From Wikipedia, the free encyclopedia - View original article
|PDB structures||RCSB PDB PDBe PDBsum|
|Gene Ontology||AmiGO / EGO|
|PDB structures||RCSB PDB PDBe PDBsum|
|Gene Ontology||AmiGO / EGO|
Structure of alkaline phosphatase.
Alkaline phosphatase (ALP, ALKP, ALPase, Alk Phos) (EC 220.127.116.11) 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.
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. 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.
Gifted with perhaps the best visual acuity, birds have avascular retina, supplemented with another organ, pecten oculi to compensate for vascular supply line of retina. Although chicken retina is rich in alkaline phosphatase activity, yet pecten oculi has more than three-times the alkaline phosphatase activity vis a vis retina. While, alkaline phosphatase activity of retina significantly goes down upon constant exposure of birds to light; on the contrary, it goes up significantly in pecten oculi upon the same light exposure. Alkaline phosphatase activity was therefore, implicated in transport of nutrients across cell membrane of epithelial cells of pecten oculi into vitreous humour, to be absorbed by retina, so as to compensate for defficient nutrient supply in avascular bird retina. Avascular retina is considered advantageous for allowing sharp image of light on bird retina, which, in human eye is obliterated, due to the presence of blood vessels.
Typical use in the lab for alkaline phosphatases includes removing phosphate monoester to prevent self-ligation.
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).
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.
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, 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. 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 phophatases produced by some bacteria but these bacteria should not be present in milk.
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).
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:
Normal ALP levels in adults are approximately 20 to 140 IU/L, 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. 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. 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.
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 and active form of rickets, as well as in the following diseases:
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 (LAP) is found within white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions.