Origin of the biochemical term, Pi (inorganic phosphate)

Origin of the biochemical term, Pi (inorganic phosphate)

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I would like to know when the term Pi (inorganic phosphate) was introduced in the representation of biochemical reactions, how it was originally defined, and the justification given then for using it rather than an individual species of phosphate.

(I would also be interested in the current justification, but that's probably another question.)

Let me provide some background to my question. Phosphoric acid (H3PO4) has three ionizations, which produce successively the species: dihydrogen phosphate (H2PO4-), monohydrogen phosphate (HPO42-) and orthophosphate (PO43-). At pH 7.4, according to the Wikipedia entry on phosphate, the main species are the mono- and di-hydrogen phosphates (61% and 39% respectively). The term Pi must have been introduced in the 1950s at latest (perhaps before the war), at a time when there would have been little knowledge of the nature of the species involved in reactions involving phosphate - certainly not at the active sites of enzymes.

One of the reasons I am curious to know how the term was introduced is the extent to which it persists in 21st century biochemical text books, where it would seem that many authors either do not know or do not care to explain to their readers why they are still using it at a time when much more is known about the reaction mechanisms. Neither of two well-known texts explain the different ionizations of phosphate, and give only parenthetical definitions in terms of a single species - different in each case: Berg et al. referred to Pi as orthophosphate, whereas Nelson and Cox's, Lehninger Principles of Biochemistry referred to it as HPO42-.

Acknowledgement: This question was provoked by the SE-Biology question - Where is the H+ ion in this step of glycolysis coming from?

This terminology is at least as old as September 1944 when Enzymatic Synthesis of Acetyl Phosphate Journal of Biological Chemistry 155, 55-70 was published by Lipmann, which says:

Inorganic phosphate, referred to as Pi, was estimated colorimetrically

See also the definition of "inorganic phosphate" and "orthophosphate" from this 1943 University of Wisconsin Thesis:

Compound: Inorganic phosphate or orthophosphate

Definition: Phosphate whose calcium salt is insoluble in water-alcohol mixtures under the conditions to be described. E.G. NaH2PO4

In other words, "orthophosphate" was a generic term for mono, di, or tri basic phosphate. It did not have the narrower meaning attributed in the OP.

2: Overview of Phosphate Groups

  • Contributed by Tim Soderberg
  • Emeritus Associate Professor of Chemistry at University of Minnesota Morris

Phosphate is everywhere in biochemistry. As we were reminded in the introduction to this chapter, our DNA is linked by phosphate:

The function of many proteins is regulated - switched on and off - by enzymes which attach or remove a phosphate group from the side chains of serine, threonine, or tyrosine residues.

Countless diseases are caused by defects in phosphate transferring enzymes. As just one example, achondroplasia, a common cause of dwarfism, is caused by a defect in an enzyme whose function is to transfer a phosphate to a tyrosine residue in a growth-related signaling protein.

Finally, phosphates are excellent leaving groups in biological organic reactions, as we will see many times throughout the remainder of this book.

Clearly, an understanding of phosphate chemistry is central to the study of biological organic chemistry. We'll begin with an overview of terms used when talking about phosphates.

I. Introduction

Phosphorus is an essential nutrient for all living organisms, representing one of the nine macronutrients present in large quantities in plant tissues. Phosphorus is taken up by plants in the form of inorganic phosphate (Pi for a list of abbreviations used in this article, see Table 1) (Fig. 1a). Pi is an essential building block for many cellular components, such as nucleic acids and membranes, is a major component of molecules that function as the energy currency of the cell, and is an important signaling molecule. Hence, Pi deficiency can affect a wide range of biological processes, ultimately affecting plant growth and development (Rouached et al., 2010 ).

Abbreviation Definition
CHAD C-terminal conserved α-helical domain
CK Casein kinase
InsP Inositol polyphosphate
IP6K Inositol hexakisphosphate kinase
N Nitrogen
NUDTs Nudix hydrolase family
P Phosphorus
PAP Purple acid phosphatase
PH domain Pleckstrin homology domain
PHO pathway Phosphate signal transduction pathway (yeast)
Pi Inorganic phosphate
PIP Phosphoinositide
PolyP Inorganic polyphosphate
PP-InsP Inositol pyrophosphate
PPIP5K Diphosphoinositol pentakisphosphate kinase
PPK PolyP kinase
PPN Endopolyphosphatase
pppGpp Guanosine pentaphosphate
PPX Exopolyphosphatase
PSR Phosphate starvation response (plants)
TTM Triphosphate tunnel metalloenzyme
VTC Vacuolar transporter chaperone

Plants store Pi in their vacuoles and relocate it to the cytosol when intracellular Pi concentrations are low (Liu et al., 2016 ). Some plant tissues, such as seeds and fruits, can store Pi in the form of phytic acid (inositol hexakisphosphate, InsP6 Fig. 1b) (Secco et al., 2017 ). Phytic acid is one of several inositol multiphosphorylated compounds present in plants, and these compounds share the D-myo-inositol ring-bearing ester phosphate group at one or more positions. While InsP6 may represent a storage form of Pi in plants, higher phosphorylated inositol pyrophosphates act as signaling molecules in plants (Laha et al., 2015 Wild et al., 2016 Zhu et al., 2019 ) and many other eukaryotes (Azevedo & Saiardi, 2017 ).

Inorganic polyphosphates (polyPs) exist in many prokaryotes and eukaryotes, and form a major store of Pi, for example, in yeast (Urech et al., 1978 ). PolyPs form linear chains that vary in length from 3 to c. 1000 Pi units and are linked by energy-rich phosphoanhydride bonds (Fig. 1c). Depleting inositol pyrophosphates (PP-InsPs) in yeast results in a massive decrease in polyP concentrations (Lonetti et al., 2011 ). This suggests that Pi, InsP and polyP metabolism are functionally connected, especially in sessile, soil-living organisms. However, it is presently unclear whether polyPs exist in plants and whether they contribute to Pi metabolism and storage. Here, we critically review our current knowledge concerning polyPs in plants, and the roles of PP-InsPs in Pi sensing and cell signaling.


The phosphate ion has a molar mass of 94.97 g/mol, and consists of a central phosphorus atom surrounded by four oxygen atoms in a tetrahedral arrangement. It is the conjugate base of the hydrogen phosphate ion H(PO
4 ) 2−
, which in turn is the conjugate base of the dihydrogen phosphate ion H
2 (PO
4 ) −
, which in turn is the conjugate base of orthophosphoric acid, H
3 PO
4 .

Many phosphates are soluble in water at standard temperature and pressure. The sodium, potassium, rubidium, caesium, and ammonium phosphates are all water-soluble. Most other phosphates are only slightly soluble or are insoluble in water. As a rule, the hydrogen and dihydrogen phosphates are slightly more soluble than the corresponding phosphates.

Equilibria in solution Edit

In water solution, orthophosphoric acid and its three derived anions coexist according to the dissociation and recombination equilibria below [3]

Equilibrium Dissociation constant Ka [4] pKa
H3PO4 ⇌ H
2 PO −
4 + H +
Ka1 = [ H +
] [ H
2 PO −
4 ] / [ H
3 PO
4 ] ≈ 7.5 × 10 −3
pKa1 = 2.14
2 PO −
4 ⇌ HPO 2−
4 + H +
Ka2 = [ H +
] [ HPO 2−
4 ] / [ H
2 PO −
4 ] ≈ 6.2 × 10 −8
pKa2 = 7.20
HPO 2−
4 ⇌ PO 3−
4 + H +
Ka3 = [ H +
] [ PO 3−
4 ] / [ HPO 2−
4 ] ≈ 2.14 × 10 −13
pKa3 = 12.37

Values are at 25 °C and 0 ionic strength.

The pKa values are the pH values where the concentration of each species is equal to that of its conjugate bases. At pH 1 or lower, the phosphoric acid is practically undissociated. Around pH 4.7 (mid-way between the first two pKa values) the dihydrogen phosphate ion, [H
2 PO
4 ] −
, is practically the only species present. Around pH 9.8 (mid-way between the second and third pKa values) the monohydrogen phosphate ion, [HPO
4 ] 2−
, is the only species present. At pH 13 or higher, the acid is completely dissociated as the phosphate ion, (PO
4 ) 3−

This means that salts of the mono- and di-phosphate ions can be selectively crystallised from aqueous solution by setting the pH value to either 4.7 or 9.8.

In effect, H
3 PO
4 , H
2 (PO
4 ) −
and H(PO
4 ) 2−
behave as separate weak acids because the successive pKa differ by more than 4.

Phosphate can form many polymeric ions such as pyrophosphate, (P
2 O
7 ) 4−
, and triphosphate, (P
3 O
10 ) 5−
. The various metaphosphate ions (which are usually long linear polymers) have an empirical formula of (PO
3 ) −
and are found in many compounds.

Biochemistry of phosphates Edit

In biological systems, phosphorus can be found as free phosphate anions in solution (inorganic phosphate) or bound to organic molecules as various organophosphates.

Inorganic phosphate is generally denoted Pi and at physiological (homeostatic) pH primarily consists of a mixture of [HPO
4 ] 2−
and [H
2 PO
4 ] −
ions. At a neutral pH, as in the cytosol (pH = 7.0), the concentrations of the orthophoshoric acid and its three anions have the ratios

Thus, only [H
2 PO
4 ] −
and [HPO
4 ] 2−
ions are present in significant amounts in the cytosol (62% [H
2 PO
4 ] −
, 38% [HPO
4 ] 2−
). In extracellular fluid (pH = 7.4), this proportion is inverted (61% [HPO
4 ] 2−
, 39% [H
2 PO
4 ] −

Inorganic phosphate can be present also as of pyrophosphate anions [P
2 O
7 ] 4−
, which can give orthophosphate by hydrolysis:

Organic phosphates are commonly found in the form of esters as nucleotides (e.g. AMP, ADP, and ATP) and in DNA and RNA. Free orthophosphate anions can be released by the hydrolysis of the phosphoanhydride bonds in ATP or ADP. These phosphorylation and dephosphorylation reactions are the immediate storage and source of energy for many metabolic processes. ATP and ADP are often referred to as high-energy phosphates, as are the phosphagens in muscle tissue. Similar reactions exist for the other nucleoside diphosphates and triphosphates.

Bones and teeth Edit

An important occurrence of phosphates in biological systems is as the structural material of bone and teeth. These structures are made of crystalline calcium phosphate in the form of hydroxyapatite. The hard dense enamel of mammalian teeth consists of fluoroapatite, a hydroxy calcium phosphate where some of the hydroxyl groups have been replaced by fluoride ions.

Medical and biological research uses Edit

The medicinal type (salt) of phosphorus is phosphate. Some phosphates, which help cure many urinary tract infections, are used to make urine more acidic. To avoid the development of calcium stones in the urinary tract, some phosphates are used. [5] For patients who are unable to get enough phosphorus in their daily diet, phosphates are used as dietary supplements, usually because of certain disorders or diseases. [5] Injectable phosphates can only be handled by a health care provider. [5]

Plant metabolism Edit

Plants take up phosphorus through several pathways: the arbuscular mycorrhizal pathway and the direct uptake pathway.

Hyperphosphatemia, or high level of phosphates in blood, is associated with elevated mortality in the general population. Hyperphosphatemia is generally caused by phosphate additives, that is phosphates that are added to food preparations, as phosphates that are naturally present in food are not completely absorbed by the gastro-intestinal tract. Phosphates induce vascular calcification, and a high concentration of phosphates in blood was found to be a predictor of cardiovascular events. [6]

Phosphates are commonly used as additives in industrially processed food and fast food. Fast food and ready-to-eat processed foods are the main contributors of the rising consumption of phosphate among the population. Phosphates additives are also commonly found in flavoured soft drinks as well as certain diary products. [6]

Geological occurrence Edit

Phosphates are the naturally occurring form of the element phosphorus, found in many phosphate minerals. In mineralogy and geology, phosphate refers to a rock or ore containing phosphate ions. Inorganic phosphates are mined to obtain phosphorus for use in agriculture and industry. [2]

The largest global producer and exporter of phosphates is Morocco. Within North America, the largest deposits lie in the Bone Valley region of central Florida, the Soda Springs region of southeastern Idaho, and the coast of North Carolina. Smaller deposits are located in Montana, Tennessee, Georgia, and South Carolina. The small island nation of Nauru and its neighbor Banaba Island, which used to have massive phosphate deposits of the best quality, have been mined excessively. Rock phosphate can also be found in Egypt, Palestine, Western Sahara, Navassa Island, Tunisia, Togo, and Jordan, countries that have large phosphate-mining industries.

Phosphorite mines are primarily found in:

In 2007, at the current rate of consumption, the supply of phosphorus was estimated to run out in 345 years. [7] However, some scientists thought that a "peak phosphorus" will occur in 30 years and Dana Cordell from Institute for Sustainable Futures said that at "current rates, reserves will be depleted in the next 50 to 100 years". [8] Reserves refer to the amount assumed recoverable at current market prices, and, in 2012, the USGS estimated 71 billion tons of world reserves, while 0.19 billion tons were mined globally in 2011. [9] Phosphorus comprises 0.1% by mass of the average rock [10] (while, for perspective, its typical concentration in vegetation is 0.03% to 0.2%), [11] and consequently there are quadrillions of tons of phosphorus in Earth's 3 * 10 19 ton crust, [12] albeit at predominantly lower concentration than the deposits counted as reserves from being inventoried and cheaper to extract if it is assumed that the phosphate minerals in phosphate rock are hydroxyapatite and fluoroapatite, phosphate minerals contain roughly 18.5% phosphorus by weight and if phosphate rock contains around 20% of these minerals, the average phosphate rock has roughly 3.7% phosphorus by weight.

Some phosphate rock deposits, such as Mulberry in Florida, [13] are notable for their inclusion of significant quantities of radioactive uranium isotopes. This syndrome is noteworthy because radioactivity can be released into surface waters [14] in the process of application of the resultant phosphate fertilizer (e.g. in many tobacco farming operations in the southeast US).

In December 2012, Cominco Resources announced an updated JORC compliant resource of their Hinda project in Congo-Brazzaville of 531 Mt, making it the largest measured and indicated phosphate deposit in the world. [15]

Mining Edit

The three principal phosphate producer countries (China, Morocco and the United States) account for about 70% of world production.


The first plant phytase was found in 1907 from rice bran [3] [4] and in 1908 from an animal (calf's liver and blood). [4] [5] In 1962 began the first attempt at commercializing phytases for animal feed nutrition enhancing purposes when International Minerals & Chemicals (IMC) studied over 2000 microorganisms to find the most suitable ones for phytase production. This project was launched in part due to concerns about mineable sources for inorganic phosphorus eventually running out (see peak phosphorus), which IMC was supplying for the feed industry at the time. Aspergillus (ficuum) niger fungal strain NRRL 3135 (ATCC 66876) was identified as a promising candidate [6] as it was able to produce large amounts of extracellular phytases. [7] However, the organism's efficiency was not enough for commercialization so the project ended in 1968 as a failure. [6]

Still, identifying A. niger led in 1984 to a new attempt with A. niger mutants made with the relatively recently invented recombinant DNA technology. This USDA funded project was initiated by Dr. Rudy Wodzinski who formerly participated in the IMC's project. [6] This 1984 project led in 1991 to the first partially cloned phytase gene phyA (from A. niger NRRL 31235) [6] [8] and later on in 1993 to the cloning of the full gene and its overexpression in A. niger. [6] [9]

In 1991 BASF began to sell the first commercial phytase produced in A. niger under the trademark Natuphos which was used to increase the nutrient content of animal feed. [6]

In 1999 Escherichia coli bacterial phytases were identified as being more effective than A. niger fungal phytases. [6] [10] [11] Subsequently, this led to the animal feed use of this new generation of bacterial phytases which were superior to fungal phytases in many aspects. [6]

Four distinct classes of phytase have been characterized in the literature: histidine acid phosphatases (HAPS), beta-propeller phytases (BPPs), purple acid phosphatases (PAPs), [2] and most recently, protein tyrosine phosphatase-like phytases (PTP-like phytases). [12]

Histidine acid phosphatases (HAPs) Edit

Most of the known phytases belong to a class of enzyme called histidine acid phosphatases (HAPs). HAPs have been isolated from filamentous fungi, bacteria, yeast, and plants. [1] All members of this class of phytase share a common active site sequence motif (Arg-His-Gly-X-Arg-X-Pro) and have a two-step mechanism that hydrolyzes phytic acid (as well as some other phosphoesters). [2] The phytase from the fungus Aspergillus niger is a HAP and is well known for its high specific activity and its commercially marketed role as an animal feed additive to increase the bioavailability of phosphate from phytic acid in the grain-based diets of poultry and swine. [13] HAPs have also been overexpressed in several transgenic plants as a potential alternative method of phytase production for the animal feed industry [14] and very recently, the HAP phytase gene from E. coli has been successfully expressed in a transgenic pig. [15]

Β-propeller phytases Edit

β-propeller phytases make up a recently discovered class of phytase. These first examples of this class of enzyme were originally cloned from Bacillus species, [2] but numerous microorganisms have since been identified as producing β-propeller phytases. The three-dimensional structure of β-propeller phytase is similar to a propeller with six blades. Current research suggests that β-propeller phytases are the major phytate-degrading enzymes in water and soil, and may play a major role in phytate-phosphorus cycling. [16]

Purple acid phosphatases Edit

A phytase has recently been isolated from the cotyledons of germinating soybeans that has the active site motif of a purple acid phosphatase (PAP). This class of metalloenzyme has been well studied and searches of genomic databases reveal PAP-like sequences in plants, mammals, fungi, and bacteria. However, only the PAP from soybeans has been found to have any significant phytase activity. The three-dimensional structure, active-site sequence motif and proposed mechanism of catalysis have been determined for PAPs. [ citation needed ]

Protein tyrosine phosphatase-like phytases Edit

Only a few of the known phytases belong to a superfamily of enzymes called protein tyrosine phosphatases (PTPs). PTP-like phytases, a relatively newly discovered class of phytase, have been isolated from bacteria that normally inhabit the gut of ruminant animals. [17] All characterized PTP-like phytases share an active site sequence motif (His-Cys-(X)5-Arg), a two-step, acid-base mechanism of dephosphorylation, and activity towards phosphrylated tyrosine residues, characteristics that are common to all PTP superfamily enzymes. [18] [19] Like many PTP superfamily enzymes, the exact biological substrates and roles of bacterial PTP-like phytases have not yet been clearly identified. The characterized PTP-like phytases from ruminal bacteria share sequence and structural homology with the mammalian PTP-like phosphoinositide/-inositol phosphatase PTEN, [12] and significant sequence homology to the PTP domain of a type III-secreted virulence protein from Pseudomonas syringae (HopPtoD2). [20]

Substrate specificity Edit

Most phytases show a broad substrate specificity, having the ability to hydrolyze many phosphorylated compounds that are not structurally similar to phytic acid such as ADP, ATP, phenyl phosphate, fructose 1,6-bisphosphate, glucose 6-phosphate, glycerophosphate and 3-phosphoglycerate. Only a few phytases have been described as highly specific for phytic acid, such as phytases from Bacillus sp., Aspergillus sp., E. coli [21] and those phytases belonging to the class of PTP-like phytases [18]

Pathways of phytic acid dephosphorylation Edit

Phytic acid has six phosphate groups that may be released by phytases at different rates and in different order. Phytases hydrolyze phosphates from phytic acid in a stepwise manner, yielding products that again become substrates for further hydrolysis. Most phytases are able to cleave five of the six phosphate groups from phytic acid. Phytases have been grouped based on the first phosphate position of phytic acid that is hydrolyzed. The Enzyme Nomenclature Committee of the International Union of Biochemistry recognizes three types of phytases based on the position of the first phosphate hydrolyzed, those are 3-phytase (EC, 4-phytase (EC, and 5-phytase (EC To date, most of the known phytases are 3-phytases or 4-phytases, [21] only a HAP purified from lily pollen [22] and a PTP-like phytase from Selenomonas ruminantium subsp. lactilytica [20] have been determined to be 5-phytases.

Phytic acid and its metabolites have several important roles in seeds and grains, most notably, phytic acid functions as a phosphorus store, as an energy store, as a source of cations and as a source of myo-inositol (a cell wall precursor). Phytic acid is the principal storage forms of phosphorus in plant seeds and the major source of phosphorus in the grain-based diets used in intensive livestock operations. The organic phosphate found in phytic acid is largely unavailable to the animals that consume it, but the inorganic phosphate that phytases release can be easily absorbed. Ruminant animals can use phytic acid as a source of phosphorus because the bacteria that inhabit their gut are well characterized producers of many types of phytases. However, monogastric animals do not carry bacteria that produce phytase, thus, these animals cannot use phytic acid as a major source of phosphorus and it is excreted in the feces. [23] However, human—especially vegetarians and vegans due to increased gut microbiome adaptation—can have microbes in their gut that can produce phytase that break down phytic acid. [24]

Phytic acid and its metabolites have several other important roles in Eukaryotic physiological processes. As such, phytases, which hydrolyze phytic acid and its metabolites, also have important roles. Phytic acid and its metabolites have been implicated in DNA repair, clathrin-coated vesicular recycling, control of neurotransmission and cell proliferation. [25] [26] [27] The exact roles of phytases in the regulation of phytic acid and its metabolites and the resulting role in the physiological processes described above are still largely unknown and the subject of much research.

Phytase has been reported to cause hypersensitivity pneumonitis in a human exposed while adding the enzyme to cattle feed. [28] [29]

Phytase is produced by bacteria found in the gut of ruminant animals (cattle, sheep) making it possible for them to use the phytic acid found in grains as a source of phosphorus. [30] Non-ruminants (monogastric animals) like human beings, dogs, pigs, birds, etc. do not produce phytase. Research in the field of animal nutrition has put forth the idea of supplementing feed with phytase so as to make available to the animal phytate-bound nutrients like calcium, phosphorus, minerals, carbohydrates, amino acids and proteins. [31] In Canada, a genetically modified pig called Enviropig, which has the capability to produce phytase primarily through its salivary glands, was developed and approved for limited production. [32] [33]

Phytase is used as an animal feed supplement – often in poultry and swine – to enhance the nutritive value of plant material by liberation of inorganic phosphate from phytic acid (myo-inositol hexakisphosphate). Phytase can be purified from transgenic microbes and has been produced recently in transgenic canola, alfalfa and rice plants. [34]


JANE E. AUBIN , . JOHAN N.M. HEERSCHE , in Cellular and Molecular Biology of Bone , 1993

1 Alkaline Phosphatase (APase EC

Alkaline phosphatase is an ectoenzyme that can hydrolyze monophosphate esters at a high pH optimum (for general review, see Wuthier and Register, 1984 Harris, 1989 Whyte, 1989 ). Structural studies have indicated that APase interacts with specific phospholipids on the osteoblast plasma membrane (for review, see Cross, 1987 Ferguson and Williams, 1988 Low, 1989a , b ). Alkaline phosphatase belongs to a growing class of cell surface proteins (including Thy-1 and N-CAM) that are covalently bound to phosphatidyl inositol (PI) phospholipid complexes in the plasma membrane. Thus, membrane-bound APase can be released from cells (e.g., the ROS and UMR cell lines) by PI-specific phospholipase C ( Noda et al., 1987 Turksen and Aubin, 1991 ).

Although to date the precise physiological role of this enzyme in bone is not known, APase activity is present when cells become recognized as preosteoblast and osteoblast, but it is absent from the osteocyte ( Doty and Schofield, 1976 ). It is generally accepted that as the specific activity of APase in a population of bone cells increases there is a corresponding shift to a more differentiated state, and the level of osteoblastic APase has been routinely used in in vitro experiments as a relative marker of osteoblast differentiation ( Rodan and Rodan, 1984 ). Recently, a number of studies have suggested that PI-linked proteins may be involved in transmembrane signaling (for review, see Saltier et al., 1989 Low, 1989a , b ). Therefore, it is interesting to speculate that APase may play a role in the regulation of osteoblast differentiation. A more commonly ascribed function for APase is in mineralization however, controversy over its precise role in this process still exists (for a recent discussion, see Heersche et al., 1990 ). In keeping with the histological observation that APase is already present on preosteoblastic cells prior to their assuming the cuboidal shape typically associated with the mature osteoblast, a variety of recent studies also suggest that APase expression appears in differentiating osteoblastic cells prior to expression of the matrix molecule osteocalcin ( Bronckers et al., 1987 Aronow et al., 1990 Owen et al., 1990 ).

Illustrating metabolic sources and sinks of phosphate

What therefore might be the cellular sources of phosphates, and can they now be precisely identified? We systematically investigated the central carbon derived metabolic pathways (as obtained from KEGG [Ogata et al., 1999]) to identify possible cellular phosphate sources and sinks, using an example of yeast metabolic networks. From this comprehensive analysis, trehalose, serine, chorismate, inositol, and glycerol synthesis, all of which are accompanied by the release of Pi, represent the likely examples of phosphate sources by satisfying all required criteria as defined earlier (Figure 3C). Note here that none of these metabolites – trehalose, serine, chorismate, inositol or glycerol – contain phosphates. Instead, all of these metabolites are made via intermediate reactions where Pi is released, illustrating this idea of what a Pi source might be (and that this need not be a phosphate-containing metabolite). Trehalose is a major ‘storage’ carbohydrate that accumulates in cells at very high concentrations (Chester, 1963 François and Parrou, 2001 Stewart et al., 1950), and a recent report provides strong evidence of it being a phosphate-restoration source (Gupta et al., 2019 Gupta and Laxman, 2020). Trehalose is made from the sugar phosphates, glucose-6-phosphate (G6P) and UDP-glucose in two steps, where the last step (catalyzed by Tps2) results in trehalose formation and the release of one molecule of Pi (Figure 3C). In glucose replete conditions, these two steps are involved in futile cycling of trehalose during glycolysis, and trehalose synthesis allows proper glycolytic flux by maintaining phosphate balance (van Heerden et al., 2014b van Heerden et al., 2014a). Pertinently, dampening the PHO response (which mimics phosphate limitation), results in carbon (glucose) flux rerouted towards trehalose synthesis, and away from the pentose phosphate pathway (PPP) and glycolysis (Gupta et al., 2019 Gupta and Laxman, 2020). This is largely governed by mass action, with no changes in glycolytic/PPP enzyme amounts required. Multiple lines of evidence indicate that rerouting metabolic flux towards trehalose restores the Pi balance (Gupta et al., 2019). Further, if the last step in trehalose synthesis (which releases phosphate) is removed (tps2Δ cells), this decreases the cellular phosphate pools (Gupta et al., 2019). Taken together, all these data satisfy the criteria for trehalose to be a phosphate source. A related, predictive case can be made for glycerol, which is synthesized during the lower arm of glycolysis and is accompanied by the release of Pi. Glycerol biosynthesis from dihydroxyacetone phosphate (DHAP) occurs in two steps, where the first step results in the synthesis of glycerol-3-phosphate (catalyzed by Gpd1 and Gpd2), and the second step results in the synthesis of glycerol and the release of one Pi molecule by the action of glycerol-3-phosphatases (Hor2 and Rhr2) (Austin and Mayer, 2020 Figure 3C). In line with the role of glycerol as a phosphate source, glycerol levels are elevated in response to phosphate limitation (Kazemi Seresht et al., 2011). Moreover, consistent with a role of trehalose and glycerol syntheses in phosphate restoration, the metabolic phosphatases involved in these biochemical conversions, trehalose-6-phosphate synthase/phosphatase and glycerol-3-phosphatases, respectively, are also transcriptionally upregulated during phosphate limitation (Ogawa et al., 2000). This suggests an amplified response (through the transcriptional induction of these enzymes) as a way to mobilize metabolic reserves of Pi. Thus, when phosphates are transiently limited, glucose-6-phosphate and dihydroxyacetone phosphate will reroute away from glycolysis and the PPP, and toward trehalose and glycerol respectively. While this can restore phosphate balance, it will also result in a reduction of the overall glycolytic and PPP flux.

Three other major biosynthetic processes derived from glucose metabolism result in abundant products and also release phosphate. Serine biosynthesis from 3-phosphoglycerate (3PG) occurs in three steps, and the last step releases one Pi molecule. Chorismate synthesis (a precursor for aromatic amino acids and the vitamins, p-aminobenzoate and p-hydroxybenzoate) requires one molecule of erythrose-4-P (E4P) (from the pentose phosphate pathway) and two molecules of phosphoenolpyruvate (PEP), and occurs in seven steps. Here, three molecules of Pi are released (Figure 3C). The prediction therefore is that flux towards these molecules will increase when phosphates are limiting. Indeed, consistent with this prediction, independent studies with phosphate starvation have all observed that amino acid amounts strongly increase (Boer et al., 2010 Gupta et al., 2019), along with reduced metabolites of the PPP and glycolysis. These data therefore are consistent with these metabolites acting as phosphate sources. Finally, a predictive case can be made for myo-inositol (inositol) biosynthesis as a source of phosphate. Inositol is perhaps the most abundantly present stereoisomer in yeast and mammalian cells (Desfougères et al., 2019). Inositol biosynthesis from glucose-6-phosphate (G6P) occurs in two steps. In the first step, Ino1 converts glucose-6-phosphate to inositol-1-phosphate. The second step is catalyzed by inositol monophosphatases (Inm1 and Inm2), and inositol-1-phosphate is converted to inositol and one molecule of Pi is released (Figure 3C). Although the inositol biosynthetic pathway is relatively poorly studied in yeast, and there is insufficient metabolic data in this (Pi relevant) context, inositol monophosphatase, Inm1, is upregulated under low phosphate conditions (Kazemi Seresht et al., 2011). Therefore, we can predict that this inositol synthesis, accompanied by Pi release, can help restore internal Pi levels. To summarize, phosphate sources can modulate the overall carbon flux distribution in a metabolic network depending on the internal phosphate levels, and will thereby determine the metabolic state of the cell.

What then are the sinks of phosphates? Nucleoside-, di-, tri-phosphates, NAD(P) + , inositol pyrophosphates, and polyphosphates (PolyP) are all putative phosphate sinks (and not sources). Is there evidence that is consistent with the plausibility of these metabolites as phosphate sinks? Going by the earlier described criteria of a sink, these metabolites are abundant in cells (Belenky et al., 2007 Ermakova et al., 1981 Gakière et al., 2018 Kukko and Saarento, 1983 Ljungdahl and Daignan-Fornier, 2012 Pestov et al., 2004 Rao et al., 1998). A second criterion is that upon phosphate limitation, their levels will reduce due to both decreased synthesis and increased degradation (mobilization), and this will release phosphate. The case for nucleotides as phosphate sinks: as sinks, the synthesis of these molecules should decrease in response to phosphate limitation, they should have a slow turnover rate, and should accumulate in abundance. These criteria appear to be well-satisfied for nucleotides (mono-, di-, tri phosphates). Phosphate limitation results in a rapid decrease in both their synthesis and steady-state levels (Ashihara et al., 1988 Boer et al., 2010 Klosinska et al., 2011). In a recent study, the reduction in (but not absence of) PHO gene expression (phosphate transporters, phosphatases etc.), which mimics phosphate limitation, also revealed reduced de novo nucleotide synthesis. This was not due to reduced amounts of nucleotide biosynthesis enzymes, but instead was governed by mass action (Gupta et al., 2019). Further, phosphate limitation also increases the amounts of 5’-, 3’-nucleotidases and nucleotide pyrophosphatase enzymes, which thereby increases nucleotide degradation (and will allow phosphate release) (Alipanah et al., 2018 Hammond et al., 2003 Kennedy et al., 2005 Misson et al., 2005 Rittmann et al., 2005 Shimano and Ashihara, 2006 Uhde-Stone et al., 2003 Wasaki et al., 2006 Yin et al., 2007). Phm8, a nucleotidase, is transcriptionally induced (a longer term response) upon carbon, nitrogen, and Pi starvation (Xu et al., 2013). The relavance of these nucleotidases (in the context of Pi sinks) will become clearer in the following paragraph.

Using very similar analyses, the phosphate moiety of NAD(P) + and its intermediate metabolites also become potential phosphate sinks. Although direct estimates of these molecules during phosphate limitation have not been made, we can predict that there will be reduced synthesis of these metabolites, as they utilize nucleotide triphosphates as their phosphate donor, and nucleotide synthesis itself decreases under these conditions. Indeed, consistent with this prediction, during low Pi conditions, pyridine nucleotides, NAD + and NAD + intermediates (NMN and NaMN) are broken down by specific nucleotidases, and this will also release phosphate (Bogan and Brenner, 2010). Indeed, somewhat overlooked reports suggest connections between phosphate signaling and NAD(P) + metabolism (Bogan and Brenner, 2010 Kato and Lin, 2014).

In this context of nucleotides, as well as NAD(P) + molecules as Pi sinks, two relevant points emerge. In an insightful review, Bogan and Brenner summarize that ribonucleoside monophosphates (which are potential substrates of 5’-nucleotidases) come from both salvage and de novo synthesis. The 5’-nucleotidases themselves allow the reverse reactions of nucleoside kinases (and therefore oppose salvage pathways, and nucleotide generation) (Bogan and Brenner, 2010). These opposing substrate cycles, and the relative activity of phosphorylation/dephosphorylation, determine how much nucleoside monophosphates (and eventually triphosphates) are present, or if they will be converted to nucleosides. A more recent review points to the fact that upon extreme Pi starvation, as a ‘last resort’ cells induce enzymes that release Pi from nucleotides, since it would otherwise be illogical for cells to deplete its pools of nucleotides, which are critical for future cell division (Austin and Mayer, 2020). These points are all entirely consistent with nucleotides (and NAD related molecules) being Pi sinks, which will serve to eventually release Pi if phosphate levels within cells are not restored.

Finally, inositol pyrophosphates and polyphosphates are likely phosphate sink candidates. Inositol pyrophosphates have the highest proportion of phosphate groups for any metabolite, containing more phosphates than carbon atoms (Saiardi, 2012), and they regulate the expression of PHO related transcripts in yeast (Auesukaree et al., 2005 Lee et al., 2008 Lee et al., 2007), suggesting cross-talk between inositol pyrophosphate metabolism and phosphate signaling. Polyphosphates (polyP) are linear polymers of many phosphate residues, ranging from few to hundreds, and are linked by the same high-energy phosphoanhydride bonds that are found in ATP. PolyPs are mainly stored inside the vacuole, with small pools also found in cytosol, nucleus and mitochondria (Gerasimaitė and Mayer, 2016 Saiardi, 2012). Evidence from some organisms (bacteria, yeasts) suggest that polyphosphates are effective phosphate reservoirs, and upon phosphate starvation, these stores are degraded by the action of exo- and endo-polyphosphatases to restore phosphate (Brown and Kornberg, 2008 Kulaev et al., 1999 Kulaev and Kulakovskaya, 2000 Shirahama et al., 1996). We would therefore predict these metabolites to collectively be intracellular phosphate sinks, with inositol phosphates and polyphosphates likely to be the major sinks. All these are now directly testable predictions.

Two broad points emerge when we define phosphate sources and sinks in this way. First, we reiterate a close coupling of glucose metabolism with internal phosphate homeostasis. Altering flux toward different arms of glucose metabolism – glycolysis, the pentose phosphate pathway, and trehalose/glycogen synthesis – will therefore result in substantial changes in phosphate release/utilization (as shown earlier in Figure 3C). Second, we can now with some confidence predict key metabolic state changes (based on metabolic flux through these defined sources and sinks) due to a transient phosphate limitation (or ‘squeeze’). Primarily, trehalose, aromatic amino acids/chorismate, serine, glycerol, and inositol (phosphate sources) will all increase, while nucleotides, polyphosphates and (some) inositol pyrophosphates (phosphate sinks) will decrease. Flux through glycolysis and the pentose phosphate pathway will correspondingly decrease. Amounts of these hallmark metabolites therefore represent a concise metabolic signature of a phosphate-limited cell. In summary, this conceptualization of the phosphate sources and sinks together with their biochemical reactions illustrate the importance of these metabolites in maintaining the overall phosphate levels in the cell. Second, mass-action driven metabolic processes play a fundamental role in phosphate homeostasis by regulating flux through the key phosphate-related metabolic nodes. Collective evidence therefore indicates that there is an intimate relationship and interdependence between phosphate balance and carbon metabolism.

What is an Inorganic Phosphate

An inorganic phosphate is a salt of phosphoric acid. Here, a phosphate group is connected to a metal cation. The phosphate atom is in the center surrounded by four oxygen atoms that are chemically bonded to the phosphorous atom. The phosphate group has an overall negative charge of -3. Therefore, it can form monobasic, dibasic and tribasic salts. The phosphate group has a tetrahedral arrangement.

Inorganic phosphates can be found naturally. Usually, these compounds can be found as salts of group 1 elements such as sodium, potassium, calcium, etc. There are two types of inorganic phosphate compounds: orthophosphates and condensed phosphates.

Figure 2: Diammonium Phosphate is an Inorganic Phosphate

Orthophosphates are reactive phosphate compounds. These are the simplest compounds among other phosphates and are composed of one phosphate unit. Hence these are also called as monophosphates. Condensed phosphates are composed of more than one phosphate unit.

Inorganic phosphates are also widely used as fertilizers. For example, Superphosphate and Triple super phosphate are common fertilizer substances.

Catalytic site nucleotide and inorganic phosphate dependence of the conformation of the epsilon subunit in Escherichia coli adenosinetriphosphatase

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1 (ECF1) has been found to be ligand-dependent, as measured indirectly by the activation of the enzyme that occurs on protease digestion, or when followed directly by monitoring the cleavage of this subunit using monoclonal antibodies. The cleavage of the epsilon subunit was fast in the presence of ADP alone, ADP + MG2+, ATP + EDTA, or AMP-PNP, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site(s). The half-maximal concentration of Pi required in the presence of ADP + Mg2+ to protect the epsilon subunit from cleavage by trypsin was 50 microM, which is in the range measured for the high-affinity binding of Pi to F1. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Mg2+ + Pi, the epsilon subunit cross-linked to beta in high yield. With ATP + EDTA or ADP + Mg2+ (no Pi), the yield of the beta-epsilon cross-linked product was much reduced. We conclude that the epsilon subunit undergoes a conformational change dependent on the presence of Pi. It has been found previously that binding of the epsilon subunit to ECF1 inhibits ATPase activity by decreasing the off rate of Pi [Dunn, S. D., Zadorozny, V. D., Tozer, R. G., & Orr, L. E. (1987) Biochemistry 26, 4488-4493]. This reciprocal relationship between Pi binding and epsilon-subunit conformation has important implications for energy transduction by the E. coli ATP synthase.


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. 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. [10]

The precise structure and function of the four isozymes (Int in E.coli) are solely geared to supply a source of inorganic phosphate when the environment lacks this metabolite. The four enzymes are dependent upon the location of the tissue expression. The four sites of tissue expression are the Intestinal AlP, Placental ALP, Germ Cell ALP and Liver/Bone/Kidney ALP. [11] The inorganic phosphates produced by these isozymes are then bound to carrier proteins which deliver the inorganic phosphates to a specific high-affinity transport system, known as the Pst system, which transports phosphate across the cytoplasmic membrane. [12]

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) [13] for translocation into the cytosol. [14] 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. [15]

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. [16] 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. [16] Alkaline phosphatase has a Km of 8.4 x 10 −4 . [17]

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 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 °C ALP has the uncommon characteristic of reverse denaturation. Due to this, while ALP 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. [10]

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. [18] 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 important information about enzymatic activity. [19] 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. [20]

The optimal pH for the activity of the E. coli enzyme is 8.0 [21] while the bovine enzyme optimum pH is slightly higher at 8.5. [22] Alkaline phosphatase accounts for 6% of all proteins in derepressed cells. [17]

Intragenic complementation Edit

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. [23] When particular mutant versions of alkaline phosphatase were combined, the heterodimeric enzymes formed as a result exhibited a higher level of activity than would be expected based on the relative activities of the parental enzymes. These findings indicated that the dimer structure of E.coli alkaline phosphatase allows cooperative interactions between the constituent monomers that can generate a more functional form of the holoenzyme.

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. [24] Typical uses in the lab for alkaline phosphatases include removing phosphate monoesters to prevent self-ligation, which is undesirable during plasmid DNA cloning. [25]

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. (CIP) (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. [26]
  • 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. [27]

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. [28]

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). [29]

There is a positive correlation between serum bone alkaline phosphatase (B-ALP) levels and bone formation in humans, although its use as a biomarker in clinical practice is not recommended. [30]

Ongoing research Edit

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. [31]

  • Alkaline phosphatase (AP) affects the inflammatory responses in patients with Chronic kidney disease and is directly associated with Erythropoiesis stimulating agent resistant anemia. [32]
  • Intestinal alkaline phosphatase (IAP) and the mechanism it uses to regulate pH and ATP hydrolysis in rat duodenum. [33]
  • Testing the effectiveness of the inhibitor and its impact on IAP in acute intestinal inflammation as well as explore the molecular mechanisms of IAP in "ameliorating intestinal permeability." [34]

Dairy industry Edit

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 failed pasteurization. [35] [36]

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, [37] 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. [38] 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 Edit

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. [11] Phosphate is another inhibitor which competitively inhibits alkaline phosphatase. [39]

Another known example of an alkaline phosphatase inhibitor is [(4-Nitrophenyl)methyl]phosphonic acid. [40]

In metal contaminated soil, alkaline phosphatase are inhibited by Cd (Cadmium). In addition, temperature enhances the inhibition of Cd on ALP activity, which is shown in the increasing values of Km. [41]

Physiology Edit

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. [42] Humans and most other mammals contain the following alkaline phosphatase isozymes:

    – intestinal (molecular weight of 150 kDa) – tissue-nonspecific (expressed mainly in liver, bone, and kidney) – placental (Regan isozyme)
  • ALPG – germ cell

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. [4]

Intestinal alkaline phosphatase Edit

Intestinal alkaline phosphatase (IAP) is secreted by enterocytes, and seems to play a pivotal role in intestinal homeostasis and protection [43] [44] as well as in suppressing inflammation [45] via repression of the downstream Toll-like receptor (TLR)-4-dependent and MyD88-dependent inflammatory cascade. [46] It dephosphorylates toxic/inflammatory microbial ligands like lipopolysaccharides (LPSs), [47] unmethylated cytosine-guanine dinucleotides, flagellin, and extracellular nucleotides such as uridine diphosphate or ATP. Dephosphorylation of LPS by IAP can reduce the severity of Salmonella tryphimurium and Clostridioides difficile infection restoring normal gut microbiota. [47] Thus, altered IAP expression has been implicated in chronic inflammatory diseases such as inflammatory bowel disease (IBD). [47] [48] It also seems to regulate lipid absorption [49] and bicarbonate secretion [50] in the duodenal mucosa, which regulates the surface pH.

In cancer cells Edit

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. [51]

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. [52] 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. [53] This explains why the addition of sodium butyrate show increased activity of alkaline phosphatase in the cancer cells of the human colon. [52] 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. [54] [55] 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. [56]

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).

ALP is much more elevated in metastatic prostate cancer cells than non-metastatic prostate cancer cells. [57] High levels of ALP in prostate cancer patients is associated with a significant decrease in survival. [57]

Levels are also elevated in people with untreated coeliac disease. [58] 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. [59]

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. [60]

Elevated levels Edit

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. [61] Placental alkaline phosphatase is elevated in seminomas [62] and active forms of rickets, as well as in the following diseases and conditions: [63]

Lowered levels Edit

The following conditions or diseases may lead to reduced levels of alkaline phosphatase: [65]

    , an autosomal recessive disease women receiving estrogen therapy because of aging
  • 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
  • Steroid treatment
  • Colitis

In addition, oral contraceptives have been demonstrated to reduce alkaline phosphatase. [66]

Prognostic uses Edit

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. [67]

Leukocyte alkaline phosphatase Edit

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.

  • Higher levels are seen in the physiological response, the leukemoid reaction, and in pathologies that include mature white blood cells, such as polycythemia vera (PV), essential thrombocytosis (ET), and in primary myelofibrosis (PM).
  • Lower levels are found in pathologies that involve undeveloped leukocytes, such as chronic myelogenous leukemia[68] (CML), paroxysmal nocturnal hemoglobinuria (PNH) and acute myelogenous leukaemia (AML).

Structure and properties Edit

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.

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. [4] 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. [69]

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