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PubMed1990

Paradoxical digestion of myo-inositol phosphates by alkaline phosphatase at low pH.

The ability of bovine intestinal alkaline phosphatase (0.1-10 units/ml) to cleave myo-inositol bound phosphate moieties was examined. Paradoxically the digestion was optimal for a number of isomers at pH 5-7. It is possible that digestion at higher pH (9-10) does not proceed at maximal rates due to a conformation of the myo-inositol phosphate molecule which stabilizes the molecule against enzymatic attack. Alkaline phosphatase activity did not require the addition of added divalent cations. Moreover, several divalent cations, particularly zinc, were found to have a marked inhibitory effect. Further studies into this phenomenon suggested that some divalent cations can form insoluble complexes with myo-inositol phosphates, particularly those possessing a number of phosphate moieties, preventing the action of degradative enzymes. On the basis of these experiments we conclude that phosphate moieties can be removed from myo-inositol using relatively low concentrations of alkaline phosphatase as long as optimal incubation conditions are selected. This includes the use of a slightly acidic incubation media without the addition of divalent cations, particularly zinc.

Alkaline Phosphatase↗
PubMed2005

Changes of serum alkaline phosphatase activity in dry and lactational cows.

Serum alkaline phosphatase (ALP) activities were measured during dry and lactational periods to investigate the influence of lactation on serum ALP activity in cows. Higher levels of serum ALP activity were seen in lactational periods than in dry periods. The serum activities of bone-specific ALP (BALP), liver ALP (LALP), tartrate resistant acid phosphatase (TRAP) and aspartate aminotransferase also increased in lactational periods. ALP activities in the bone extract and in whey were decreased at similar rates by the addition of lectin. Moreover, since the ALP band in whey was observed to have the same migration in polyacrylamide gel (PAG) disk electrophoresis as that of the bone extract, analysis of ALP isoenzymes by lectin affinity or PAG disk electrophoresis could not distinguish ALP originating from the mammary gland from that of bone. In this study, it was clear that the increased level of serum ALP activity was due to increases of BALP and LALP in lactational periods. However, the extent of the influence of ALP originating from the mammary glands on serum ALP activity was unknown. Judging from changes of BALP and TRAP activities in the serum and the correlation between the both, it was guessed that ALP originating from the mammary glands influenced serum ALP activity.

Acid Phosphatase↗
PubMed1978

Activity and subcellular distribution of mouse uterine alkaline phosphatase during pregnancy and pseudopregnancy.

The biochemical assay of alkaline phosphatase in the uterus of mice revealed a 13-fold increase in the activity of the enzyme during the peri-implantation period with a relatively short-lived maximum being reached on Day 7 of pregnancy. A similar increase in the activity of the enzyme occurred between Days 5 and 7 of pseudopregnancy in uterine horns of mice receiving a deciduoma-inducing stimulus on Day 4. The presence of factors in uterine homogenates which could possibly modify the activity of the enzyme could not be detected. Low and unaltered levels of alkaline phosphatase activity were found in the blood serum of mice between Days 1 and 10 of pregnancy, suggesting that no appreciable entry of the uterine enzyme into the circulation occurs in this species. Alkaline phosphatase activity in the uterine homogenates was associated with particulate fractions sedimented during centrifucation at at 500, 10,000 and 105,000 g, and only about 29% of the activity was associated with the cytosol fraction. The subcellular distribution of the enzyme activity was the same in decidualized and non-decidualized horns. The association of the enzyme with the particulate material in uterine cells was considered to involve lipoprotein membranes because treatment of homogenates with 0.2% (v/v) Triton X-100 solubilized the enzyme without affecting its catalytic activity.

Alkaline Phosphatase↗
PubMed1983

Dibutyryl cAMP-inducible alkaline phosphatase in animal cell plasma membranes: fluorescence detection of mutant clones on polyester cloth.

We have developed a rapid screening assay that allows us to estimate the alkaline phosphatase content of mouse L-M cell colonies immobilized on polyester cloth. This permitted the identification and isolation of two mutant clones with increased constitutive alkaline phosphatase activity and six clones that fail to express this activity when treated with dibutyryl cyclic AMP. Both of the strains with increased constitutive activity have basal enzymatic activities that are 6- to 7-fold higher than the activity of the parental strain. The extents to which the cyclic nucleotide further induces alkaline phosphatase in these two strains are different, however, indicating that they represent two classes of mutants. Studies using amino acids and synthetic peptides as alkaline phosphatase inhibitors suggest that only one alkaline phosphatase isoenzyme predominates, in both the parental and the mutant cell lines, with or without induction by cyclic nucleotide. Comparison to mouse tissues indicates that our cell lines express an isozyme resembling that found in kidney and bone. The six clones that fail to express alkaline phosphatase activity when treated with dibutyryl cyclic AMP also have extremely low basal levels of the enzyme. All of these mutant strains continue to synthesize protein when treated with dibutyryl cyclic AMP and undergo growth cessation and morphological changes in the presence of this agent. Thus, the mutations all appear to affect factors specific to the expression of alkaline phosphatase activity rather than factors that affect general cellular responsiveness or permeability to dibutyryl cyclic AMP. The characterization of these strains may help elucidate mechanisms of eukaryotic membrane protein biogenesis, enzyme induction, and regulation of gene expression by cyclic nucleotides.

Alkaline Phosphatase↗
PubMed1968

The reversible inactivation of pig kidney alkaline phosphatase at low pH.

1. Pig kidney alkaline phosphatase is inactivated by treatment with acid at 0 degrees . 2. Inactivated enzyme can be partially reactivated by incubation at 30 degrees in neutral or alkaline buffer. The amount of reactivation that occurs depends on the degree of acid treatment; enzyme that has been inactivated below pH3.3 shows very little reactivation. 3. Studies of the kinetics of reactivation indicate that the process is greatly accelerated by increasing temperature and proceeds by a unimolecular mechanism. The reactivated enzyme has electrophoretic and gel-filtration properties identical with those of non-treated enzyme. 4. The results can be best explained by assuming that a lowering of the pH causes a reversible conformational change of the alkaline phosphatase molecule to a form that is no longer enzymically active but is very susceptible to permanent denaturation by prolonged acid treatment. A reactivation mechanism involving sub-unit recombination seems unlikely.

Alkaline Phosphatase↗
PubMed1988

Diagnostic value of alkaline phosphatase isoenzyme separation by affinity electrophoresis in the dog.

Affinity electrophoresis, using wheat germ lectin, was used to separate the alkaline phosphatase isoenzymes in the sera of 150 dogs with alkaline phosphatase values greater than or equal to 150 IU/L. The method provided clearer separation of the liver, bone and steroid-induced alkaline phosphatase isoenzymes commonly observed in canine serum, compared to conventional cellulose acetate electrophoresis. The dogs were divided into four patient groups determined by previous corticosteroid treatment, evidence of elevated endogenous corticosteroid levels, age and alanine aminotransferase values. The isoenzyme pattern of each patient was qualitatively assessed. The isoenzyme pattern most frequently observed was greater than 50% steroid induced alkaline phosphatase, which was present in 76 of 150 dogs. This pattern was observed in 18 of 22 dogs receiving corticosteroid therapy, two of three dogs with hyperadrenocorticism, and in dogs with a variety of other diagnoses. The majority of immature dogs (12 of 20) had an isoenzyme pattern consisting of greater than 50% bone. The majority of dogs with active hepatocellular injury (16 of 27) had greater than 50% liver isoenzyme. The isoenzyme pattern was not specific for certain diseases, therefore the diagnostic usefulness is limited. However the isoenzyme result is useful in some cases to determine which further diagnostic tests are indicated, and to determine the source of alkaline phosphatase elevation.

Adrenal Cortex Hormones↗
PubMed1978

Serum alkaline phosphatase (Al-Pase) isozyme in gastric and colonic cancer (using a simple thin layer polyacrylamide gel electrophoresis).

Using the simple thin layer polyacrylamide gel electrophoresis, serum alkaline phosphatase could be separated 5 isozyme bands in various digestive diseases, consisting of 54 cases of gastric cancer, 11 of colonic cancer, 12 of hepatoma, 4 of cholangioma, 14 of pancreatic cancer, 81 of benign hepatobilliary diseases, 13 of cancers of other organs and 61 of control. The obtained results were as follows: 1) The electrophoretic analysis of serum alkaline phosphatase showed the specific band remaining at the origin, already reported as "alkaline phosphatase O", in primary and metastatic cancer of the liver and cholelithiasis. On the contrary, alkaline phosphatase O was never found in gastric and colonic cancer without cholelithiasis. On the contrary, alkaline phosphatase O was never found in gastric and colonic cancer without cancerous metastasis to the liver, and it was also inclined to be positive with the progress of liver metastasis among them. 2) Intestinal alkaline phosphatase was usually found in higher frequency in blood group B and O than in the others, and it was apt to disappear in gastric or colonic cancer with an exacerbation of its cancerous lesions. 3) Heat-stable alkaline phosphatase was found in 10% of gastric or colonic cancer, all of which were histologically proved to be well differentiated adenocarcinoma.

Adenocarcinoma↗
PubMed2002

Function assignment to conserved residues in mammalian alkaline phosphatases.

We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k(cat) and increased K(m) when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxyl-terminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in k(cat), a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors l-Phe and l-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.

Alkaline Phosphatase↗
PubMed1975

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.

Alkaline Phosphatase↗
PubMed1982

Colonic and small intestinal alkaline phosphatase. A histochemical and biochemical study.

Colonic and intestinal alkaline phosphatase were studied histoenzymatically and biochemically in normal mucosae from eight hemicolectomy specimens. Histochemically, the most intense reaction was found in the small intestine, followed by vascular endothelium and colonic epithelium, respectively. The reaction was stronger in frozen sections than in acetone-fixed specimens, weakest in formalin-fixed tissues, and best when cold fixatives and low melting point paraffin were used. In the small intestine, the reaction was most marked on the villus tips. In the colon, the crypt necks showed the strongest reaction although good staining of luminal epithelium was found with more prolonged incubation. The addition of phenylalanine to the substrate medium resulted in selective inhibition of epithelial staining, whereas levamisole selectively inhibited the vascular reaction. On cellulose acetate electrophoresis, colonic and intestinal alkaline phosphatase showed slightly different rates of migration suggesting the presence of different isoenzymes. Quantitative analysis indicated that the intestinal enzyme expressed in U/g wet tissue was 7.7 times the colonic enzyme in amount. It is concluded that despite its relatively low biochemical activity, alkaline phosphatase can be demonstrated histochemically in colonic epithelium if tissues are processed at low temperatures and if the appropriate fixatives and adequate times are used.

Alkaline Phosphatase↗
PubMed1980

Intestinal alkaline phosphatase: an immunoprecipitation method for the determination in feces.

An immunoprecipitation method for the determination of human intestinal alkaline phosphatase (I-AP) in feces is described using anti-human I-AP IgG. Antibodies to human I-AP are monospecific after absorption with human placental alkaline phosphatase and show no cross-reactions with human liver alkaline phosphatase or alkaline phosphatase from E. coli. Normal controls demonstrate a logarithmic normal distribution of I-AP activity in feces. The standard deviation of the intra- as well as of the inter-assay variation is 11.7%. This method represents a simple quantitative non-invasive in vivo assay for brush border damage under experimental and/or clinical conditions.

Alkaline Phosphatase↗
PubMed1990

A monoclonal antibody against the surface of osteoblasts recognizes alkaline phosphatase isoenzymes in bone, liver, kidney, and intestine.

Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the osteoblastic lineage. One antibody, SB-1, reacts in frozen sections with a family of cells in bone, liver, kidney, and intestine which are identically stained by the histochemical substrate for alkaline phosphatase. In this report, biochemical and immunochemical evidence is presented to indicate that SB-1 is directed against an epitope on alkaline phosphatase which is shared by isoenzymes in a variety of chick tissues. In a solid-phase assay system, high dilutions (1:10(5] of ascites fluid were found to give significant binding of SB-1 to alkaline phosphatase extracted from chick limb or intestine. Partial purification of intestinal alkaline phosphatase on a Sepharose CL-6B column results in the co-elution of alkaline phosphatase enzyme activity and antibody-binding material; this indicates that SB-1 recognizes intestinal alkaline phosphatase rather than an impurity in the crude preparation. Furthermore, Western immunoblots of chick calvarial bone extract electrophoresed on a 5-20% SDS-polyacrylamide gel show that SB-1 reacts with a single 155 kD band which also is stained by the alkaline phosphatase histochemical substrate. In a similar set of experiments, SB-1 reacts with an intestinal alkaline phosphatase isoenzyme whose molecular weight is approximately 185 kD. From these studies, we conclude that SB-1 is specifically reactive with alkaline phosphatase isoenzymes present in bone, liver, kidney, cartilage, and intestine. The reactive epitope is stable to SDS denaturation, not associated with the active site of the enzyme, and dependent on disulfide bonds which impart secondary structure to the protein.

Alkaline Phosphatase↗
PubMed1987

Effect of bromelain on alkaline phosphatases of intestinal and non-intestinal tissues and serum.

Human alkaline phosphatases extracted with butanol from liver, kidney and placenta, and from foetal and adult small intestine each contain fragments with molecular masses within the range of approximately 8 kDa to 20 kDa which can be removed by digestion with bromelain. However, in the case of adult intestine, this fragment (which is presumed to represent a membrane-binding domain) can only be demonstrated in tissue extracted immediately after removal at operation. Similar fragments are also present in foetal intestinal phosphatase in amniotic fluid, and in liver and bone alkaline phosphatases recovered from serum. Again, however, adult intestinal phosphatase from serum differs in the absence of the bromelain-sensitive fragment. These observations indicate differences in the ways in which intestinal and non-intestinal alkaline phosphatases gain access to the circulation, and also have implications for structural studies on intestinal phosphatase extracted post mortem from adult tissue.

Adult↗
PubMed1989

A hybrid Escherichia coli alkaline phosphatase formed on proteolysis.

Cleavage of bacterial alkaline phosphatase by trypsin at the R-11, A-12 bond of both subunits results in changes in the structure and function of the enzyme as previously reported (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1985) J. Biol. Chem. 260, 7557-7561). A hybrid dimer has been formed by cleaving the R-11, A-12 bond of only one of the two subunits. This enzyme species has been purified and characterized to investigate subunit interactions of this hybrid dimeric enzyme species. Subunit interactions were observed using various methods to study functional and structural properties of the enzyme. In a kinetic study the T-2/A-12 hybrid enzyme was found to have a Vmax similar to the A-12 fully trypsin-modified enzyme. On exposure to EDTA the hybrid was found to lose activity at essentially the same rate as the A-12 enzyme presumably as a consequence of loss of metal ions required for function. On adding metal ions back to the apoenzyme form, activity of the hybrid was reconstituted to a degree similar to that of the native enzyme whereas the activity of the A-12 enzyme was reconstituted to a much lesser extent. The Tm of the hybrid measured by differential scanning calorimetry was closer to the value obtained for the A-12 enzyme than the T-2 enzyme but circular dichroic spectra indicated secondary structural features of the hybrid different from both symmetrical forms of the enzyme. These results provide evidence for strong subunit interactions in the alkaline phosphatase dimer.

Alkaline Phosphatase↗
PubMed1977

The effect of increased crypt cell proliferation on the activity and subcellular localization of esterases and alkaline phosphatase in the rat small intestine.

The activity and ultrastructural localization of alkaline phosphatase and esterase has been studied in normal rat intestine and after the increased crypt cell proliferation that occurs during recovery after 400 rad X-irradiation. Alkaline phosphatase activity is not present in crypt cells of normal intestine, but becomes apparent after the cell has migrated on to the villus. The enzyme is localized in the microvilli, along the lateral cell membranes and in dense bodies. Its activity increases 10 to 15-fold from the base to the tip of the villus. Morphometric analysis of the cell structures where this enzyme is localized reveals no marked changes in their relative proportions during crypt cell development. The expansion of the proliferative cell compartment along the whole length of the crypt which occurs during recovery after irradiation (72 hr after 400 rad X-irradiation) results in a marked reduction of alkaline phosphatase activity in the lower 10-15 cell positions at the base of the villus. During subsequent migration of these cells, the activity increases with cell age but normal values are not attained. From a morphometric analysis it was found that the ultrastructural development is similar to that in controls. These results suggest that during cell maturation, normal values for alkaline phosphatase activity are only attained after 10-12 hr period of maturation in a non-proliferative state and only after the cell has migrated on to the functional villus compartment. In normal intestine, esterase activity shows a 3-fold increase from the bottom to the tip of the crypt and 3 to 4-fold increase during migration up to the middle of the villus. Enzyme activity is localized in the endoplasmic reticulum, the dense bodies and the perinuclear space. Morphometric analyses reveal a 2 to 3-fold increase in the absolute size of these subcellular compartments during crypt cell differentiation and a 2-fold increase at the crypt-villus junction. The relative sizes increase 1-5-fold during crypt cell differentiation and at the time of transition of the cells on to the villus. Increased crypt cell proliferation after irradiation leads to a marked decrease in esterase activity both in crypts and villi. Morphometric analyses of electron micrographs indicate that these changes in activity are not related to any changes in the subcellular structures in which the enzyme is localized. It appears that the normal development of esterase activity depends both on the functional state of the cell and its localization in the crypt or villus.

Alkaline Phosphatase↗
PubMed1975

A new direct lead technique for histochemical demonstration of alkaline phosphatase activity.

A lead method for demonstrating alkaline phosphatase is described. The method is based on direct precipitation of lead as lead phosphatase at pH 9.5, the pH optimum of the enzyme. Stable incubation medium was achieved by using tartrate, instead of maleate, as chelating for lead. The method was found to be suitable for visualization of alkaline phosphatase in different types of tissues.

Alkaline Phosphatase↗
PubMed1980

Immunohistochemical localization of alkaline phosphatase in the chicken epiphyseal growth cartilage.

Alkaline phosphatase of chicken epiphyseal cartilage has been localized by two immunohistochemical methods. Double layer immunofluorescence and peroxidase anti-peroxidase (PAP) methods gave similar results. Alkaline phosphatase in epiphyseal cartilage is extracellular as well as intracellular in the localization. Extracellular reaction was strongest in the lower layers of growth plate and the most intense reaction was noted in the pericellular lacunae of hypertrophic chondrocytes. Also intracellular immunoreaction was noticed through the whole growth plate.

Alkaline Phosphatase↗
PubMed1977

Spectral studies of the interactions of Escherichia coli alkaline phosphatase with 4-(4-aminophenylazo)-phenylarsonic acid.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is reversibly inhibited by a variety of phenylarsonic acids, including some N-haloacetylated derivatives. The inhibition is of the competitive type, and Ki values are reported. The action on the enzyme of one of the arsonate inhibitors, the azo dye, 4-(4-aminophenylazo)-phenylarsonic acid was studied in detail, using spectrophotometric and kinetic methods. The azo dye binds more strongly to E. coli alkaline phosphatase than do the other arsonates. Spectrophotometric titration indicates the presence of a single, strong dye-binding site on the enzyme dimer molecule in the concentration range covered. In 0.1 M Tris - HCl buffer pH 8.0, 25 degrees C K diss for the dye - enzyme complex is 1.50 - 10(-5) M as determined by spectrophotometric titration. This value is in good agreement with the Ki = 1.30 - 10(-5) M obtained from kinetic measurements. The dye can be displaced from alkaline phosphatase by phosphate and competitive inhibitor 2-aminoethyl phosphonate. These results indicate that the dye binds with its arsonic acid group to the anion binding site of the active site of the enzyme. The binding of the dye to the native enzyme is associated with a red shift in the visible spectrum of the dye. It seems that the aromatic portion of the dye interacts with a hydrophobic region close to the anion binding site. The spectrum of the dye is not changed in the presence of the apoenzyme. When zinc is added to an apoenzyme-dye solution, the spectral changes of the dye depend on both the ratio of zinc per apoenzyme and the pH. The presence of Mg2+ had no effect on the observed phenomenon.

Alkaline Phosphatase↗