|Alkaline phosphatase E. coli K12|
|Ribbon model of the E. coli alkaline phosphatase dimer , according to PDB 1ALK|
AP, ALP, basic phosphatase
Existing structure data: 1aja , 1ajb , 1ajc , 1ajd , 1alh , 1ali , 1alj , 1alk , , 1anj 1ani, 1anj , 1b8j , 1ed8 , 1ed9 , 1elx , 1ely , 1elz , , 1ew8 1ew2, 1ew8 , 1ew9 , 1hjk , 1hqa , 1k7h , 1kh4 , 1kh5 , , 1kh9 1kh7, 1kh9 , 1khj , 1khk , 1khl , 1khn , 1shn , 1shq , , 1urb 1ura, 1urb , 1y6v , 1y7a , 1zeb , 1zed , 1zef , 2anh , , 2ga3 2g9y, 2ga3 , 2glq
|EC, category||126.96.36.199 , hydrolase|
|Substrate||Phosphate monoester + H 2 O|
|Products||Alcohol + phosphate|
Alkaline phosphatase ( AP , ALP , bone- specific also Ostase ) is the name for enzymes that hydrolyze phosphoric acid esters . Alkaline phosphatases remove phosphate groups ( dephosphorylation ) from many types of molecules such as proteins , nucleotides and alkaloids . They work most effectively at an alkaline pH .
Alkaline phosphatases are found in almost all living things, with the exception of a few plants. An increased laboratory value in humans can indicate a disease process such as biliary obstruction , internal organ damage, broken bones , a tumor or osteoporosis / osteomalacia ( rickets in children ).
The alkaline phosphatase hydrolyzes phosphoric acid esters to phosphate and alcohols, with protons being formed.
This enzyme works only in weakly alkaline solution with a pH optimum of 9.8. If the enzyme were added to an unbuffered solution, the reaction would come to a standstill because of the release of protons. In vitro it can therefore only be followed in the presence of a buffer with a suitable pH value.
Alkaline phosphatase is a cytoplasmic enzyme that occurs in almost all tissues and that plays an important role in metabolic dephosphorylation reactions. So far, five tissue-specific isoenzymes have been found, the activity optimum of which is at a pH of 9.8. In the case of liver and biliary dysfunction, skeletal disorders, some tumors and hyperthyroidism, the activity of the AP in the serum increases. The enzymatic activity is determined by the optical test.
In recombinant DNA technology, AP is used to remove terminal phosphate groups from the 5 'or 3' end or from both positions.
Even microorganisms have ALP or ALP-like enzymes.
The ALP consists of at least 500 amino acids or more, although the number can vary. The reactive ALP is shorter because it is shortened in the post-translational modification (PTM) and sugar molecules are added accordingly. This process is called glycosylation because this is how the organ-specific ALP isoenzyme is created.
Human tissue-unspecific ALP are particularly sensitive to the inhibitors levamisole and L- homoarginine and are inactivated at temperatures below 60 ° C. Levamisole is a non-competitive inhibitor of alkaline phosphatase. It also inhibits germ cell ALP and tissue-unspecific ALP. The inhibition is increased by higher concentrations of N-ethylaminoethanol and substrate. L- phenylalanine is a non-competitive inhibitor that specifically binds to the phosphoseryl intermediate of placental and small intestinal ALP. 5'-AMP inhibits placental ALP. Leucine is a non-competitive inhibitor of placental ALP, germ cell ALP, GAP. Imidazole is an inhibitor of placental, small bowel AP. Various organic phosphates are inhibitors, as are some reaction products.
|inhibition||Liver AP||Bone AP||Intestinal AP||Placenta AP|
|Phenylalanine inhibition (%)||0-10||0-10||75||75|
|Inhibition by homoarginine (%)||78||78||5||5|
|Inhibition by heat inactivation (%)||50-70||90-100||50-60||0|
The bacterial enzyme
In Gram-negative bacteria, the alkaline phosphatases are located in the periplasmic space. They are comparatively thermally stable .
The human enzyme
There are 15 different isoenzymes in humans . Four of these isoenzymes come from different genes (small intestine AP, placenta AP, germ cell AP and tissue-unspecific AP). The tissue-unspecific AP are glycosylated differently depending on the tissue , i.e. that is, they contain different sugar chains. This creates further isoenzymes (liver AP, bone AP, kidney AP).
The highest concentration of AP is found in humans in descending order in the intestinal mucosa, placenta, kidney and bone cells, and the liver.
Before the glycerol-3-phosphate is introduced into the citric acid cycle, the phosphate residue is split off at the third 3-C atom. This is where the ALP works.
Placental and placental-like alkaline phosphatase consists of 513 amino acids, with a 98% agreement. In contrast, the ALP in the liver, kidneys and bones consist of 507 amino acids. However, there are gaps in the amino acid sequences, giving a degree of agreement of 50 to 60%.
The ALP development arose from the non- tissue -specific (TNAP, tissue non- specific AP ) and the three tissue-specific ALPs. The three tissue-specific isoenzymes, IAP, GCAP and PLAP, emerged from these. An increased ALP level in the blood serum indicates a pathological disorder in the organism. In contrast, the ALP level in the human organism decreases as a result of natural aging. Basically, both growing children and women in the last trimester of pregnancy have higher ALP values. However, this is normal and does not provide a general indication of an illness.
Pregnant women and children have a higher value, as their bone formation, especially in the fetus, is not yet complete; the same applies to children until the end of puberty. Here, the released phosphoric acid is attached to the bone matrix as phosphates, which, as hydroxyapatite , gives it strength.
All of these enzymes are measured as "alkaline phosphatase" in standard blood tests and can provide information about existing diseases of the liver and skeleton.
The placental isoform (PLAP) is normally produced by placental syncytiotrophoblasts . Their determination is recommended for seminomas . The half-life in blood serum is less than three days.
For measurements at 37 ° C according to IFCC :
- Infants 110-590 IU / l
- Infants 110-550 IU / l
- Schoolchildren 130–700 IU / l
- Women 55-147 IU / l
- Men 62-176 IU / l
Alkaline phosphatases are present in large quantities in the skeletal system, in the liver parenchyma and in the bile duct epithelia . Values that are too high can be the cause B. in diseases of the liver , gallbladder , thyroid or pancreas . The value of the AP is also usually increased in the case of bone diseases such as osteomalacia , Paget's disease , rickets , bone metastases , hyperparathyroidism or fractures . One of the most common causes of an increase in AP is malignant tumors that have metastasized to the bones (bone metastases).
Basically, both growing children and women in the last trimester of pregnancy have higher AP values; however, this is normal and does not provide a general indication of a disease.
Too low an alkaline phosphatase content can be found e.g. B. in the rare hereditary disease hypophosphatasia ; in addition, as an accompanying symptom of a vitamin C deficiency ( scurvy ), as a result of a bypass operation, in the case of an underactive thyroid ( hypothyroidism ), Wilson's disease , zinc deficiency, severe anemia , magnesium deficiency and when taking contraceptives .
In CML, there are decreased alkaline phosphatase values in the granulocytes .
Application in biology
In biochemistry , alkaline phosphatase is used in conjunction with a chromogenic substrate for various detection methods (staining):
- Antibody staining in Western Blot for the detection of proteins
- in Northern Blot and Southern Blot for the detection of RNA or DNA
- in in situ hybridization for the detection of RNA
BCIP in connection with NBT is often used as a chromogenic substrate, which is converted by the alkaline phosphatase into a blue indigo dye.
In molecular biology , the alkaline phosphatase from calf intestines ( Calf Intestine Alkaline Phosphatase ) and from shrimp ( Shrimp Alkaline Phosphatase ) is used to dephosphorylate linear DNA.
The most common alkaline phosphatases are:
- Bacterial Alkaline Phosphatase (BAP), from Escherichia coli
- Calf Intestine Alkaline Phosphatase (CIAP), from calf intestines
- Shrimp Alkaline Phosphatase (SAP), from Arctic Shrimp ( Pandalus borealis )
Alkaline phosphatases were previously measured in Bodansky units .
Application in the dairy industry
Alkaline phosphatase is often used in the dairy industry as a method of detection of successful pasteurization. The most heat-resistant microorganism in milk, Mycobacterium paratuberculosis, denatures at temperatures below those of AP. If no AP activity is detected in the sample , the product has been successfully pasteurized. The measurement takes place in an alkaline medium at 37 ° C with disodium phenyl phosphate as substrate. If active AP is present in the sample, phenol is released, which is determined photometrically after reaction with 2,6-dibromoquinine-1,4-chlorimide ( Gibb's reagent ) and converted to the AP activity.
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- Alkaline phosphatase on ExPASy.org
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- Stability in blood samples published by the WHO (PDF file; 292 kB)
- HN Fernley, PG Walker: Studies on alkaline phosphatase. In: Biochem J. 104 (3), Sep 1967, pp. 1011-1018. PMC 1271245 (free full text)
- Stephen P. Coburn, J. Dennis Mahuren, Manas Jain, Yvonne Zubovic, Jacobo Wortsman: Alkaline Phosphatase (EC 188.8.131.52) in Serum Is Inhibited by Physiological Concentrations of Inorganic Phosphate. In: The Journal of Clinical Endocrinology & Metabolism. 83, 1998, p. 3951, doi: 10.1210 / jcem.83.11.5288 .
- K. McDougall, C. Plumb, WA King, A. Hahnel: Inhibitor Profiles of Alkaline Phosphatases in Bovine Preattachment Embryos and Adult Tissues. In: Journal of Histochemistry & Cytochemistry. 50, 2002, p. 415, doi: 10.1177 / 002215540205000312 .
- ↑ Uniprot: http://www.uniprot.org/
- ^ S. Iino, L. Fishman: The effect of sucrose and other carbohydrates on human alkaline phosphatase isoenzyme activity. In: Clinica Chimica Acta . Volume 92, Issue 2, March 1, 1979, pp. 197-207.
- ↑ a b A. Kozlenkov, T. Manes, MF Hoylaerts, JL Millán: Function assignment to conserved residues in mammalian alkaline phosphatases. In: The Journal of biological chemistry. Volume 277, Number 25, June 2002, pp. 22992-22999, doi: 10.1074 / jbc.M202298200 . PMID 11937510 .
- ↑ a b c W. H. Fishman: Perspectives on alkaline phosphatase isoenzymes. In: The American journal of medicine. Volume 56, Number 5, May 1974, pp. 617-650. PMID 4596648 .
- ^ Robert B. McComb: Alkaline Phosphatase. Springer Science & Business Media, 2013, ISBN 978-1-4613-2970-1 , p. 419.
- ^ H. Harris: The human alkaline phosphatases: what we know and what we don't know. In: Clinica Chimica Acta . Volume 186, Number 2, January 1990, pp. 133-150. PMID 2178806 (Review).
- ^ MH Le Du, JL Millan: Structural evidence of functional divergence in human alkaline phosphatases. In: The Journal of biological chemistry. Volume 277, Number 51, December 2002, pp. 49808-49814, doi: 10.1074 / jbc.M207394200 . PMID 12372831 .
- ^ WH Lee, CY Loo et al.: Osteoblast response to the surface of amino acid-functionalized hydroxyapatite. In: Journal of biomedical materials research. Part A. Volume 103, number 6, June 2015, pp. 2150–2160, doi: 10.1002 / jbm.a.35353 . PMID 25346517 .
- ↑ Reinhard Matissek, Gabriele Steiner, Markus Fischer: Food analysis. 5th edition. Springer Verlag, 2013, ISBN 978-3-642-34828-0 , p. 418.