Alkaline phosphatase

In gram-negative bacteria, such as Escherichia coli, alkaline phosphatase is located in the periplasmic space, external to the inner cell membrane and within the peptidoglycan portion of the cell wall. Since the periplasmic gap is more prone to environmental variation than the inner cell, alkaline phosphatase is suitably resistant to inactivation, denaturation, or degradation. This characteristic of the enzyme is uncommon to many other proteins. 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. 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. for translocation into the cytosol, 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. 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. −4.--> 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. 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. 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. Alkaline phosphatase accounts for 6% of all proteins in derepressed cells. 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. ==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. Typical uses in the lab for alkaline phosphatases include removing phosphate monoesters to prevent self-ligation, which is undesirable during plasmid DNA cloning. 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. In addition, it is useful in detoxifying mediated lipopolysaccharide inflammatory responses thus a drug for lipopolysaccharide mediated disease. • 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. • 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. 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. 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 due to the elevated catalytic activity of ALP enzyme. 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). There is a positive correlation between serum bone alkaline phosphatase levels (= BAP) and bone formation in humans, although its use as a biomarker in clinical practice is not recommended. 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 the enzyme. Therefore, its presence is ideal for indicating failed pasteurization. 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, 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. 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. 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. Phosphate is another inhibitor which competitively inhibits alkaline phosphatase. Another known example of an alkaline phosphatase inhibitor is [(4-Nitrophenyl)methyl]phosphonic acid. 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. ==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. Humans and most other mammals contain the following alkaline phosphatase isozymes: • 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. Intestinal alkaline phosphatase Intestinal alkaline phosphatase is secreted by enterocytes, and seems to play a pivotal role in intestinal homeostasis and protection as well as in suppressing inflammation via repression of the downstream Toll-like receptor (TLR)-4-dependent and MyD88-dependent inflammatory cascade. It dephosphorylates toxic/inflammatory microbial ligands like lipopolysaccharides (LPSs), It also seems to regulate lipid absorption and bicarbonate secretion in the duodenal mucosa, which regulates the surface pH. Since the 1960s intestinal alkaline phosphatase has been utilized in drug delivery. As it cleaves phosphate substructures from drugs, auxiliary agents, and even from the surface of nanocarriers, this enzyme enables the design of drug delivery systems that can alter their properties in the body on demand. The solubility of many drugs can be substantially improved by the design of phosphate prodrugs. On the intestinal mucosa the phosphate substructures are cleaved off by alkaline phosphatase and the drug is absorbed. Furthermore, anionic nanocarriers exhibiting bioinert properties can alter their surface to interactive once having reached the intestinal epithelium as due to an alkaline phosphatase triggered cleavage of anionic phosphate groups from their surface charge converts to cationic improving for instance cellular uptake. 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. 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. 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. This explains why the addition of sodium butyrate show increased activity of alkaline phosphatase in the cancer cells of the human colon. but 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 alkaline phosphatase levels can occur if the bile ducts are obstructed. 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). The level of alkaline phosphatase is much more elevated in metastatic prostate cancer cells than non-metastatic prostate cancer cells. High levels of ALP in prostate cancer patients is associated with a significant decrease in survival. Lowered levels of the level of alkaline phosphatase are less common than elevated levels. The source of elevated levels can be deduced by obtaining serum levels of γ-glutamyltransferase. Concomitant increases of alkaline phosphatase with γ-glutamyltransferase should raise the suspicion of hepatobiliary disease. 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. Elevated levels As of 2020, normal ALP levels were "not well defined", and there tend to be variations by sex and racial background, and by age, with children and adolescents having markedly higher levels. There are many possible explanations for elevated ALP. When the cause is unclear, isoenzyme studies using electrophoresis can confirm the source of the increase. Skelphosphatase (which is localized in osteoblasts and extracellular layers of newly synthesized matrix) is released into circulation by a yet unclear mechanism. Placental alkaline phosphatase is elevated in seminomas and active forms of rickets, as well as in the following diseases and conditions: • Biliary obstruction • Bone conditions • Osteoblastic bone tumors • Osteomalacia • Osteoporosis • Hepatitis • Mononucleosis • Cirrhosis • Acute cholecystitis • Myelofibrosis • Leukemoid reaction • Congestive heart failure • Lymphoma • Paget's disease • Sarcoidosis • Hyperthyroidism • Hyperparathyroidism • Myocardial infarction • Cholangitis • Ischemic cholangiopathy • Pregnancy Lowered levels The following conditions or diseases may lead to reduced levels of alkaline phosphatase: • Hypophosphatasia, a genetic disorder • Women receiving estrogen therapy for menopausal symptoms • Estrogen-containing oral contraceptives • Men with recent heart surgery, malnutrition, magnesium deficiency, or severe anemia • Children with achondroplasia and 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 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. • Higher than typical levels are seen in the physiological response, the leukemoid reaction, and in pathologies that include mature white blood cells, such as polycythemia vera, essential thrombocytosis, and in primary myelofibrosis. • Lower than typical levels are found in pathologies that involve undeveloped leukocytes, such as chronic myelogenous leukemia (CML), paroxysmal nocturnal hemoglobinuria and 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. == See also ==