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      Specific Immunoassays for Placental Alkaline Phosphatase As a Tumor Marker

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          Abstract

          Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57–71) of hPLAP (ICA-PEP assay); the working range was 0.1–11 U/L and cutoff value was 0.2 U/L EA for nonsmokers. The intra- and interassay coefficients of variation were 3.7%–6.5% (ICA-PLAP assay) and 9.0%–9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes.

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          Epitopes on protein antigens: misconceptions and realities.

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            Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity.

            Human placental alkaline phosphatase (PLAP) is one of three tissue-specific human APs extensively studied because of its ectopic expression in tumors. The crystal structure, determined at 1.8-A resolution, reveals that during evolution, only the overall features of the enzyme have been conserved with respect to Escherichia coli. The surface is deeply mutated with 8% residues in common, and in the active site, only residues strictly necessary to perform the catalysis have been preserved. Additional structural elements aid an understanding of the allosteric property that is specific for the mammalian enzyme (Hoylaerts, M. F., Manes, T., and Millán, J. L. (1997) J. Biol. Chem. 272, 22781-22787). Allostery is probably favored by the quality of the dimer interface, by a long N-terminal alpha-helix from one monomer that embraces the other one, and similarly by the exchange of a residue from one monomer in the active site of the other. In the neighborhood of the catalytic serine, the orientation of Glu-429, a residue unique to PLAP, and the presence of a hydrophobic pocket close to the phosphate product, account for the specific uncompetitive inhibition of PLAP by l-amino acids, consistent with the acquisition of substrate specificity. The location of the active site at the bottom of a large valley flanked by an interfacial crown-shaped domain and a domain containing an extra metal ion on the other side suggest that the substrate of PLAP could be a specific phosphorylated protein.
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              The human alkaline phosphatases: what we know and what we don't know.

              A review of the human alkaline phosphatases dealing specifically with (1) the gene loci, (2) characterization and discrimination of the various enzymes, (3) polymorphism at the enzyme level, (4) cDNA and gene structures, (5) membrane binding, (6) the carbohydrate moieties, (7) hypophosphatasia, (8) alkaline phosphatases in malignancies, (9) function.
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                Author and article information

                Journal
                J Biomed Biotechnol
                JBB
                Journal of Biomedicine and Biotechnology
                Hindawi Publishing Corporation
                1110-7243
                1110-7251
                2006
                8 August 2006
                : 2006
                : 56087
                Affiliations
                1Centro de Genética Molecular e Pesquisa do Câncer em Crianças (CEGEMPAC), Rua Agostinho Leão Júnior, 400 Alto da Glória, Curitiba, PR, CEP 80030-110, Brazil
                2Division of Pediatric Hematology and Oncology, Department of Pediatrics, Federal University of Paraná, Curitiba, PR, CEP 80060-000, Brazil
                3Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 6097, 06560 Valbonne Sophia Antipolis, France
                4Meharry Medical College, Nashville, TN 37208, USA
                5Center for Research and Production of Immunoglobulins (CPPI), Rua Targino da Silva s/n, Piraquara, PR, CEP 83302-160, Brazil
                6St. Jude Children's Research Hospital, Department of Hematology and Oncology and International Outreach Program, 332 North Lauderdale, Memphis, TN 38105, USA
                7Research Institute Pelé Pequeno Príncipe (IPPP), Avenida Silva Jardim, 1632 Água Verda, Curitiba, PR, CEP 80250-200, Brazil
                Author notes
                *Bonald C. Figueiredo: bonald@ 123456ufpr.br
                Article
                10.1155/JBB/2006/56087
                1559920
                17489017
                5fb0234a-1fdf-4a5d-af76-db99f8add2fc
                Copyright © 2006 Sérvio T. Stinghen et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 23 December 2005
                : 1 June 2006
                : 6 June 2006
                Categories
                Research Article

                Molecular medicine
                Molecular medicine

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