of 1 pmol of phenol/mm at 37’ C. Purification procedure Extraction Normal human liver was cut into small pieces, and homogenized in a Polytron homogenizer in 3 vol of 20 mmol/l Tris-HCl buffer, pH 7.5, containing 2 mmol/l MgCl, and 0.025 mmol/l ZnCl, (buffer A). n-Butanol was added to the homogenate to a final concentration to 300 ml/l, and the mixture was stirred for 2 h at room temperature. After centrifugation at 6000 X g for 20 min at 4OC, the water phase containing the enzyme was collected. Acetone precipitation Cold acetone ( - 20 o C) was added’ to the extracted enzyme solution to give a final concentration of 500 ml/l. The precipitate, which contained most of the enzyme, was immediately collected by centrifugation at 15 000 X g for 20 min at - 10 o C, and dissolved in buffer A. DEAE-cellulose chromatography The enzyme solution was applied to a column (3.0 x 28 cm) of DEAE-cellulose previously equilibrated with buffer A. The column was washed with 30 mmol/l NaCl in buffer A, and developed with 2000 ml of a linear gradient of 30 to 120 mmol/l of NaCl in buffer A. The active fractions were pooled and concentrated. The enzyme sample was adjusted to a Phenyl-Sepharose column chromatography final concentration of 2.7 mol/l NaCl in buffer A with 5 mol/l NaCl in buffer A. Then, the sample was applied to a phenyl-Sepharose column (2.0 x 25 cm) which had been equilibrated with buffer A containing 2.7 mol/l NaCl. Stepwise elution was carried out with 240 ml of NaCl at 2.7 mol/l in buffer A and 460 ml of NaCl at 2.5 mol/l in buffer A. Fractions of 10 ml were collected at a flow rate of 25 ml/h. The fractions of eluate with activity were concentrated by ultrafiltration, and dialyzed against 50 mmol/l Tris-HCl, pH 7.5, containing 2 mmol/l MgCl, and 0.025 mmol/l ZnCl,.
319
TABLE I Purification of human liver alkaline phosphatase Procedure
Total protein (mg)
Total act (U)
Spec act (KU/g protein)
Purification factor
Yield (W
Crude homogenate 30% (v/v) butanol extract 50% (v/v) acetone precipitate DEAE-cellulose Phenyl-Sepharose Sephadex G-200
163000
3260
0.020
1
100
17800
2310
0.130
6.50
71
2290 1160 814 620
0.295 5.80 502 1510
14.8 290 25 100 75 500
70 36 25 19
7750 200 1.62 0.411
Fig 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 10 (*g of enzyme stained for protein. Electrophoresis was carried out as described in the ‘Methods’.
320
Sephadex G-200 chromatography Chromatography was performed in 50 mmol/l Tris-HCl buffer, pH 7.5, containing 2 mmol/l MgCl, and 0.025 mmol/l ZnCl, on Sephadex G-200 (2.5 X 90 cm) at a flow rate of 12 ml/h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out by the method of Weber et al [15]. Protein was stained with Coomassie Brilliant Blue R. Results Table I summarizes the purification of human liver AP. The specific activity of the final preparation was 1510 KU/g protein, which represents an increase of about 75 500-fold over that of the crude homogenate. The yield was 19%. The purified enzyme gave a single protein band on electrophoresis on sodium dodecyl sulfatepolyacrylamide gel (Fig. 1). Figure 2 shows the elution profile of the enzyme from the phenyl-Sepharose column. In this step, the active enzyme was eluted from the column with 2.5 mol/l NaCl in buffer A, and the peak of AP activity was obtained with 2 column volumes of elution buffer. Higher concentrations of NaCl removed inactive protein. This salt-mediated hydrophobic chromatography achieved 87-fold purification of the enzyme (increase from 5.80 to 502 KU/g protein). This step, in which the yield was 70%, was the most effective of the purification steps. Neither intestinal nor placental AP was eluted with 2.5 mol/l NaCl in buffer A. The peak of intestinal AP activity was eluted at 3.9 column volumes in 1.75 mol/l NaCl in buffer A, and the peak of placental AP activity was eluted at 3.6 column
Fraction
number
Fig. 2. Elution profile of human liver alkaline phosphatase from a phenyl-Sepharose column (2.0~25 cm). Fractions of 10 ml were collected at a flow rate of 25 ml/h. Enzyme activity (o), protein (0). Conditions of elution buffers are presented as concentrations of NaCl in buffer A at the top of the figure.
321
(mol/l)
C”,,ccntrati<,n
_ 4
0.3-
$
0.2-
2
0.1
ci e
NnCI
,,I
,,ut*rr
n
--1.25-
(a)
.i > .rl -I :
of
w-2.0--1.75--1.5
-
0
--I (b)
0.10
-
zo.05 2
-
Oo
IO
20
30
40
Fraction
50
60
70
00
90
number
Fig. 3. Elution profile of human intestinal (a) and placental (b) alkaline phosphatases from a phenyl-sepharose column (1.4 x 10 cm). Fractions of 5 ml were collected at a flow rate of 20 ml/h. Enzyme activity (0). Conditions of elution buffers are presented as concentrations of NaCl in buffer A at the top of the figure.
volumes in 1.25 mol/l NaCl in buffer A. Therefore, the human AP isozymes, placental, intestinal and liver APs, were eluted in order of their hydrophobicities. Figure 3 shows the elution profiles of human intestinal and placental APs on hydrophobic chromatography on phenyl-Sepharose. Discussion of human In the present purification procedure, based on the hydrophobicity liver AP as a glycoprotein [16-181, we developed a simple, efficient method for its purification. This principle was at first applied in purification of human placental AP on L-phenylalanine-Sepharose [19]. Since then, several other methods for purification of AP based on its hydrophobicity have been reported. For example, the APs from Dictyostelium discoideum [20] and Sinclair swine melanoma [21] were purified by phenyl-Sepharose column chromatography, and AP from thermophilic Actinomycetes was purified by chromatographies on octyl-Sepharose and pentyl-Sepharose columns [22]. But phenyl-Sepharose chromatography has not previously been used to efficient purification of human liver AP. Seiffer et al [lo] used this material for repeated chromatography just after the n-butanol extraction step to isolate two forms of human liver AP [23]. They were the first to use a phenyl-Sepharose column for purification of human liver AP, but in their method recovery was sacrificed for purity. In contrast, our NaCl-mediated phenyl-Sepharose chromatography method was effective and increased the specific activity 87-fold, with 70% recovery of AP activity. In NaCl at 2.5 mol/l in buffer A, human liver AP is in weak adsorption
322
equilibrium with the column, the peak of activity is eluted after two column volumes of eluent and is separated from various other proteins. Our procedure for purification of liver AP consists of only 5 steps including n-butanol extraction and acetone precipitation. In contrast, conventional procedures for purification of human liver AP require 6 or more steps. In these procedures, affinity chromatography on concanavalin A-Sepharose [5] or phosphonic acid-Sepharose [8] is most effective in increasing the specific activity (17.4-fold and 51-fold, respectively), and the final specific activities are 650 KU/g protein and 1300 KU/g protein, respectively, with p-nitrophenyl phosphate as substrate. The specific activity of the enzyme purified by our procedure was 1510 KU/g protein with phenyl phosphate as substrate. This specific activity is comparable with reported values of 1360 KU/g protein after a 6-step purification procedure [7] and 1450 KU/g protein after a 9-step procedure [4]. Our method using phenyl-Sepharose is very effective not only for purification of human AP but also for its separation from intestinal and placental APs. References 1 Moss DW. Alkaline phosphatase isoenzymes. Clin Chim 1982;28:2007-2016. 2 Seargeant LE, Stinson RA. Evidence that three structural genes code for human alkaline phosphatase. Nature (London) 1979;281:152-154. MJ, Hamilton A, Sussman H. Comparison of human alkaline phosphatase isoenzymes. 3 McKenna Biochem J 1979;181:67-73. purified by affinity chromatography, 4 Latner AL, Hodson AW. Human liver alkaline phosphatase ultracentrifugation and polyacrylamide gel electrophoresis. Biochem J 1976; 159:697-705. LE, Stinson RA. Affinity purification and some molecular-properties of 5 Trepanier JM, Seargeant human liver alkaline phosphatase. Biochem J 1976;155:653-660. and some properties of human liver alkaline phos6 Komoda T, Sakagishi Y. Partial purification phatase. Biochim Biophys Acta 1976;438:138-152. OH. Human alkaline phosphatases. I. Purification and some 7 Gerbitz KD, Kolb HJ, Wieland structural properties of the enzyme from human liver. Hoppe-Seyler’s Z Physiol Chem 1977;358:435-446. LE, Stinson RA. Affinity elution from a phosphonic acid-Sepharose derivative in the 8 Seargeant purification of human liver alkaline phosphatase. J Chromatogr 1979;173:101-108. LE. Comparative studies of pure alkaline phosphatases from five human 9 Stinson RA, Seargeant tissues. Clin Chim Acta 1981;110:261-272. in human liver 10 Seiffer UB, Sied WH, Welsch GJ, Oremek G. Multiple forms of alkaline phosphatases tissue. Clin Chim Acta 1984;144:17-27. MIE, Leijnse B. Hydrophobic properties of alkaline phosphatases. Int J 11 Wulkan RW, Huijskes-Heins Biochem 1986;18:1045-1051. 12 Okochi T, Seike H, Saeki K, Sumikawa K, Yamamoto T, Higashino K. A novel alkaline phosphatase isozyme in human adipose tissue. Clin Chim Acta 1987;162:19-27. of microgram quantities of protein 13 Bradford MM. A rapid and sensitive method for the quantitation utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-254. M, Kang K, Takahashi Y, Yamamura Y. Studies on a variant alkaline 14 Higashino K, Hashinotsume phosphatase in sera of patients with hepatocellular carcinoma. Clin Chim Acta 1972;40:67-81. of molecular weights by electrophoresis on SDS15 Weber K, Pringle JR, Osborn M. Measurement acrylamide gel. Methods Enzymol 1972;26c:3-27. 16 Robinson JC, Pierce JE. Differential action of neuraminidase on human serum alkaline phosphatase. Nature (London) 1964;204:473-473.
323 17 Moss DW, Eaton RH, Smith JK, Whitby LG. Alteration in the electrophoretic mobility of alkaline phosphatase after treatment with neuraminidase. Biochem J 1966;98:32c-33~. 18 Ghosh NK. Purification and molecular properties of placental and intestinal alkaline phosphatase. Ann NY Acad Sci 1969;166:604-640. 19 Doellgast GJ, Fishman WH. ~~fi~tion of human placental alkaline phosphatase. B&hem J 1974;141:103-112. 20 Armant DR, Rutherford CL. Copurification of alkaline phosphatase and 5’-AMP specific nucleotidase in Dictyosfelium discoideum. J Biol Chem 1981;256:12710-12718. 21 Williams MV, Hook RR. Properties of an alkaline phosphatase from Sinclair swine melanoma. J Invest Dermatol 1984;82:526-531. 21 Iny D, Sofer J, Pinsky A. isolation of a thermopbili~ alkaline phosphatase by either hydrophobic or Pro&n red Sepharose c~omato~aphy. J Chromatogr 1986;3~:437-~2. 23 Simon FR, Sutherland E. Hepatic alkaline phosphatase isoenzymes: isolation, characterization and differential alteration. Enzyme 1977;22:80-90.