Phosphatase, Alkaline, Human, Bone (BAP)

Nomenclature: Orthophosphoric-monoester phosphorylase (alkaline optimum); EC 3.1.3.1\n\nAlkaline phosphatases (APs) are highly ubiquitous enzymes, present in all species from bacteria to man. In humans, APs are encoded by a multi-gene family composed of four loci; i.e., tissue-nonspecific AP, also called bone/liver/kidney AP, (Weiss et al, J. Biol. Chem., 264,12002-12010, 1988), intestinal (Henthorn et. al., J.Biol. Chem., 263, 12020-12027, 1988). The sequence and complexity of the AP genes from other vertebrates and lower species are now being elucidated. The biological function of AP isozymes is still unknown. In vitro, the enzymes behave as phosphohydrolases at a characteristically alkaline pH optimum but they can also function as phosphotransferases at neutral pH. The use of phosphate acceptor molecules (diethanolamine, tris, 2-amino-2-methyl-1-propanol) in the buffered substrate solutions increases the reaction rates and, thus, the sensitivity of assays based on AP determinations. The following scheme summarizes the general reaction catalyzed by APs: \n\nThe active site of APs contains two Zn atoms and one Mg atom which are essential for catalysis (Kim & Wyckoff, J. Mol. Biol., 218, 449-464, 1991). The isozymes are homodimeric glycoproteins ranging between 120-180 kD in molecular weight depending on the source and glycosylation status. APs are bound to the exterior surface of the cytoplasmic membrane by a phosphatldyllnositol glycan (PGI) moiety (Low & Saltiel, Science, 239, 268-275, 1988) and the presence or absence of intact and partially degraded anchor confer different degrees of hydrophobicity to serum AP isozymes and to purified AP preparations. This PGI moiety can be detached from the protein by treatment in vitro with endogenous membrane-embedded phospholipase-C as well as with phospholipase-D circulating in plasma (Malik & Low, Biochem. J., 240, 519-527, 1986). Isozymes are generally offered in their hydrophilic form (phosphatidyllnositol anchor removed) which eliminates non-specific binding in enzyme-linked immunoassay. \n\nAPs are uniquely inhibited stereo-specifically by certain L-amino acids and peptides (Fishman & Sie, Enzymolgia, 41: 141-167, 1971; Hoylaerts et al., Biochem J., 286, 23-30, 1992), like L-phenylalanine, L-leucine, L-homoarginine, and these inhibitors are often used in the discrimination and specific quantitation of AP isozymes in the clinical laboratory (Green et al., Enzymologia, 41, 9-26, 1971; Makayama et al., Clin. Chim Acta, 30, 546-548, 1970). Human serum normally contains a mixture of organ-specific AP isozymes mainly derived from liver, bone, intestine and placenta (Fishman, Am. J. Med., 56, 617-650, 1974). The determination of serum levels of AP and the tissular origin of increased amounts has clinical significance in the follow-up of patients with a variety of afflictions. For example, in Paget's, disease and elevated skeletal AP in serum reflects an increase in bone formation rate (Farley et al., J. Biol. Chem., 225, 4680, 1980). Similarly, serum levels of liver-derived AP are indicators of colestasis or tumor masses (Winkelman et al., Clin Chim. Acta, 219, 697-699, 1968; Wahren et al., Int. J. Cancer, 24, 749-753, 1979; Vergote et al., Obstet. Gynecol., 69, 228-232, 1987). \n\nPurified APs have numerous applications in biotechnology, including their use to dephosphaorylate proteins and DNA samples and their use as detection and signal amplification enzymes. Purified AP can be coupled to biological molecules, i.e., antibodies or other ligand proteins, to be used in sensitive enzymes-linked assays.\n\nProtein: (same/more than) 90%\n\nUnit Definition:\nThe amount of enzyme which liberates one micromole of p-nitrophenol from p-nitrophenyl-phosphate per minute at 37 degrees C, pH 10.15 in Tris buffer.\n\nStorage and Stability:\nLyophilized powder may be stored at -20 degrees C. Stable for 12 months at -20 degrees C. Reconstitute by adding sterile ddH2O or PBS. Reconstituted product is stable for 6 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.\nActivity: ~1-10U/mg solid

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SPECIFICATIONS

Catalog Number

P4071-13

Size

1mg

Applications

ELISA

Form

Supplied as a lyophilized powder. Reconstitute with sterile dH2O or PBS.

References

1. Weiss et al,.J. Biol. Chem., 264,12002-12010, 1988. 2. Henthorn et. al., J.Biol. Chem., 263, 12020-12027, 1988. 3. Kim & Wyckoff, J. Mol. Biol., 218, 449-464, 1991. 4. Low & Saltiel, Science, 239, 268-275, 1988. 5. Malik & Low, Biochem. J., 240, 519-527, 1986. 6. Fishman & Sie, Enzymolgia, 41: 141-167, 1971. 7. Hoylaerts et al., Biochem J., 286, 23-30, 1992. 8. Green et al., Enzymologia, 41, 9-26, 1971. 9. Makayama et al., Clin. Chim Acta, 30, 546-548, 1970. 10. Fishman, Am. J. Med., 56, 617-650, 1974.11. Farley et al., J. Biol. Chem., 225, 4680, 1980. 12. Winkelman et al., Clin Chim. Acta, 219, 697-699, 1968. 13. Wahren et al., Int. J. Cancer, 24, 749-753, 1979. 14. Vergote et al., Obstet. Gynecol., 69, 228-232, 1987.