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1.
Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines. 总被引:4,自引:0,他引:4
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The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line. 相似文献
2.
Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta. 相似文献
3.
1. A heat-stable alkaline phosphatase was purified from Penaeus japonicus, with a final specific activity of 21,280 U/mg of protein. 2. In polyacrylamide-gel electrophoresis under non-denaturing conditions, the purified shrimp alkaline phosphatase was found to have an identical molecular size and surface charge as the human placental enzyme. 3. By using SDS-PAGE, the monomers of shrimp alkaline phosphatase were discovered to have a Mr 55,000 but those of human placental enzyme with a Mr 70,000. Deglycosylation decreases the Mr values of the subunits to 33,000 for shrimp alkaline phosphatase. 4. The purified alkaline phosphatase from shrimp was recovered with both the attachment sites for sialic acids and phosphatidylinositol. 5. The shrimp alkaline phosphatase has an isoelectric point (pI) of 7.6 and the human placental enzyme has a pI of 4.8. 相似文献
4.
Human liver alkaline phosphatase, purification and partial sequencing: homology with the placental isozyme 总被引:2,自引:0,他引:2
E Garattini J C Hua Y C Pan S Udenfriend 《Archives of biochemistry and biophysics》1986,245(2):331-337
Human liver alkaline phosphatase (AP) has been purified to homogeneity. The enzyme has a molecular weight of 150,000 in its native state and consists of two identical subunits of Mr 75,000. After treatment with endoglycosidase F the molecular weight is reduced to 50,000 indicating a high degree of glycosylation. The amino-terminal sequence up to 22 residues was found to be Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Lys-Tyr-(Ala)-Arg-Asp-Gln-Ala-Gln-?- Thr-Leu-Lys-Tyr. The amino-terminal portions of human and bovine liver AP are identical. The amino termini of the human liver and human placental AP isozymes have appreciable homology. Conformationally the amino termini are very similar. 相似文献
5.
Three monoclonal antibodies with distinct antigenic specificities were examined by electron microscopy for their binding to three common genetic variants (SS, FS, and FF) of human placental alkaline phosphatase. In the reaction with the monoclonal antibody H5, all three variants of human placental alkaline phosphatase preferentially formed circular immune complexes composed of two antibodies and two enzyme molecules. In separate reactions with the F11 and B2 monoclonal antibodies, the SS variant formed circular complexes and the FS variant formed Y-shaped complexes composed of one antibody and two enzyme molecules, whereas the FF variant scarcely reacted. These results confirm immunochemical data showing that H5 binds to both S and F subunits with similar affinities, whereas F11 and B2 bind the S subunit with markedly higher affinity than they do the F subunit. Furthermore, the formation of circular complexes in the reaction of the mixture of the two antibodies, F11 and B2, with FS molecules suggests that these two antibodies bind to different sites on the S subunit. Therefore, the F and S subunits differ from one another at more than one site. This is the first indication that alleles of human placental alkaline phosphatase may result from more than just single point mutations in the gene encoding them. 相似文献
6.
The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum. 总被引:1,自引:1,他引:0
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E M Bailyes R N Seabrook J Calvin G A Maguire C P Price K Siddle J P Luzio 《The Biochemical journal》1987,244(3):725-733
1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane. 相似文献
7.
Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis. 总被引:9,自引:7,他引:2
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Two secreted alkaline phosphatase proteins were purified from cultures of Bacillus subtilis JH646MS. The two proteins showed slight differences in subunit molecular weight, substrate specificity, and charge characteristics. A total of 62% of the first 22 amino-terminal amino acids were identical. Both sequences showed conservation of structural features identified in Escherichia coli and human alkaline phosphatases. One alkaline phosphatase was a monomer and the other was a dimer. Southern analysis of genomic DNA with degenerative oligomers based on the amino acid sequences suggest that there are two structural genes for alkaline phosphatase in the genome of B. subtilis. 相似文献
8.
Y Yokota 《Journal of biochemistry》1978,83(5):1293-1298
Alkaline phosphatase of cultured rat ascites hepatoma cells has been purified by butanol extraction, DEAE-cellulose column chromatography, gel filtration through Sephadex G-200, concanavalin A-Sepharose affinity chromatography, and polyacrylamide gel electrophoresis. Affinity chromatography confirmed the glycoprotein nature of alkaline phosphatase from cultured rat ascites hepatoma cells. Electrophoresis on polyacrylamide gels of various concentrations indicated a molecular weight of 290,000. The molecular weight of the subunit was estimated to be 72,000 by SDS-polyacrylamide gel electrophoresis. These findings suggest that alkaline phosphatase of cultured rat ascites hepatoma cells is a tetramer with a subunit molecular weight of 72,000. 相似文献
9.
J C Hua E Garattini Y C Pan J D Hulmes M Chang L Brink S Udenfriend 《Archives of biochemistry and biophysics》1985,241(2):380-385
Bovine liver alkaline phosphatase has been purified to homogeneity by procedures that include reverse-phase HPLC. The pure enzyme has an apparent Mr of 160,000 and is composed of what appears to be two identical monomers of Mr 82,000. About 80% of the material yielded the amino-terminal sequence Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln. The minor component was extended at the amino terminus by two residues that have not yet been identified, i.e., ?-?-Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln. 相似文献
10.
Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring 总被引:8,自引:0,他引:8
N Takami S Ogata K Oda Y Misumi Y Ikehara 《The Journal of biological chemistry》1988,263(6):3016-3021
The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase. 相似文献
11.
Rat intestinal alkaline phosphatase is a dimeric enzyme with identical subunits and thus possesses two presumably identical active sites. Binding studies with Pi and l-phenylalanine and pre-steady-state “burst” titrations confirm the existence of two active sites per molecule of enzyme. The sites appear to be nonequivalent with respect to Pi binding, both at low pH, where an enzyme (E)-Pi covalent complex is formed, and at high Pi, where an E-Pi noncovalent complex predominates. The binding affinity of the first site is 100-fold greater than that of the second, i.e., there is negative cooperativity. The Ki value for competitive inhibition of substrate hydrolysis by Pi corresponds to the higher affinity site. The negative cooperativity appears not to be an artifact resulting from contaminating Pi in the purified enzyme preparation. l-Phenylalanine does not bind to the enzyme unless Pi is present, as expected from the previously proposed mechanism of uncompetitive inhibition by the amino acid. No negative cooperativity is seen in l-phenylalanine binding, but the number of moles of amino acid bound at saturation depends on the degree of saturation by Pi The enzyme is also inhibited uncompetitively by NADH, which can compete with l-phenylalanine for the same site on alkaline phosphatase. 相似文献
12.
Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity. 相似文献
13.
5-Bromo-2'-deoxyuridine (BrdUrd) stimulated the biosynthesis and hence increased the activity of placental alkaline phosphatase in choriocarcinoma cells. While BrdUrd had no effect on the rate of degradation or processing of placental alkaline phosphatase, it increased the rate of phosphatase synthesis. The stimulation of enzyme activity could be completely accounted for by the increase in alkaline phosphatase protein. Both control and BrdUrd-induced cells contained polypeptides of 61,500 and 64,500 Da, identified as the precursor and fully processed forms of placental alkaline phosphatase monomer. The half-life of this enzyme monomer in both control and BrdUrd-treated cells was estimated to be 36 h. BrdUrd induced a specific increase in the placental alkaline phosphatase mRNA leading to the observed enhancement of biosynthesis. The continued rise in alkaline phosphatase biosynthesis in BrdUrd-induced cells following BrdUrd removal indicated that this analog acted by incorporation into DNA. 相似文献
14.
J L Millán 《The Journal of biological chemistry》1986,261(7):3112-3115
The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced. 相似文献
15.
E Ezra R Blacher S Udenfriend 《Biochemical and biophysical research communications》1983,116(3):1076-1083
Two forms of human placental alkaline phosphatase have been purified to homogeneity utilizing high performance liquid chromatography. Both have the same amino acid composition but they differ in their carbohydrate substituents. Sequence data indicate that the two forms are identical for the first forty two residues from the amino terminus are presented. 相似文献
16.
C Pratt 《Journal of bacteriology》1980,143(3):1265-1274
17.
The effect of procaine hydrochloride, an anesthetic known to alter membrane structure, on the induced formation of alkaline phosphatase, a periplasmic enzyme, in Escherichia coli was investigated. Procaine hydrochloride specifically arrested the appearance of active alkaline phosphatase while permitting the induction of another enzyme, beta-galactosidase, which is internally localized. Evidence has been obtained to show that procaine hydrochloride does not arrest synthesis of inactive monomer subunits of the enzyme, indicating that the drug interferes in the conversion of monomer subunits to an active dimer enzyme. 相似文献
18.
19.
The activities of hybrid dimers of alkaline phosphatase containing two chemically modified subunits have been investigated. One hybrid species was prepared by dissociation and reconstitution of a mixture of two variants produced by chemical modification of the native enzyme with succinic anhydride and tetranitromethane, respectively. The succinyl-nitrotyrosyl hybrid was separated from the other members of the hybrid set by DEAE-Sephadex chromatography and then converted to a succinyl-aminotyrosyl hybrid by reduction of the modified tyrosine residues with sodium dithionite. A comparison of the activities of these two hybrids with the activities of the succinyl, nitrotyrosyl and aminotyrosyl derivatives has shown that either the subunits of alkaline phosphatase function independently or if the subunits turnover alternately in a reciprocating mechanism, then the intrinsic activity of each subunit must be strongly dependent on its partner subunit. 相似文献
20.
Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions. 总被引:18,自引:8,他引:10
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Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein. 相似文献