Summary: Alkaline phosphatase
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Alkaline phosphatase Edit Wikipedia article
| Alkaline phosphatase | |||||||||
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 Ribbon diagram (rainbow colored, N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase.[1] |
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| 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 | ||||||||
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| Alkaline phosphatase | |||||||||
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 Structure of alkaline phosphatase.[1] |
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| Identifiers | |||||||||
| Symbol | Alk_phosphatase | ||||||||
| Pfam | PF00245 | ||||||||
| InterPro | IPR001952 | ||||||||
| SMART | SM00098 | ||||||||
| PROSITE | PDOC00113 | ||||||||
| SCOP | 1alk | ||||||||
| SUPERFAMILY | 1alk | ||||||||
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Alkaline phosphatase (ALP, ALKP) (EC 3.1.3.1) 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.[2]
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[edit] 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.[3] 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.[4] 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.[5]
The optimal pH for the activity of the E. coli enzyme is 8.0[6] while the bovine enzyme optimum pH is slightly higher at 8.5.[7]
[edit] Use in research
Typical use in the lab for alkaline phosphatases includes removing phosphate monoester to prevent self ligation.[8]
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 heat, a useful feature in some applications.
- 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.
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).[9]
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).[10] 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.
[edit] 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).[11]
[edit] Human
[edit] 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 â intestinal
- ALPL â tissue non-specific (liver/bone/kidney)
- ALPP â placental (Regan isozyme)
[edit] Diagnostic use
The normal range is 20 to 140 IU/L.[12] High ALP levels can show that the bile ducts are obstructed.[13] 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). Levels are also elevated in people with untreated Coeliac Disease.[14]
Lowered levels of ALP are less common than elevated levels.
[edit] 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[15] and active form of Rickets.
[edit] 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[16]
[edit] 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[17] (CML), paroxysmal nocturnal hemoglobinuria (PNH) and acute myelogenous leukaemia (AML).
[edit] See also
[edit] References
- ^ a b 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): 449â64. doi:10.1016/0022-2836(91)90724-K. PMID 2010919.
- ^ Tamás L, Huttová J, Mistrk I, Kogan G (2002). "Eï¬ect of Carboxymethyl Chitin-Glucan on the Activity of Some Hydrolytic Enzymes in Maize Plants". Chem. Pap. 56 (5): 326â329.
- ^ T. HORIUCHI, S. HORIUCHI, D. MIZUNO (may 1959). "A Possible Negative Feedback Phenomenon controlling Formation of Alkaline Phosphomonoesterase in Escherichia coli". Nature 183 (30 May 1959): 1529â1530. doi:10.1038/1831529b0.
- ^ James W. Ammerman and Farooq Azam (mar 1985). "Bacterial 5'-Nucleotidase in Aquatic Ecosystems: A Novel Mechanism of Phosphorus Regeneration". Science 183 (4692): 1338â1340. doi:10.1126/science.227.4692.1338.
- ^ Wanner, Barry L.; Patrick Latterell (August 7, 1980). "Mutants Affected in Alkaline Phosphatase Expression: Evidence for Multiple Positive Regulators of the Phosphate Regulon in Escherzchza Coli". Genetics 96: 353â366. Retrieved 19 September 2012.
- ^ Garen A, Levinthal C (March 1960). "A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase". Biochim. Biophys. Acta 38: 470â83. doi:10.1016/0006-3002(60)91282-8. PMID 13826559.
- ^ Harada M, Udagawa N, Fukasawa K, Hiraoka BY, Mogi M (February 1986). "Inorganic pyrophosphatase activity of purified bovine pulp alkaline phosphatase at physiological pH". J. Dent. Res. 65 (2): 125â7. doi:10.1177/00220345860650020601. PMID 3003174.
- ^ Maxam AM, Gilbert W (1980). "Sequencing end-labeled DNA with base-specific chemical cleavages". Meth. Enzymol. Methods in Enzymology 65 (1): 499â560. doi:10.1016/S0076-6879(80)65059-9. ISBN 978-0-12-181965-1. PMID 6246368.
- ^ Appendix E: Stem Cell Markers . In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2009 [cited Monday, January 02, 2012] Available at <http://stemcells.nih.gov/info/scireport/appendixe>
- ^ Aschaffenburg R, Mullen JEC (1949). "A rapid and simple phosphatase test for milk". Journal of Dairy Research 16: 58â67. doi:10.1017/S0022029900005288.
- ^ Alkaline Phosphatase Why It Is Done from Everday Health.com. Retrieved October 15,2012.
- ^ "MedlinePlus Medical Encyclopedia: ALP isoenzyme test".
- ^ ALP: The Test
- ^ Preussner, Harold T (March 1998). "Detecting coeliac disease in your patients". American Family Physician 57 (5): 1023â1034.
- ^ 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): 3244â7. PMID 7093962.
- ^ 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): 871â8. doi:10.1515/CCLM.1998.153. PMID 9877094.
- ^ Ian M. Hann; Owen P. Smith (26 September 2006). Pediatric hematology. Wiley-Blackwell. pp. 763â. ISBN 978-1-4051-3400-2. Retrieved 5 November 2010.
[edit] Further reading
- Coleman JE (1992). "Structure and mechanism of alkaline phosphatase". Annu Rev Biophys Biomol Struct 21: 441â83. doi:10.1146/annurev.bb.21.060192.002301. PMID 1525473.
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This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
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Internal database links
| Similarity to PfamA using HHSearch: | Sulfatase Metalloenzyme |
External database links
| PANDIT: | PF00245 |
| PRINTS: | PR00113 |
| PROSITE: | PDOC00113 |
| Pseudofam: | PF00245 |
| SCOP: | 1alk |
| SYSTERS: | Alk_phosphatase |
This tab holds annotation information from the InterPro database.
InterPro entry IPR001952
This entry represents alkaline phosphatases (EC) (ALP), which act as non-specific phosphomonoesterases to hydrolyse phosphate esters, optimally at high pH. The reaction mechanism involves the attack of a serine alkoxide on a phosphorus of the substrate to form a transient covalent enzyme-phosphate complex, followed by the hydrolysis of the serine phosphate. Alkaline phosphatases are found in all kingdoms of life, with the exception of some plants. Alkaline phosphatases are metalloenzymes that exist as a dimer, each monomer binding metal ions. The metal ions they carry can differ, although zinc and magnesium are the most common. For example, Escherichia coli alkaline phosphatase (encoded by phoA) requires the presence of two zinc ions bound at the M1 and M2 metal sites, and one magnesium ion bound at the M3 site [PUBMED:15938627]. However, alkaline phosphatases from Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity [PUBMED:11910033].
In mammals, there are four alkaline phosphatase isozymes: placental, placental-like (germ cell), intestinal and tissue-nonspecific (liver/bone/kidney). All four isozymes are anchored to the outer surface of the plasma membrane by a covalently attached glycosylphosphatidylinositol (GPI) anchor [PUBMED:17520090]. Human alkaline phosphatases have four metal binding sites: two for zinc, one for magnesium, and one for calcium ion. Placental alkaline phosphatase (ALPP or PLAP) is highly polymorphic, with at least three common alleles [PUBMED:11124260]. Its activity is down-regulated by a number of effectors such as l-phenylalanine, 5'-AMP, and by p-nitrophenyl-phosphonate (PNPPate) [PUBMED:15946677]. The placental-like isozyme (ALPPL or PLAP-like) is elevated in germ cell tumours. The intestinal isozyme (ALPI or IAP) has the ability to detoxify lipopolysaccharide and prevent bacterial invasion across the gut mucosal barrier [PUBMED:18292227]. The tissue-nonspecific isozyme (ALPL) is, and may play a role in skeletal mineralisation. Defects in ALPL are a cause of hypophosphatasia, including infantile-type (OMIM:241500), childhood-type (OMIM:241510) and adult-type (OMIM:146300). Hhypophosphatasia is an inherited metabolic bone disease characterised by defective skeletal mineralisation [PUBMED:17719863].
This entry also contains the related enzyme streptomycin-6-phosphate phosphatase (EC) (encoded by strK) from Streptomyces species. This enzyme is involved in the synthesis of the antibiotic streptomycin, specifically cleaving both streptomycin-6-phosphate and, more slowly, streptomycin-3-phosphate [PUBMED:1654502].
Gene Ontology
The mapping between Pfam and Gene Ontology is provided by InterPro. If you use this data please cite InterPro.
| Molecular function | phosphatase activity (GO:0016791) |
| Biological process | metabolic process (GO:0008152) |
Domain organisation
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
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Pfam Clan
This family is a member of clan Alk_phosphatase (CL0088), which contains the following 9 members:
Alk_phosphatase DUF1501 DUF229 Metalloenzyme PglZ Phosphodiest Phosphoesterase Sulfatase Sulfatase_CAlignments
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the NCBI sequence database, and our metagenomics sequence database. More...
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Meta (602) |
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| RP15 (299) |
RP35 (560) |
RP55 (845) |
RP75 (1070) |
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| PP/heatmap | 1 | |||||||
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| Seed (10) |
Full (3561) |
Representative proteomes | NCBI (2985) |
Meta (602) |
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| RP15 (299) |
RP35 (560) |
RP55 (845) |
RP75 (1070) |
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| Gzipped | ||||||||
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
External links
MyHits provides a collection of tools to handle multiple sequence alignments. For example, one can refine a seed alignment (sequence addition or removal, re-alignment or manual edition) and then search databases for remote homologs using HMMER3.
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This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
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Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
Curation
| Seed source: | Prosite |
| Previous IDs: | alk_phosphatase; |
| Type: | Domain |
| Author: | Finn RD |
| Number in seed: | 10 |
| Number in full: | 3561 |
| Average length of the domain: | 329.70 aa |
| Average identity of full alignment: | 28 % |
| Average coverage of the sequence by the domain: | 81.45 % |
HMM information
| HMM build commands: |
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 23193494 -E 1000 --cpu 4 HMM pfamseq
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| Model details: |
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| Model length: | 421 | ||||||||||||
| Family (HMM) version: | 15 | ||||||||||||
| Download: | download the raw HMM for this family |
Species distribution
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Interactions
There is 1 interaction for this family. More...
Alk_phosphataseStructures
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the Alk_phosphatase domain has been found. There are 200 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.
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