Alkaline phosphatase

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Alkaline phosphatase
1ALK.png
Ribbon diagram (rainbow colored, N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase.[1]
Identifiers
EC number3.1.3.1
CAS number9001-78-9
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / EGO
 
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Alkaline phosphatase
1ALK.png
Ribbon diagram (rainbow colored, N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase.[1]
Identifiers
EC number3.1.3.1
CAS number9001-78-9
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / EGO
Alkaline phosphatase
PDB 1alk EBI.jpg
Structure of alkaline phosphatase.[1]
Identifiers
SymbolAlk_phosphatase
PfamPF00245
InterProIPR001952
SMARTSM00098
PROSITEPDOC00113
SCOP1alk
SUPERFAMILY1alk

Alkaline phosphatase (ALP, ALKP) (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[edit]

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 comparatively resistant to inactivation, denaturation, and degradation, and also has a higher rate of activity. Although the purpose of the enzyme is not fully resolved, the simple hypothesis that it is a means for the bacteria to generate free phosphate groups for uptake and use is supported by the fact that alkaline phosphatase is usually produced by the bacteria only during phosphate starvation and not when phosphate is plentiful.[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 in the wild.[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[edit]

Typical use in the lab for alkaline phosphatases includes removing phosphate monoester to prevent self ligation.[8]

Common alkaline phosphatases used in research include:

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).[9]

One common use in the dairy industry is as a marker of pasteurisation in cows' milk. This molecule is denatured by elevated temperatures found during pasteurisation, and can be tested for via colour change of a para-Nitrophenylphosphate substrate in a buffered solution (Aschaffenburg Mullen Test).[10] Raw milk would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised milk should show no change. There are of course exceptions to this in the case of heat-stable alkaline phophatases produced by some bacteria.

Inhibitors[edit]

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 blocked by levamisole. Heating for ~2 hours at 65°C inactivated most isoenzymes except Placental isoforms (PALP and SEAP).[11]

Human[edit]

Physiology[edit]

In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta. Humans and most other mammals contain the following alkaline phosphatase isozymes:

Diagnostic use[edit]

Normal ALP levels in adults are approximately 20 to 140 IU/L,[12] though 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.[13] 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.[14] Lowered levels of ALP are less common than elevated levels.

Elevated levels[edit]

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 ("bone burns, liver lasts"). Placental alkaline phosphatase is elevated in seminomas[15] and active form of Rickets.

Lowered levels[edit]

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:

Leukocyte alkaline phosphatase[edit]

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

See also[edit]

References[edit]

  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". Chem. Pap. 56 (5): 326–329. 
  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. 
  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. 
  8. ^ 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. 
  9. ^ "Appendix E: Stem Cell Markers". Stem Cell Information. National Institutes of Health, U.S. Department of Health and Human Services. Retrieved 2013-09-24. 
  10. ^ 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. 
  11. ^ Alkaline Phosphatase Why It Is Done from Everday Health.com. Retrieved October 15, 2012.
  12. ^ "MedlinePlus Medical Encyclopedia: ALP isoenzyme test". 
  13. ^ ALP: The Test
  14. ^ Preussner, Harold T, HT (March 1998). "Detecting coeliac disease in your patients". American Family Physician 57 (5): 1023–1034. PMID 9518950. 
  15. ^ 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. 
  16. ^ 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. 
  17. ^ Arceci RJ, Hann IM, Smith OP, ed. (2006). Pediatric hematology (3rd ed.). Wiley-Blackwell. p. 763. ISBN 978-1-4051-3400-2. 

External links[edit]

Further reading[edit]