Alkaline phosphatase

Alkaline phosphatase

Ribbon diagram (rainbow-color, N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase.[1]
Identifiers
EC number 3.1.3.1
CAS number 9001-78-9
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Alkaline phosphatase

Structure of alkaline phosphatase.[1]
Identifiers
Symbol Alk_phosphatase
Pfam PF00245
InterPro IPR001952
SMART SM00098
PROSITE PDOC00113
SCOP 1alk
SUPERFAMILY 1alk

Alkaline phosphatase (ALP, ALKP, ALPase, Alk Phos) (EC 3.1.3.1) or basic phosphatase[2] is a homodimeric protein enzyme of 86 kilodaltons, containing two zinc atoms crucial to its catalytic function per monomer, and is optimally active at alkaline pH environments.[3] As its name indicates, ALP functions best under alkaline pH environments and has the physiological role of dephosphorylating compounds. The enzyme is found across a plethora of organisms, prokaryotes and eukaryotes alike, with the same general function but in different structural forms suitable to the environment they function in. In humans for example, it is found in many forms depending on its origin within the body - it plays an integral role in metabolism within the liver and development within the skeleton. Due to its widespread prevalence in these areas, its concentration in the bloodstream is used by diagnosticians as a biomarker in helping determine diagnoses such as hepatitis or osteomalacia.[4] 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, gender, blood type and whether an individual is pregnant or not. Additionally, abnormal levels of Alkaline phosphatase in the blood could indicate issues relating to the liver, gall bladder or bones. Kidney tumors, infections as well as malnutrition has also shown abnormal level of alkaline phosphatase in blood.[5]

Bacterial

In Gram-negative bacteria, such as Escherichia coli (E.coli), alkaline phosphatase is located in the periplasmic space, external to the inner cell membrane and within the peptidoglycan portion of the cell wall. With the periplasmic gap being 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.[6]

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. 

While the outer membrane of E.coli contains porins that are permeable to phosphorylated compounds, the inner membrane does not. Then, an issue arises in how to transport such compounds across the inner membrane and into the cytosol. Surely, with the strong anionic charge of phosphate groups along with the remainder of the compound they are very much immiscible in the nonpolar region of the bilayer. The solution arises in cleaving the phosphate group away from the compound via ALP. In effect, along with the concomitant compound the phosphate was bound to, this enzyme yields pure inorganic phosphate which can be ultimately targeted by the phosphate-specific transport system (Pst system)[7] for translocation into the cytosol.[8] As such, the main purpose of dephosphorylation by alkaline phosphatase is to increase the rate of diffusion of the molecules into the cells and inhibit them from diffusing out.[9]

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.[10] 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, whereas the Mg site doesn’t appear to be close enough to directly partake in the hydrolysis mechanism, however, it may contribute to the shape of the electrostatic potential around the active center.[10] Alkaline Phosphatase has a Km of 8.4 x 10^-4.[11]

Alkaline phosphatase in E.coli is uncommonly soluble and active within elevated temperature conditions such as 80 degrees Celsius. 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 ALP 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 degrees Celsius ALP has the uncommon characteristic of reverse denaturation. Due to this, while ALP ultimately denatures at about 90 degrees 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 desphosphorylates 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 spectophotometry to determine important information about 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 depressed cells.[11]

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.[17] Typical uses in the lab for alkaline phosphatases include removing phosphate monoesters to prevent self-ligation, which is undesirable during plasmid DNA cloning.[18]

Common alkaline phosphatases used in research include:

Human-intestinal ALPase shows around 80% homology with bovine intestinal ALPase, which holds true their shared evolutionary origins. That same bovine enzyme has more than 70% homology with human placental enzyme. However, the human intestinal enzyme and the placental enzyme only share 20% homology despite their structural similarities.[21]

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.

Because 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).[22]

Ongoing research

Current researchers are looking into the increase of tumor necrosis factor-α and its direct effect on the expression of alkaline phosphatase in vascular smooth muscle cells as well as how alkaline phosphatase (AP) affects the inflammatory responses and may play a direct role in preventing organ damage.[23]

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.[27][28]

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,[29] 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.[30] 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 (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 inactivates most isoenzymes except placental isoforms (PALP and SEAP).[31] Phosphate is another inhibitor which competitively inhibits alkaline phosphatase.[32]

Another known example of an alkaline phosphatase inhibitor is [(4-Nitrophenyl)methyl]phosphonic acid.[33]

Human

Physiology

In humans, alkaline phosphatase is present in all tissues throughout the entire 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.[34] Humans and most other mammals contain the following alkaline phosphatase isozymes:

Alkaline Phosphatase in cancer cells

Studies show that the alkaline phosphatase protein found in cancer cells has similar characteristics to that found in non-malignant body tissues and that the protein originates from the same gene in both the malignant and the non-malignant cells. One study tested the structural comparison between the alkaline phosphatase proteins found in liver giant-cell carcinoma and non-malignant placental cells. In this study, an alkaline phosphatase that was immunochemically similar to placental alkaline phosphatase was purified from metastases of giant-cell carcinoma of the lung and its physical and chemical properties were determined. Thereafter, these were compared with purified placental alkaline phosphatase. The results showed great similarity in both based on evaluations of NH2-terminal sequence, peptide map, subunit molecular weight, and isoelectronic point. Overall, this study strongly supports the supposition that the alkaline phosphatase protein in both tumor and non-malignant placental cells are derived from the same gene.[35]

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.[36] 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.[37] This explains why the addition of sodium butyrate show increased activity of alkaline phosphatase in the cancer cells of the human colon.[36] In addition, this further supports the theory that alkaline phosphatase enzyme activity is actually present in cancer cells.

In another study, choriocarcinoma cells were grown in the presence of 5-bromo-2’-deoxyuridine (BrdUrd) 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 enzyme induction. The evidence shows that there is in fact activity of alkaline phosphatase in tumor cells, but it is minimal and needs to be enhanced. Results from this study further indicate that activities of this enzyme vary among the different choriocarcinoma cell lines and that the activity of the alkaline phosphatase protein in these cells is lower than in the non-malignant placenta cells.[38][39] 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 ALP levels can occur if the bile ducts are obstructed.[40] 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.[41] 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.[42]

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.[43]

Elevated levels

If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using electrophoresis can confirm the source of the ALP. Skelphosphatase (which is localized in osteoblasts and extracellular layers of newly synthesized matrix) is released into circulation by a yet unclear mechanism.[44] Placental alkaline phosphatase is elevated in seminomas[45] and active forms of rickets, as well as in the following diseases and conditions:[46]

Lowered levels

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:

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.[49]

Leukocyte alkaline phosphatase

Leukocyte alkaline phosphatase (LAP) is found within mature white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions.

Structure and properties

Essentially, an alkaline phosphatase is homodimeric enzyme, meaning it is formed with two molecules. Three metal ions are contained in the catalytic sites, which are two Zn atoms and one Mg atom, both types are crucial enzymatic activity to occur. The enzymes will 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 when comparing mammalian and bacterial alkaline phosphate, however mammalian alkaline phosphatase has higher specific activity and Km values thus a lower affinity; have a more alkaline pH optimum; showcase a lower heat stability; are typically membrane bound and are inhibited by l-amino acids and peptides via a means of uncompetitive mechanism. These properties noticeably contrast among different mammalian alkaline phosphatase isozymes and therefore showcase a difference in in vivo functions.

Alkaline phosphatase have homology in a large number of other enzymes and compose part of a superfamily of enzymes with several overlapping catalytic aspects 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.[51]

See also

References

  1. 1 2 PDB: 1ALK​: Kim EE, Wyckoff HW (March 1991). "Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis". J. Mol. Biol. 218 (2): 449–64. PMID 2010919. doi:10.1016/0022-2836(91)90724-K.
  2. ↑ Tamás L, Huttová J, Mistrk I, Kogan G (2002). "Effect of Carboxymethyl Chitin-Glucan on the Activity of Some Hydrolytic Enzymes in Maize Plants" (PDF). Chem. Pap. 56 (5): 326–329.
  3. ↑ Ninfa; Ballou; Benore (2010). Biochemistry and Biotechnology. USA: John Wiley & Sons, INC. pp. 229–230. ISBN 978-0-470-08766-4.
  4. ↑ "Alkaline Phosphatase Level Test (ALP)". Healthline. Retrieved 2017-05-07.
  5. ↑ "Alkaline Phosphatase Level Test (ALP)". Healthline. Retrieved 2017-05-15.
  6. 1 2 Schlesinger; Barrett (June 9, 1965). "The Reversible Dissociation of the Alkaline Phosphatase of Escherichia coli" (PDF). THE JOURNAL OFBIOLOGICALCHEMISTRY. 240: 4285–4290.
  7. ↑ Rao, N. N.; Torriani, A. (1990-07-01). "Molecular aspects of phosphate transport in Escherichia coli". Molecular Microbiology. 4 (7): 1083–1090. ISSN 0950-382X. PMID 1700257.
  8. ↑ Willsky; Malamy; Bennett (1973). "Inorganic Phosphate Transport in Escherichia coli: Involvement of Two Genes Which Play a Role in Alkaline Phosphatase Regulation". Journal of Bacteriology. 113: 529–539.
  9. ↑ Horiuchi T, Horiuchi S, Mizuno D (May 1959). "A possible negative feedback phenomenon controlling formation of alkaline phosphomonoesterase in Escherichia coli". Nature. 183 (4674): 1529–30. PMID 13666805. doi:10.1038/1831529b0.
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  12. ↑ Ammerman JW, Azam F (March 1985). "Bacterial 5-nucleotidase in aquatic ecosystems: a novel mechanism of phosphorus regeneration". Science. 227 (4692): 1338–40. PMID 17793769. doi:10.1126/science.227.4692.1338.
  13. ↑ Biolabs, New England. "p-Nitrophenyl Phosphate (PNPP) | NEB". www.neb.com. Retrieved 2017-05-15.
  14. ↑ Wanner BL, Latterell P (October 1980). "Mutants affected in alkaline phosphatase, expression: evidence for multiple positive regulators of the phosphate regulon in Escherichia coli". Genetics. 96 (2): 353–66. PMC 1214304 Freely accessible. PMID 7021308.
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  17. ↑ W, MANDECKI; J, TOMAZICALL; A, SHALLCROSS; J, TOMAZIC-ALLEN. "Mutant Escherichia coli alkaline phosphatase enzymes - having amino acid changes to increase specific activity while retaining thermal stability". Retrieved 1 May 2016.
  18. ↑ Maxam AM, Gilbert W (1980). "Sequencing end-labeled DNA with base-specific chemical cleavages". Meth. Enzymol. Methods in Enzymology. 65 (1): 499–560. ISBN 978-0-12-181965-1. PMID 6246368. doi:10.1016/S0076-6879(80)65059-9.
  19. ↑ Birkett, Donald J.; Done, James; Neale, Francis C.; Posen, Solomon (1966-01-01). "Serum Alkaline Phosphatase In Pregnancy: An Immunological Study". The British Medical Journal. 1 (5497): 1210–1212. JSTOR 25407775. doi:10.1136/bmj.1.5497.1210.
  20. ↑ Benham, Frances J.; Harris, Harry (1979-01-01). "Human Cell Lines Expressing Intestinal Alkaline Phosphatase". Proceedings of the National Academy of Sciences of the United States of America. 76 (8): 4016–4019. JSTOR 69758. PMC 383967 Freely accessible. PMID 291061. doi:10.1073/pnas.76.8.4016.
  21. ↑ Hua, Jia-Cheng; Berger, Joel; Pan, Yu-Ching E.; Hulmes, Jeffrey D.; Udenfriend, Sidney (1986-01-01). "Partial Sequencing of Human Adult, Human Fetal, and Bovine Intestinal Alkaline Phosphatases: Comparison with the Human Placental and Liver Isozymes". Proceedings of the National Academy of Sciences of the United States of America. 83 (8): 2368–2372. JSTOR 27284. PMC 323298 Freely accessible. PMID 3458202. doi:10.1073/pnas.83.8.2368.
  22. ↑ "Appendix E: Stem Cell Markers". Stem Cell Information. National Institutes of Health, U.S. Department of Health and Human Services. Retrieved 2013-09-24.
  23. ↑ Jody A. Charnow, ed. (April 16, 2010). "Alkaline Phosphatase May Be a Marker of Inflammation in CKD Patients". Renal and Urology News.
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  29. ↑ "BS EN ISO 11816-1:2013 - Milk and milk products. Determination of alkaline phosphatase activity. Fluorimetric method for milk and milk-based drinks". Retrieved 23 August 2016.
  30. ↑ Aschaffenburg R, Mullen JE (1949). "A rapid and simple phosphatase test for milk". Journal of Dairy Research. 16 (1): 58–67. doi:10.1017/S0022029900005288.
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  32. ↑ Iqbal, J (2011) "An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases" J Anal. Biochem. 414, 226-231
  33. ↑ C.R. Ganellin, David J. Triggle - 1996
  34. ↑ I, Reiss; D, Inderrieden; K, Kruse (Sep 1996). "Measurement of skeletal specific alkaline phosphatase in disorders of calcium metabolism in childhood". Monatsschrift Kinderheilkunde. 144 (9): 885–890. doi:10.1007/s001120050054.
  35. ↑ Greene, Patricia J.; Sussman, Howard H. (1973-01-01). "Structural Comparison of Ectopic and Normal Placental Alkaline Phosphatase". Proceedings of the National Academy of Sciences of the United States of America. 70 (10): 2936–2940. JSTOR 63137. PMC 427142 Freely accessible. PMID 4517947. doi:10.1073/pnas.70.10.2936.
  36. 1 2 Herz, Fritz; Schermer, Alexander; Halwer, Murray; Bogart, Lee H. (1981-09-01). "Alkaline phosphatase in HT-29, a human colon cancer cell line: Influence of sodium butyrate and hyperosmolality". Archives of Biochemistry and Biophysics. 210 (2): 581–591. PMID 7305346. doi:10.1016/0003-9861(81)90224-1.
  37. ↑ Paskova, Lenka; Smesny Trtkova, Katerina; Fialova, Barbora; Benedikova, Andrea; Langova, Katerina; Kolar, Zdenek (2013-08-01). "Different effect of sodium butyrate on cancer and normal prostate cells". Toxicology in Vitro. 27 (5): 1489–1495. PMID 23524101. doi:10.1016/j.tiv.2013.03.002.
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  40. ↑ "ALP: The Test - Alkaline Phosphatase". Retrieved 23 August 2016.
  41. ↑ Preussner, Harold T, HT (March 1998). "Detecting coeliac disease in your patients". American Family Physician. 57 (5): 1023–1034. PMID 9518950.
  42. ↑ Vroon, David. "Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition".
  43. ↑ "Alkaline phosphatase: Liver Function Test - Viral Hepatitis". www.hepatitis.va.gov. Retrieved 2016-05-02.
  44. ↑ l, Karaca (Feb 1999). "What do we know about serum alkaline phosphatase activity as a biochemical bone formation marker?". Biochemical Archives. 15 (1): 1–4. Retrieved 1 May 2016.
  45. ↑ Lange PH, Millan JL, Stigbrand T, Vessella RL, Ruoslahti E, Fishman WH (August 1982). "Placental alkaline phosphatase as a tumor marker for seminoma". Cancer Res. 42 (8): 3244–7. PMID 7093962.
  46. ↑ Dugdale, David C. "ALP-bloodtest:MedlinePlus Medical Encyclopedia". MedlinePlus. Retrieved 2014-02-26.
  47. ↑ P, FOUCAULT; MH, FOUCAULT; B, KUCHAREWICZ; F, BUREAU; M, ALIX; MA, DROSDOWSKY (1991). "BONE AND TOTAL ALKALINE-PHOSPHATASE MEASUREMENTS IN AN OSTEOPOROTIC POPULATION". Annales De Biologie Clinique. 49 (9): 477–481. PMID 1789501. Retrieved 2 May 2016.
  48. ↑ Schiele F, Vincent-Viry M, Fournier B, Starck M, Siest G (November 1998). "Biological effects of eleven combined oral contraceptives on serum triglycerides, gamma-glutamyltransferase, alkaline phosphatase, bilirubin and other biochemical variables". Clin. Chem. Lab. Med. 36 (11): 871–8. PMID 9877094. doi:10.1515/CCLM.1998.153.
  49. ↑ Robinson, David; Sandblom, Gabriel; Johansson, Robert (Jan 2008). "Prediction of survival of metastatic prostate cancer based on early serial measurements of prostate specific antigen and alkaline phosphatase". Journal of Urology. 179 (1): 117–122. PMID 17997442. doi:10.1016/j.juro.2007.08.132. Retrieved 2 May 2016.
  50. ↑ Arceci RJ, Hann IM, Smith OP, eds. (2006). Pediatric hematology (3rd ed.). Wiley-Blackwell. p. 763. ISBN 978-1-4051-3400-2.
  51. ↑ Millán, José Luis (2017-05-22). "Alkaline Phosphatases". Purinergic Signalling. 2 (2): 335–341. ISSN 1573-9538. PMC 2254479 Freely accessible. PMID 18404473. doi:10.1007/s11302-005-5435-6.

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