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PubMed1988

Cytophotometric analysis of alkaline phosphatase and 5'-nucleotidase activity in regenerating rat liver after partial hepatectomy.

Alkaline phosphatase and 5'-nucleotidase activities were analysed cytophotometrically in cryostat sections of female rat liver after partial hepatectomy. Alkaline phosphatase activity increased rapidly after operation up to a maximum seven-fold rise at 24 h in comparison with sham operated or control rats. There was no indication of preferential localization of alkaline phosphatase activity in either periportal or pericentral areas at any time point in control rats, sham operated rats or hepatectomized rats. Microscopical observation revealed that (a) all alkaline phosphatase activity was present at the bile canalicular surface of hepatocytes and (b) hepatocytes in mitosis did not show any increase in activity. These findings indicate that the high alkaline phosphatase activity after partial hepatectomy is not involved primarily in proliferation processes because cell division mainly takes place periportally. It may be needed for enhanced bile secretion by conversion of intracellular phosphorylcholine into choline which can be transported into the bile. The intracellular phosphorylcholine level is high after operation due to changes in phospholipid metabolism. 5'-Nucleotidase appeared to be three times higher pericentrally than periportally under normal conditions. Partial hepatectomy caused a 40 per cent decrease in activity in pericentral areas and only a small decrease periportally. It has been suggested that 5'-nucleotidase plays a role in breakdown of messenger RNA and its activity in control liver could be considerably lower periportally because plasma protein synthesis mainly takes place in this area.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1962

Distribution of alkaline phosphatase in the serum proteins in hypophosphatasia.

A patient with hypophosphatasia and her relatives, including an affected brother, were studied with regard to serum alkaline phosphatase levels, phosphoethanolamine excretion, leucocyte alkaline phosphatase, blood group genes, and alkaline phosphatase distribution in the serum protein fractions. The pattern of alkaline phosphatase distribution in the serum protein fractions was normal both in the clinically affected patients and in their relatives. Thus the electrophoretic mobility of serum alkaline phosphatases is not altered in hypophosphatasia. The mere presence of phosphoethanolamine in the urine was of no help in detecting heterozygotes for the hypophosphatasia gene(s), as this substance was also found in control urines.

Alkaline Phosphatase↗
PubMed2002

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies.

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.

Alkaline Phosphatase↗
PubMed1987

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase.

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.

Adenocarcinoma↗
PubMed1980

Characterization of equine alkaline phosphatase isoenzymes based on their electrophoretic mobility by polyacrylamide gel disc electrophoresis.

Alkaline phosphatase isoenzymes of equine tissues, peritoneal fluid, and serum were characterized by their electrophoretic mobilities, using polyacrylamide gel disc electrophoresis. The alkaline phosphatase isoenzymes in liver, kidney, spleen, small intestine, placenta, bone, small colon, and large colon tissue samples were extracted and separated by electrophoresis. The resulting isoenzyme mobilities and spectrophotometric scans were evaluated for their tissue specificity and for their possible use in determining the tissue contribution of alkaline phosphatase to serum and peritoneal fluid. The sensitivity of the tissue alkaline phosphatase to heating at 56 C was also determined. The isoenzymes of all the tissue extracts, except small intestine and large colon, were distinguishable from one another by their specific electrophoretic mobilities and patterns and by their sensitivity to heating at 56 C. The mobilities and patterns of small intestine and large colon isoenzyme extracts were not different enough to allow making a differentiation, but the heat resistance of small intestine did distinguish it from large colon. The tissue sources of alkaline phosphatase in the serum of healthy adult horses, pregnant mares, and normal foals and in the peritoneal fluid of healthy horses were determined by characterization of the mobilities of their isoenzymes.

Alkaline Phosphatase↗
PubMed1976

An active site peptide from human placental alkaline phosphatase.

A preparation of human placental alkaline phosphatase was labelled covalently at the active site with [32P]orthophosphate. Treatment with trypsin gave essentially one radioactive peptide, the active site peptide, of approximately 2300 molecular weight. Dansylation of the peptide showed that the amino-terminal residue was glycine. After acid hydrolysis the only radioactively-labelled amino acid present was serine phosphate. The amino acid composition was similar to those compositions reported for active site peptides from other alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1999

Alkaline phosphatase production during sporulation of Bacillus cereus.

Cell-bound alkaline phosphatase of Bacillus cereus was produced during vegetative growth and sporulation in a complex medium. Addition of glucose repressed the sporulation process and the amount of enzyme synthesized increased. The time course of alkaline phosphatase production is very similar in both sporulating and non-sporulating cells. Irrespective of sporulation, alkaline phosphatase level shows a peak of activity in the exponential phase, and another in the stationary phase of growth. This preliminary data indicates differences between B. cereus, and B. subtilis in alkaline phosphatase characteristics.

Alkaline Phosphatase↗
PubMed1993

The adverse effect of glycation of human serum albumin on its preservative activity in the freeze-drying and accelerated degradation of alkaline phosphatase.

The effects of a glycated protein additive on the stability of freeze-dried biological standards were studied using alkaline phosphatase as a model. Alkaline phosphatase was formulated with artificially glycated albumin, freeze-dried, sealed into glass ampoules and subjected to accelerated degradation studies at temperatures from -20 to 56 degrees C. Alkaline phosphatase, freeze-dried without an additive in neutral buffer, lost over 95% of its activity, but when freeze-dried with human serum albumin it retained approximately 70% of the initial activity. Both deliberately glycated and native albumin protected approximately 70% of the initial activity on freeze-drying and this protection was maintained during storage of the freeze-dried product at temperatures of 20 degrees C or below for up to 16 weeks. At 37 degrees C or above, alkaline phosphatase activity was lost in a time- and temperature-related manner with changes appearing in the SDS-PAGE gels and FPLC chromatograms but, with the artificially glycated albumin formulations, this loss of activity and the changes in the gels and chromatograms happened earlier and at lower temperatures. Formulations with trehalose at 1% w/v and 15% w/v, but without albumin, preserved some 40% of alkaline phosphatase activity following freeze-drying. Further, approximately half of that activity was maintained after 16 weeks' storage at all temperatures up to 56 degrees C by the 15% trehalose without albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed2006

Crystal structure of alkaline phosphatase from the Antarctic bacterium TAB5.

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.

Alkaline Phosphatase↗
PubMed1995

A variant alkaline phosphatase found in a case of gastric carcinoma with super bone scan.

A rare case of gastric carcinoma associated with increased serum variant alkaline phosphatase activities is presented. A 54 year old man had extremely high serum alkaline phosphatase activity (18,607 U/l) with normal calcium and phosphate concentrations. His bone scintigram showed abnormal findings, 'super bone scan'. He was diagnosed as having Borrmann type 4 gastric carcinoma with diffuse bone metastases by examinations of the upper gastrointestinal tract and iliac bone biopsy. The alkaline phosphatase isozyme of this patient was of the bone type as measured by cellulose acetate membrane electrophoresis and the placenta/bone type by agarose gel electrophoresis, respectively. Immunoelectrophoresis and the immunoprecipitation method using monoclonal antibodies against various alkaline phosphatase isozymes, however, showed that his serum alkaline phosphatase had the liver type antigenicity. Furthermore, it had a larger molecular size and different sugar chains compared with the common liver type alkaline phosphatase. These findings suggest that a unique variant alkaline phosphatase was produced by gastric cancer cells, which is possibly an explanation for the high serum alkaline phosphatase activities in this patient.

Alkaline Phosphatase↗
PubMed1994

A simple in vitro method to detect singlet oxygen and to compare photodynamic activity using alkaline phosphatase.

A simple, sensitive and reliable in vitro method based on photodynamic inactivation of alkaline phosphatase to detect singlet oxygen and for evaluating relative photosensitizing efficiencies of photosensitizers such as hematoporphyrin (Hp) and phthalocyanines has been developed and compared with photobleaching of p-nitroso dimethyl aniline (RNO) and photooxidation of L-tryptophan. Inactivation of alkaline phosphatase is dependent both on light fluence and sensitizer concentration. Scavengers like mannitol and azide anion indicated the involvement of singlet oxygen in the deactivation of alkaline phosphatase, since azide anion provided concentration dependent protection whereas mannitol had no effect and that compared to ordinary water, photoinactivation of alkaline phosphatase was three times higher in 65% D2O. Alkaline phosphatase appears to be resistant to free radical attack (particularly to OH radicals) since hydrogen peroxide alone or in presence of ferrous ions did not reduce the enzyme activity and mannitol or azide anion gave no significant protection when alkaline phosphatase was irradiated with Co-60 gamma rays up to 2 K Gy. With the present method using red light, the chloroaluminium phthalocyanine sulphonates prepared by sulphonation showed higher and the corresponding condensation product lower photodynamic activity; Hp being intermediate and Mn- and Gd-phthalocyanines had no photodynamic activity.

Alkaline Phosphatase↗
PubMed1995

Tumor necrosis factor alpha facilitates nuclear actions of retinoic acid to regulate expression of the alkaline phosphatase gene in preosteoblasts.

This study examines the molecular mechanisms of interaction between tumor necrosis factor alpha (TNF alpha) and retinoic acid on the expression of the alkaline phosphatase gene by rat clonal preosteoblastic cells. In this cell line, alkaline phosphatase mRNA was not constitutively expressed but was progressively induced by treatment with 1 microM retinoic acid, detectable by 6 h. Combining retinoic acid with 0.6 nM TNF alpha resulted in alkaline phosphatase mRNA appearing by 2 h, as well as enhanced expression above that observed with retinoic acid alone at 6, 12, and 24 h. Nuclear run-on analysis showed constitutive transcription of the alkaline phosphatase gene in control and TNF alpha-treated cells. At 4 h, retinoic acid, alone or combined with TNF alpha, increased alkaline phosphatase gene transcriptional rate by 2-fold. However, at 24 h, while no retinoic acid effect was retained, retinoic acid plus TNF alpha resulted in a 5-fold increase in alkaline phosphatase transcriptional rate. Examination of the distribution of nuclear alkaline phosphatase mRNA demonstrated that pre-spliced precursor mRNA was localized to the nuclear matrix in control and all treatment groups. Retinoic acid caused a time-dependent accumulation of mature, spliced alkaline phosphatase mRNA located in the non-matrix and cytoplasmic fractions, implying a post-transcriptional action of retinoic acid in nuclear processing and nucleocytoplasmic transport. Adding TNF alpha with retinoic acid greatly enhanced this effect, which was observed after 4 h, prior to any detectable interaction between TNF alpha and retinoic acid on gene transcription. In sharp contrast, only a negligible amount of nuclear processing occurred in control and TNF alpha-treated cells. This study reveals distinct interactions between TNF alpha and retinoic acid at post-transcriptional as well as transcriptional levels to regulate expression of the alkaline phosphatase gene in preosteoblasts.

Alkaline Phosphatase↗
PubMed1983

A kinetic study of rat salivary gland alkaline phosphatase and its inhibition by cadmium.

Alkaline phosphatase, confined to myoepithelial cells and blood capillaries in rat submandibular salivary gland (SSG), may participate in the regulation of salivary flow. To determine whether the alkaline phosphatase of SSG has unique properties, comparative kinetic and inhibition studies on enzymes from SSG, intestine and kidney were performed. The Km values (at optimal pH for each tissue) of 0.34, 0.55 and 0.49 mM with p-nitrophenylphosphate for the enzymes from SSG, kidney and small intestine respectively were similar. However, in the presence of cadmium the Ki values of 0.08 and 0.12 microM for the enzymes from SSG and kidney respectively were different from the value of 1.86 microM for the enzyme from small intestine. Differences in Ki values suggest differences in biochemical properties between the enzyme from small intestine and that isolated from SSG or kidney.

Acetates↗
PubMed1996

Utilization of the bone/liver alkaline phosphatase activity ratio in blood plasma as an indicator of ascorbate deficiency in salmonid fish.

The goal of this study was to test the hypothesis that the ratio of liver to bone alkaline phosphatase in blood plasma reflects the ascorbate status in scurvy-prone teleost fish (rainbow trout [Oncorhynchus mykiss]). The studies focused on finding a method for distinguishing bone alkaline phosphatase present in blood plasma from other alkaline phosphatase isoforms. We tested temperature optima and thermostability of liver, kidney, gill cartilage, and intestinal alkaline phosphatases. We did not observe differences among liver, bone, and kidney enzymes with respect to temperature optima and thermostability. We partially purified alkaline phosphatase from juvenile rainbow trout vertebrae and liver using n-butanol solubilization and ammonium sulfate fractionation. We found a difference between bone alkaline phosphatase, which precipitated in 0%-20% ammonium sulfate saturation, and liver enzyme, which required 40%-50% ammonium sulfate saturation to precipitation. We conducted a series of urea inactivation studies on partially purified enzymes from liver and vertebrae. Urea differentially inhibited the enzymes with t 1/2 = 1.1 and 0.4 min, for bone and liver, respectively. Subsequently, we subjected blood plasma alkaline phosphatase to urea inhibition, and using regression analysis we calculated the ratio of liver to bone alkaline phosphatase. We found that thus obtained ratios of bone enzyme in blood plasma correlated with liver ascorbate concentration. Bone alkaline phosphatase declined in ascorbate deficiency 10-fold, whereas low ascorbate status resulted in a 3.5-fold decrease. In order to draw a general conclusion on the linearity of the response of blood plasma/bone alkaline phosphatase as an indicator of ascorbate deficiency in fish, further studies must include analysis of individual fish followed in the process of developing avitaminosis.

Alkaline Phosphatase↗
PubMedDate not supplied

Bile acid-dependent secretion of alkaline phosphatase in rat bile.

The role of bile acids in the biliary secretion of alkaline phosphatase was studied. Rats with external bile fistulae were drained for 4 hr. After 2 hr, bile acid secretion fell progressively. Alkaline phosphatase secretion also decreased progressively during the period of drainage, suggesting that secretion of the two components was related. Each rat was then given an i.v. infusion of the taurine conjugate of either cholate, chenodeoxycholate, or ursodeoxycholate. Alkaline phosphatase secretion increased in a dose-dependent manner as bile acid secretion was varied over and beyond the physiologic range. Each bile acid affected alkaline phosphatase secretion differently: given at 0.5 mumoles per min per 100 gm, tauroursodeoxycholate caused a 3-fold, taurocholate a 14-fold, and taurochenodeoxycholate a 75-fold increase in enzyme secretion. To determine if these findings might represent elution of canalicular enzyme by bile acids, isolated liver surface membranes were incubated with the bile acids. Like the findings in vivo, taurochenodeoxycholate was strongest and tauroursodeoxycholate weakest in removing alkaline phosphatase from the membrane. Differential centrifugation of liver surface membranes after exposure to bile acids and ultracentrifugation of bile showed that more than half of the enzyme released by the action of bile acids did not sediment at 100,000 g and, thus, could be considered soluble. These results document bile acid-dependent secretion of alkaline phosphatase in rat bile and suggest that the process involves solubilization of both membrane fragments and free enzyme from membranes lining the biliary space.

Alkaline Phosphatase↗
PubMed1991

Characterization of seminoma-derived placental-like alkaline phosphatase.

Characteristics of placental-like alkaline phosphatase (PLAP-like enzyme) in seminoma was studied. By use of lectin affinity chromatography, PLAP-like enzyme in seminoma revealed extra sugar chains compared to placental alkaline phosphatase (PLAP), indicating heterogeneity of the carbohydrate moiety. However, the glycosylation patterns were found to be essentially similar between seminoma and normal testis. On isoelectric focusing, differences in migration patterns were revealed between seminoma-derived and normal testis-derived PLAP-like enzyme as well as between PLAP-like enzyme and PLAP. The differences in charge were mainly due to differences in sialylation of the molecules. The complex pattern of PLAP-like enzyme from seminoma on isoelectric focusing was not altered by neuraminidase treatment, indicating a considerable charge heterogeneity within the population of the enzyme molecules from the tumor.

Alkaline Phosphatase↗
PubMed1986

Effect of phospholipase C on high-molecular-mass alkaline phosphatase in serum.

Electrophoresis of some serum samples on polyacrylamide gel, followed by staining for alkaline phosphatase (EC 3.1.3.1), produces a band of activity at the gel origin. This high-Mr band consists of liver membrane fragments containing alkaline phosphatase and other enzymes. Alkaline phosphatase is closely associated with phosphatidylinositol in liver plasma membranes, and we have found that phospholipase C (EC 3.1.4.3) from Bacillus cereus, known to possess some phosphatidylinositol specificity, was able to release liver alkaline phosphatase from the high-Mr band. Two preparations of phospholipase C from Clostridium perfringens, however, which has no phosphatidylinositol specificity, had no effect on the alkaline phosphatase activity in the high-Mr band.

Adult↗
PubMed1976

Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability.

Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme.

Alkaline Phosphatase↗