Alkaline phosphatase: Wikis

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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 number 3.1.3.1
CAS number 9001-78-9
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures
Gene Ontology AmiGO / EGO
Alkaline phosphatase
Identifiers
Symbol Alk_phosphatase
Pfam PF00245
InterPro IPR001952
SMART SM00098
PROSITE PDOC00113
SCOP 1alk

Alkaline phosphatase (ALP) (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. The optimal pH for the activity of the E. coli enzyme is 8.0[2] while the bovine enzyme optimum pH is slightly higher at 8.5.[3]

It is sometimes used synonymously as basic phosphatase.[4]

Contents

Bacterial

In 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 actual purpose of the enzyme is still not fully understood, 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 only produced by the bacteria during phosphate starvation and not when phosphate is plentiful.[citation needed] 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. 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.

Use in research

The most common alkaline phosphatases used in research are:

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.

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).[5] 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.

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Inhibitors

All mammalian alkaline phosphatase isoenzymes except placental (PALP and SEAP) are inhibited by homoarginine and similarly 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).

Human

Physiology

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:

  • ALPI – intestinal
  • ALPL – tissue non-specific (liver/bone/kidney)
  • ALPP – placental (Regan isozyme)

Diagnostic use

The normal range is 20 to 140 IU/L.[6] High ALP levels can show that the bile ducts are blocked.[7] Levels are significantly higher in children and pregnant women. Also, elevated ALP indicates that there could be active bone deposition occurring as ALP is a by product of osteoblast activity (such as the case in Paget's disease of bone).

Lowered levels of ALP are less common than elevated levels.

Elevated levels

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.[8]

Lowered levels

Other notes

Leukocyte alkaline phosphatase (LAP) is found within white blood cells. White blood cell levels of LAP can help in the diagnosis of certian conditions. Higher levels are seen in polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PM), and the leukemoid reaction. Lower levels are found in chronic myelogenous leukemia (CML) and paroxysmal nocturnal hemoglobinuria (PNH).

See also

References

  1. ^ 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. ^ 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. PMID 13826559. 
  3. ^ 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. PMID 3003174. http://jdr.sagepub.com/cgi/pmidlookup?view=long&pmid=3003174. 
  4. ^ 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. http://www.chempap.org/papers/565a326.pdf. 
  5. ^ Aschaffenburg R, Mullen JEC (1949). "A rapid and simple phosphatase test for milk". Journal of Dairy Research 16: 58–67. 
  6. ^ "MedlinePlus Medical Encyclopedia: ALP isoenzyme test". http://www.nlm.nih.gov/MEDLINEPLUS/ency/article/003497.htm. 
  7. ^ ALP: The Test
  8. ^ 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. 

Further reading


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