Alkaline phosphatase isozymes

Further studies (38) in vivo and in vitro showed that the new enzymes produced by the cross between E. coli and S. marcescens were a dimer containing one monomer of the E. coli type and one monomer of the S. marcescens type. By crossing E. coli with an S. marcescens mutant that produced very little alkaline phosphatase, it was shown that the alkaline phosphatase isozymes produced gave the same tryptic peptide map as the E. coli wild-type enzyme with the exception of two new peptides. 

Pio. 18. Starch-gel pattern of placental alkaline phosphatase isozymes in the final steps of the purification (a) variant A and B in the best alcohol fractions before Sephadex-gel filtration, (b) variant A after removing B in the Sephadex-gel column, and (c) crystalline alkaline phosphatase (variant B). 

Other studies have reported raised urinary levels of alkaline phosphatase in a wide variety of renal disease (A2, A14, R22, W4). The patterns of distribution of alkaline phosphatase isozymes in the urines of patients having higher blood levels of this enzyme closely resemble those in the circulation. Urine enzyme may also originate from the prostate (S25). 

Summary of the gene nomenclature, accession numbers, common names, tissue distribution, and function, if known, for the human and mouse alkaline phosphatase isozymes... 

Ishino, Y., Shinagawa, H., Makino, K., et al. (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169, 5429. 

Phosphatases are numerous and important enzymes (see also Chapt. 2). They are classified as phosphoric monoester hydrolases (phosphatases, EC 3.1.3), phosphoric diester hydrolases (phosphodiesterases, EC 3.1.4), triphosphoric monoester hydrolases (EC 3.1.5), diphosphoric monoester hydrolases (pyrophosphatases, EC 3.1.7), and phosphoric triester hydrolases (EC 3.1.8) [21] [63]. Most of these enzymes have a narrow substrate specificity restricted to endogenous compounds. However, some of these enzymes are active toward xenobiotic organophosphorus compounds, e.g., alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), aryldialkylphosphatase (para-oxonase (PON1), EC 3.1.8.1) and diisopropyl-fluorophosphatase (tabunase, somanase, EC 3.1.8.2) [64 - 70]. However, such a classification is far from definitive and will evolve with further biochemical findings. Thus, a good correlation has been found in human blood samples between somanase and sarinase activities on the one hand, and paraoxonase (PON1) type Q isozyme concentrations on the other [71]. 

The non-specific alkaline phosphatases present in bone and calcifying cartilage have several properties in common. The ATPases concerned in the formation of different hard tissues seem to be isozymes. It could be shown that two enzymes capable of degrading ATP exist. One of them can be inhibited by levamisole and R 8231 and is probably identical with non-specific alkaline phosphates. The activity of the other enzyme, tentatively named Ca-ATPase , is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The Ca-ATPase is unaffected by ouabain and ruthenium red. It may be speculated that the Ca-ATPase is concerned with the transmembranous transport of Ca2+-ions to the mineralization front229. ... 

Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three bands (38, 39, 41, 43, 69), four (44) and five (70) equally spaced bands have been found. 

Schlesinger and Anderson (44) separated the isozymes on starch-gel and DEAE-cellulose at various times after 14C-amino acids were added to a culture of E. coli synthesizing alkaline phosphatase. They found that initially most of the counts appeared in isozyme I, but later they appeared mainly in isozymes II and III. In another experiment where only enough radioactive amino acid was added to allow for 5 min of synthesis of radioactive protein, they found that the label is initially found in isozymes I and II and later in isozymes II and III. These experiments establish that the monomers of isozyme I are precursors for isozymes II and III. 

Isoenzymes or isozymes are enzymes from a single species that have the same kind of enzymatic activity but differ in chemical structure. In addition, they may differ in quantitative characteristics such as possessing different Km s with the same substrate and may differ in response to temperature and effectors. Isozymes of more than 100 enzymes have been demonstrated in humans. The most important of these for diagnostic purposes are the isozymes of LDH, CK, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, and aldolase. These have been exploited for differential organ diagnosis. 

Heterogeneity of alkaline phosphatase in human and mouse intestine (M34, M35), calf intestine (B13), liver (M33), and kidney (B46) has been reported in the literature, but the separation of the individual components, especially the slow- and fast-moving isozymes completely free from each other has not been achieved. It may be reasonable to suggest that in each organ several variants may be expected, but their biochemical significance is basically the same. 

The resolution of the isozymes of human alkaline phosphatase in normal individuals by starch-gel electrophoresis was systematically studied in 1961 by Boyer, who observed a characteristic alkaline phosphatase pattern similar in pregnancy sera and in placenta (B38). With regard to placenta, recent work (H5, R15, R16) has indicated genetic variation of placental alkaline phosphatase in human placenta when the starch-gel electrophoresis is carried out at two different pH s (8.6 and 6.0). Other tissues could not be differentiated by their starch-gel patterns by Boyer (B38, B39). 

In the pregnant subject, the placenta and intestine become the two major sources of serum alkaline phosphatase. Like intestine, placenta is a rich source of alkaline phosphatase and it enriches the outermost cell membranes of the microvilli of the syncytiotrophoblast, which is bathed by the maternal circulation. The properties of the placental enzyme differ sufficiently from those of the intestinal isozyme to permit their differentiation. ... 

Millan LL, Manes T (1988) Seminoma-derived Nagao isozyme is encoded by a germcell alkaline phosphatase gene. Proc Natl Acad Sci U S A 85 3024-3028... 

Bossi M, Hoylaerts MF, MiUan JL (1993) Modifications in a flexible surface loop modulate the isozyme-specific properties of mam-maUan alkaline phosphatases. J Biol Chem 268 25409-25416... 

Hailing Linder C, Englund UH, Narisawa S et al (2013) Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum. Bone 53(2) 399 08... 

Alkaline phosphatase (AP orthophosphoric monoester phospho-hydrolase, EC 3.1.3.1) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a broad spectrum of substrates in vitro [1]. Their broad snbstrate specificity and localization on the outside leaf of the cytoplasmic membrane suggests potential involvement in numerons extracellular processes. In humans, four isozymes of APs have been identified. One of them, tissue-nonspecific alkaline phosphatase (TNAP), is ubiquitously expressed, demonstrating especially high level of expression in bone, liver and kidney tissues. Three other isozymes demonstrate tissue-specific... 

Interestingly, all these compounds are inactive against mouse lAPs, therefore, unsuitable for studies of lAP function in mouse models. The objectives of the current studies were the identification of small-molecule modulators of mouse lAP and, potentially, identification of novel selective scaffolds of human lAP. To this end, we applied and optimized the chemiluminescent assays that were successfully utilized for screening human lAP, TNAP, and FLAP and developed a novel assay for mouse lAP, analogous to the assays of other isozymes. These assays utilize CDP-star, a substrate of alkaline phosphatases specifically invented for and commonly utilized in blotting techniques [19, 20]. Development and utilization of the prototype plate-reader enzymatic assay for TNAP isozyme with CDP-star substrate was previously described in detail elsewhere [21]. 

Serum isozymes following different types of injuries. The multimolecular forms of enzymes, called isozymes, may be identified using combinations of suitable gel electrophoresis and histochemistry. The changes in naphthylamidase and alkaline phosphatase following burns most likely indicate an alteration of liver metabolism and may persist for several weeks (A5). A rise in plasma alkaline phosphatase, but of kidney origin, can be found after limb ischemia in the rabbit. 

Strong labeling for the osseous isozyme of alkaline phosphatase has been used in the past to distinguish extraskeletal osteosarcoma from other pleomorphic sarcomas. The major drawback of this marker is that it must be assessed using cryostat sections or imprint smears paraffin sections are unsuitable for study. 

Besman, M., Coleman, J.E. (1985) Isozymes of bovine intestinal alkaline phosphatase./. Biol. Chem. 260, 11190-11193. 

Basal cells of the rabbit trachea and bronchus have fairly high specificity for alkaline phosphatase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, this enzyme is considered to be a useful marker for basal cells (Inayama et al. 1995). 

In another study (Herold et al., 1975), the only adverse effect reported in rats given tretinoin orally at a dose of 0.4 or 10 mg/kg/day for 90 days was elevation of serum alkaline phosphatase. Although no attempt was made to identify the isozymes, the elevated values of alkaline phosphatase were attributed to osteoblastic hyperactivity. Evidence for bone remodeling was seen only at the higher dose (Cahn et al., 1975). 

Although a major adverse effect of the retinoids is an increase in serum alkaline phosphatase, an association of this effect with fractures has not been unequivocally established. There have been no studies demonstrating that the increase in the activity of the serum enzyme is due to an increase in bone isozyme and there has been no clear-cut association between the incidence of fractures and serum alkaline phosphatase activity. 

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