Clinica Chimicu Acta, 186 (1989) 133-150 Elsevier

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CCA 04559

The human alkaline phosphatases: what we know and what we don’t know Harry Harris Universi~ of Penmylvani~ School of Medicine, Department of Human Genetics, Philadelphia, PA (USA) (Received 14 June 1989; accepted 19 June 1989) Key work

Liver/bone/kidney;

Placental; Intestinal; Placental-like; Polymorphism; DNA; Malignancy

A review of the human alkaline phosphatases dealing specifically with (1) the gene loci, (2) characterization and discrimin ation of the various enzymes, (3) polymorphism at the enzyme level, (4) cDNA and gene structures, (5) membrane binding, (6) the carbohydrate moieties, (7) hypophosphatasia, (8) alkaline phosphatases in malignancies, (9) function.

Introduction

During the last few years, remarkable advances in our knowledge of the human ALPS have occurred. We now know very much more than we did just 3 or 4 years ago. But much still remains obscure. In this paper I would like to outline what I think we now know with reasonable confidence. I will also take the opportunity of indicating some of the outstanding problems. Many of these will be addressed and some perhaps resolved during the course of this meeting. But many will still remain for the future. The gene loci

The ALPS are glycoproteins and we now know that there are at least four gene loci encoding their protein moieties. (a) The L/B/K locus [l] determines the so-called liver/bone/kidney or ‘tissue non-specific’ ALP and is expressed in virtually all tissues [2]. It shows particularly Correspondence to: Dr. H. Harris, University of Pennsylvania, School of Medicine, Department Human Genetics, Rm. 181 John Morgan Bldg., Philadelphia, PA 19104, USA.

0009-8981/89/$03.50

Q 1989 Elsewier Science Publishers B.V. (Biomedical Division)

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high activity in mineralizing bone where it is mainly local&d in the plasma membrane of osteoblastic cells [3,4). (b) The intestinal locus [5] is expressed in the intestinal mucosa. Here it is localised to the brush borders of the epithelial cells [a]. (c) The placental locus [7], determines placental ALP and is characteristically expressed in placenta after about the 12th wk of gestation [8,9] where it occurs in very large amounts in the syncitiotrophoblast by term [2]. It is also expressed, but only in relatively very small amounts, in lung and in cervix [2,10]. (d) The placental-like locus [ll] determines so-called ‘placental-like’ ALP. This is very similar, but not identical, in its structure and properties to placental ALP. In the healthy individual it has been found, but only in very small amounts, in testis and in thymus [2,12,13]. The reasons for the characteristic differences in the expression of the various loci in different tissues, and indeed in different cells within the same tissue, are not known. And this problem of tissue differentiation is a central issue for future work. It is possible that other loci exist. For example, fetal and adult forms of intestinal ALP have been found [14,15]. But it is not known whether this is because the protein moieties of the two forms are encoded by distinct loci, or whether the differences between the two forms are the consequence of differences in splicing or in processing at the mRNA or protein level. The intestinal, placental and the placental-like loci are closely linked and located near the end of the long arm of chromosome no. 2 (q34-q37) [16]. In contrast the L/B/K locus is located near the end of the short arm of chromosome no. 1 (p36.1-~34) [17]. 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. Thennostability The placental and placental-like ALPS are remarkably thermostable. They may be heated at 65 o C for an hour or more without loss of activity [2,12,18]. In contrast the intestinal and L/B/K ALPS are rapidly inactivated under these conditions (Table I). However, the intestinal ALPS are somewhat more thermostable than the L/B/K ALPS 119,201. It has also been shown that in serum, liver ALP is slightly, though significantly, more thermostable than bone ALP 1211. Inhibition studies Various low molecular weight substances show differential inhibition of the different ALPS [2,12,22-311. Table II summ&s the effects with five inhibitors

135 TABLE I Relative thermostabilities of human ALPS Human ALP

56°C(min)B

65”C(mitQB

L/B/R Intestinal Placental and Plac-like

7.4 > 60.0

1.0 6.5 z 60.0

a Time in minutes required to give 50% inactivation of different human ALPS at 56°C and 65°C.

which have been extensively used. Thus placental, placental-like and intestinal ALPS are about 30 times more sensitive to inhibition with L-phenylalanine (Phe) than the L/B/K ALPS. In contrast, the L/B/K ALPS are very much more sensitive to inhibition with L-homoarginine (Har) than are placental, placental-like or intestinal 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) characteristically 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. It should be noted that these various inhibitors are stereospecific and uncompetitive.

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 ALPS [2,12,32,33]. In Ouchterlony double diffusion tests, a continuous precipitin line is seen with the placental-like ALP, but there is spurring with intestinal ALP. Complementary results are obtained with antisera raised against intestinal ALP or L/B/K ALP [32-341. These findings demonstrate that some, though not all, of the antigenic determinants detected on placental ALPare also present on intestinal ALP, but the

TABLE II Effects of various inhibitors on different Human ALPS Inhibitor

ALP a L/B/R

Intestinal

Placental

Plac.-like

L-PhenylaIanine (Phe) L-Homoarginine (Har) L-Phenylalanylglycylglycine (Pgg) L-Leucine (Leu) Levamisole (Leva)

31 2.1 30.6 13.1 0.03

0.8 40 3.7 3.6 6.8

1.1 > 50 0.1 5.7 1.7

0.8 36 2.9 0.6 2.7

a Concentrations (mmol/l) of various inhibitors required to produce 50% inhibition of different human ALPS under standardized conditions.

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and placental ALPS in 73 amniotic fluids obtained at different times of gestation.

placental and placentaMike ALPS are immunologically very similar. Recently, however, a polyclonal antiserum that reacts with placental-like ALP but not with placental ALP, has been obtained by immunizing rabbit with a small peptide synthesized to correspond to a region where the aminoacid sequences of the two enzymes had been shown to differ by two aminoacid substitutions [ll]. Extensive studies with monoclonal antibodies raised against each of the different ALPS, have confirmed these general relationships and have provided a wealth of detailed information about the antigenic determinants present on each of the different molecular forms [35-451. For example, 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 enzymes [39,40,45]. Combinations of these various biochemical and immunological techniques have been used to devise methods which give precise analytical information 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. The point is well illustrated by the analyses of ALPS in 73 amniotic fluids obtained at different times during pregnancy, shown in Fig. 1 1461.Remarkable changes in ALP composition occur. Until about 21 wk of gestation most of the ALP activity is due to intestinal ALP which, as shown by electrophoretic studies, is of the fetal intestinal type and presumably

therefore derived from the fetal intestine. Although there is much variation from sample to sample, on average about 80% of the total ALP activity between 15 and 20 wk of gestation is intestinal ALP. The remainder is L/B/K ALP (on average about 16%) and placental ALP (about 3%). At about 20-21 weeks of gestation a sharp fall in intestinal ALP occurs and from then on during the rest of gestation only trace amounts of intestinal ALP are seen. The sharp fall in intestinal ALP at about 20-21 wk of gestation is associated with a marked decline in total ALP activity. However, from about 25 wk onward the total ALP activity progressively increases due to increasing L/B/K ALP and placental ALP. The fetal intestinal ALP which predominates in amniotic fluids in the early part of gestation is presumably derived from desquamated fetal intestinal epithelium cells which enter the amniotic fluid with defecated meconium. The sharp fall in intestinal ALP in amniotic fluid at about 21 weeks gestation coincides with the development of the fetal anal sphincter which prevents further movement of meconium into the amniotic fluid. Polymorphism

at the enzyme level

In the early 196Os, my colleagues and I, working in London, started a long-term study with the aim of finding out the extent of polymorphism among human enzymes [47]. We used the method of starch gel electrophoresis and developed a series of highly specific stains to identify different enzymes. Our approach was to examine each of the enzymes in a random series of unrelated individuals and to see which enzymes showed electrophoretic polymorphism. It turned out that enzyme polymorphism in human populations is a very much more frequent phenomenon than was, at that time, thought to be the case. Placental ALP was one of the enzymes we studied quite early on in this project. It was found to be highly polymorphic. If one takes a random series of placentas, as they come off the production line, one can readily identify six common electrophoretic phenotypes and we showed that these could be attributed to the various heterozygous and homozygous combinations of three common alleles at an autosomal locus [48,49]. However, it soon became clear as we examined increasing numbers of placentas that many so-called rare electrophoretic variants also occur. Thus, in a series of > 3000 placentas, some 15 different rare variant phenotypes were recognized [50]. They could be attributed to a series of rare alleles in heterozygous combination in most cases, with one or another of the three common alleles. Although individually rare, these heterozygotes together appeared to occur in at least 3% of the general population. This, for various technical reasons, is almost certainly an underestimate of the level of heterozygosity for rare alleles at the placental ALP locus. Rare alleles determinin g electrophoretically detectable enzyme variants occur at many other enzyme loci. But their abundance at the placental ALP locus is quite unusual. In the early 1970s we examined this point in the large mass of data we had collected, using a variety of tissue sources and essentially the same electrophoretic methods, over the’ previous 10 years. There were extensive data on a wide range of

138 TABLE III Heterozygosity for rare alleles at the placental ALP locus compared with 42 other enzyme loci

Number of loci Sample size (range 400-11000) Number of heterozygotes for rare alleles per thousand individuals

Placental ALP locus

Other loci

1 3815

42 2619 a

35.4 b

1.14 =

a Average. b For technical reasons, this is thought to be a minimal value. The true value was estimated to be close to 50. ’ Average weighted by sample size.

enzymes, other than placental ALP, coded by some 42 different loci [50]. These loci could be classified into two groups according to whether there were two or three common alleles, the so-called polymorphic loci, or only one common allele, the so-called non-polymorphic loci. A common allele for this purpose was defined as one with an allele frequency > 0.01. There were 12 so-called polymorphic loci and 30 non-polymorphic loci. Rare variant alleles had been found in both groups. The average heterozygosity for rare alleles was about the same in the two groups (1.10% for the polymorphic loci, and 1.16 for the non-polymorphic loci). The average heterozygosity in the total series was 1.14% (Table III). This is in marked contrast to what is found with placental ALP where the average heterozygosity for rare alleles is at least 35 per thousand and may be as high as 50%. Thus, the placental ALP locus appears to be very unusual in this respect. It is noteworthy that no allelic variants have been identified in either the intestinal or L/B/K ALPS by enzyme electrophoresis. Electrophoretic population surveys of placental-like ALP are difficult to carry out. However studies with monoclonal antibodies suggest that this enzyme, like placental ALP, is polymorphic though the details remain to be clarified [39]. In this connection, it is relevant that in a recent comparison of the aminoacid sequences of two of the common placental ALPS determined from their cDNAs, as many as 7 different aminoacid substitutions were identified, rather than the one or two that might have been expected [51]. The reason for the remarkable degree of allelic diversity expressed at the enzyme level in placental and perhaps placental-like ALPS is not known. It may involve an unusual degree of intragenic crossing over or gene conversion, or an unusually high single base change mutation rate. Whatever the phenomenon involved, it seems likely that it also accounts for the remarkable number of aminoacid substitutions between two common alleles. The elucidation of this problem in population genetics is an important one for future work.

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cDNA and gene stn~ctures In the last few years, a considerable amount of work has been devoted to defining the structures of the genes and the corresponding cDNAs of the four main types of human ALP; L/B/K, intestinal, placental and placental-like [1,5,7,11, 51-561. A wealth of detailed information has been assembled and it would not be practical to review all this in any detail. So I will simply confine myself to listing some of the salient points that have so far emerged. The sequences of the coding regions of the different genes are all very similar in length (1572-1602 bp). They each include at their 5’ ends, a short sequence coding for a signal peptide (17-21 aminoacids) which is cleaved off in protein synthesis leaving mature polypeptide chains 507-513 aminoacids long (Table IV). The placental and placental-like aminoacid sequences each comprise 513 amino acids with a positional identity of 98%. There are 12 aminoacid substitutions scattered throughout the molecule. In the case of intestinal ALP the mature polypeptide predicted from its cDNA sequence comprises 509 aminoacids and, after allowing for small gaps shows about 87% positional identity with both the placental and placental-like sequences. The aminoacid sequence of the mature L/B/K ALP polypeptide is somewhat different. It comprises 507 aminoacids but, after allowing for gaps, there is only about 50-60% positional identity with the other three ALPS. These findings as well as the DNA findings discussed below, tell us much about the evolutionary history of the four genes. They appear to have evolved from a common ancestral gene by a series of successive gene duplications. A rough outline of the deduced evolutionary tree is shown in Fig. 2. This conforms with earlier conclusions based on immunochemical studies. We do not yet know, with any certainty, the times in evolution when the several gene duplications occurred and this is a major problem for the future. Studies of the four genes at the DNA level have revealed an important difference between the L/B/K ALP gene on the one hand and the intestinal, placental and

TABLE IV Predicted aminoacid sequences of human ALP polypeptide subunits (derived from cDNA sequences of coding regions) ALP

Signal peptide

ALP polypeptide

L/B/K

17 19 21 18

507 509 513 513

Intestinal Placental PlacentaHike

% Positional identity (after allowing for’ ‘gaps’) Placental v Placental-like ALP Intestinal v placental or placentaHike ALP L/B/K v intestinal, placental or placental-like ALP

98% 87% 50-6041

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Fig. 2. Diagram showing the postulated evolutionary relationships of the human liver/bone/kidney (L/B/K), intestinal, placental and placental-like genes.

placental-like ALP genes on the other (Fig. 3). The L/B/K gene appears to be at least five times longer than each of the other three genes and has 12 exons compared with 11 in each of the other genes. The additional exon is at the 5’ end in the non-coding region. The overall difference in length is due to very much longer introns in the L/B/K ALP gene. The introns in the intestinal, placental and placental-like genes are all quite small (74-425 bp).

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Fig. 3. Diagram showing a comparison of the structures of the human L/B/K, intestinal, placental and placental-like alkaline phosphatase genes. The vertical bars represent the exons. 1. The L/B/K ALP gene is at least 5 xlarger than any of the other ALP genes. 2. The protein coding regions of each of the ALP genes are interrupted by introns at analogous positions. 3. The L/B/K gene contains an untranslated exon at the 5’ end which is not found in the other ALP genes.

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However, the introns interrupt the protein coding regions in closely analogous positions in all four of the ALP genes. A promoter region has been identified in each of the four genes and there is currently much interest in finding out how differences in sequence in this region may play a role in determining the differences in the expression of the various genes in different tissues. However at present the situation is unclear. Other sequences in the gene that may be involved in tissue-specific expression, are also being investigated. These are obvious important topics for future work. It should be noted that restriction fragment length polymorphisms have been described at both the L/B/K [57,58] and the placental ALP loci [59&O]. The allelic variation at the placental ALP locus using restriction enzymes does not stand out as being particularly unusual in the same way as does the allelic variation at the placental ALP locus discovered by enzyme electrophoresis. However, the sizes of the population samples studied are still relatively small. Membrane binding

The ALPS are now known to be members of a rather diverse group of membrane proteins which are anchored to cell membrane lipid bilayers by a phosphatidylinositol-glycan moiety attached to the carboxyl-terminus of the protein [61-671. The term PI-G tailed proteins has been suggested for this class of proteins and the biogenesis and transfer of the PI-G structure has been called glycosylphosphatidylinositolation (glypiation). So-called ‘soluble’ forms of ALP also occur, e.g. in serum. They are derived from the membrane bound forms by the action of a specific phospholipase C or D, the latter being particularly abundant in normal serum [67a,67b]. The ‘soluble’ forms are dimers, but the membrane bound forms are probably tetramers [68]. The detailed processes involved in glypiation and the detailed structures of the phosphatidyl-glycans, are still somewhat uncertain, However, there is currently much active work going on and I expect we will hear about the present situation during the course of this meeting. It is thought that co- or post-translationally a hydrophobic, carboxyl-terminal region of the nascent protein is removed and the glycolipid is attached to the carboxyl-terminus. In the case of human placental ALP a hydrophobic 29-residue carboxyl-terminal aminoacid sequence is removed and the phosphatidyl-inositol glycan is apparently linked to the alpha-carboxyl group of the now terminal aspartic acid via an ethanolamine residue [67,69]. Carbohydrate moieties

Much less is known about the structures of the carbohydrate moieties of these glycoproteins than of the protein moieties, though the structure of a single asparagine-linked sugar chain in human placental ALP has recently been reported [70]. However, the use of serial lectin affinity columns [71] for the analysis of the carbohydrate structures has produced some very interesting results which may be of general significance. Elution profiles by this technique were obtained for liver, bone,

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kidney, duodenum and placenta in both human and rat [72]. Not surprisingly, it was found that in humans there were differences between the elution profiles obtained for placental, intestinal and the L/B/K ALPS. But there were also clear differences between the elution profiles for liver, bone and kidney ALPS. Furthermore, the liver, bone, kidney and jejunal ALP elution profiles for human tissues, closely resembled those for the corresponding tissues in the rat. Thus the patterns of ALP glycosylation appear to be organ specific and not species specific; an important result, which may well also be the case for other glycoproteins. The placental ALP elution profiles were different in the two species, which is not surprising, since in the rat the enzymic properties and other characteristics of the ALP in placenta are the same as the L/B/K ALPS and quite different from those for human placental ALP. Hypophosphatasia

Hypophosphatasia is a typical ‘inborn error of metabolism’ characterized by defective osteogenesis due to failure or partial failure in bone mineralization and by a gross deficiency of L/B/K ALP in all tissues and in serum 173-761. In contrast, intestinal and placental ALPS are unaffected [77-791. There is also a considerable increase in the urinary excretion of phosphoethanolamine [74,80-821, and of urinary inorganic pyrophosphate [83]. A marked elevation of pyridoxal-5’-phosphate in the serum is also found [84]. Thus these three metabolites appear to be natural substrates of L/B/K ALP. It has been suggested that inorganic pyrophosphate controls the mineralization process in bone [85]. Its accumulation in hypophosphatasia might therefore be the immediate cause of the defect in bone mineralization. Hypophosphatasia shows much variation in clinical expression, and it is customary to classify the patients into three main forms: perinatal and infantile; childhood; and adult. However, there is no very sharp division between the different forms. In perinatal and infantile hypophosphatasia there are severe bony deformities which are often recognized in-utero by ultrasonography or h-ray. Most of these patients die shortly after birth or within the next few months [74-761. The childhood form is milder and is characterized by rickets-like deformities and premature shedding of the deciduous teeth [76,86,87]. The so-called adult form may become manifest by pseudofractures and the development of bony deformities in adult life although there is often a history of rickets and premature shedding of deciduous teeth in childhood, followed by some years of apparently normal growth [88-901. The condition appears to be inherited as an autosomal recessive. Despite the variability among families affected siblings within a family tend to show very similar clinical manifestations [76], so that the condition appears to be genetically heterogeneous, the affected patients either being heterozygous for different mutant alleles or homozygous for one or another of them. Heterozygotes for the normal allele and one of the mutant alleles are in most cases clinically unaffected. However they show reduced levels of L/B/K ALP in their serum, which on average amounts to about half the level seen in normal subjects in the same age group. But there is much

143

variation in serum ALP levels among both the heterozygotes and among the normal controls, resulting in overlap of the two distributions. The heterozygotes also exhibit a small but significant increase in urinary phosphoethanolamine and some increase in serum pyridoxal-5’-phosphate. Recently, in my own laboratory, we have been studying hypophosphatasia at the mRNA and DNA level. Two interesting results have emerged so far. 1. In one study [91], ALP activity and mRNA were assayed quantitatively in cultured fibroblasts from a series of 14 unrelated patients with infantile hypophosphatasia and from 12 healthy controls each < 2 years old. In the controls there was a high correlation between the ALP activity and the amount of mRNA. In the

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patients, the ALP activity was, as expected, virtually absent. But the mRNA levels were similar or in some cases higher than in the controls (Fig. 4). The sizes of the mRNAs were electrophoretically indistinguishable from those of the controls. The findings suggest that most cases of hypophosphatasia are the consequence of point mutations or of small electrophoretically undetectable deletions. 2. In another study [92], cDNA from a patient with a lethal infantile form of hypophosphatasia, the offspring of second cousin parents who came from an isolated inbred community in Nova Scotia, was shown to have a single base pair mutation. This predicted an aminoacid substitution (threonine for alanine) at aminoacid position 162 in the ALP protein. The patient was homozygous and both parents as well as other relatives, heterozygous for this mutant allele. The mutation, when introduced into an otherwise normal L/B/K ALP cDNA by in vitro mutagenesis, was sufficient to abolish ALP enzyme expression after transfection of the cDNA into mammalian cells, but it produced a stable, cross-reacting protein. The mutation was not found in about 20 other unrelated hypophosphatasia cases, emphasizing the heterogeneity of this condition. Clearly the extension of this type of study to other patients with hypophosphatasia should greatly clarify the genetics of the condition. ALPS in malignancies

The discovery by Fishman and his colleagues of the ‘Regan’ isozyme in a patient with disseminated lung cancer in the late 196Os, inaugurated a field of enquiry into the appearance of ALPS in malignancy [93]. The ‘Regan’ isoenzyme corresponds to placental ALP, but shortly after its discovery another ALP with slight though significant differences in its properties was identified in certain malignancies. This was called the ‘Nagao’ isozyme and is now known to correspond to placental-like ALP [25]. Finally the ‘Kasahara’ isoenzyme was identified in certain hepatomas [94,95]. This turned out to correspond to intestinal ALP. The ‘Regan’ isozyme has been reported in malignancies of lung, gastrointestinal tract, ovary, uterus, and in other tissues [96-981. However only some of the patients with these malignancies produce the enzyme. The ‘Nagao’ enzyme has also been reported in a variety of tumors [25,27,100]. It also occurs in only a proportion of the patients with a particular tumor. An exception to this is seminoma, which almost invariably produces large amounts of the ‘Nagao’ isozyme [101,102]. The ‘Kasahara’ isozyme has mainly been found in hepatoma [103] but occasionally appears to be produced by other tumors [104,105]. The reasons why some malignancies, though by no means all, express significant amounts of ALPS which have either not been detected in the normal tissue from which the tumor originated, or at most are found in only very small amounts in the normal tissue, are still largely obscure. Quite probably different causes operate in different tumors. In some cases it is possible that the malignant process activates in a cell the expression of an ALP gene not normaRy expressed, or amplifies the expression of a gene normally expressed at only a very low level. In other cases the tumor may represent inordinate clonal proliferation of certain cells that in the

145

normal tissue express that particular ALP to a much higher degree than most other cells in the tissue. This latter possibility is almost certainly the explanation for the large amounts of placental-like ALP found in seminomas. Clearly much work will be required in the future to clarify these matters. Function It is remarkable

that despite studies of these various enzymes over more than is known, with any certainty, about their biological functions in the normal organism. Perhaps the only exception to this statement comes from the work on hypophosphatasia which clearly indicates that normal L/B/K ALP on the cell membrane of osteoblasts is necessary for bone mineralization. However, it is quite unclear what L/B/K ALP is doing on the cell surface of most other cells in the body. Intestinal ALP occurs in high concentrations in the brush borders of intestinal epithelial cells. This has suggested that it might be concerned with the active absorption of metabolites from the small intestine such as inorganic phosphate. However, a variety of experiments aimed at establishing this point have in general failed to give clear or consistent results [106-1091. Another plausible suggestion is that the presence of ALP on the luminal surface of the intestinal epithelial cells catalyzes the hydrolysis of phosphate esters which are not normally absorbed in the gut, so that the non-phosphorylated moiety and the inorganic phosphate released are available for absorption by the epithelial cells [109,110]. However this possibility requires further substantiation. Placental ALP occurs in large amounts in the plasma membrane of the syncitiotrophoblast lining the microvilli and therefore represents an interface between the maternal blood and the fetus [ill]. This again suggests that it may be concerned with the active transport of some metabolite or metabolites from the mother to the fetus. However there is as yet no direct evidence for this. It is also unclear whether the marked allelic diversity of placental ALP has any selective significance and if so whether it plays a role in the maintenance of the polymorphism in different populations. The placental-like ALP in testis appears to be localized to the cell membrane of immature germ cells. However as yet we have no idea what its presumably special biological function may be. Thus, there is much to be found out about these fundamental questions in the future. four decades,

so little

Acknowledgements

This work has been supported by National Institutes of Health Grant GM 27018 and March of Dimes Grant 858. References 1 Weiss MJ, Ray K, Henthom PS, Lamb B, Kadesch T, Harris H. Structure of the human liver/bone/kidney alkaline phosphatase gene. J Biol Chem 1988;263:12002-12010.

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The human alkaline phosphatases: what we know and what we don't know.

A review of the human alkaline phosphatases dealing specifically with (1) the gene loci, (2) characterization and discrimination of the various enzyme...
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