http://myjms.mohe.gov.my/index.php/bsk
Copyright © 2021 Faculty of Health Science UKM. All right reserved Artikel Ulasan/Review Article
Role of Alkaline Phosphatase: An Overview
Peranan Alkali Fosfatase: Gambaran keseluruhan
VANITHA MARIAPPAN & KUMUTHA MALAR VELLASAMY
ABSTRACT
Phosphatases are extracellular enzymes that catalyze the hydrolysis of phospho-ester bonds inorganic phosphate- containing substrates and release inorganic phosphate in the arrangement of orthophosphates that able to be consumed by organisms. In the current years, there has been a major escalation in the number of studies and publications focusing on alkaline phosphatases (ALPs). This expansion reveals more complexity of the functions of this enzyme. This review highlights multiple roles played particularly on ALP in relation to human health, aquaculture, bacteria, and mammals. Continuous research on this multifaceted enzyme will undeniably offer better insight into understanding the overall function in every living organism.
Keywords: Alkaline phosphatase, phosphorylation, isoenzyme, phosphomonoesterase, multifaceted enzyme
ABSTRAK
Fosfotase adalah enzim luar selular yang memangkinkan hidrolisis ikatan fosfat-ester dalam substrat yang mengandungi organik fosfat dan melepaskan fosfat tak-organik dalam bentuk ortofosfates yang boleh diguna pakai oleh organisma. Beberapa tahun kebelakangan ini, terdapat peningkatan besar dalam jumlah kajian dan penerbitan yang memfokuskan pada alkali fosfatase (ALP). Pengembangan ini mendedahkan kerumitan fungsi enzim ini.
Ulasan ini menyoroti pelbagai peranan yang dimainkan terutamanya pada ALP berkaitan dengan kesihatan manusia, akuakultur, bakteria, dan mamalia. Penyelidikan berterusan mengenai enzim pelbagai aspek ini pasti akan memberikan gambaran yang lebih baik untuk memahami fungsi keseluruhan dalam setiap organisma hidup.
Kata kunci: alkali fosfatase, fosforilasi, isoenzim, fosfomonoesterase, enzim pelbagai segi
INTRODUCTION
Phosphatase is a crucial enzyme for all living organisms. It provides key enzymatic function involved in healthy cellular function, immune activation, and skeletal maintenance throughout the whole human body.
The term ‘phosphatase’ actually refers to a group of an enzyme that hydrolyzes phosphomonoesters and releasing free phosphate. Both specific and unspecific phosphatase exists (Vincent and Crowder 1995). The phosphatases are vital for the regulation of numerous metabolic processes that come about by phosphorylation and dephosphorylation, collectively with kinases (Sparks and Brautigan 1986). The phosphatases are found profusely in prokaryotic and eukaryotic species, as it regularly occurs in a number of isoforms (Moss 1992). One of the key roles of these systems is the enzyme transfer mechanism involving ATP and also important in its role in the formation of nucleic acid molecules, as a critical link in the mechanism for the regulation of enzymatic activity, and as a means of signal transcription within cells and between cells. The cleavage of protein phosphate monoesters is carried out
by a family of enzymes known as the protein phosphatases, which are responsible for the regulation of numerous biological processes (McComb et al.
1979).
Alkaline (EC 3.1.3.1) (ALP) and acid phosphatase (EC 3.1.3.2) (ACP) are similar to the carboxylesterases, a group of broadly disseminated enzymes, different somewhat with the source of the enzyme (Dixon and Webb 1979). The enzymes act on a varied series of monoesters of orthophosphoric acid, such as glycerol 1-phosphate and glycerol 2-phosphate (Dixon and Webb 1979). However, the enzymes do not react to diesters or triesters phosphoric. There is a major distinction between the two groups with regards to sulfur-containing esters (Dixon and Webb 1979). The ALP can hydrolase S-substituted monoesters of phosphorothioic acid, the ACPs appears to require oxygen in the linkage being spilled. On the other hand, ACP can hydrolase O-substituted monoesters of phosphorothioic acid, such as O-4- nitrophenyl thiophosphate (Dixon and Webb 1979). This may indicate that two-free-hydroxyl groups on the phosphate radical are required for the ALP enzyme activity.
ACPs act on single charged phosphate groups under
‘acidic’ condition and ALPs act on the double charged phosphate groups under ‘alkaline’ conditions.
Phosphatase is stimulated by hormones, cyclic AMP, vitamins A and D, calcitonin, parathyroid hormone, and steroids (McComb et al. 1979).
THE SPECIFICITY OF ALKALINE AND ACID PHOSPHATASE
Historically, the difference between acid and alkaline phosphatase rested on the simple observation that enzymes could be separated, which had optimal rates in completely different pH ranges in which these enzymes are active (Neumann 1968). Since the addition or deletion of various peptide groups with alteration of common enzymatic core might be expected to alter the effect of pH on the optimal rate, the enzymes should be examined for more fundamental differences in the
reaction mechanism. The hydrolysis of the O-substitute monoesters of phosphorothioic acid (ROPO2 SKH; R= - CH3, -nitrophenyl) not only does not occur but also O- p-nitrophenol thiophosphate was a potent inhibitor of ALP. ACP able to hydrolyze O-substituted monoesters phosphorothioic acid, however, did not hydrolyze S- substituted monoesters of phosphorothioic acid although under the identical condition (Boyer 1970).
ALKALINE PHOSPHATASE
There are thousands of different phosphatases widely distributed throughout nature. Of these, the most predominant and best understood are the ALP. ALP is also known as alkaline phosphomonoesterase and catalyzes the hydrolysis of many orthophosphoric monoester phosphohydrolase at alkaline pH to generate alcohol and orthophosphate as shown in Fig. 1 (McComb et al. 1979).
FIGURE 1 Schematic diagram of the alkaline phosphatase hydrolytic reaction.
ALP is a group of cell-membrane associated enzymes with hydrolase activity that acts on a variety of phosphatase substrates and it is the most studies among the phosphate ester hydrolases (McComb et al. 1979). In eukaryotes, the protein is normally bound to the cytoplasmic membrane by a modified amino acid, glycosyl-phosphatidylinositol (GPI-), in the proximity of the carboxyl membrane where wide-ranging transport takes place and specify that ALP is intricate in fundamental biological processes (Kim and Wyckof 1991). ALP removes 5’ phosphate groups from DNA, RNA, and proteins. In addition to this phosphatase activity, the enzyme can also catalyze certain trans- phosphorylation activity whereby a phosphoryl residue is transferred directly from a phosphate ester to acceptor alcohol (Trowsdale et al. 1990). In other words, the enzyme behaves as phosphohydrolases at a characteristically alkaline pH optimum and they also function as phosphotransferase at neutral pH.
ALP occurs in cell membranes throughout most of the organs and tissues of the body. The major organs
where high ALP levels is found are usually traced in the osteoblast cell of the bone, liver cells, intestinal mucosa, spleens, kidneys, placentas and bile ducts, which could hydrolyze nucleotides, hexosephosphates and glycerophosphate. The comparative dispersal of the activity of the enzyme has been described by Fernley (1971) as intestinal mucosa = placenta > kidney = bone
> liver = lung = spleen. Besides in the different organs, ALP is also found in invertebrates, fungi, bacteria, fishes, plants and mammals. In mammals, there are four different types of ALP isoenzymes which are known as intestinal, placental, placenta-like (germ cell type) and tissues non-specific (denoted to as the bone/ kidney/
liver) type (Trowsdale et al. 1990).
ROLE OF ALKALINE PHOSPHATASE
HEALTHY CELLULAR FUNCTION
Many cellular processes are controlled by phosphorylation-induced conformational deviations of
proteins. The grade of phosphorylation is resulted by the antagonist actions of phosphatases. This dephosphorylation is crucial since it permits the reversible nature, necessary in the regulation of biological systems (Vincent and Crowder 1995).
Phosphates are involved in the normal function of cell replication, including mitosis and metaphase, whereas abnormal phosphates function has been associated with the transformation of cells leading to tumor formation.
Phosphatases may also be involved in modulating the activity of a tumor suppressor protein called retinoblastoma protein. Phosphatase is also involved in cell adhesion (Vincent and Crowder 1995). Phosphatase appears to be a regulator of protein synthesis and insulin regulation and is involved in other metabolic processes, including glycogen storage (Vincent and Crowder 1995). Phosphatase is involved in the transport of thiamine, calcium, sodium, potassium, inorganic phosphate, fat, carbohydrates, proteins, and water (McComb et al. 1979). Phosphatase acts on the main muscle protein, myosin, causing the contraction of skeletal, cardiac, and smooth muscle (Vincent and Crowder 1995).
HEALTHY BONE FORMATION AND BONE MAINTENANCE
Phosphatase was speculated to be responsible for liberating phosphate esters leading to the deposition of calcium phosphate salts in human bone. In the 1920s, Robinson observed large quantities of phosphatase in bone, especially growing bone (1923). In the same period, Kay demonstrated that serum ALP activity in many bone disorders was irregularly associated with the severity of disease (Kay 1929). Since that discovery, the use of phosphatase measures in bone diseases has grown considerably and remains a practical diagnostic tool for several bone diseases, including osteoporosis (McComb et al. 1979). For instance, in Paget’s disease, cells that usually construct bone, or attempt to restore the bone obliteration in disease, become rich in ALP. The additional phosphatase spills over into blood, where it shows up as elevated plasma ALP (Whyte 1994).
HEALTHY LIVER AND KIDNEY FUNCTION
Metabolic disease, such as type I glycogen storage disease result because of abnormal phosphatase activity (Rubin and Farber 1995). In this disease, the deficiency of a key phosphatase can lead to hypoglycemia, hyperlipidemia, massive hepatomegaly, and gout. The synthesis of glycogen is also dependent on phosphate.
Likewise, defects in glycogen metabolism lead to several disorders, including diabetes mellitus and hyperglycemia (Vincent and Crowder 1995).
HEALTHY IMMUNE FUNCTION
During some phagocytic processes, granule-bound enzymes including ALP are released into the surrounding environment. Leukocytes in the host that are organized to wall off or engulf invading organisms largely demonstrate this increased ALP activity.
Pregnancy also causes leukocytes ALP to rise, probably in response to estrogens and progestogens (McComb et al. 1979). Phosphatase is involved in the signal transduction pathway that results in T cell (Vincent and Crowder 1995) activation, and also in T cell and B cell proliferation and maturation. It is also involved in the activation of gamma-interferons phosphatase causes dephosphorylation of potential toxins (McComb et al.
1979).
HEALTHY REPRODUCTIVE FUNCTION
ALP is associated with the placenta and pregnancy as demonstrated by Coryn’s (1934) finding of ALP in maternal blood during pregnancy and this has been confirmed repeatedly by other researchers (Cayla and Fabre 1935; Meranze et al. 1937). Placental ALP is a heat-stable enzyme existing at great levels in the placenta. A small quantity of this isoenzyme can be traced in the normal sera. The increase of ALP enzyme activity has been the focus of significant assumptions.
The earliest theories incorporated amplified osteoblastic activity in the pregnant mother and the passage of fetal osteoblastic ALP enzyme onto the maternal flow (Ebbs and Scott 1940). While few researchers have suggested that placental origin of the additional ALP (Klees and Frenzel 1960; Kubli 1961).
IN ESCHERICHIA COLI
Most of the experiments on ALP have been carried out using Escherichia coli. The overproducing mutant of E.
coli CW3747 can easily produce ALP from large cultures as well as increase solubility due to the pack of covalent modifications such as glycosylation or fatty acid attachment. The E. coli ALP has taken its place as the prototype of this class of phosphate (Coleman and Gettins 1983; Vincent et al. 1992). ALP can occur in three forms that have been designated isoenzymes mono, di and tri.
Although at first dismissed as impurities in the preparation, it was subsequently shown that these forms differ by the presence of NH2-terminal arginine residue on the subunit of isoenzymes representing a heterodimer of the two types of chains. The relative proportion of each isoenzyme depends on the growth condition of the cell. ALP is a metalloenzyme of identical subunits containing two classes of zinc (II) ions as well as magnesium ions as a cofactor. The active site of these
enzymes contains a serine residue. As the enzyme is devoid of thiol groups and noncovalent bonds, the half- associate the subunit scystinyl residues are in the form of an intra-chain disulfide bond. E. coli ALP is synthesized on polysomes bound to the inner membrane of the cell and is secreted to its last position in the periplasmic space.
IN AQUATIC ECOSYSTEMS
According to the study done by Bleich (1997) bacteria and primary producers such as seaweed are the two major sources of ALP. In the estuaries, they found that the activities of ALP are affected by the anoxic condition, phosphate activity, sedimentation, growth of algal mats, and addition of organics matters. Negative effects on ALP are due to anoxic condition and phosphate.
In aquatic ecosystems, ALP is responsible for the regeneration of phosphorus. This phosphorus cannot be generated through biological processes but only can be recycled through ALP activity (Chrost 1991). Algal mats found on the bottom of the estuary are beneficial to the microbial community (Bleich 1997). Increasing organic matters to the bacterial community shows an increase in the bacterial population. There will be a higher demand for phosphate if there are more bacteria and it means less availability in the estuary, so recycling becomes necessary, and more ALP is produced.
ALPs in aquatic ecosystems play an important role in cell phosphate metabolism, which is linked to the absorption of phosphate and calcium for H2) and the biomineralisation process in aquatic organisms (Xiao et al. 2002). This enzyme plays a significant role in the mineralization of aquatic animal skeletons (Olsen et al.
1991). High levels of ALP in the blood of a fish, indicates that the fish is either sick or has been fed with inducers like p-nitronyl phosphate.
The ALP activity is found to be involved in the regulation of the osmoregulatory of the blue crab, Callinectes sapiduc (Lovett et al. 1994). Studies on ALP from fishes and poikilotherms are not abundant.
However, this offers a foundation for kinetic and structural comparisons with the well-studied ALP enzymes from warm-blooded animals and also bacteria (Asgeirsson et al. 1995). In order to distinguish the effects of low-body temperature or cold-blooded animals such as fishes, exhibit molecular and physiological adaptation range is actually helpful to upsurge the rate of physiological processes (Asgeirsson et al. 1995). Several comparative studies on the mixed form have done with the ALP from rainbow trout, eel, carp, and catfish (Yora and Sakagishi 1986) and additionally, the thermal properties of crude extracts have also been studied (Gelman et al. 1989 &1992).
Following that, Olsen et al. (1991) reported the purification and properties of ALP from shrimp.
IN MAMMALS
A large amount of ALP can be found in different organs such as the liver, placenta, bone, intestine, kidney, and lungs of mammals. ALP is synthesized by hepatocytes and secreted into the bile duct to aid in intestinal digestion of phospholipids in the liver (Rubin and Farber 1995). Where else in the intestine, it is associated with cells of the brush border (Coleman and Gettins 1983a) activity along the intestinal tract is variable. In the bone, ALP is found in hypertrophic cartilage cells, osteoblasts, osteocytes and the matrix vesicle of the lamellar membrane while in the kidney in the brush border microvilli of renal tubules (Fernley 1971). The kidney cortex is much richer in phosphatase than the medulla. In the lungs, ALP is found in the apical plasma membrane of type II alveolar pneumocytes (Goldfischer et al. 1976). Apparently these isoenzymes are also dimeric but glycosylated and have larger subunits (McCombs et al. 1979). In placental tissues, the enzyme is located at the surface of the trophoblastic syneytium, and in the liver, is it adjacent to the bile.
CONCLUSION
This review has cited numerous articles that could be considered as old or outdated, from the 1960s, 1970s, 1980s, and 1990s. Indeed, the research and studies on APL goes way back, nearly 100 years ago when Robinson (1923) initially stated ‘a bone enzyme freeing phosphate’. This eventually triggered several questions;
1) even after nearly 100 years, how much do we know overall about the roles and functions of ALPs? 2) is there more implication of ALPs that we still didn’t know about? The recent attention given in ALPs research from all aspects and fields might provide more insights into understanding ALP.
REFERENCES
Asgeirsson, B., Hartemik, R. & Chlebowski. J. F. 1995.
Alkaline Phosphatase from Atlantic Cod (Gadus Morhua). Kinetic and Structural Properties Which Indicate Adaptation to Low Temperatures. Comp.
Biochem. Physoil. 110B: 315-329.
Bleich, N. A. 1997. Mating Systems among Cichlid Fishes. London: Chapman and Hall.
Boyer, P.D. 1970. The Enzyme: Structure and Control.
3rd Edition. New York: Academic Press.
Cayla, J. & Fabre, F. 1935. Serum Phosphatase during Pregnancy. C. R. Seances Soc. Biol. 120: 748-750.
Chrost, R. J. 1991. Microbial Enzymes in Aquatic Environments. USA: Springer-Verlag.
Coleman, J. E. & Gettins, P. 1983. Alkaline Phosphatase, Solution, Structure and Mechanism.
Adv. Enzymol. 55: 381-414.
Coleman, J. E. & Gettins, P. 1983 Molecular Proteins and Mechanism of Alkaline Phosphatase. In Metal Ions in Biology. New York: John Wiley.
Dixon, M. & Webb, E. C. 1979. Enzymes 3rd Edition.
USA: Longman.
Ebbs, J. H. & Scott, W. A. 1940. Blood Phosphatase in Pregnancy an Indication of Twins: A Preliminary Report. Am. J. Obstet. Gynecol. 39: 1043-1044.
Fernley, H. N. 1971. Mammalian Alkaline Phosphatase.
In P. D. Boyer (Ed.) The Enzyme 3rd Edition. New York: Academic Press.
Gelman, A., Mokady, S. & Cogan, U. 1989. The Thermal Properties of Intestinal Alkaline Phosphatase of Three Kind of Deep-Water Fish.
Comp. Biochem. Physiol. 94B: 113-116.
Gelman, A., Cogan, U., & Mokady, S. 1992. The Thermal Properties of Fish Enzymes as a Possible Indicator of the Temperature Adaptation Potential of the Fish. Comp. Biochem. Physiol. 101B: 205- 208.
Goldfischer, S., Jognson, A. B. & Morecki, R. 1976.
Hypophosphatasia: A Cytochemical Study of Phosphatase Activities. Lab. Invest. 35:55-62.
Kay, H. D. 1929. Phosphatase in Growth and Disease of Bone. Br. J. Exp. Pathol. 10(4): 253 – 256.
Klees, E. & Frenzel, G. 1960. On the Problem of Increase in Serum Alkaline Phosphatase. Activity in Labor and in Puerperium. Klin. Wochenschr.
1(38): 540-543.
Komodo, T. & Sakagishi, Y. 1976. Partial Purification and Some Properties of Human Liver Alkaline Phosphatase. Biochim. Biophys. Acta. 438: 138- 152.
Kim, E. E. & Wyckoff, H. W. 1989. Structure of Alkaline Phosphatases. Clin. Chim. Acta. 186: 175- 188.
Kubli, F. 1961. Transaminase, Lactic Dehydrogenase and Alkaline Phosphatase in Late Pregnancy, during Labor and in Toxicosis. Gynaecol. 151:72-4.
Lovett, D. T., Towle, D. W. & Faris, J. E. 1994.
Salinity-Sensitive Alkaline Phosphatase Activity in Gills of Blue Crab, Callinectes sapidus, Rathbun.
Comp. Biochem. Physiol. 109B: 163-173.
McComb, R. B., Bowers, Jr. G. N. & Posen, S. 1979 Alkaline Phosphatase. Ney York: Plenum Press.
Moss, D. W. 1992. Perspectives in Alkaline Phosphatase of Animal Tissues. Biochem J. 57:
2486-2492.
Meranze, T., Meranze, D. R. & Rothman, M. M. 1937.
Blood Phosphatase in Pregnancy. Am. J. Obstet.
Gynecol. 33:444-450
Neumann, H. 1968. Substrate Selectivity in the Action of Alkaline and Acid Phosphatase. J. Biol. Chem.
243: 4671-4676.
Olsen, R. L., Overbo, K. & Myrnes, B. 1991. Alkaline Phosphatase from the Hepatopancreas of Shrimp (Pandalus Borealis): A Dimeric Enzyme with Catalytically Active Subunit. Comp. Biochem.
Physoil. 99B: 755-761.
Robison, R. 1923. The Possible Significance of Hexosephosphoric Esters in Ossification. Biochem.
J. 17: 286 - 293.
Rubin, A. H. & Wilson, I. B. 1971. The Enzyme. New York: Academic Press.
Sparks, J. W. & Brautigan, D. L. 1986. Molecular Basis for Substrate Specificity of Protein Kinase and Phosphatases. Int. J. Biochem. 18: 497-504.
Trowsdale, J., Martin, D., Bicknell, D. & Campbell, I.
1990. Alkaline Phosphatase. Biochem. Soc. Trans.
18: 178-180.
Vincent, J., Crowder, M. & Averill, B. A. 1992.
Hydrolysis of Phosphatase Monoesters: A Biological Problem with Multiple Chemical Solutions. Trends Biochem. Sci. 17: 105-110.
Vincent, J. & Crowder, M. 1995. Phosphatase in Cell Metabolism and Signal Transduction: Structure, Function and Metabolism of Action. Austin, Texas:
RG Landes Company.
Whyte, M. P. 1994. Why Does the Doctor Measure Your Blood Alkaline Phosphatase Level? In A Patient’s Guide to Paget’s Diseases of Bone. New York: The Paget’s Disease Foundation, Inc.
Xiao, R., Xie, L. P., Lin, J. Y., Li, C. H., Chen, Q. X., Zhou, H. M. & Zhang, R. Q. 2002. Purification and Enzymatic Characterization of Alkaline Phosphatase from Pinctada fucata. J. Mol. Cat.
Enzym. 17B: 65-74.
Yora, T. & Sakagishi, Y. 1986. Comparative Biochemical Study of Alkaline Phosphatase Isozymes in Fish, Amphibians, Reptiles, Birds and Mammals. Comp. Biochem. Physiol. 85B: 649-658.
Vanitha Mariappan
Centre of Toxicology and Health Risk Studies (CORE) Faculty of Health Sciences
Universiti Kebangsaan Malaysia
Jalan Raja Muda Abdul Aziz
50300 Wilayah Persekutuan Kuala Lumpur Malaysia
Kumutha Malar Vellasamy
Department of Medical Microbiology Faculty of Medicine
University of Malaya
Jalan Profesor Diraja Ungku Aziz
50603 Wilayah Persekutuan Kuala Lumpur Malaysia
Corresponding author: Vanitha Mariappan E-mail: [email protected]
Tel: +603 9289 7131 Fax: -
Received: 25 February 2021 Revised: 10 May 2021
Accepted for publication: 19 May 2021
