Alkaline phosphatase
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The enzyme alkaline phosphatase (ALP, alkaline phenyl phosphatase) is a
The level of alkaline phosphatase in the blood is checked through the ALP test, which is often part of routine blood tests. The levels of this enzyme in the blood depend on factors such as age, sex, or blood type.[2] Blood levels of alkaline phosphatase also increase by two to four times during pregnancy. This is a result of additional alkaline phosphatase produced by the placenta and the liver.[3][4] Additionally, abnormal levels of alkaline phosphatase in the blood could indicate issues relating to the liver, gall bladder or bones. Kidney tumors and infections as well as malnutrition have also shown abnormal level of alkaline phosphatase in blood.[2] Alkaline phosphatase levels in a cell can be measured through a process called "The scoring method". A blood smear is usually taken and stained to categorize each leukocyte into specific "leukocyte alkaline phosphatase indices". This marker is designed to distinguish leukocytes and determine different enzyme activity from each sample's extent of staining.[5]
Bacterial
In
The precise structure and function of the isozyme in E. coli is solely geared to supply a source of inorganic phosphate when the environment lacks this metabolite. The inorganic phosphates are then bound to carrier proteins which deliver the inorganic phosphates to a specific high-affinity transport system, known as the phosphate-specific transport system, which transports phosphate across the cytoplasmic membrane.[7]
While the outer membrane of E. coli contains porins that are permeable to phosphorylated compounds, the inner membrane does not. An issue arises in how to transport such compounds across the inner membrane and into the cytosol. The strong anionic charge of phosphate groups along with the remainder of the compound make phosphorylated compounds very much immiscible in the nonpolar region of the bilayer. The solution arises in cleaving the phosphate group away from the compound via ALP.[8] for translocation into the cytosol,[9] The main purpose of dephosphorylation by alkaline phosphatase is to increase the rate of diffusion of phosphorylated molecules into the cells while inhibiting them from diffusing out.[10]
Alkaline phosphatase is a zinc-containing dimeric enzyme with the MW: 86,000 Da, each subunit containing 429 amino acids with four cysteine residues linking the two subunits.[11] Alkaline phosphatase contains four Zn ions and two Mg ions, with Zn occupying active sites A and B, and Mg occupying site C, so the fully active native alkaline phosphatase is referred to as (ZnAZnBMgC)2 enzyme. The mechanism of action of alkaline phosphatase involves the geometric coordination of the substrate between the Zn ions in the active sites.[11]
Alkaline phosphatase in E. coli is uncommonly soluble and active within elevated temperature conditions such as 80 °C. Due to the kinetic energy induced by this temperature the weak hydrogen bonds and hydrophobic interactions of common proteins become degraded and therefore coalesce and precipitate. However, upon dimerization of alkaline phosphatase, the bonds maintaining its secondary and tertiary structures are effectively buried such that they are not affected as much at this temperature. Furthermore, even at more elevated temperatures such as 90 °C alkaline phosphatase has the unusual characteristic of reverse denaturation. Due to this, although it ultimately denatures at about 90 °C it has the added ability to accurately reform its bonds and return to its original structure and function once cooled back down.[6]
Alkaline phosphatase in E. coli is located in the periplasmic space and can thus be released using techniques that weaken the cell wall and release the protein. Due to the location of the enzyme, and the protein layout of the enzyme, the enzyme is in solution with a smaller amount of proteins than there are in another portion of the cell. [12] The proteins' heat stability can also be taken advantage of when isolating this enzyme (through heat denaturation). In addition, alkaline phosphatase can be assayed using p-nitrophenyl phosphate. A reaction where alkaline phosphatase dephosphorylates the non-specific substrate, p-nitrophenyl phosphate in order to produce p-nitrophenol (PNP) and inorganic phosphate. PNP's yellow color, and its λmax at 410 allows spectrophotometry to determine enzymatic activity.[13] 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.[14]
The optimal pH for the activity of the E. coli enzyme is 8.0[15] while the bovine enzyme optimum pH is slightly higher at 8.5.[16] Alkaline phosphatase accounts for 6% of all proteins in derepressed cells.[17]
Heterodimeric Alkaline Phosphatase
When multiple copies of a polypeptide encoded by a gene form an aggregate, this protein structure is referred to as a multimer. When a multimer is formed from polypeptides produced by two different mutant alleles of a particular gene, the mixed multimer may exhibit greater functional activity than the unmixed multimers formed by each of the mutants alone. In such a case, the phenomenon is referred to as intragenic complementation. E. coli alkaline phosphatase, a dimer enzyme, exhibits intragenic complementation.[18]
Use in research
By changing the amino acids of the wild-type alkaline phosphatase enzyme produced by Escherichia coli, a mutant alkaline phosphatase is created which not only has a 36-fold increase in enzyme activity, but also retains thermal stability.[19] Typical uses in the lab for alkaline phosphatases include removing phosphate monoesters to prevent self-ligation, which is undesirable during plasmid DNA cloning.[20]
Common alkaline phosphatases used in research include:
- Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis). This phosphatase is easily inactivated by spiking up the temperature, a useful feature in some applications.
- Calf-intestinal alkaline phosphatase (CIP) typically useful in molecular cloning, where DNA undergoes recombination and no new DNA is made.[21] In addition, it is useful in detoxifying mediated lipopolysaccharide inflammatory responses thus a drug for lipopolysaccharide mediated disease.[22]
- Placental alkaline phosphatase (PLAP) 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). It presents certain characteristics like heat stability, substrate specificity, and resistance to chemical inactivation.[23]
- Human-intestinal alkaline phosphatase. The human body has multiple types of alkaline phosphatase present, which are determined by a minimum of three gene loci. Each one of these three loci controls a different kind of alkaline phosphatase isozyme. However, the development of this enzyme can be strictly regulated by other factors such as thermostability, electrophoresis, inhibition, or immunology.[24]
Human-intestinal alkaline phosphatase shows around 80% homology with the bovine intestinal enzyme which holds true their shared evolutionary origins. That same bovine enzyme has more than 70% homology with human placental enzyme. However, the human liver enzyme and the placental enzyme only share 20% homology despite their structural similarities.[25]
Alkaline phosphatase has become a useful tool in
Another important use of alkaline phosphatase is as a label for enzyme immunoassays due to the elevated catalytic activity of ALP enzyme.[27]
Undifferentiated
There is a positive correlation between serum bone alkaline phosphatase levels and bone formation in humans, although its use as a biomarker in clinical practice is not recommended.[29]
Dairy industry
Alkaline phosphatase is commonly used in the dairy industry as an indicator of successful pasteurization. This is because the most heat stable
Pasteurization verification is typically performed by measuring the fluorescence of a solution which becomes fluorescent when exposed to active alkaline phosphatase. Fluorimetry assays are required by milk producers in the UK to prove alkaline phosphatase has been denatured,[32] as p-Nitrophenylphosphate tests are not considered accurate enough to meet health standards.
Alternatively the color change of p-nitrophenylphosphate as substrate in a buffered solution (Aschaffenburg Mullen Test) can be used.[33] Raw milk would typically produce a yellow coloration 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 phosphatases produced by some bacteria, but these bacteria should not be present in milk.[34]
In addition, the Scharer Rapid Phosphatase test investigates the presence and the absence of ALP enzymes in raw milk. This test is advantageous since a full and complete pasteurization inactivates the ALP enzymes, resulting in no color change. However, positive test results in changing the raw milk color into blue, thus indicating the presence of ALP enzymes. The test works by cleaving off a phosphate group from the substrate disodium phenyl phosphate, causing a release of phenol group and indicating positive test results observed visually and by spectrophotometer. [35]
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.[36] Phosphate is another inhibitor which competitively inhibits alkaline phosphatase.[37]
Another known example of an alkaline phosphatase inhibitor is [(4-Nitrophenyl)methyl]phosphonic acid.[38]
In metal contaminated soil, alkaline phosphatase are inhibited by Cd (Cadmium). In addition, temperature enhances the inhibition of Cd on the enzyme activity, which is shown in the increasing values of Km.[39]
Human
Physiology
In humans, alkaline phosphatase is present in all tissues throughout the body, but is particularly concentrated in the liver, bile duct, kidney, bone, intestinal mucosa and placenta. In the serum, two types of alkaline phosphatase isozymes predominate: skeletal and liver. During childhood the majority of alkaline phosphatase are of skeletal origin.[40] Humans and most other mammals contain the following alkaline phosphatase isozymes:[41]
- ALPI – intestinal (molecular mass of 150 kDa)
- ALPL – tissue-nonspecific (expressed mainly in liver, bone, and kidney)
- ALPP– placental (Regan isozyme)
- ALPG – germinal tissues
Four genes encode the four isozymes. The gene for tissue-nonspecific alkaline phosphatase is located on chromosome 1, and the genes for the other three isoforms are located on chromosome 2.[42]
Intestinal Alkaline Phosphatase
Intestinal alkaline phosphatase is secreted by
In Cancer Cells
Studies show that the alkaline phosphatase protein found in cancer cells is similar to that found in nonmalignant body tissues and that the protein originates from the same gene in both. One study compared the enzymes of liver metastases of giant-cell lung carcinoma and nonmalignant placental cells. The two were similar in NH2-terminal sequence, peptide map, subunit molecular weight, and isoelectronic point.[54]
In a different study in which scientists examined alkaline phosphatase protein presence in a human colon cancer cell line, also known as HT-29, results showed that the enzyme activity was similar to that of the non-malignant intestinal type. However, this study revealed that without the influence of sodium butyrate, alkaline phosphatase activity is fairly low in cancer cells.[55] A study based on sodium butyrate effects on cancer cells conveys that it has an effect on androgen receptor co-regulator expression, transcription activity, and also on histone acetylation in cancer cells.[56] This explains why the addition of sodium butyrate show increased activity of alkaline phosphatase in the cancer cells of the human colon.[55] In addition, this further supports the theory that alkaline phosphatase enzyme activity is actually present in cancer cells.[57]
In another study, choriocarcinoma cells were grown in the presence of 5-bromo-2'-deoxyuridine and results conveyed a 30- to 40-fold increase in alkaline phosphatase activity. This procedure of enhancing the activity of the enzyme is known as
Also, the level of alkaline phosphatase increases if there is active bone formation occurring, as the enzyme is a byproduct of osteoblast activity (such as the case in Paget's disease of bone).[57]
The level of alkaline phosphatase is much more elevated in metastatic prostate cancer cells than non-metastatic prostate cancer cells.[61] High levels of ALP in prostate cancer patients is associated with a significant decrease in survival.[61]
Levels are also elevated in people with untreated
Some diseases do not affect the levels of alkaline phosphatase, for example, hepatitis C. A high level of this enzyme does not reflect any damage in the liver, even though high alkaline phosphatase levels may result from a blockage of flow in the biliary tract or an increase in the pressure of the liver.[64]
Elevated Levels
As of 2020, normal ALP levels were "not well defined", and there tend to be variations by sex and racial background,[65] and by age, with children and adolescents having markedly higher levels.[66]
There are many possible explanations for elevated ALP. When the cause is unclear,
- Biliary obstruction
- Bone conditions
- Osteoblastic bone tumors
- Osteomalacia
- Osteoporosis[70]
- Hepatitis
- Mononucleosis[71]
- Cirrhosis
- Acute cholecystitis
- Myelofibrosis
- Leukemoid reaction
- Congestive heart failure
- Lymphoma
- Paget's disease
- Sarcoidosis
- Hyperthyroidism
- Hyperparathyroidism
- Myocardial infarction
- Cholangitis
- Ischemic cholangiopathy
- Pregnancy[4]
- High doses of estrogens[4][72][73]
Lowered Levels
This section needs more primary sources. (April 2024) |
The following conditions or diseases may lead to reduced levels of alkaline phosphatase:
- Hypophosphatasia, a genetic disorder
- Women receiving menopausal symptoms
- Estrogen-containing oral contraceptives
- Men with recent magnesium deficiency, or severe anemia
- Children with congenital iodine deficiency
- Children after a severe episode of enteritis
- Pernicious anemia
- Aplastic anemia
- Wilson's disease
- Hypothyroidism
- Zinc deficiency
- Malnutrition
- Steroid treatment
- Colitis
- Thyroid disease[74]
Prognostic Uses
Measuring alkaline phosphatase (along with prostate specific antigen) during, and after six months of hormone treated metastatic prostate cancer was shown to predict the survival of patients.[75]
Leukocyte Alkaline Phosphatase
Leukocyte alkaline phosphatase is found within mature white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions.[76]
- Higher than typical levels are seen in the physiological response, essential thrombocytosis, and in primary myelofibrosis.
- Lower than typical levels are found in pathologies that involve undeveloped leukocytes, such as acute myelogenous leukaemia.
Structure and Properties
Alkaline phosphatase is homodimeric enzyme, meaning it is formed with two molecules. Three metal ions, two Zn and one Mg, are contained in the catalytic sites, and both types are crucial for enzymatic activity to occur. The enzymes catalyze the hydrolysis of monoesters in phosphoric acid which can additionally catalyze a transphosphorylation reaction with large concentrations of phosphate acceptors. While the main features of the catalytic mechanism and activity are conserved between mammalian and bacterial alkaline phosphate, mammalian alkaline phosphatase has a higher specific activity and Km values thus a lower affinity, more alkaline pH optimum, lower heat stability, and are typically membrane bound and are inhibited by l-amino acids and peptides via a means of uncompetitive mechanism. These properties noticeably differ between different mammalian alkaline phosphatase isozymes and therefore showcase a difference in in vivo functions.[78]
Alkaline phosphatase has homology in a large number of other enzymes and composes part of a superfamily of enzymes with several overlapping catalytic aspects and substrate traits. This explains why most salient structural features of mammalian alkaline are the way they are and reference their substrate specificity and homology to other members of the nucleoside pyrophosphatase/phosphodiesterase family of isozyme.[42] Research has shown a relationship between members of the alkaline phosphatase family with aryl sulfatases. The similarities in structure indicate that these two enzyme families came from a common ancestor. Further analysis has linked alkaline phosphates and aryl sulfatases to a larger superfamily. Some of the common genes found in this superfamily, are ones that encode phosphodiesterases as well as autotoxin.[79]
See also
References
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Further reading
- Coleman JE (1992). "Structure and mechanism of alkaline phosphatase". Annual Review of Biophysics and Biomolecular Structure. 21: 441–83. S2CID 34764597.
External links
- Alkaline phosphatase at Lab Tests Online