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PubMed1985

Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA.

A human term (third trimester) placental alkaline phosphatase (PLAP; EC 3.1.3.1) cDNA was isolated from a human placental lambda gt11 cDNA library. The expression library was screened by using rabbit antibodies against PLAP and oligonucleotide probes. DNA sequence analysis of a positive clone with an insert of 2.7 kilobase pairs allowed us to predict the complete amino acid sequence of PLAP (530 residues), which coincided with the reported 42 N-terminal amino acid sequence of PLAP except at position 3. Contrary to the previous supposition that there was no amino acid sequence homology between PLAP and Escherichia coli alkaline phosphatase (471 residues), we found 30% overall homology, with regions of strong homology including the putative active site and the metal-binding sites. The 44-residue C-terminal extension of PLAP has a stretch of 17 hydrophobic amino acids, which presumably anchors the protein to the plasma membrane, a change perhaps necessary for the transition from a bacterial periplasmic enzyme to a mammalian membrane-associated enzyme. We have also localized PLAP-related DNA sequences mainly on chromosome 2 and to a lesser degree on chromosome 17. It seems likely therefore that the PLAP gene resides on chromosome 2 and other member(s) of the alkaline phosphatase family may exist (on this chromosome and) on chromosome 17.

Alkaline Phosphatase↗

PubMed1974

The action of secretin and pancreozymin on small-intestinal alkaline phosphatase.

The hormones secretin and cholecystokinin-pancreozymin (CCK) have been shown to release brush-border alkaline phosphatase into the small-intestinal lumen in the human subject. In contrast to the disaccharidases, large amounts of alkaline phosphatase are present in human duodenal juice. The range has been established in normal subjects. Following the intravenous administration of both secretin and CCK-pancreozymin there is a large rise in the output of alkaline phosphatase in human duodenal juice. These rises are also present in patients with complete obstruction of the common bile and pancreatic ducts, and since the pancreatic juice of man contains negligible amounts of alkaline phosphatase, it is clear that both hormones must cause small-intestinal alkaline phosphatase to be released into duodenal juice. The isoenzyme characteristics of bile, small-intestinal, and pancreatic alkaline phosphatase have been established, and isoenzyme studies used to confirm this new action of secretin and CCK-pancreozymin.

Alkaline Phosphatase↗

PubMed1978

Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma.

Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.

Alkaline Phosphatase↗

PubMed1986

Regulation of alkaline phosphatase activity in rat hepatoma cells. Effects of serum proteins, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide.

Alkaline phosphatase activity in rat hepatoma cells (R-Y121B) cultured in a monolayer at 0.5% serum was enhanced by serum, bovine serum albumin, casein and gamma-globulin, but ovalbumin, polyvinylpyrrolidone, dexamethasone, insulin and dibutyrylcyclic AMP showed little effect on alkaline phosphatase activity. In addition, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide also increased the enzyme activity, although the incorporation of [14C]leucine into cellular proteins was almost completely inhibited in the presence of these cytotoxic substances. When R-Y121B cell homogenates were incubated at 37 degrees C, alkaline phosphatase activity increased in a pH-dependent manner: the maximal increase was observed at pH 7.1. The magnitudes of the increase differed among cell homogenates and a 4- to 10-fold increase was observed. Alkaline phosphatase in R-Y121B cells was apparently heat-stable, but that in the cells obtained from various treatments was heat labile and the latter activity decreased to less than 50% of the initial activity after 15 min of incubation at 56 degrees C. Alkaline phosphatase in the control and also in the treated cells was more sensitive to L-homoarginine than L-phenylalanine. The Lineweaver-Burk plot showed that the increases in the enzyme activity were accompanied by changes not only in V but also in Km for alkaline phosphatase reaction. Finally, it has been suggested that the increases in alkaline phosphatase activity under various conditions are due to the conversion of the molecule with a low enzyme activity to the molecule with a high enzyme activity in R-Y121B cells.

Alkaline Phosphatase↗

PubMed1965

ACID INACTIVATION OF AND INCORPORATION OF PHOSPHATE INTO ALKALINE PHOSPHATASE FROM ESCHERICHIA COLI.

1. Alkaline phosphatase of Escherichia coli undergoes below pH 6.0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6.0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3.5 is rapidly recovered when the enzyme is taken back to pH 8.0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn(2+), although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme.

Alkaline Phosphatase↗

PubMed1981

The cyclic AMP-mediated induction of alkaline phosphatase in mouse L-cells.

A cell surface-localized glycoprotein that exhibits alkaline phosphatase activity was induced by treatment of mouse L-cell cultures with dibutyryl cyclic AMP. Treatment of cells with 1.5 mM dibutyryl cyclic AMP for a period of 7 days resulted in a approximately 2000-fold increase in the specific activity of the enzyme. Enzyme induction was dependent upon de novo RNA and protein biosynthesis since this induction was completely suppressed when actinomycin D (0.5 microgram/ml) or cycloheximide (5 microgram/ml) was administered with dibutyryl cyclic AMP. Further, the overall rates of incorporation of either [3H]glucosamine or [3H]leucine into macromolecules were identical in the presence or absence of dibutyryl cyclic AMP. Alkaline phosphatase was immunotitrated in 0.5% Triton X-100-solubilized cell extracts with antisera prepared against purified native enzyme and the results indicated that dibutyryl cyclic AMP stimulated that de novo synthesis of the enzyme. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis of specifically immunoprecipitated protein from cells incubated with either [35S]methionine or [6-3H]glucosamine demonstrated that dibutyryl cyclic AMP induced a 76,000-dalton glycoprotein that was characterized as alkaline phosphatase by its identity with native alkaline phosphatase that had been labeled with 32P in its active site. Electrophoretic analysis of specifically immunoprecipitated translation products from an in vitro protein-synthesizing system supplemented with L-cell RNA isolated from uninduced and cAMP-induced cells indicated that dibutyryl cyclic AMP induced the production of alkaline phosphatase-specific mRNA. These results suggest that dibutyryl cyclic AMP directly or indirectly influences the regulation of transcription of the alkaline phosphatase gene in L-cells.

Alkaline Phosphatase↗

PubMed1989

Chemical modification and composition of tetrameric isozyme K of alkaline phosphatase from harp seal intestinal mucosa.

1. The carbohydrate content of isozyme K of alkaline phosphatase (EC 3.1.3.1) from harp seal intestinal mucosa was examined. The presence of N-acetylglucosamine, N-acetylgalactosamine and considerable amounts of mannose residues was shown. 2. The amino acid content of seal alkaline phosphatase was determined. A high extent of homology (85%) between bovine and seal alkaline phosphatases was demonstrated. 3. By chemical modification lysine, dicarboxylic acids, arginine and tyrosine residues of tetrameric seal alkaline phosphatase are located near or at the active site. By contrast, the modification of either thiol or imidazole groups resulted in no alterations of the enzyme activity. 4. It has been demonstrated that inorganic phosphate is an inhibitor of alkaline phosphatase and entirely prevents the enzyme inactivation with succinic anhydride.

Alkaline Phosphatase↗

PubMed2002

[Effect of different environmental factors on the activities of digestive enzymes and alkaline phosphatase of Macrobrochium nipponense].

The activities of digestive enzymes and alkaline phosphatase from the hepatopancreas of Macrobrochium nipponense were determined under different environmental factors (calcium concentrations 20 mg.L-1, 35 mg.L-1, 60 mg.L-1, 80 mg.L-1, 150 mg.L-1; salinity 7@1000, 14@1000, pH 7.6, 8.8, 9.8). The results showed that higher Ca2+ concentration could enhance the pepsin activity, but inhibit the trysin-like activity in hepatopancreas of M. nipponense. The activities of pepsin, trysin-like, alkaline phosphatase in hepatopancreas of M. nipponense were higher under salinity of 14@1000 than under salinity of 7@1000 and 20@1000. It showed that the activities of digestive enzymes and alkaline phosphatase of shrimp increased gradually with increasing pH value from 7.6 to 9.8.

Acid Phosphatase↗

PubMed1969