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REVIEW ARTICLE
Alkaline Phosphatase: An Overview
Ujjawal Sharma •Deeksha Pal •Rajendra Prasad
Received: 21 August 2013 / Accepted: 11 November 2013 / Published online: 26 November 2013
ÓAssociation of Clinical Biochemists of India 2013
Abstract Alkaline phosphatase (ALP; E.C.3.I.3.1.) is an
ubiquitous membrane-bound glycoprotein that catalyzes the
hydrolysis of phosphate monoesters at basic pH values.
Alkaline phosphatase is divided into four isozymes
depending upon the site of tissue expression that are Intes-
tinal ALP, Placental ALP, Germ cell ALP and tissue non-
specific alkaline phosphatase or liver/bone/kidney (L/B/K)
ALP. The intestinal and placental ALP loci are located near
the end of long arm of chromosome 2 and L/B/K ALP is
located near the end of the short arm of chromosome 1.
Although ALPs are present in many mammalian tissues and
have been studied for the last several years still little is known
about them. The bone isoenzyme may be involved in mam-
malian bone calcification and the intestinal isoenzyme is
thought to play a role in the transport of phosphate into
epithelial cells of the intestine. In this review, we tried to
provide an overview about the various forms, structure and
functions of alkaline phosphatase with special focus on liver/
bone/kidney alkaline phosphatase.
Keywords Enzymes Isoenzymes Alkaline
phosphatase L/B/K alkaline phosphatase Liver
alkaline phosphatase Intestinal alkaline phosphatase
Placental alkaline phosphatase
Introduction
Alkaline phosphatases [ALP; orthophosphoric monoester
phosphohydrolase (alkalineoptimum) EC 3.1.3.1] are plasma
membrane-bound glycoproteins [1,2]. These enzymes are
widely distributed in nature, including prokaryotes and
higher eukaryotes [3–6], with the exception of some higher
plants [7]. Alkaline phosphatase forms a large family of
dimeric enzymes, usually confined to the cell surface [8,9]
hydrolyzes various monophosphate esters at a high pH opti-
mum with release of inorganic phosphate [10,11].
Mammalian alkaline phosphatases (ALPs) are zinc-
containing metalloenzymes encoded by a multigene family
and function as dimeric molecules. Three metal ions
including two Zn
2?
and one Mg
2?
in the active site are
essential for enzymatic activity. However, these metal ions
also contribute substantially to the conformation of the
ALP monomer and indirectly regulate subunit–subunit
interactions [12].
Isoforms of Alkaline Phosphatase and Their
Distribution
Human ALPs can be classified into at least four tissue-
specific forms or isozyme mainly according to the speci-
ficity of the tissue to be expressed, termed as placental
alkaline phosphatase (PLALP or Regan isozyme), Intesti-
nal alkaline phosphatase (IALP), liver/bone/kidney alka-
line phosphatase (L/B/K ALP), germ cell ALP (GCALP or
NAGAO isozyme) [13]. The enzyme products of at least
three ALP gene loci (placental, intestinal and L/B/K) [14–
16] are distinguishable in man by a variety of structural,
biochemical and immunologic methods [17–19].
Placental Alkaline Phosphatase
The human placental ALP gene was mapped to chromo-
some 2 [20]. A homology of 87 % is found with the IAP
U. Sharma D. Pal R. Prasad (&)
Department of Biochemistry, Postgraduate Institute of Medical
Education and Research, Chandigarh, India
e-mail: fateh1977@yahoo.com
123
Ind J Clin Biochem (July-Sept 2014) 29(3):269–278
DOI 10.1007/s12291-013-0408-y
gene. There are, however, differences at their carboxyl
terminal end [21]. Placental ALP is a heat stable enzyme
present at high levels in the placenta. A trace amount of
this isoenzyme can be detected in normal sera [22]. Part of
the serum placental-type activity originates from neutro-
phils. The placental ALP gene can be re-expressed by
cancer cells as the Regan isoenzyme. Placental ALP is a
polymorphic enzyme, with up to 18 allelozymes resulting
from point mutations, in contrast to the other ALP isoen-
zymes [23].
Intestinal Alkaline Phosphatase
The gene encoding for intestinal ALP (IAP) is a member of
the gene family mapping to the long arm of chromosome 2
[24]. IAP is partially heat-stable isozyme present at high
levels in intestinal tissue. In contrast to the other ALP
isoenzymes, the carbohydrate side-chains of IAP are not
terminated by sialic acid [25]. Distinct IALPs can be iso-
lated from fetal and adult intestinal tissue, with the fetus
forming a sialylated isoenzyme in contrast to the adult. The
fetal and adult forms differ not only in the carbohydrate
content but also in the protein moiety itself, suggesting that
a separate ALP gene locus may exist in humans during
fetal development. This embryonic gene can be reex-
pressed (in a modified form) by cancer cells and is desig-
nated as Kasahara isoenzyme [26].
Germ Cell Alkaline Phosphatase
The gene encoding for germ-cell ALP (GCAP, placental-
like ALP) was also mapped to chromosome 2 [27]. It is
heat-stable isozyme present at low levels in germ cells [2]
embryonal and some neoplastic tissues [28,29]. It encodes
testis/thymus ALP and can be expressed in the placenta at
low levels [30]. GCAP in testis appears to be localized to
the cell membrane of immature germ cells and, like the
other ALP isoenzymes, is attached to the cell membrane by
means of a phosphatidyl-inositol-glycan anchor. Like the
placental ALP gene, it can be reexpressed by cancer cells
(or NAGAO isozyme) [31].
Liver/Bone/Kidney Alkaline Phosphatase
The heat-labile isozyme represents the liver/bone/kidney or
tissue nonspecific (TNSALP) form [1,2]. It is expressed in
many tissues throughout the body and is especially abun-
dant in hepatic, skeletal, and renal tissue. Slight differences
in electrophoretic mobility and thermo stability between
the L/B/K ALPs from various tissues are attributed to
differences in post-translational modification, although it is
possible that their protein moieties are encoded by separate
but related genes [19].
Liver/bone/kidney or tissue-nonspecific alkaline phos-
phatase (TNSALP) is encoded as a single genetic locus,
mapped to the short arm of chromosome 1 [32,33]. Ko-
moda and Sakagishi [25] postulated a hypothesis regarding
the physiological role of the sugar moieties in ALPS: they
would protect the enzyme from rapid removal from the
circulation through binding by the asialoglycoprotein
receptors of the liver.
The evolution of the ALP gene family has presumably
involved the duplication of a primordial tissue-nonspecific
ALP gene to create the TNSALP gene and an intermediate
IAP gene, followed by additional duplications of the latter
to create intestinal, placental, and germ-cell ALP genes.
Only humans and great apes have placental ALP; all other
mammals have IAP [34].
Structure of the Gene
Alkaline phosphatase is a membrane-bound metalloenzyme
that consists of a group of isoenzymes. Each isoenzyme is a
glycoprotein encoded by different gene loci [35]. It is
believed that all of the human ALP genes evolved from a
single ancestral gene. Figure 1shows a rough outline of the
deduced evolutionary tree of ALP [19].
Three ALP genes at chromosome 2q34–37 are expres-
sed in essentially a tissue-specific manner and produce a
placental, placental-like and intestinal ALP isoenzyme
(PALP, PLALP and IALP respectively). The fourth ALP
gene that is L/B/K ALP maps to the distal short arm of
human chromosome 1, bands p34–p36.1 encodes a family
of proteins [9,35,36]. Differential glycosylation of
TNSALP gives rise to tissue-specific isoforms that differ
from one another only by post-translational modification;
these secondary ALP isoenzymes are present throughout
the body, but individually are most abundant in hepatic,
skeletal and renal tissue [37]. Accordingly, they are col-
lectively called liver/bone/kidney or tissue-nonspecific
ALP (TNSALP) [4].
Types and chromosomal locations of the ALP gene with
their accession numbers are shown in Table 1. The L/B/K
gene appears to be at least five times longer than each of
the other three genes. The overall difference in length is
due to very much longer introns in the L/B/K ALP gene
(Fig. 2). The introns in the intestinal, placental and pla-
cental-like genes are all quite small (74–425 bp). The
complete cDNA sequence of L/B/K ALP is known (Fig. 3)
and the gene consists of 12 exons, compared with 11 in
each of the other genes. The coding exons are 2–12. The
additional exon is at the 50end in the non-coding region.
Exons 2–12 are contained within 25 kb of DNA. The dis-
tance between exons 1 and 2 is at least an additional 25 kb
270 Ind J Clin Biochem (July-Sept 2014) 29(3):269–278
123
of DNA. Thus, the entire gene is comprised of at least
50 kb of DNA [38].
The sequences at the 50and 30ends of each intron are
in agreement with the consensus sequence for intron-
exon boundaries of other eukaryotic genes. All introns
begin with the dinucleotide GT and end with AG. Intron
number 1, at least 25 kb in length, interrupts the 50
untranslated sequence 105 bp upstream of the initiation
methionine codon. All other introns interrupt the gene
within protein coding regions. Exon 12, about 1,025 bp,
contains 263 nucleotides of coding sequence, the termi-
nation codon, and the entire 30untranslated region. At
the end of exon 12, there are putative 30-mRNA pro-
cessing signals that are commonly found in other
eukaryotic genes; the mRNA cleavage/polyadenylation
site is flanked by the sequence AATAAA about 12 bp
upstream, and a G/T-rich region about 12 bp
downstream.
Characterization and Discrimination of the ALPs
Many different biochemical and immunological methods
have been used to discriminate between and selectively
assay the different ALPS at the enzyme and protein
level. Three general methods have proved particularly
useful: thermostability studies; differential inhibition with
various aminoacids, small peptides and other low
molecular weight substances; and immunologic methods
[39].
Fig. 1 Illustration showing the
postulated evolutionary
relationships of the human liver/
bone/kidney, intestinal,
placental and placental-like
genes [18]
Table 1 Nomenclature of human ALP isozymes and gene including chromosomal location, gene size and accession numbers
Gene Protein name Common name Chromosomal location Accession no.
ALPL TNAP Tissue-nonspecific alkaline phosphatase; TNSALP;
liver/bone/kidney type AP
Chr1: 21581174–21650208 NM_000478
ALPP PLAP Placental alkaline phosphatase; PLALP Chr2: 233068964–233073097 NM_001632
ALPI IAP Intestinal alkaline phosphatase; IALP Chr2: 233146369–233150245 NM_001631
ALPP2 GCAP Germ cell alkaline phosphatase; GCALP Chr2: 233097057–233100922 NM_031313
Fig. 2 Relationship between exon organization and polypeptide
structure of the L/B/K ALP gene. L/B/K ALP gene exons 1–12 are
shown as large rectangles. Untranslated regions are indicated by
green colour. The signal peptide at the amino terminus and the
hydrophobic stretch of amino acids at the carboxyl terminus in exons
2and 12, respectively, are shown in yellow. Regions which comprise
the active pocket that are conserved in intestinal ALP, placental ALP,
and E. coli ALP are shown as follows: small rectangles above the
exons indicate conserved units of amino acid sequence which exist as
discrete units of secondary structure in E. coli ALP (black for &
sheets, white for a-helices); the open circles indicate metal ligands,
and the closed circles indicate residues that directly interact with
incoming substrate
Ind J Clin Biochem (July-Sept 2014) 29(3):269–278 271
123
Thermostability
The intestinal and L/B/K ALPs are rapidly inactivated at
temperature [65 °C (Table 2). In contrast, placental and
placental-like ALPS are remarkably thermostable. They
may be heated at 65 °C for an hour or more without loss of
activity. However, the intestinal ALP is somewhat more
thermostable than the L/B/K ALP. It has also been shown
that in serum, liver ALP is slightly, though significantly,
more thermostable than bone ALP [39].
Inhibition Studies
Various low molecular weight substances show differential
inhibition of the different ALPs. Table 3summaries the
effects with five inhibitors which have been extensively
Fig. 3 DNA sequence and deduced amino acid sequence of the L/B/
K ALP cDNA. Numbers preceded by ?or–refer to amino acid
positions. All other numbers refer to nucleotide positions. Asterisks
occur at 10-base intervals. Amino acids -17 to -1 comprise a putative
signal peptide. A vertical line precedes amino acid ?1, the amino-
terminal residue found in the mature protein. Amino acid residues that
have been determined by protein sequence analysis of purified liver
ALP are underlined. Five potential N-linked glycosylation signals,
Asn-Xaa-Thr/Ser, are boxed. A 12-bp direct repeat in the 30
untranslated region of the cDNA is labeled by arrows. A single
poly(A) addition signal AATAAA is underlined twice [10]
272 Ind J Clin Biochem (July-Sept 2014) 29(3):269–278
123
used. The L/B/K ALPs are more sensitive to inhibition with
L-homoarginine (Har) than placental, placental-like or
intestinal ALPs. In contrast, placental, placental-like and
intestinal ALPS are about 30 times more sensitive to
inhibition with L-phenylalanine (Phe) than the L/B/K
ALPs. r,-Phenylalanyl-glycyl-glycine (Pgg) gives sharp
differential inhibition between placental, intestinal and
L/B/K ALPs. It also differentiates between placental ALP
and placental-like ALP, which with this inhibitor more
nearly resembles intestinal ALP. L-Leucine (Leu) charac-
teristically gives much stronger inhibition with placental-
like ALP than with the other ALPs. Levamisole (Leva) is a
particularly potent inhibitor of L/B/K ALP, but has little
inhibitory effect on the other ALPs [39].
Immunologic Studies
Antisera raised in rabbits against purified placental ALP
cross-react with placental- like ALP and intestinal ALP,
but not with L/B/K ALP. Complementary results are
obtained with antisera raised against intestinal ALP or L/B/
K ALP. These findings demonstrate that some, though not
all, of the antigenic determinants detected on placental
ALP are also present on intestinal ALP, but the placental
and placental like ALPs are immunologically very similar.
Some but not other monoclonals, raised against placental
and intestinal ALPs react with both ALPs and some,
though not other, monoclonals differentiate the placental
and placental-like ALP. Combinations of these various
biochemical and immunological techniques have been used
to devise methods which give precise analytical informa-
tion about the quantities of each of the ALPs when they are
present together in a tissue extract or body fluid such as
serum or amniotic fluid.
L-Phenylalanyl-glycyl-glycine (Pgg) gives sharp differ-
ential inhibition between placental, intestinal and L/B/K
ALPs. Leva is particularly a potent inhibitor of L/B/K
ALP, but has little inhibitory effect on other ALPs. It
should be noted that these various inhibitors are stereo-
specific and uncompetitive [19].
Homology Between Different Isoforms
The complete amino acid sequences of ALP proteins are
now known (Fig. 4). A computer-assisted comparison of
E. coli (471 amino acids) [40], human placental (535 amino
acids) [41] and human L/B/K ALP (524 amino acids)
precursor proteins is shown in Fig. 4. Amino acid positions
that are identical in all three proteins, or in the two human
proteins, are depicted in boxed (Fig. 4). Gaps have been
introduced into the protein sequences to maximize align-
ment of homologous regions.
At the amino acid level, the tissue-specific ALP isoen-
zymes are 86–98 % identical to one another [9,42], but
52–56 % identical when compared with TNSALP [4,35].
IALP, PALP and GCALP are highly homologous
with [90 % identical amino acid sequences, whereas
TNSALP is significantly more diverse. At the DNA level,
L/B/K and placental ALP are 60 % homologous in the
coding regions but no homology is detected between the
cDNA in the 50and 30untranslated regions. As expected,
there is less homology between E. coli and mammalian
ALPs. Thus, E. coli ALP is 25 % homologous to L/B/K
ALP and 29 % homologous to placental ALP over the
47 % amino acids of the E. coli enzyme [37].
Several areas are highly conserved in all three ALP
polypeptides. These are the same regions detected by
Millan, 1986 [27] and Kam et al. 1985 [43] in their com-
parisons of placental and E. coli ALPs. These areas rep-
resent conservation of amino acids that comprise the active
site region in the E. coli ALP [44]. There are also several
regions that are conserved only between the human L/B/K
and placental ALPs, presumably representing functions of
mammalian ALPs not present in E. coli. Two N-linked
glycosylation signals at homologous sites occur in the L/B/
K and placental ALPs, though the L/B/K enzyme contains
three additional glycosylation signals that are absent in
placental ALP.
Table 2 Relative thermostabilities of human ALPs [39]
Human ALP 56 °C (min) 65 °C (min)
L/B/K 7.4 1.0
Intestinal [60.0 6.5
Placental and plac-like – [60.0
Time in minute required to give 50 % inactivation of different human
ALPs at 56 °C and 65 °C
Table 3 Effects of various inhibitors on different Huaman ALPs [19]
Inhibitors ALP
L/B/K
ALP
Intestinal Placental Plac-
like
L-Plenylalanine (Phe) 31 0.8 1.1 0.8
L-Homoarginine (Har) 2.7 40 [50 36
L-Phenylalanineglycylglycine
(Pgg)
30.6 3.7 0.1 2.9
L-Leucine (Leu) 13.1 3.6 5.7 0.6
Levamisole (Leva) 0.03 6.8 1.7 2.7
Concentrations (nmol/l) of various inhibitors required to produce 50 %
inhibition of different human ALPs under standardized conditions
Ind J Clin Biochem (July-Sept 2014) 29(3):269–278 273
123
Molecular Modeling of L/B/K ALP Protein
Two zinc atoms are present in the active site and one
calcium atom is present in the metal-binding site. Calcium
is the natural ion that binds to the metal-binding domain.
The effects of calcium on ALP activity should be recon-
sidered including in the analysis of the presence of calcium
site. The precise biological role of calcium in TNSALP
remains to be addressed. It is very interesting to observe
that with the evolution and the specialization of the enzyme
function, new features have been added: in E. coli, where
there is no skeleton to mineralize, there is no calcium site
in its ALP [9].
The model of TNSALP shows that the active site valley
located on both sides of the active site contains a large
number of polar residues. Thus, the hydrophobic residues,
Trp168, Tyr169, and Tyr206 are surrounded by ionic res-
idues. A basic residue, Arg433, is present close to the
Fig. 4 Comparison of the amino acid sequences of E. coli (E), human
placental (P), and human L/B/K (L) ALP precursor proteins. Gaps
that have been introduced into the sequences to maximize pairing of
homologous amino acids are indicated by -Amino acid ?1
corresponds to the first residue in each of the mature proteins. Amino
acids that are identical in all three proteins or in the two human
proteins are boxed. Amino acids are shown in the single-letter code
[10]
274 Ind J Clin Biochem (July-Sept 2014) 29(3):269–278
123
active site. The hydrophobic pocket is not conserved in
TNSALP. However, the tyrosine, which enters in the active
site of the other monomer (Tyr367 in PLALP), is con-
served in TNSALP (Tyr371). This reinforces the idea that
Tyr371 may contribute to the allosteric properties shared
by the two enzymes. All residues that are essential for the
hydrolytic activity of the bacterial and the other mamma-
lian phosphatases are preserved in TNSALP, but those that
confer substrate specificity are different.
The structural features that comprise the N-terminal
helix involved in the dimer interface, the 76 residues of the
calcium-binding-domain residues 211–289), and the inter-
facial ‘‘crown-domain’’ formed by the insertion of a
60-residue segment (371–431) from each monomer occur
in TNSALP. Within the crown domain, a unique surface
loop not present in the E. coli enzyme that extends from
amino acids 400–430. This loop has been shown to play an
important role in defining the conformation and stability of
the ALP molecule. The loop is also partially responsible
for the interaction of ALPs with extracellular matrix pro-
teins, such as collagen. The TNSALP model shows that
this loop is highly accessible and located at the very tip of
the crown domain. This loop is responsible for the unique
property of mammalian ALPs of being uncompetitively
inhibited by a number of amino acids and small peptides
(Fig. 5)[11].
Intracellular Calcium and L/B/K ALP
The three dimensional structural model of human TNSALP
was proposed by Mornet in 2001 [9]. According to this
model, one of the feature that differentiate ALP of mam-
mals from that of E. coli is the acquisition of a calcium
binding site during evolution in addition to two zinc and
one magnesium binding sites indispensable for ALP
activity. Human alkaline phosphatase has four metal
binding sites -two for zinc, one for magnesium, and one for
calcium ion. Calcium helps to stabilize a large area that
includes loops 210–228 and 250–297 [45]. The calcium
atom in TNSALP is assumed to be coordinated by four
amino acid residues (Glu218, Phe273, Glu274 and Asp289)
and a water molecule. Calcium binding is crucial for the
proper folding and correct assembly of newly synthesized
TNSALP molecule. It has been demonstrated that loss of
calcium binding potency has a deleterious effect on bio-
synthesis of the TNSALP molecule. It might result in
misfolded ALP molecule. There is increasing evidence that
many misfolded proteins are retained in the ER or moved
from cis-golgi to the ER as part of the quality control
system, thus permitting only properly folded and assem-
bled proteins to move to their final destination However,
the physiological importance of this calcium binding site of
TNSALP remains obscure [46].
Physiological Functions of ALP
Since its first description by Suzuki and colleagues [47]in
1907, alkaline phosphatase (ALP) has been investigated
continuously and extensively. But little is known regarding
the physiological function of ALPs in most tissues except
that the bone isoenzyme has long been thought to have a
role in normal skeletal mineralization [48]. The natural
substrates for TNSALP appear to include at least three
phosphor compounds: phosphoethanolamine (PEA), inor-
ganic pyrophosphate (PPi), and pyridoxal-50-phosphate
(PLP), as evidenced by increased plasma and/or urinary
levels of each in subjects with hypophosphatasia [49,50],
but this is uncertain. Indeed, a variety of mechanisms have
been proposed to explain the role of ALP in bone miner-
alization [51]. However, apart from its role in normal bone
mineralization, the other functions of L/B/K remains
obscure both in physiological and neoplastic conditions.
Alkaline Phosphatase in Health and Diseases
The activity of liver and bone alkaline phosphatases in
serum has been applied extensively in routine diagnosis.
Values for each isoenzyme in healthy individuals of dif-
ferent ages are reported together with results obtained in
various diseases. Data from normal subjects shows that
bone alkaline phosphatase contributes about half the total
alkaline phosphatase activity in adults. The normal serum
range of alkaline phosphatase is 20 to 140U/L. The enzyme
alkaline phosphatase is an important serum analyte and its
elevation in serum is correlated with the presence of bone,
liver, and other diseases [52]. High ALP levels can show
that the bile ducts are obstructed. Levels are significantly
higher in children and pregnant women. Also, elevated
ALP indicates that there could be active bone formation
occurring as ALP is a byproduct of osteoblast activity
(such as the case in Paget’s disease of bone) or a disease
that affects blood calcium level (hyperparathyroidism),
vitamin D deficiency, or damaged liver cells [53]. Levels
are also elevated in people with untreated Celiac Disease
[54]. Placental alkaline phosphatase is elevated in semi-
nomas [55]. Lowered levels of ALP are less common than
elevated levels. Some conditions or diseases such as
hypophosphatasia, postmenopausal women receiving
estrogen therapy because of osteoporosis, men with recent
heart surgery, malnutrition, magnesium deficiency, hypo-
thyroidism, severe anemia, children with achondroplasia
Ind J Clin Biochem (July-Sept 2014) 29(3):269–278 275
123
and cretinism, children after a severe episode of enteritis,
pernicious anemia, aplastic anemia, chronic myelogenous
leukemia, wilson’s disease may lead to reduced levels of
alkaline phosphatase. In addition, the drugs such as oral
contraceptives have been demonstrated to reduce alkaline
phosphatase [56].
Deficiency in TNSALP leads to hypophosphatasia
(HPP), an inborn error of metabolism characterized by
epileptic seizures in the most severe cases, caused by
abnormal metabolism of pyridoxal-50-phosphate (the pre-
dominant form of vitamin B6) and by hypomineralization
of the skeleton and teeth featuring rickets and early loss of
teeth in children or osteomalacia and dental problems in
adults caused by accumulation of inorganic pyrophosphate
(PPi) [57]. Subjects with hypophosphatasia have general-
ized deficiency of TNSALP activity and suffer from
defective bone mineralization (rickets or osteomalacia), yet
placental and intestinal ALP isoenzyme activity is normal.
The most severe cases are lethal in infancy, with virtually
complete absence of L/B/K ALP in all tissues [58]. Severe
forms of the disease are transmitted as an autosomal
recessive trait. Identification of a missense mutation in the
TNSALP gene in one typical case of the severe perinatal
(lethal) form of hypophosphatasia established this link
between TNSALP and skeletal mineralization [59,60].
Several studies have indicated the involvement of ALPs in
cellular events such as the regulation of protein phosphory-
lation, cell growth, apoptosis and cellular migration during
embryonic development. ALP genes are regulated by dis-
tinct signals as shown by clear differences in their expression
profiles [2]. Ectopic expression of ALPs have been associ-
ated with a variety of human cancers. The expression pattern
of ALP isozymes are altered in malignant tissues, for
example, PALP and GCALP are over expressed in cells
derived from breast cancer and choriocarcinoma, respec-
tively. PALP is a marker of cancer of ovary, testis, lung, and
gastrointestinal tract. Plasma TNALP levels can indicate the
presence of osteosarcomas, Paget’s disease and osteoblastic
bone metastates [36]. Enhanced expression of IALP has also
been reported in hepatocellular carcinoma [61]. The aberrant
expression of ALP genes in cancer [11,62] has led to the
suggestion that ALP isozymes may be involved in tumori-
genesis [6]. ALPL itself represents a new tumor suppressor
gene homozygously inactivated in meningiomas [63].
Higher ALP activities reported in breast cancer patients [64].
Finally, recent study proposes a new role for TNSALP
in the toxic effect of extracellular tau protein. The extra-
cellular tau remains in a dephosphorylated state. Hyper-
phosphorylated tau protein, the main component of
intracellular neurofibrillary tangles present in the brain of
Alzheimer’s disease (AD) patients, plays a key role in
progression of the disease. An increase in TNSALP activity
together with increase in protein and transcript levels were
detected in Alzheimer’s disease patients as compared to
healthy controls [56].
References
1. McComb RB, Bowers GN, Posen S. Alkaline phosphatase. New
York: Plenum Publishing Corp; 1979.
2. Tsai LC, Hung MW, Chen YH, Su WC, Chang GG, Chang TC.
Expression and regulation of alkaline phosphatases in human
breast cancer MCF-7 cells. Eur J Biochem. 2000;267:1330–9.
3. Chang TC, Wang JK, Hung MW, Chiao CH, Tsai LC, Chang GG.
Regulation of the expression of alkaline phosphatase in a human
breast cancer cell line. Biochem J. 1994;303:199–205.
Fig. 5 A ribbon diagram of
L/B/K ALP protein structure.
(http://biochem.dental.upenn.
edu/)
276 Ind J Clin Biochem (July-Sept 2014) 29(3):269–278
123
4. Whyte MP, Landt M, Ryan LM, Mulivor RA, Henthorn PS,
Fedde KN, Mahuren JD, Coburn SP. Alkaline phosphatase: pla-
cental and tissue-nonspecific isoenzymes hydrolyze phosphoeth-
anolamine, inorganic pyrophosphate, and pyridoxal 50-phosphate
substrate accumulation in carriers of hypophosphatasia corrects
during pregnancy. J Clin Invest. 1995;95:1440–5.
5. Calhau C, Martel F, Hipolito-Reis C, Azevedo I. Effect of P-
glycoprotein modulators on alkaline phosphataseactivity in cul-
tured rat hepatocytes. Cell Physiol Biochem. 2000;10:195–202.
6. Sadeghirishi A, Yazdanparast R. Plasma membrane homing of
tissue nonspecific alkaline phosphatase under the influence of
3-hydrogenkwadaphnin, an anti-proliferative agent from Dend-
rostellera lessertii. Acta Biochim Pol. 2007;54:323–9.
7. Shigenari A, Ando A, Baba R, Vaniamoti T, Katsuoka Y, Inoko
H. Characterization of alkaline phosphatase genes expressed in
seminoma by cDNA cloning. Cancer Res. 1998;58:5079–82.
8. Benham F, Cottell DC, Franks M, Wilson PD. Alkaline phos-
phatase activity in human bladder tumor cell lines. J Histochem
Cytochem. 1977;25:266–74.
9. Mornet E, Stura E, Lia-Baldin AS, Stigbrand T, Menez A, Le Du
MH. Structural evidence for a functional role of human tissue non
specific alkaline phosphatase in bone mineralization. J Biol
Chem. 2001;276:31171–8.
10. Weiss MJ, Henthorn PS, Lafferty MA, Slaughter C, Raducha M,
Harris H. Isolation and characterization of a cDNA encoding a
human liver/bone/kidney-type alkaline phosphatase. Proc Natl
Acad Sci. 1986;83:7182–6.
11. Sharma U, Singh SK, Pal D, Khajuria R, Mandal AK, Prasad R.
Implication of BBM lipid composition and fluidity in mitigated
alkaline phosphatase activity in renal cell carcinoma. Mol Cell
Biochem. 2012;369:287–93.
12. Hoylaerts MF, Manes T, Millan JL. Mammalian alkaline phos-
phatases are allosteric enzymes. J Biol Chem. 1997;272:22781–7.
13. Sligbrand T. Present status and future trends of human alkaline
phosphatases. Prog Clin Biol Res. 1984;166:3–14.
14. Henthorn PS, Raducha M, Fedde KN, Lefferty MA, Whyte MP.
Different missense mutations at the tissue nonspecific alkaline
phosphatase genes locus in autosomal recessively inherited forms
of mild and severe hypophosphatasia. Proc Natl Acad Sci.
1992;89:9924–8.
15. Millan JL, Fishman WH. Biology of human alkaline phosphatase
with special reference to cancer. Crit Rev Clin Lab Sci.
1995;32:1–39.
16. Muller H, Yamazaki M, Michigami T, Kageyama T, Chonau E,
Schneider P, Ozono K. Asp361Val mutant of alkaline phospha-
tase found in patients with dominantly inherited hypophospha-
tasia inhibits the activity of the wild-type enzyme. J Clinical
Endocrinol Metabolism. 2000;85:743–7.
17. Mulivor RA, Plotkin LI, Harris H. Developmental change in
human intestinal alkaline phosphatase. Ann Hum Genet.
1978;42:1–13.
18. McKenna MJ, Hamilton TA, Sussman HH. Comparison of
human alkaline phosphatase isoenzymes. structural evidence for
three protein classes. Biochem J. 1979;181:67–73.
19. Harris H. The harvey lectures: series 76. New York: Academic;
1986. p. 95–123.
20. Raimondi E, Talarico D, Moro L, et al. Regional mapping of the
human placental alkaline phosphatase gene (ALP) to 2q37 by
in situ hybridization. Cyrogenet Cell Genet. 1988;47:98–9.
21. Henthom PS, Raducha M, Hadesch T, et al. Sequence and
characterization of the human intestinal alkaline phosphatase
gene. J Biol Chem. 1988;263:12011–9.
22. Vergote IB, Abeler VM, Bormer OP, et al. CA125 and placental
alkaline phosphatase as serum tumor markers in epithelial ovar-
ian carcinoma. Tumor Biol. 1992;13:168–74.
23. Fishman WH, Inglis NR, Green S, et al. Immunology and bio-
chemistry of regan isoenzyme of alkaline phosphatase in human
cancer. Nature. 1968;219:697–9.
24. Griffin CA, Smith M, Henthorn PS, et al. Human placental and
intestinal alkaline phosphatase genes map to 2q34–q37. Am J
Hum Genet. 1987;41:1025–34.
25. Komoda T, Sakagishi Y. The function of the carbohydrate moiety
and alteration of carbohydrate composition in human alkaline
phosphatase isoenzymes. Biochim Biophys Acta.
1978;523:395–406.
26. Higashino K, Muratani K, Hade T, et al. Gene structure of
alkaline phosphatases: purification and some properties of the fast
migrating alkaline phosphatase in FL-amnion cells (the Kasahara
isoenzyme) and its cDNA cloning. Clin Chim Acta.
1989;186:151–64.
27. Millan JL. Molecular cloning and sequence analysis of human
placental alkaline phosphatase. J Biol Chem. 1986;261:3112–5.
28. Millan JL, Manes R. Seminoma-derived nagao isozyme is
encoded by a germ cell alkaline phosphatase gene. Proc Natl
Acad Sci. 1988;85:3024–8.
29. Hofmann MC, Jeltsch W, Brecher J, Walt H. Alkaline phospha-
tase isozymes in human testicular germ cell tumors, precancerous
stage, and three related cell lines. Cancer Res. 1989;49:4696–700.
30. Povinelli CM, Knoll BJ. Trace expression of the germ-cell
alkaline phosphatase gene in human placenta. Placenta.
1991;12(663):8.
31. Fishman WH. Clinical and biological significance of an isoen-
zyme tumor marker-PLAP. Clin Biochem. 1987;20:387–92.
32. Smith M, Weiss MJ, Griffin CA, et al. Regional assignment of the
gene for human liver/bone/kidney alkaline phosphatase to chro-
mosome 1 p36.1–p34. Genomics. 1988;2:139–43.
33. Orimo H. The mechanism of mineralization and the role of
alkaline phosphatase in health and disease. J Nippon Med Sch.
2010;77:4–12.
34. Fishman WH. Alkaline phosphatase isozymes: recent progress.
Clin Biochem. 1990;23:99–104.
35. Weiss MJ, Ray K, Henthorn PS, Lamb B, Kadesch T, Harris H.
Structure of the human liver/bone/kidney alkaline phosphatase
gene. J Biol Chem. 1988;263:12002–10.
36. Du MHL, Milla JL. Structural evidence of functional divergence
in human alkaline phosphatases. J Biol Chem.
2002;277:49808–14.
37. Moss DW. Perspectives in alkaline phosphatase research. Clin
Chem. 1992;38:2486–92.
38. Kiledjian M, Kadesch T. Post-transcriptional regulation of the
human liver/bone/kidney alkaline phosphatase gene. J Biol
Chem. 1991;266:4207–13.
39. Harris H. The human alkaline phosphatases: what we know and
what we don’t know. Clin Chim Acta. 1990;186:133–50.
40. Bradshaw RA, Cancedda F, Ericsson LH, Neumann PA, Piccoli
SP, Schlesinger MJ, Shriefer K, Walsh KA. Amino acid sequence
of Escherichia coli alkaline phosphatase. Proc Natl Acad Sci.
1981;78:3473–7.
41. Henthorn PS, Raducha M, Edwards YN, Weiss MJ, Slaughter C,
Lafferty MA, Harris H. Nucleotide and amino acid sequences of
human intestinal alkaline phosphatase: close homology to placental
alkaline phosphatase. Proc Natl Acad Sci. 1984;84:1234–8.
42. Weiss MJ, Cole DEC, Ray K, Whyte MP, Lafferty MA, Mulivor
RA, Harris H. A missense mutation in the human liver/bone/
kidney alkaline phosphatase gene causing a form of lethal
hypophosphatasia. Proc Natl Acad Sci. 1988;85:7666–9.
43. Kam W, Clauser E, Kim YS, Kan YW, Rutter WJ. Cloning,
sequencing, and chromosomal localization of human term pla-
cental alkaline phosphatase cDNA. Proc Natl Acad Sci.
1985;82:8715–9.
Ind J Clin Biochem (July-Sept 2014) 29(3):269–278 277
123
44. Sowadski JM, Handschumacher MD, Murthy HMK, Foster BA,
Wyckoff HW. Refined structure of alkaline phosphatase from
Escherichia coli at 2.8 A resolution. J Mol Biol. 1985;186:417–33.
45. Llinas P, Masella M, Stigbrand T, Menez A, Stura EA, Le Du
MH. Structural studies of human alkaline phosphatase in complex
with strontium: implication for its secondary effect in bones.
Protein Sci. 2006;15:1691–700.
46. Yoko I, Keiichi K, Masahiro I, Yoshihiro A, Shoji K, Kimimitsu
O. Tissue-nonspecific alkaline phosphatase with an Asp
289
?Val
mutation fails to reach the cell surface and undergoes proteosome-
mediated degradation. J Biochem. 2003;134:63–70.
47. Suzuki U, Yoshimura K, Takashi M. Uber ein enzyme¯phytasek
das anhydro-oxy-methylen-diphosphorsaure spaltet. Bull Coll
Agri Tokyo Imp Univ. 1907;7:503–12.
48. Wuthier RE, Register T. Role of alkaline phosphatase, a poly-
functional enzyme, in mineralizing tissues. In: Butler WT, editor.
Chemistry and biology of mineralized tissues. Birmingham:
EBSCO Media; 1985. p. 113–24.
49. Whyte MP. Physiological role of alkaline phosphatase explored
in hypophosphatasia. Ann N Y Acad Sci. 2010;1192:190–200.
50. Zhu T, Gan YH, Liu H. Functional evaluation of mutations in the
tissue-nonspecific alkaline phosphatase gene. Chin J Dent Res.
2012;15:99–104.
51. Moss DW. Aspects of the relationship between liver, kidney and
bone alkaline phosphatase. In: Stigbrand T, Fishman WH, editors.
Human alkaline phosphatases. New York: Alan R Liss; 1984.
p. 79–86.
52. Epstein E, Kiechle FL, Artiss JD, Zak B. The clinical use of
alkaline phosphatase enzymes. Clin Lab Med. 1986;6:491–505.
53. Rodan GA, Rodan SB. In: Peck WA, editor. Advances in bone
and mineral research annual II. Amsterdam: Excerpta Medica;
1984. p. 244–85.
54. Preussner HT. Detecting coeliac disease in your patients. Am
Fam Physician. 1998;57:1023–34.
55. Lange PH, Millan JL, Stigbrand T, Vessella RL, Ruoslahti E,
Fishman WH. Placental alkaline phosphatase as a tumor marker
for seminoma. Cancer Res. 1982;42:3244–7.
56. Schiele F, Vincent-Viry M, Fournier B, Starck M, Siest G. Bio-
logical effects of eleven combined oral contraceptives on serum
triglycerides, gamma-glutamyltransferase, alkaline phosphatase,
bilirubin and other biochemical variables. Clin Chem Lab Med.
1998;36:871–8.
57. Buchet R, Milla
´n JL, Magne D. Multisystemic functions of
alkaline phosphatases. Methods Mol Biol. 2013;1053:27–51.
58. Mueller HD, Stinson RA, Mohyuddin F, Milne JK. Isoenzymes of
alkaline phosphatase in infantile hypophosphatasia. J Lab Clin
Med. 1983;102:24–30.
59. Sultana S, Al-Shawafi HA, Makita S, Sohda M, Amizuka N,
Takagi R, Oda K. An asparagine at position 417 of tissue-non-
specific alkaline phosphatase is essential for its structure and
function as revealed by analysis of the N417S mutation associated
with severe hypophosphatasia. Mol Genet Metab. 2013;109:282–8.
60. Chang KC, Lin PH, Su YN, Peng SS, Lee NC, Chou HC, Chen
CY, Hsieh WS, Tsao PN. Novel heterozygous tissue-nonspecific
alkaline phosphatase (TNAP) gene mutations causing lethal peri-
natal hypophosphatasia. J Bone Miner Metab. 2012;30:109–13.
61. Usoro NI, Omabbe MC, Usoro CAO, Nsonwu A. Calcium,
inorganic phosphates, alkaline and acid phosphatase activities in
breast cancer patients in Calabar, Nigeria. African Health Sci.
2010;10:9–13.
62. Prasad R, Lambe S, Kaler P, Pathania S, Kumar S, Attari S, Singh
SK. Ectopic expression of alkaline phosphatase in proximal
tubular brush border membrane of human renal cell carcinoma.
Biochim et Biophy acta. 2005;1741:240–5.
63. Niedermayer I, Feiden W, Henn W, Steilen-Gimbel H, Steudel
WI, Zang KD. Loss of alkaline phosphatase activity in menin-
giomas: a rapid histochemical technique indicating progression-
associated deletion of a putative tumor suppressor gene on the
distal part of the short arm of chromosome 1. J Neuropathol Exp
Neurol. 1997;56:879–86.
64. Kim JM, Kwon CH, Joh JW, Park JB, Ko JS, Lee JH, Kim SJ,
Park CK. The effect of alkaline phosphatase and intrahepatic
metastases in large hepatocellular carcinoma. World J Surg
Oncol. 2013;11:40.
278 Ind J Clin Biochem (July-Sept 2014) 29(3):269–278
123
