Roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated calcification. Effect of selective release of membrane-bound alkaline phosphatase and treatment with isosmotic pH 6 buffer

The roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated mineralization have been studied by selectively releasing the enzyme from a wide variety of matrix vesicle preparations using treatment with a bacterial phosphatidylinositol-specific phospholipase C and by demineralization of the vesicles using isosmotic pH 6 buffer. Following depletion of 50-90% of the alkaline phosphatase activity or treatment with citrate buffer, the vesicles were tested for their ability to accumulate 45Ca2+ and 32Pi from a synthetic cartilage lymph. Removal of alkaline phosphatase by phospholipase C treatment caused two principal effects, depending on the matrix vesicle preparation. In rapidly mineralizing vesicle fractions which did not require organic phosphate esters (Po) to accumulate mineral ions, release of alkaline phosphatase had only a minor effect. In slowly mineralizing vesicles preparations or those dependent on Po substrates for mineral ion uptake, release of alkaline phosphatase caused significant loss of mineralizing activity. The activity of rapidly calcifying vesicles was shown to be dependent on the presence of labile internal mineral, as demonstrated by major loss in activity when the vesicles were decalcified by various treatments. Ion uptake by demineralized vesicles or those fractionated on sucrose step gradients required Po and was significantly decreased by alkaline phosphatase depletion. Uptake of Pi, however, was not coupled with hydrolysis of the Po substrate. These findings argue against a direct role for alkaline phosphatase as a porter in matrix vesicle Pi uptake, contrary to previous postulates. The results emphasize the importance of internal labile mineral in rapid uptake of mineral ions by matrix vesicles.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Purification and characterization of phytase from rat intestinal mucosa.

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8 or 3.1.3.26) was purified from rat intestinal mucosa. The purified enzyme preparation exhibited two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular masses of 70 kDa and 90 kDa. Rabbit antisera prepared against the 90K subunit cross-reacted with the 70K subunit on immunoblotting. The peptide maps of the 70K and 90K subunits were similar, and the N-terminal amino acid sequences of the two subunit proteins were almost identical. Treatments to remove sugar moieties from the proteins showed that the two subunit proteins had different oligosaccharide chains, although the difference in their molecular masses was not due to the difference in their oligosaccharide compositions. The purified enzyme also showed activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1), but the properties of the two enzyme activities were different; the optimum pH for phytase activity was 7.5, while that for alkaline phosphatase was 10.4. Phytase activity did not necessarily require divalent cations, while Mg2+ was essential for alkaline phosphatase activity. Phenylalanine, a specific inhibitor of intestine-type alkaline phosphatase had no effect on the phytase activity.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

Alterations in the hepatic enzymes following experimental lead poisoning

An investigation on the influence of lead toxicity on some of the hepatic enzymes was studied in rats both after a shorter interval of 15 d and after longer intervals of 60 and 90 d. Three different doses of lead as 5, 10, and 50 mg/kg body wt were administered orally on every alternate day. Whereas significant inhibition of succinic dehydrogenase was seen following lead poisoning, the activity acid and alkaline phosphatase increased with lead intoxication. The histoarchitecture of the liver was grossly intact. Liver accumulated less lead compared to kidney at 60 and 90 d.     

Extracellular phosphatases of Chlamydomonas reinhardi and their regulation.

Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.     

The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Production of phosphatates by fungi isolated from desert soils

Twelve fungal cultures isolated from Indian desert soils belonging toAspergillus, Penicillium, Acrophialophora andAlternaria were found to produce both acid and alkaline phosphatases in liquid medium, their amounts varying from culture to culture. Maximum production of these enzymes was observed withA. niger. In general, acid phosphatase activity was much higher as compared to alkaline phosphatase. The optimum incubation period for the production of these enzymes was found to be 14 d and thereafter it started declining. There was a significant and positive correlation between biomass production and acid phosphatase activity but not with alkaline phosphatase.     

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.     

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.     

Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C.

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.     

Increased plasma pyridoxal-5′-phosphate when alkaline phosphatase activity is reduced in moderately zinc-deficient rats

It is generally believed that the zinc metalloenzyme alkaline phosphatase is required to hydrolyze phosphorylated forms of vitamin B-6 prior to their use. To test this hypothesis, rats were fed a liquid diet containing either adequate or moderately low zinc during gestation and lactation. Zinc deficiency was produced in dams evidenced by significant reductions in zinc concentration of plasma (49%), liver (25%), and femur (24%), and plasma alkaline phosphatase activity (48%). Plasma pyridoxal-5′-phosphate (PLP), which significantly increased (61%) in these same rats, was negatively correlated (r=−0.74,P<0.02) with plasma alkaline phosphatase activity. Maternal liver PLP concentration was unaffected by zinc status. The zinc and vitamin B-6 relationship seen in dams was less observable in offspring. Stimulation of erythrocyte alanine aminotransferase activity by exogenously added PLP in vitro tended to be higher in both moderately zinc-deficient mothers and their offspring, but the difference was not significant. Our results support the hypothesis that alkaline phosphatase activity is required for the hydrolysis of plasma PLP. Our results also suggest that zinc status as alkaline phosphatase activity should be defined in an individual if plasma PLP is to be used as an indicator of vitamin B-6 status.     

Determinants of variation in plasma alkaline phosphatase activity: a twin study

Plasma alkaline phosphatase activity has been measured in 204 pairs of twins aged 18-34. Estimated genetic variance was the same in men and women, after exclusion of the youngest men, but environmental variance was greater in men so that the heritability was .90 +/- .02 in the women and .71 +/- .05 in the 19-34-year-old men. About 15% of the genetic variance was associated with the ABO blood group polymorphism, which is known to affect intestinal alkaline phosphatase. Differences in plasma alkaline phosphatase activity between normal people may be mainly due to genetically determined proportions of isoenzymes of differing stability.     

Relationship of serum and saliva calcium, phosphorus and alkaline phosphatase with dry mouth feeling in menopause

doi: 10.1111/j.1741‐2358.2012.00619.x Relationship of serum and saliva calcium, phosphorus and alkaline phosphatase with dry mouth feeling in menopause Objectives: The aim of this study was to compare serum and saliva calcium, phosphorus and alkaline phosphatase of menopausal women with/without dry mouth (DM) feeling. Background: The composition of saliva in menopause women with/without DM feeling is different. Some of these differences are in hormones that are related to bone turnover. Methods: A case–control study was carried out on 60 selected menopausal women aged 45–79 years with or without DM feeling (30 as case, 30 as control), conducted at the Clinic of Oral Medicine, Tehran University of Medical Sciences. The phosphorus concentration was measured by photometrical measurement of the blue colour formed after the addition of ammonium molybdate and stannous chloride; calcium was measured by Arsenazo reaction; and alkaline phosphatase by the pNPP‐AMP method. Statistical analysis of Student’s t‐test was used. Results: The mean serum phosphorus and alkaline phosphatase, stimulated and unstimulated saliva calcium and alkaline phosphatase levels were significantly higher in the menopausal women suffering from DM. There were no significant differences between groups regarding saliva phosphorus and serum calcium concentration. Conclusion: Calcium, phosphorus and alkaline phosphatase appear associated with DM feeling in menopause.     

Pyridoxal 5'-phosphate disappearance from perfused rat jejunal segments: correlation with perfusate alkaline phosphatase and water absorption

The relative effects of perfusate alkaline phosphatase activity and net water absorption on 2 microM pyridoxal 5'-phosphate (PLP) luminal disappearance from rat jejunum were studied in a single-pass, in vivo perfused intestinal segment model. Perfusate consisted of unlabeled PLP in buffer (pH = 7.4). Net water flux was monitored by inclusion of [3H]polyethylene glycol. PLP was measured by the [14C]tyrosine apodecarboxylase assay. Single and multiple regression analysis of results during perfusion of 2 microM PLP in Krebs bicarbonate buffer demonstrated no correlation between perfusate alkaline phosphatase activity and net water absorption and significant correlations between PLP luminal disappearance and both perfusate alkaline phosphatase activity and net water absorption. Correlation for the latter was improved when disappearance results were corrected for variations in perfusate alkaline phosphatase activity. When perfusate buffers were selected to yield divergent rates of net water absorption, the one associated with greater net water absorption was also associated with greater PLP disappearance. That this could not be explained by changes in perfusate alkaline phosphatase activity was demonstrated both by assessment of the rate of decay of PLP added in vitro to exited perfusate incubated at 37 degrees C and by measurement of alkaline phosphatase activity under conditions defined by the buffers using a modified spectrophotometric assay. Conclusions were: (1) In vivo PLP luminal disappearance correlates significantly with both perfusate alkaline phosphatase activity and net water absorption; (2) these two factors appear to act as independent variables; and (3) future studies on PLP intestinal absorption will need to take both of these variables into account in the interpretation of results.     

The development of phosphomonesterases in the testes of prepuberal chicks.

1. The biochemical development and histochemical localisation of phosphomonoesterases in the testes of prepuberal chicks have been studied. 2. Maximum acid phosphatase activity was observed at 12 weeks with a decrease in enzyme activity after this age, whereas alkaline phosphatase activity fluctuated with age. 3. Acid phosphatase activity in chicks was similar to that of the cockerel in being tartarate-insensitive. 4. There was a low level of significant correlation between acid phosphatase activity and testes weight. 5. Both alkaline and acid phosphatase activities were observed in the basement membrane of the seminiferous tubules, and acid phosphatase activity also in the various spermatogenic elements. 6. The results suggest that acid phosphatase is more involved in spermatogenesis, and more widely distributed than alkaline phosphatase in testicular tissue during testicular development.     

A highly sensitive assay method for human placental alkaline phosphatase involving a monoclonal antibody bound to a paper disk

A monoclonal antibody which is specific for human placental alkaline phosphatase and does not cross-react at all with intestinal alkaline phosphatase was prepared, and a procedure for the determination of placental alkaline phosphatase activity in serum was developed involving this monoclonal antibody bound to a paper disk. The minimum amount of placental alkaline phosphatase detectable by this method is 0.0025 King-Armstrong unit. Good correlation with the heat-treatment method was obtained. Therefore this proposed method can be used as a routine clinical test for the determination of serum placental alkaline phosphatase.     

The effect of doxycycline on the intestinal alkaline phosphatase (EC 3.1.3.1) activity in rat. A quantitative study

The doxycycline effect on the alkaline phosphatase activity in the small intestinal glandular epithelium in rat was examined, using the interferometric technique. The antibiotic caused a statistically significant fall of the alkaline phosphatase activity and the enzyme was found to decrease with consecutive doses of the drug.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver: lack of sensitivity to alkaline phosphatase when compared with that of glucocorticoid receptor

Rat hepatic cytosol was treated with alkaline phosphatase in order to determine if dephosphorylation altered the ability of Ah receptor to bind 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD). Glucocorticoid receptor was studied for comparison. As previously had been shown in other laboratories, treatment of cytosol with purified alkaline phosphatase dramatically reduced the subsequent ability of glucocorticoid receptor to bind hormone. However, alkaline phosphatase had no effect on the ability of Ah receptor to bind [3H]TCDD. If either glucocorticoid receptor or Ah receptor was occupied by its ligand prior to exposure to alkaline phosphatase there was no loss in ligand binding capacity. Crude alkaline phosphatase (containing some protease activity) substantially reduced the ability of glucocorticoid receptor to bind hormone and shifted the sedimentation position of the glucocorticoid receptor from approximately 8 S to approximately 2 S. Crude alkaline phosphatase did not reduce the ability of Ah receptor to bind [3H]TCDD and did not alter sedimentation of the 9 S [3H]TCDD. Ah receptor complex. Although the Ah receptor appears to be a member of the steroid receptor superfamily, the lack of effect of alkaline phosphatase on Ah receptor (compared to the sensitivity of glucocorticoid receptor) highlights another significant difference in molecular characteristics between the Ah receptor and the receptors for steroid hormones.