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Secreted Alkaline Phosphatase (SEAP)

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Secreted alkaline phosphatase (SEAP)

SEAP is a hydrolyase which derives from the human placental alkaline phosphatase and is secreted into the medium by the cell.

Usage and Biology

Phosphatases

Phosphatases cleavage phosphordiester bonds and release anorganic phosphate groups. Since water is required for this type of reaction, phospatases belong to hydrolases and are divied in two large subfamilies depending on their needed pH for the greatest activity. This group of enzymes is widespread in nature and play an important role in signalling pathways, cell proliferation and osteostasis.

Acid phospatases

Despite human acid phosphatases share their maximum turnover in a low pH range, they differ in sequence length, mass, tissue or chromosom origin. The lysosomal acid phosphatase, prostatic acid phosphatase, erythrocytic acid phosphatase, macrophage acid phosphatase, and osteoclastic acid phosphatase are examples of clinical relevance. Human acid phosphatases in general are expressed at low concentrations but changes are observed in several diseaes like prostate cancer or Gaucher's diseas [1].

alkaline phospatases

In general human alkaline phosphatases are built up of four segments: tissue non-specific alkaline phosphatases, germ cell alkaline phosphatases, intestinal alkaline phosphatases and placental alkaline phosphatases [2]. They need a high pH to catalyze reactions. Structural Studies in E. COLI helped to solve the reaction mechanism and to prove its conservation in mammalian alkaline phospatases. The active centrum contains two zinc and one magnesium ion [3]. Step one is the activation of a conserved serine by a zinc atom, followed by the formation of a covalent phosphoserine intermediate and release of phosphate or its transfer to a phosphate acceptor [4]. Unlike procaryotic versions, mammallian alkaline phosphatases are uncompetitively inhibited by some L-amino acids like leucine, phenylalanine, tryptophane or homoarginie [5].

Secreted human placental alkaline phosphatase

The human placental alkaline phosphatase is one of three tissue specific alkaline phosphatases. It shows high sequence similarity with all remaining human alkaline phophatases [6]. It is thought that this protein plays a role in cell divison in both normal and transferred cells [7]. Due to the phosphatidylinositol-glycan-anchor, the human placental alkaline phosphatase is a membrane-bound protein. The hydrophobic moiety is added postranslationally after removing 29 amino acids from the C-Terminus. A single substitute in the previous hydrophobic domain by a charged residue prevents addition of the phosphatidylinositol-glycan-anchor and leads to the secreated human placental alkaline phosphatase (SEAP) [9].

SEAP is an ideal reporter protein and a mighty tool for eucaryotic promotor studies [9] and part of many commercial kits. The big advantage is its self-excretion into the medium. It can be easily measured with para-Nitrophenylphosphate as a substrate. Cleaveging the phosphate group leads to the formation of para-nitrophenol, which can be detected with light of 405 nm wave length. Regarding to the reaction kinetics, the measured activity in the medium is linear proportional to changes of inracellular protein and mRNA levels. The whole procedure takes just about 60 minutes and is very precisely. It 's suitable to detect substances in a concentrastion range of 1 ng - 1 µg/ml. The accuratest SEAP assays to date use chemiluminescent substrates such as 1,2-dioxetane CSPD and are also fast and easy to perform.

References

[1] Acid phosphatases, H Bull, P G Murray, D Thomas, A M Fraser, P N Nelson, Mol Pathol. 2002 April; 55(2): 65–72.
[2] Structural evidence of functional divergence in human alkaline phosphatases, Marie-Hélène Le Du, Jose Luis Millan, J Biol Chem. 2002 December 20; 277(51): 49808–49814.
[3] Structural studies of human placental alkaline phosphatase in complex with functional ligands., Paola Llinas, Enrico A. Stura, André Ménez, Zoltan Kiss, Torgny Stigbrand, José Luis Millán, Marie Hélène Le Du, J Mol Biol. 2005 July 15; 350(3): 441–451.
[4] 3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity., L. Chen, D. Neidhart, W. M. Kohlbrenner, W. Mandecki, S. Bell, J. Sowadski, C. Abad-Zapatero, Protein Eng. 1992 October; 5(7): 605–610.
[5] Organ-specific inhibition of human alkaline phosphatase isoenzymes of liver, bone, intestine and placenta; L-phenylalanine, L-tryptophan and L homoarginine., W. H. Fishman, H. G. Sie, Enzymologia. 1971 September 30; 41(3): 141–167. [6] Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity., M. H. Le Du, T. Stigbrand, M. J. Taussig, A. Menez, E. A. Stura, J Biol Chem. 2001 March 23; 276(12): 9158–9165.
[7] Profile of placental alkaline phosphatase expression in human malignancies: effect of tumour cell activation on alkaline phosphatase expression., A. A. Dabare, A. M. Nouri, H. Cannell, T. Moss, A. K. Nigam, R. T. Oliver, Urol Int. 1999; 63(3): 168–174.
[8] Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol- glycan-anchored protein to a secreted protein., J Cell Biol. 1992 February 1; 116(3): 799–807.
[9] Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells., J. Berger, J. Hauber, R. Hauber, R. Geiger, B. R. Cullen, Gene. 1988 June 15; 66(1): 1–10.
Sequence and Features