Comp. Biochem. Physiol. Vol. 96B, No. 4, pp. 787-789, 1990
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press pie
Printed in Great Britain
A COMPARATIVE STUDY OF ALKALINE PHOSPHATASES AMONG HUMAN PLACENTA, BOVINE MILK, HEPATOPANCREASES OF SHRIMP PENAEUS M O N O D O N (CRUSTACEA- DECAPODA) AND CLAM MERETRIX LUSORIA (BIVALVIA: VENEIDAE): TO OBTAIN AN ALKALINE PHOSPHATASE WITH IMPROVED CHARACTERISTICS AS A REPORTER NIN-NIN CHUANG* a n d BEI-CHIAYANG Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, R. of China (Received 19 January 1990) Absa-act--1. Alkaline phosphatases were purified from human placenta, bovine milk, shrimp and clam with a final spec. act. of 67,000, 32,000, 22,000 and 15,000 U/mg of protein respectively. 2. The alkaline phosphatase from Meretrix lusoria is unique with its thermostability at 65°C for 30 min; whereas the remaining enzymes studied, including the human placental alkaline phosphatase, are inactivated and have negligible activities. 3. The alkaline phosphatase from Penaeus monodon can be differentiated by its pH optimum at 9.0; the remaining enzymes studied have their optimal pH at 10.0. 4. The alkaline phosphatases from shrimp and clam are proposed to be applied as "reporters" in the study of mammalian cells.
INTRODUCTION
MATERIALS AND METHODS
Alkaline phosphatases (EC 3.1.3.1) are intrinsic plasma membrane enzymes found on the membranes of almost all animal cells. Based on structural and genetic studies, in man these isoenzymes may be divided into three distinct groups: placental, intestinal, and hepatic/renal/skeletal (Moss, 1982; Seargeant and Stinson, 1979). Recently, the studies with peptide "mapping" and N-terminal sequence analysis indicate that these isoenzymes are the products of the same gene locus and the placental alkaline phosphatase is the latest mutated form (Knoll et al., 1988; Hua et al., 1986; Garratini et al., 1986). Furthermore, a significant similarity of nucleotide sequence was found between regions of the placental alkaline phosphatase and the Escherichia coli enzyme (Knoll et al., 1988). The aim of the present study is to prepare the purified alkaline phosphatases from species with different evolutionary development, such as Bos taurus, Penaeus monodon and Meretrix lusoria, and use them in investigating the nature and extent of differences from the human placental enzyme. It is hoped that from this comparison, an index could be born which can be applied to obtain a suitable alkaline phosphatase for the application as a reporter in genetic engineering. Recent studies of transcription control elements have been greatly facilitated by linking them to readily assayable reporter genes; expression of the reporter gene is used as an indication of transcription activity. *Author to whom correspondence should be addressed. CBPB 96/4~K
787
Materials All reagents used were of the highest grade available commercially. Reagents for isoelectric focusing were from Pharmacia, Sweden.
Experimental animals Shrimps (Penaeus monodon, 2 kg) and clams (Meretrix lusoria, 2 kg) collected offTaiwain, were held at 18°C for less than 3 days in a recirculating sea-water system. Hepatopancreases were dissected out immediately after death. Human placenta were removed from the body not more than 1 hr after delivery and stored at - 2 0 ° C for up to a week at most before use. Bovine (Bos taurus) milk (1 l) was freshly obtained from the Department of Animal Husbandry at National Taiwan University. Milk was placed on ice immediately after being removed.
Purification of alkaline phosphatase Alkaline phosphatase was purified with essentially the same procedure as described by Meyer et al. (I 982) but with modifications as mentioned previously (Chuang, 1990). The enzyme preparation from human placenta has a spec. act. of 6700 U/mg of protein (Chuang, 1987), the enzyme from bovine milk has a spec. act. of 32,000 U/mg of protein, that from Penaeus monodon has a spec. act. of 22,000 U/mg of protein, and the enzyme from Meretrix lusoria has a spec. act. of 15,000 U/rag of protein.
lsoelectric focusing Isoelectric focusing was carried out essentially as described by Moss and Edwards (1984) using ampholytes with a pH range of 3.0-10.0. For some experiments, restricted ranged ampholytes (pH range 4.0-6.0 and 5.0-8.0) were employed for better resolution ofisoelectric forms. Focusing was performed at a constant voltage (200 V) for 18-21 hr.
788
N I N - N I N CHUANG a n d BEI-CHIA YANG
Assay of alkaline phosphatase Alkaline phosphatase activity was measured at 37°C with the use of 10 mM 4-nitrophenyl phosphate in 0.75 M 2-amino-2-methyl-l-propanol/HC1 buffer, pill0.3. One unit of enzyme activity = 1/zmol substrate hydrolyzed/min. Enzyme activity after isoelectric focusing was determined by incubating the gels at 37°C in 2 mg of fl-naphthylic acid phosphate (Sigma F-7375) per ml, and 1 mg of Fast Blue BB (Sigma N-0250) per ml in 60 mM borate buffer/pH 9.7 for 30 rain to stain the activity bands. Assay of protein Bovine serum albumin was served as the standard in the measurement of proteins. The amount of protein was determined by the Lowry method (1951).
1001
5D
'ID e I
45 °
R E S U L T S AND D I S C U S S I O N
I
150°
L
610o
155 °
I
65°C
Temperature
The key features of a useful reporter gene are that its activity can be easily assayed and its endogenous activity in the appropriate cells is low. Human placental alkaline phosphatase (Henthorn et al., 1988), bacterial fl-galactosidase (An et al., 1982), human growth hormone (Seldon et al., 1986) and firefly luciferase (De Wet et al., 1987) are the best examples of reporter genes. In the application of alkaline phosphatase as a reporter, 4-nitrophenyl phosphate is used as the substrate and the yellow product, nitrophenol, which is easily detected by spectrophotometry, makes alkaline phosphatase prevail over others in cost and non-radioactivity contamination. In view of the other requirements for a good reporter gene, human placental alkaline phosphatase is not quite content in limited expression of endogenous activity. Notwithstanding the fact that human placental alkaline phosphatase can be differentiated from the other two isoenzyme groups, intestinal and hepatic/renal/skeletal, by its resistance to heating at 65°C, at which non-placental phosphatases are rapidly inactivated (Moss, 1982; Seargeant and Stinson, 1979; Neale et al., 1965), the evidence that the widespread presence of human placental alkaline phosphatase in the lung (Goldstein et al., 1980), cervix (Goldstein et al., 1982) and milk (Chuang, 1987), and also in the patients' sera with ulcerative colitis, familial polposis, cirrhosis (Stolbach et al., 1976) and carcinoma (Nathanoson and Fishman, 1971) reinforces the need of searching for a thermostable alkaline phosphatase with more limited expression in mammalian tissues. In the present study, we propose two alkaline phosphatases to be applied and used as a reporter in genetic engineering, one is the enzyme from Meretrix lusoria with its extreme thermostability, and the other is the enzyme from Penaeus monodon with its unique optimal pH at 9.0. When purified alkaline phosphatases from human placenta, bovine milk, Penaeus monodon and Meretrix lusoria are subjected to the comparative analysis of thermostability, no enzyme activity was lost upon preincubation at 50°C for l0 rain (Fig. 1). In particular, the alkaline phosphatase from Meretrix lusoria was found to retain 50% of the original activity after incubation at 65°C for 30 min; whereas 90% of the enzyme activity was inactivated in human placental alkaline phosphatase (Fig. 2). That is, the
Fig. I. Heat inactivation of alkaline phosphatase. The stability of the enzyme was investigated by incubating them at 45-65°C for I0 rain prior to assaying with 4-nitrophenyl phosphate. Alkaline phosphatase purified from human placenta (©), bovine milk (O), Penaeus monodon (ff]) and Meretrix lusoris ( . ) were incubated at indicated temperature for 10rain and the remaining enzyme activity was measured. Activities are expressed as percentages of the initial total activity. alkaline phosphatase from Meretrix lusoria is unique at therrnostability. Alkaline phosphatase shows species speciality at isoelectric points. The enzyme from Penaeus monodon was discovered to have an isoelectric point (pI) of 6.9 _+ 0.1 (Fig. 3C), the enzyme from Meretrix lusoria having a pI of 6.0 _+ 0.1 (Fig. 3D), the bovine milk enzyme having a pI of 5.5 _+ 0.1 (Fig. 3B), and the placental alkaline phosphatase having a more acidic pI, 4.8 +_ 0.1 (Fig. 3A). In the study of optimal pH, the purified alkaline phosphatase from Penaeus
% IO0
50
m ¢g n" i
t
10
20
) 30
Time
Fig. 2. Heat inactivation of alkaline phosphatase from Meretrix lusoria. The stability of the purified clam enzyme ( I ) was investigated by incubating them at 65°C for 10-30 min prior to assaying with 4-nitrophenyl phosphate. For comparison, the human placental alkaline phosphatase was included (C)). Activities are expressed as percentages of the initial total activity.
Alkaline phosphatases A
'
,
n,
I
B
I
'
,
C
I
I
I
i
D
n
I
i
I
,
n
I
I
REFERENCES
An G., Hidaka K. and Siminovitch L. (1982) Expression of bacterial fl-galactosidase in animal cells. Mol. Cell. Biol. 2, 1628-1632, Chuang N.-N. (1987) Alkaline phosphatase in human milk: a new heat-stable enzyme. Clin. Chim. Acta 169,
i
I
I
i
,
i
i
i
I
'm
I
I
I
l
3
4
5
6
7
8
9
10
Fig. 3. Isoelectric points of alkaline phosphatase. Purified alkaline phosphatase from human placenta (A), bovine milk (B), Penaeus monodon (C) and Meretrix lusoria (D) were subjected to isoelectric focusing. For each sample, triplicate gels were measured.
monodon has an optimal pH of the enzyme activity at
9.0, which is different from those of enzymes obtained from human placenta, bovine milk and Meretrix lusoria (Fig. 4); the remaining enzymes, including the alkaline phosphatase from Penaeus japonicus (Chuang, 1990), shared the same optimal pH for enzyme activity at 10.0. Accordingly, the alkaline phosphatase from Penaeus monodon can be differentiated from the other studied enzymes by its pH optimum. Further studies on comparing primary structures of alkaline phosphatases are being undertaken. Acknowledgements--Financial support for this work was
provided by the National Science Council and Academia Sinica, Republic of China.
lOU
50
.-
@ rr
0 3
8
8
7
8
8
10
789
11
pH
Fig. 4. Effect of pH on the alkaline phosphatase activity. Alkaline phosphatase purified from human placenta (C)), bovine milk (O), Penaeus monodon (i"-I) and Meretrix lusoria ( i ) were assayed in the reaction mixture consisting of 0.1% Triton X-100, 1 mM MgCI2 and 100mM buffer of succinate--sodium hydroxide (pH 3-6) or Tris-malate (pH 6-9). Activities are expressed as percentages of the initial activity.
165-174.
Chuang N.-N. (1990) A heat-stable alkaline phosphatase from Penaeus japonicus Bate (Crustacea: Decapoda). Comp. Biochem. Physiol. 95, 165-169. De Wet J. R., Wood K. V., DeLuca M., Helinski D. R. and Subramani S. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725-737. Garanini E., Hua J.-C., Pan Y.-E. and Udenfriend S. (1986) Human liver alkaline phosphatase, purification and partial sequencing: homology with the purified isozyme. Arch. Biochem. Biophys. 245, 331-337. Goldstein D. J., Blasca L. and Harris H. (1980) Placental alkaline phosphatase in non-malignant human cervix. Proc. natn. Acad. Sci. USA 77, 4226-4228. Goldstein D. J., Rogers C. and Harris H. (1982) Evolution of alkaline phosphatases in primates. Proc. natn. Acad. Sci. USA 79, 879-883. Gorman C. M., Moffat L. F. and Howard B. H. (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044-1051. Henthorn P., Zervos P., Raducha M., Harris H. and Kadesch T. (1988) Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression. Proc. hath. Acad. Sci. USA 85, 6342-6346. Hua J.-C., Berger J., Pan Y. E., Hulmes J. D. and Udenfriend S. (1986) Partial sequencing of human adult, human fetal and bovine intestinal alkaline phosphatases: comparison with the human placental and liver enzyme. Proc. natn. Acad. Sci. USA 83, 2368-2372. Knoll B. J., Rothblum K. N. and Longley M. (1988) Nucleotide sequence of the human placental alkaline phosphatase gene. J. biol. Chem. 263, 12,020-12,027. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Meyer L. J., Lafferty M. A., Raducha M. G., Foster C. J., Gogolin K. J. and Harris H. (1982) Production of a monoclonal antibody to human liver alkaline phosphatase. Clin. Chim. Acta 126, 109-117. Moss D. W. (1982) Alkaline phosphatase isoenzymes. Clin. Chem. 28, 2007-2016. Moss D. W. and Edwards R. K. (1984) Improved electrophoretic resolution of bone and liver alkaline phospbatases resulting from partial digestion with neuraminidase. Clin. Chim. Acta 143, 177-182. Nathanson L. and Fishman W. H. (1971) New observations on the Regan isoenzyme of alkaline phosphatase in cancer patients. Cancer 27, 1388-1397. Neale F. C., Clubb J. S., Hotchkis D. and Posen S. (1965) Heat-stability of human placental alkaline phosphatase. J. Clin. Pathol. 18, 359-363. Seargeant L. E. and Stinson R. A. (1979) Affinity elution from a phosphoric acid-Sepharose derivative in the purification of human liver alkaline phosphatase. Nature 281, 152-154. Seldon R. F., Howie K. B., Rowe M. E., Goodman H. M. and Moore D. M. (1986) Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell. Biol. 6, 3173-3179. Stolbach L. L., Inglis N. I., Lin C., Turksoy R., Fishman W. H., Marchant D. and Rule A. (1976) Onco-Developmental Gene Expression (Edited by Fishman W. H. and Sell S.), pp. 433-443. Academic Press, New York.
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press pie
Printed in Great Britain
A COMPARATIVE STUDY OF ALKALINE PHOSPHATASES AMONG HUMAN PLACENTA, BOVINE MILK, HEPATOPANCREASES OF SHRIMP PENAEUS M O N O D O N (CRUSTACEA- DECAPODA) AND CLAM MERETRIX LUSORIA (BIVALVIA: VENEIDAE): TO OBTAIN AN ALKALINE PHOSPHATASE WITH IMPROVED CHARACTERISTICS AS A REPORTER NIN-NIN CHUANG* a n d BEI-CHIAYANG Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, R. of China (Received 19 January 1990) Absa-act--1. Alkaline phosphatases were purified from human placenta, bovine milk, shrimp and clam with a final spec. act. of 67,000, 32,000, 22,000 and 15,000 U/mg of protein respectively. 2. The alkaline phosphatase from Meretrix lusoria is unique with its thermostability at 65°C for 30 min; whereas the remaining enzymes studied, including the human placental alkaline phosphatase, are inactivated and have negligible activities. 3. The alkaline phosphatase from Penaeus monodon can be differentiated by its pH optimum at 9.0; the remaining enzymes studied have their optimal pH at 10.0. 4. The alkaline phosphatases from shrimp and clam are proposed to be applied as "reporters" in the study of mammalian cells.
INTRODUCTION
MATERIALS AND METHODS
Alkaline phosphatases (EC 3.1.3.1) are intrinsic plasma membrane enzymes found on the membranes of almost all animal cells. Based on structural and genetic studies, in man these isoenzymes may be divided into three distinct groups: placental, intestinal, and hepatic/renal/skeletal (Moss, 1982; Seargeant and Stinson, 1979). Recently, the studies with peptide "mapping" and N-terminal sequence analysis indicate that these isoenzymes are the products of the same gene locus and the placental alkaline phosphatase is the latest mutated form (Knoll et al., 1988; Hua et al., 1986; Garratini et al., 1986). Furthermore, a significant similarity of nucleotide sequence was found between regions of the placental alkaline phosphatase and the Escherichia coli enzyme (Knoll et al., 1988). The aim of the present study is to prepare the purified alkaline phosphatases from species with different evolutionary development, such as Bos taurus, Penaeus monodon and Meretrix lusoria, and use them in investigating the nature and extent of differences from the human placental enzyme. It is hoped that from this comparison, an index could be born which can be applied to obtain a suitable alkaline phosphatase for the application as a reporter in genetic engineering. Recent studies of transcription control elements have been greatly facilitated by linking them to readily assayable reporter genes; expression of the reporter gene is used as an indication of transcription activity. *Author to whom correspondence should be addressed. CBPB 96/4~K
787
Materials All reagents used were of the highest grade available commercially. Reagents for isoelectric focusing were from Pharmacia, Sweden.
Experimental animals Shrimps (Penaeus monodon, 2 kg) and clams (Meretrix lusoria, 2 kg) collected offTaiwain, were held at 18°C for less than 3 days in a recirculating sea-water system. Hepatopancreases were dissected out immediately after death. Human placenta were removed from the body not more than 1 hr after delivery and stored at - 2 0 ° C for up to a week at most before use. Bovine (Bos taurus) milk (1 l) was freshly obtained from the Department of Animal Husbandry at National Taiwan University. Milk was placed on ice immediately after being removed.
Purification of alkaline phosphatase Alkaline phosphatase was purified with essentially the same procedure as described by Meyer et al. (I 982) but with modifications as mentioned previously (Chuang, 1990). The enzyme preparation from human placenta has a spec. act. of 6700 U/mg of protein (Chuang, 1987), the enzyme from bovine milk has a spec. act. of 32,000 U/mg of protein, that from Penaeus monodon has a spec. act. of 22,000 U/mg of protein, and the enzyme from Meretrix lusoria has a spec. act. of 15,000 U/rag of protein.
lsoelectric focusing Isoelectric focusing was carried out essentially as described by Moss and Edwards (1984) using ampholytes with a pH range of 3.0-10.0. For some experiments, restricted ranged ampholytes (pH range 4.0-6.0 and 5.0-8.0) were employed for better resolution ofisoelectric forms. Focusing was performed at a constant voltage (200 V) for 18-21 hr.
788
N I N - N I N CHUANG a n d BEI-CHIA YANG
Assay of alkaline phosphatase Alkaline phosphatase activity was measured at 37°C with the use of 10 mM 4-nitrophenyl phosphate in 0.75 M 2-amino-2-methyl-l-propanol/HC1 buffer, pill0.3. One unit of enzyme activity = 1/zmol substrate hydrolyzed/min. Enzyme activity after isoelectric focusing was determined by incubating the gels at 37°C in 2 mg of fl-naphthylic acid phosphate (Sigma F-7375) per ml, and 1 mg of Fast Blue BB (Sigma N-0250) per ml in 60 mM borate buffer/pH 9.7 for 30 rain to stain the activity bands. Assay of protein Bovine serum albumin was served as the standard in the measurement of proteins. The amount of protein was determined by the Lowry method (1951).
1001
5D
'ID e I
45 °
R E S U L T S AND D I S C U S S I O N
I
150°
L
610o
155 °
I
65°C
Temperature
The key features of a useful reporter gene are that its activity can be easily assayed and its endogenous activity in the appropriate cells is low. Human placental alkaline phosphatase (Henthorn et al., 1988), bacterial fl-galactosidase (An et al., 1982), human growth hormone (Seldon et al., 1986) and firefly luciferase (De Wet et al., 1987) are the best examples of reporter genes. In the application of alkaline phosphatase as a reporter, 4-nitrophenyl phosphate is used as the substrate and the yellow product, nitrophenol, which is easily detected by spectrophotometry, makes alkaline phosphatase prevail over others in cost and non-radioactivity contamination. In view of the other requirements for a good reporter gene, human placental alkaline phosphatase is not quite content in limited expression of endogenous activity. Notwithstanding the fact that human placental alkaline phosphatase can be differentiated from the other two isoenzyme groups, intestinal and hepatic/renal/skeletal, by its resistance to heating at 65°C, at which non-placental phosphatases are rapidly inactivated (Moss, 1982; Seargeant and Stinson, 1979; Neale et al., 1965), the evidence that the widespread presence of human placental alkaline phosphatase in the lung (Goldstein et al., 1980), cervix (Goldstein et al., 1982) and milk (Chuang, 1987), and also in the patients' sera with ulcerative colitis, familial polposis, cirrhosis (Stolbach et al., 1976) and carcinoma (Nathanoson and Fishman, 1971) reinforces the need of searching for a thermostable alkaline phosphatase with more limited expression in mammalian tissues. In the present study, we propose two alkaline phosphatases to be applied and used as a reporter in genetic engineering, one is the enzyme from Meretrix lusoria with its extreme thermostability, and the other is the enzyme from Penaeus monodon with its unique optimal pH at 9.0. When purified alkaline phosphatases from human placenta, bovine milk, Penaeus monodon and Meretrix lusoria are subjected to the comparative analysis of thermostability, no enzyme activity was lost upon preincubation at 50°C for l0 rain (Fig. 1). In particular, the alkaline phosphatase from Meretrix lusoria was found to retain 50% of the original activity after incubation at 65°C for 30 min; whereas 90% of the enzyme activity was inactivated in human placental alkaline phosphatase (Fig. 2). That is, the
Fig. I. Heat inactivation of alkaline phosphatase. The stability of the enzyme was investigated by incubating them at 45-65°C for I0 rain prior to assaying with 4-nitrophenyl phosphate. Alkaline phosphatase purified from human placenta (©), bovine milk (O), Penaeus monodon (ff]) and Meretrix lusoris ( . ) were incubated at indicated temperature for 10rain and the remaining enzyme activity was measured. Activities are expressed as percentages of the initial total activity. alkaline phosphatase from Meretrix lusoria is unique at therrnostability. Alkaline phosphatase shows species speciality at isoelectric points. The enzyme from Penaeus monodon was discovered to have an isoelectric point (pI) of 6.9 _+ 0.1 (Fig. 3C), the enzyme from Meretrix lusoria having a pI of 6.0 _+ 0.1 (Fig. 3D), the bovine milk enzyme having a pI of 5.5 _+ 0.1 (Fig. 3B), and the placental alkaline phosphatase having a more acidic pI, 4.8 +_ 0.1 (Fig. 3A). In the study of optimal pH, the purified alkaline phosphatase from Penaeus
% IO0
50
m ¢g n" i
t
10
20
) 30
Time
Fig. 2. Heat inactivation of alkaline phosphatase from Meretrix lusoria. The stability of the purified clam enzyme ( I ) was investigated by incubating them at 65°C for 10-30 min prior to assaying with 4-nitrophenyl phosphate. For comparison, the human placental alkaline phosphatase was included (C)). Activities are expressed as percentages of the initial total activity.
Alkaline phosphatases A
'
,
n,
I
B
I
'
,
C
I
I
I
i
D
n
I
i
I
,
n
I
I
REFERENCES
An G., Hidaka K. and Siminovitch L. (1982) Expression of bacterial fl-galactosidase in animal cells. Mol. Cell. Biol. 2, 1628-1632, Chuang N.-N. (1987) Alkaline phosphatase in human milk: a new heat-stable enzyme. Clin. Chim. Acta 169,
i
I
I
i
,
i
i
i
I
'm
I
I
I
l
3
4
5
6
7
8
9
10
Fig. 3. Isoelectric points of alkaline phosphatase. Purified alkaline phosphatase from human placenta (A), bovine milk (B), Penaeus monodon (C) and Meretrix lusoria (D) were subjected to isoelectric focusing. For each sample, triplicate gels were measured.
monodon has an optimal pH of the enzyme activity at
9.0, which is different from those of enzymes obtained from human placenta, bovine milk and Meretrix lusoria (Fig. 4); the remaining enzymes, including the alkaline phosphatase from Penaeus japonicus (Chuang, 1990), shared the same optimal pH for enzyme activity at 10.0. Accordingly, the alkaline phosphatase from Penaeus monodon can be differentiated from the other studied enzymes by its pH optimum. Further studies on comparing primary structures of alkaline phosphatases are being undertaken. Acknowledgements--Financial support for this work was
provided by the National Science Council and Academia Sinica, Republic of China.
lOU
50
.-
@ rr
0 3
8
8
7
8
8
10
789
11
pH
Fig. 4. Effect of pH on the alkaline phosphatase activity. Alkaline phosphatase purified from human placenta (C)), bovine milk (O), Penaeus monodon (i"-I) and Meretrix lusoria ( i ) were assayed in the reaction mixture consisting of 0.1% Triton X-100, 1 mM MgCI2 and 100mM buffer of succinate--sodium hydroxide (pH 3-6) or Tris-malate (pH 6-9). Activities are expressed as percentages of the initial activity.
165-174.
Chuang N.-N. (1990) A heat-stable alkaline phosphatase from Penaeus japonicus Bate (Crustacea: Decapoda). Comp. Biochem. Physiol. 95, 165-169. De Wet J. R., Wood K. V., DeLuca M., Helinski D. R. and Subramani S. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725-737. Garanini E., Hua J.-C., Pan Y.-E. and Udenfriend S. (1986) Human liver alkaline phosphatase, purification and partial sequencing: homology with the purified isozyme. Arch. Biochem. Biophys. 245, 331-337. Goldstein D. J., Blasca L. and Harris H. (1980) Placental alkaline phosphatase in non-malignant human cervix. Proc. natn. Acad. Sci. USA 77, 4226-4228. Goldstein D. J., Rogers C. and Harris H. (1982) Evolution of alkaline phosphatases in primates. Proc. natn. Acad. Sci. USA 79, 879-883. Gorman C. M., Moffat L. F. and Howard B. H. (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044-1051. Henthorn P., Zervos P., Raducha M., Harris H. and Kadesch T. (1988) Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression. Proc. hath. Acad. Sci. USA 85, 6342-6346. Hua J.-C., Berger J., Pan Y. E., Hulmes J. D. and Udenfriend S. (1986) Partial sequencing of human adult, human fetal and bovine intestinal alkaline phosphatases: comparison with the human placental and liver enzyme. Proc. natn. Acad. Sci. USA 83, 2368-2372. Knoll B. J., Rothblum K. N. and Longley M. (1988) Nucleotide sequence of the human placental alkaline phosphatase gene. J. biol. Chem. 263, 12,020-12,027. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Meyer L. J., Lafferty M. A., Raducha M. G., Foster C. J., Gogolin K. J. and Harris H. (1982) Production of a monoclonal antibody to human liver alkaline phosphatase. Clin. Chim. Acta 126, 109-117. Moss D. W. (1982) Alkaline phosphatase isoenzymes. Clin. Chem. 28, 2007-2016. Moss D. W. and Edwards R. K. (1984) Improved electrophoretic resolution of bone and liver alkaline phospbatases resulting from partial digestion with neuraminidase. Clin. Chim. Acta 143, 177-182. Nathanson L. and Fishman W. H. (1971) New observations on the Regan isoenzyme of alkaline phosphatase in cancer patients. Cancer 27, 1388-1397. Neale F. C., Clubb J. S., Hotchkis D. and Posen S. (1965) Heat-stability of human placental alkaline phosphatase. J. Clin. Pathol. 18, 359-363. Seargeant L. E. and Stinson R. A. (1979) Affinity elution from a phosphoric acid-Sepharose derivative in the purification of human liver alkaline phosphatase. Nature 281, 152-154. Seldon R. F., Howie K. B., Rowe M. E., Goodman H. M. and Moore D. M. (1986) Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell. Biol. 6, 3173-3179. Stolbach L. L., Inglis N. I., Lin C., Turksoy R., Fishman W. H., Marchant D. and Rule A. (1976) Onco-Developmental Gene Expression (Edited by Fishman W. H. and Sell S.), pp. 433-443. Academic Press, New York.
