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Alkaline phosphatase

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Alkaline phosphatase
Ribbon diagram (rainbow-color, N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase.[1]
Identifiers
EC no.3.1.3.1
CAS no.9001-78-9
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Search
PMCarticles
PubMedarticles
NCBIproteins
Alkaline phosphatase
Structure of alkaline phosphatase.[1]
Identifiers
SymbolAlk_phosphatase
PfamPF00245
InterProIPR001952
SMARTSM00098
PROSITEPDOC00113
SCOP21alk / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1aja​, 1ajb​, 1ajc​, 1ajd​, 1alh​, 1ali​, 1alj​, 1alk​, ,1anj 1ani ,1anj​, 1b8j​, 1ed8​, 1ed9​, 1elx​, 1ely​, 1elz​, ,1ew8 1ew2 ,1ew8​, 1ew9​, 1hjk​, 1hqa​, 1k7h​, 1kh4​, 1kh5​, ,1kh9 1kh7 ,1kh9​, 1khj​, 1khk​, 1khl​, 1khn​, 1shn​, 1shq​, ,1urb 1ura ,1urb​, 1y6v​, 1y7a​, 1zeb​, 1zed​, 1zef​, 2anh​, ,2ga3 2g9y ,2ga3​, 2glq​

Alkaline phosphatase (ALP, ALKP, ALPase, Alk Phos) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase.[2]

Bacterial

Alkaline phosphatase is a ubiquitous enzyme that is a dimer that contains zinc. In Gram-negative bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell membrane. Since this space is much more subject to environmental variation than the actual interior of the cell, bacterial alkaline phosphatase is resistant to inactivation, denaturation, and degradation, and contains a higher rate of activity. Although the purpose of the enzyme is not fully resolved, the simple hypothesis is that it serves to cleave phosphate groups from phosphorylated compounds facilitating transport across membranes and providing the cell with a source of inorganic phosphate at times of phosphate starvation.The main purpose of dephosphorylation by Alkaline Phosphatase is to increase the rate of diffusion of the molecules into the cells and inhibit them from diffusing out.[3] However, other possibilities exist. For instance, the presence of phosphate groups usually prevents organic molecules from passing through the membrane; therefore, dephosphorylating them may be important for bacterial uptake of organic compounds.[4] Some complexities of bacterial regulation and metabolism suggest that other, more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable to shut off alkaline phosphatase production.[5]

The optimal pH for the activity of the E. coli enzyme is 8.0[6] while the bovine enzyme optimum pH is slightly higher at 8.5.[7]

Use in research

By changing the amino acids of the wild-type alkaline phosphatase enzyme produced by Escherichia coli, a mutant alkaline phosphatase is created which not only has a 36-fold increase in enzyme activity, but also retains thermal stability.[8] Typical uses in the lab for alkaline phosphatases include removing phosphate monoesters to prevent self-ligation, which is undesirable during plasmid DNA cloning.[9]

Common alkaline phosphatases used in research include:

  • Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis). This phosphatase is easily inactivated by heat, a useful feature in some applications.
  • Calf-intestinal alkaline phosphatase (CIP)
  • Placental alkaline phosphatase (PLAP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the domain that targets for GPI membrane anchoring) - the secreted alkaline phosphatase (SEAP). It presents certain characteristics like heat stability, substrate specificity, and resistance to chemical inactivation.[10]
  • Human-intestinal alkaline phosphatase. The human body has multiple types of alkaline phosphatase present, which are determined by a minimum of three gene loci. Each one of these three loci controls a different kind of alkaline phosphatase isozyme. However,  the development of this enzyme can be strictly regulated by other factors  such as thermostability, electrophoresis, inhibition, or immunology.[11]

Human-intestinal ALPase shows around 80% homology with bovine intestinal ALPase, which holds true their shared evolutionary origins. That same bovine enzyme has more than 70% homology with human placental enzyme. However, the human intestinal enzyme and the placental enzyme only share 20% homology despite their structural similarities.[12]

Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end), thereby keeping DNA molecules linear until the next step of the process for which they are being prepared; also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.

Another important use of alkaline phosphatase is as a label for enzyme immunoassays.

Undifferentiated pluripotent stem cells have elevated levels of alkaline phosphatase on their cell membrane, therefore alkaline phosphatase staining is used to detect these cells and to test pluripotency (i.e., embryonic stem cells or embryonal carcinoma cells).[13]

Dairy industry

Main article: Pasteurization § Verification

Alkaline phosphatase is commonly used in the dairy industry as an indicator of successful pasteurization. This is because the most heat stable bacterium found in milk, Mycobacterium paratuberculosis, is destroyed by temperatures lower than those required to denature ALP. Therefore ALP presence is ideal for indicating successful pasteurization.[14][15]

Pasteurization verification is typically performed by measuring the fluorescence of a solution which becomes fluorescent when exposed to active ALP. Fluorimetry assays are required by milk producers in the UK to prove alkaline phosphatase has been denatured,[16] as p-Nitrophenylphosphate tests are not considered accurate enough to meet health standards.

Alternatively the colour change of a para-Nitrophenylphosphate substrate in a buffered solution (Aschaffenburg Mullen Test) can be used.[17] Raw milk would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised milk should show no change. There are exceptions to this, as in the case of heat-stable alkaline phosphatases produced by some bacteria, but these bacteria should not be present in milk.

Inhibitors

All mammalian alkaline phosphatase isoenzymes except placental (PALP and SEAP) are inhibited by homoarginine, and, in similar manner, all except the intestinal and placental ones are reversibly inhibited by levamisole.N-Ethylaminoethanol is a widely used buffer most specfically for AP enzymes from the intestine and placenta, due to its nature of uncompetitive activation.[18] Heating for ~2 hours at 65 °C inactivates most isoenzymes except placental isoforms (PALP and SEAP).[19] Phosphate is another inhibitor which competitively inhibits alkaline phosphatase.[20]

Human

Physiology

In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in the liver, bile duct, kidney, bone, intestinal mucosa and placenta. In the serum, two types of alkaline phosphatase isozymes predominate: skeletal and liver. During childhood the majority of alkaline phosphatase are of skeletal origin.[21] Humans and most other mammals contain the following alkaline phosphatase isozymes:

  • ALPI – intestinal (molecular weight of 150 kDa)
  • ALPL – tissue-nonspecific (liver/bone/kidney)
  • ALPP – placental (Regan isozyme)

Diagnostic use

Normal ALP levels in adults are approximately 20 to 140 IU/L,[22] but levels are significantly higher in children and pregnant women. Blood tests should always be interpreted using the reference range from the laboratory that performed the test. High ALP levels can occur if the bile ducts are obstructed.[23] Also, ALP increases if there is active bone formation occurring, as ALP is a byproduct of osteoblast activity (such as the case in Paget's disease of bone). Levels are also elevated in people with untreated coeliac disease.[24] Lowered levels of ALP are less common than elevated levels. The source of elevated ALP levels can be deduced by obtaining serum levels of gamma glutamyltransferase (GGT). Concomitant increases of ALP with GGT should raise the suspicion of hepatobiliary disease.[25]

Some diseases do not affect the levels of alkaline phosphatase, for example, hepatitis C. A high level of this enzyme does not reflect any damage in the liver, even though high alkaline phosphatase levels may result from a blockage of flow in the biliary tract or an increase in the pressure of the liver.[26]

Elevated levels

Main article: Elevated alkaline phosphatase

If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using electrophoresis can confirm the source of the ALP. Heat stability also distinguishes bone and liver isoenzymes[citation needed] ("bone burns, liver lasts"). Serum alkaline phosphatase is acquired through several sources: liver, bone, kidney, intestine, and placenta (for women). Skeletal alkaline phosphatase (which is localized in osteoblasts and extracellular layers of newly synthesized matrix) is released into circulation by a yet unclear mechanism.[27] Placental alkaline phosphatase is elevated in seminomas[28] and active forms of rickets, as well as in the following diseases and conditions:[29]

Lowered levels

The following conditions or diseases may lead to reduced levels of alkaline phosphatase:

In addition, the following drugs have been demonstrated to reduce alkaline phosphatase:

  • Oral contraceptives[31]

Leukocyte alkaline phosphatase

Leukocyte alkaline phosphatase (LAP) is found within mature white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions.

See also

References

  1. ^ a b PDB: 1ALK​: Kim EE, Wyckoff HW (March 1991). "Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis". J. Mol. Biol. 218 (2): 449–64. doi:10.1016/0022-2836(91)90724-K. PMID 2010919.
  2. ^ Tamás L, Huttová J, Mistrk I, Kogan G (2002). "Effect of Carboxymethyl Chitin-Glucan on the Activity of Some Hydrolytic Enzymes in Maize Plants" (PDF). Chem. Pap. 56 (5): 326–329.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  3. ^ Horiuchi T, Horiuchi S, Mizuno D (May 1959). "A possible negative feedback phenomenon controlling formation of alkaline phosphomonoesterase in Escherichia coli". Nature. 183 (4674): 1529–30. doi:10.1038/1831529b0. PMID 13666805.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  4. ^ Ammerman JW, Azam F (March 1985). "Bacterial 5-nucleotidase in aquatic ecosystems: a novel mechanism of phosphorus regeneration". Science. 227 (4692): 1338–40. doi:10.1126/science.227.4692.1338. PMID 17793769.
  5. ^ Wanner BL, Latterell P (October 1980). "Mutants affected in alkaline phosphatase, expression: evidence for multiple positive regulators of the phosphate regulon in Escherichia coli". Genetics. 96 (2): 353–66. PMC 1214304. PMID 7021308.
  6. ^ Garen A, Levinthal C (March 1960). "A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase". Biochim. Biophys. Acta. 38: 470–83. doi:10.1016/0006-3002(60)91282-8. PMID 13826559.
  7. ^ Harada M, Udagawa N, Fukasawa K, Hiraoka BY, Mogi M (February 1986). "Inorganic pyrophosphatase activity of purified bovine pulp alkaline phosphatase at physiological pH". J. Dent. Res. 65 (2): 125–7. doi:10.1177/00220345860650020601. PMID 3003174.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ^ W, MANDECKI; J, TOMAZICALL; A, SHALLCROSS; J, TOMAZIC-ALLEN. "Mutant Escherichia coli alkaline phosphatase enzymes - having amino acid changes to increase specific activity while retaining thermal stability". Retrieved 1 May 2016. {{cite journal}}: Cite journal requires |journal= (help)
  9. ^ Maxam AM, Gilbert W (1980). "Sequencing end-labeled DNA with base-specific chemical cleavages". Meth. Enzymol. Methods in Enzymology. 65 (1): 499–560. doi:10.1016/S0076-6879(80)65059-9. ISBN 978-0-12-181965-1. PMID 6246368.
  10. ^ Birkett, Donald J.; Done, James; Neale, Francis C.; Posen, Solomon (1966-01-01). "Serum Alkaline Phosphatase In Pregnancy: An Immunological Study". The British Medical Journal. 1 (5497): 1210–1212.
  11. ^ Benham, Frances J.; Harris, Harry (1979-01-01). "Human Cell Lines Expressing Intestinal Alkaline Phosphatase". Proceedings of the National Academy of Sciences of the United States of America. 76 (8): 4016–4019.
  12. ^ Hua, Jia-Cheng; Berger, Joel; Pan, Yu-Ching E.; Hulmes, Jeffrey D.; Udenfriend, Sidney (1986-01-01). "Partial Sequencing of Human Adult, Human Fetal, and Bovine Intestinal Alkaline Phosphatases: Comparison with the Human Placental and Liver Isozymes". Proceedings of the National Academy of Sciences of the United States of America. 83 (8): 2368–2372.
  13. ^ "Appendix E: Stem Cell Markers". Stem Cell Information. National Institutes of Health, U.S. Department of Health and Human Services. Retrieved 2013-09-24.
  14. ^ Kay, H. (1935). "Some Results of the Application of a Simple Test for Efficiency of Pasteurisation". The Lancet. 225 (5835): 1516–1518. doi:10.1016/S0140-6736(01)12532-8.
  15. ^ Hoy, W. A.; Neave, F. K. (1937). "The Phosphatase Test for Efficient Pasteurisation". The Lancet. 230 (5949): 595–598. doi:10.1016/S0140-6736(00)83378-4.
  16. ^ BS EN ISO 11816-1:2013
  17. ^ Aschaffenburg R, Mullen JEC (1949). "A rapid and simple phosphatase test for milk". Journal of Dairy Research. 16 (1): 58–67. doi:10.1017/S0022029900005288. {{cite journal}}: Cite has empty unknown parameter: |month= (help)
  18. ^ Belle, H Van. "Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans". Clinical Chemistry 1976;. v. 22: p.972–6. {{cite journal}}: |page= has extra text (help)CS1 maint: extra punctuation (link)
  19. ^ Alkaline Phosphatase Why It Is Done from Everday Health.com. Retrieved October 15, 2012.
  20. ^ Iqbal, J (2011) “An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases” J Anal. Biochem. 414, 226-231
  21. ^ I, Reiss; D, Inderrieden; K, Kruse (Sep 1996). "Measurement of skeletal specific alkaline phosphatase in disorders of calcium metabolism in childhood". MONATSSCHRIFT KINDERHEILKUNDE. 144 (9): 885–890. doi:10.1007/s001120050054. Retrieved 1 May 2016.
  22. ^ "MedlinePlus Medical Encyclopedia: ALP isoenzyme test".
  23. ^ ALP: The Test
  24. ^ Preussner, Harold T, HT (March 1998). "Detecting coeliac disease in your patients". American Family Physician. 57 (5): 1023–1034. PMID 9518950.
  25. ^ Vroon, David. "Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition".
  26. ^ "Alkaline phosphatase: Liver Function Test - Viral Hepatitis". www.hepatitis.va.gov. Retrieved 2016-05-02.
  27. ^ l, Karaca (Feb 1999). "What do we know about serum alkaline phosphatase activity as a biochemical bone formation marker?". BIOCHEMICAL ARCHIVES. 15 (1): 1–4. Retrieved 1 May 2016.
  28. ^ Lange PH, Millan JL, Stigbrand T, Vessella RL, Ruoslahti E, Fishman WH (August 1982). "Placental alkaline phosphatase as a tumor marker for seminoma". Cancer Res. 42 (8): 3244–7. PMID 7093962.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  29. ^ Dugdale, David C. "ALP-bloodtest:MedlinePlus Medical Encyclopedia". MedlinePlus. Retrieved 2014-02-26.
  30. ^ P, FOUCAULT; MH, FOUCAULT; B, KUCHAREWICZ; F, BUREAU; M, ALIX; MA, DROSDOWSKY (1991). "BONE AND TOTAL ALKALINE-PHOSPHATASE MEASUREMENTS IN AN OSTEOPOROTIC POPULATION". ANNALES DE BIOLOGIE CLINIQUE. 49 (9): 477–481. Retrieved 2 May 2016.
  31. ^ Schiele F, Vincent-Viry M, Fournier B, Starck M, Siest G (November 1998). "Biological effects of eleven combined oral contraceptives on serum triglycerides, gamma-glutamyltransferase, alkaline phosphatase, bilirubin and other biochemical variables". Clin. Chem. Lab. Med. 36 (11): 871–8. doi:10.1515/CCLM.1998.153. PMID 9877094.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  32. ^ Arceci RJ, Hann IM, Smith OP, ed. (2006). Pediatric hematology (3rd ed.). Wiley-Blackwell. p. 763. ISBN 978-1-4051-3400-2.{{cite book}}: CS1 maint: multiple names: editors list (link)

Further reading

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