Alkaline Phosphatase

Alkaline phosphatases (E.C. 3.1.3.1) belong to a family of orthophosphoric monoester phosphohydrolases that have an alkaline pH optimum. Their genes are very frequently used as a reporter gene. Alkaline phosphatase activity is most commonly detected by the hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), which, when coupled to the reduction of nitro blue tetrazolium (NBT), forms a formazan and an indigo dye that together form a strong black/purple precipitate (1). In addition, a number of fluorogenic substrates are also available (1). 4-Methylumbelliferyl phosphate (MUP) gives a blue fluorescent product upon hydrolysis, and 2-hydroxy-3-naphthoic acid-2-phenylanilide phosphate (HNPP/Fast Red TR) fluoresces with a broad emission peak between 540 and 590 nm and can be observed using either fluorescein or rhodamine filter sets. Molecular Probes Inc. have also developed a proprietary substrate called ELF-97 (Enzyme Linked Fluorescence-97), in which cleavage of the molecule converts it from a soluble phosphate to an insoluble alcohol, with an accompanying shift from weak blue fluorescence to a bright yellow fluorescence (2).

Human placental alkaline phosphatase (hpAP) is most commonly used as a reporter enzyme in nonradioactive detection systems, where the enzyme is linked to other molecular probes (eg, specific antibodies)—for example, for the detection of proteins and nucleic acids by Western blot, Southern blot, and Northern blot analysis, and most commonly in in situ hybridization. Elegant studies have also been performed in which hpAP is fused to soluble extracellular domains of receptor molecules, which are then used as probes to detect the sites of ligand production in vivo and facilitate the subsequent cloning of the ligand genes (3).

Although animals express a number of alkaline phosphatase genes, the human placental isoform has been developed as a reporter gene, because it can be distinguished from other endogenous isoforms through its high thermostability (4). Thus all background endogenous alkaline phosphatase activities, from embryonic, intestinal, and nonspecific genes, can be minimized by preheating tissue preparations up to 80°C for prolonged periods. hpAP also retains its activity following histological processing for wax imbedding and sectioning tissues. In addition, background from endogenous alkaline phosphatases can be further inhibited by the amino acids L-phenylalanine or L-homoarginine.

 hpAP has been used in a wide range of applications, including in vitro transfection studies and transgenic studies in vivo. The sensitivity of hpAP in transient expression assays is equivalent to that of chloramphenicol acetyltransferase (CAT) (see Reporter Genes). A particularly useful variant of hpAP is a cDNA encoding a secreted form of the protein (5, 6) that allows hpAP activity to be assayed by sampling tissue culture medium, giving the benefit of monitoring changes in gene expression with time. In this system, background activities are further eliminated, because the endogenous isoforms are anchored to the cell membranes and do not contribute to the activity in the culture supernatants.

hpAP is also an effective reporter gene to analyze gene expression in situ in tissue preparations. hpAP was first used in retroviral vectors to infect small numbers of cells in developing embryos as a tool to study cell fate and lineage analysis; subsequently, hpAP has been used as a robust reporter gene in transgenic mice (7). Indeed, mice that express high levels of hpAP from a ubiquitously expressed promoter thrive with no adverse effects. hpAP is particularly useful to use in combination with a second reporter gene, such as lacZ (beta-galactosidase), in dual labeling studies, as the common substrates are quite distinct and give different colored products (8).

References

1. D. A. Knecht and R. L. Dimond (1984) Anal. Biochem. 136, 180–184.

2. K. D. Larison et al. (1995) J. Histochem. Cytochem. 43, 77–83. 

3. J. G. Flanagan and P. Leder (1990) Cell 63, 185–194. 

4. P. Henthorn (1988) Proc. Natl. Acad. Sci. 85, 6342–6346. 

5. T. T. Yang, P. Sinai, P. A. Kitts, and S. R. Kain (1997) Biotechniques, 23, 1110–1114.

6. B. R. Cullen and M. H. Malim (1992) Methods Enzymol. 216, 362–368. 

7. S. E. DePrimo, P. J. Stambrook, and J. R. Stringer (1996) Transgenic Res. 5, 459–466.

8. X. Li, W. Wang, and T. Lufkin (1997) Biotechniques, 23, 874–878.