Alkaline phosphatase 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 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. 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, from a species of Arctic shrimp. This phosphatase is easily
inactivated by heat, a useful feature in some applications.
Calf-intestinal alkaline phosphatase Placental alkaline phosphatase and its C terminally
truncated version that lacks the last 24 amino acids - the secreted alkaline phosphatase
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, 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 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. Dairy Industry
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 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. Inhibitors
All mammalian alkaline phosphatase isoenzymes except placental 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.
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-nonspecific
ALPP – placental Diagnostic use
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. 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.
Concomitant increases of ALP with GGT should raise the suspicion of hepatobiliary disease.
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.
Placental alkaline phosphatase is elevated in seminomas and active form of rickets, as
well as in the following diseases: Biliary obstruction
Bone conditions Osteoblastic bone tumors
Osteomalacia Liver disease or hepatitis
Leukemia Lymphoma
Paget's disease Sarcoidosis
Hyperparathyroidism 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 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, essential thrombocytosis, primary myelofibrosis,
and the leukemoid reaction. Lower levels are found in chronic myelogenous
leukemia, paroxysmal nocturnal hemoglobinuria and acute myelogenous leukaemia.
See also Acid phosphatase
Liver function tests References
External links Alkaline phosphatase at Lab Tests Online
Further reading