Alkaline phosphatase

Alkaline phosphatase (ALP, ALKP) 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.

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 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.

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

Use in research
Typical use in the lab for alkaline phosphatases includes removing phosphate monoester to prevent self ligation.

Common alkaline phosphatases used in research include:
 * Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis)
 * Calf Intestinal Alkaline Phosphatase (CIP)
 * Placental alkaline phosphatase (PALP) 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)

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). 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
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).

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. High ALP levels can show that the bile ducts are blocked. Levels are significantly higher in children and pregnant women. Also, elevated ALP indicates that there could be active bone formation occurring as ALP is a byproduct 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.

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


 * Hypophosphatasia, an autosomal recessive disease
 * Postmenopausal women receiving estrogen therapy because of osteoporosis
 * Men with recent heart surgery, malnutrition, magnesium deficiency, hypothyroidism, or severe anemia
 * Children with achondroplasia and cretinism
 * Children after a severe episode of enteritis
 * Pernicious anemia
 * Aplastic anemia
 * Chronic myelogenous leukemia
 * Wilson's disease

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


 * Oral contraceptives

Leukocyte 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.
 * 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), paroxysmal nocturnal hemoglobinuria (PNH) and acute myelogenous leukaemia.