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Resistance to Levamisole (R12456) in heat-stable alkaline phosphatases.

The inhibitory effect of Levamisole (R12456) on human alkaline phosphatases has been studied. At a concentration of 0.1 mM it is possible to differentiate the source of the enzyme. Enzyme activity relatively resistant to Levamisole (less than 20 percent loss) is present in serum with the Regan isoenzyme, in placental extracts, in the intestinal "B" preparation and in heat-stable alkaline phosphatases "unmasked" after treatment at high ionic strength.

Alkaline Phosphatase↗

The measurement of alkaline phosphatase at nanomolar concentration within 70 s using a disposable microelectrochemical transistor.

We report a new approach to the measurement of alkaline phosphatase concentration based on the use of a disposable poly(aniline) microelectrochemical transistor. The measurement is carried out in a two cell configuration in which the poly(aniline) microelectrochemical transistor operates in acid solution and is connected to the alkaline buffer solution containing the analyte by a salt bridge. Disposable microelectrochemical transistors were reproducibly fabricated by electrochemical deposition of poly(aniline) onto photolithographically fabricated gold microband arrays. Using these devices alkaline phosphatase was detected by employing p-aminophenyl phosphate as the substrate for the enzyme and using glucose and glucose oxidase to recycle the p-aminophenol generated upon enzyme catalysed hydrolysis of the phosphate. Recycling the p-aminophenol with glucose and glucose oxidase amplified the detection of alkaline phosphatase approximately tenfold. Using this approach we obtain linear calibration curves for alkaline phosphatase up to 5 nM within 70 s on single use devices.

Alkaline Phosphatase↗

Transcriptional regulation and the effects of sodium butyrate and glycosylation on catalytic activity of human germ cell alkaline phosphatase.

Human choriocarcinoma cells, the malignant trophoblasts, synthesize germ cell alkaline phosphatase (GCAP) which shares 98% sequence identity with the placental alkaline phosphatase (AP). The two isozymes are immunologically similar but react differentially toward inhibition by L-leucine or EDTA. Administration of sodium butyrate to choriocarcinoma cells greatly increased the transcription rate of the GCAP gene, resulting in an increase in mRNA expression and enzyme biosynthesis. The butyrate-modulated AP induction was blocked by cycloheximide, suggesting that a mediator protein may be involved. Protein sequence deduced from complementary DNA analysis suggests that GCAP contains two potential sites for asparagine (N)-linked glycosylation. The marked increase in GCAP expression by butyrate in choriocarcinoma cells allowed us to study the extent of N-linked glycosylation and its role on GCAP enzyme activity. After limited tunicamycin treatment, Mr 65,000 (fully processed), Mr 58,000 (nonglycosylated), and Mr 62,000 polypeptides were synthesized by these cells in the presence of butyrate. This suggests that the Mr 62,000 product may be the singly glycosylated GCAP monomer and that both sites are glycosylated in this phosphatase. The glycosylated and nonglycosylated GCAPs, synthesized by butyrate-treated choriocarcinoma cells in the absence or presence of tunicamycin, respectively, were similarly inhibited by L-leucine or EDTA. Moreover, the specific enzyme activity of glycosylated and nonglycosylated GCAP remained unchanged, indicating that AP lacking N-linked oligosaccharide side chains was catalytically active. This is supported by the finding that nonglycosylated GCAP incorporated inorganic phosphate which binds to the active site of AP. Since the active form of AP is a homodimer, our data indicate that the glycan moieties are not required for the dimerization and catalytic activity of GCAP.

Alkaline Phosphatase↗

Activity of alkaline phosphatase in the major salivary glands of mice at various ages of postnatal life, and during pregnancy and lactation.

Activity of alkaline phosphatase in the major salivary glands of male and female mice at various ages of postnatal life, and in females during lactation was studied histochemically. Enzyme activity was not detected on the day of birth, but was found in the terminal tubules of all major salivary glands during the first postnatal week. Alkaline phosphatase activity was increasing gradually with age and a definitive enzymatic pattern was observed by the age of 6 weeks. No difference in enzyme activity was found among the major salivary glands of young adult and old animals. The parenchyma of fully differentiated submandibular glands showed clear sexually dimorphic patterns of alkaline phosphatase activity. During pregnancy, a significant increase of alkaline phosphatase activity was detected in submandibular gland. From gestation day 15 to the end of pregnancy, enzymic pattern of granular convoluted tubules of pregnant females was the same as in the adult males. Histochemical masculinization of the submandibular gland during pregnancy suggests that besides androgens also progesterone exerts masculinization of the murine submandibular salivary gland.

Aging↗

A band of alkaline phosphatase activity in the crypts of mouse duodenal epithelium.

Isolated mouse duodenal epithelium, in the form of structurally intact crypt-villus units, was used to study the distribution of alkaline phosphatase with histochemistry. The tissue was incubated on ice in the medium of Hugon and Borgers (J Histochem Cytochem 14:629, 1966), with constant stirring to ensure uniform reaction. Continuous activity was observed frm the crypt mouth to the villus tip. A single band of alkaline phosphatase activity, 2-03 cell in height, was observed in the mid-crypt region in about 80% of crypts studied. Control studies (no substrate control, no lead control, pH control, inhibitor control, inactivated enzyme control, no enzyme control, and stimulator indeed due to alkaline phosphatase. The narrow band of activity in the crypt was also observed in vibratome sections of nonfrozen tissue. When isolated epithelium was subjected to a freeze-thaw cycle and then incubated in Hugon's medium, reaction product was observed continuously from the mid-crypt region to the villus tip. This pattern was similar to that observed with frozen sections. We conclude that alkaline phosphatase is present in an active form in epithelial cells in the region of the band. In epithelial cells above the band, e.e., in the upper crypt, alkaline phosphatase is present in an inactive form which may be activated by a freeze-thaw cycle.

Alkaline Phosphatase↗

Localization of alkaline phosphatase in three gram-negative rumen bacteria.

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.

Alkaline Phosphatase↗

[Alkaline phosphatase isoenzymes in the serum of patients with renal insufficiency].

OBJECTIVE: The aim of this study was to test the utility of serum alkaline phosphatase isoenzymes determination from patients with renal insufficiency. MATERIAL AND METHODS: Serum levels of alkaline phosphatase isoenzymes were determined in a group of 58 patients: 22 of them suffering acute renal insufficiency (ARI) and 36 with chronic renal failure (CRF) undergoing regular hemodialysis, results obtained were compared from a population of 30 healthy adults. Intestinal, bone, liver, macromolecular and intestinal variant isoenzymes, were separated by electrophoresis on agarose gel and quantified using a densitometer. RESULTS: Were found a significant increase the total alkaline phosphatase activity in both pathologic groups (p < 0.05 in ARI and p < 0.01 in CRF). Isoenzymatic profiles showed: increase of the bone fraction (p < 0.05 in ARI and p < 0.001 in CRF), decrease of the liver isoenzyme (p < 0.001) in both groups, macromolecular fraction elevated in acute patients (p < 0.05) and a significant increase of the intestinal and intestinal variant isoenzyme in the chronic patients (p < 0.001). CONCLUSION: The renal insufficiency modified the normal distribution of alkaline phosphatase isoenzymes and the study of their serum levels could be an effective non-invasive marker, for the evaluation of bone disease and intestinal disorders associated with renal failure.

Adult↗

The Ala-161-->Thr substitution in Escherichia coli alkaline phosphatase does not result in loss of enzymatic activity although the homologous mutation in humans causes hypophosphatasia.

The correlation between sequence homology and catalytic importance of a specific amino acid in the E. coli and Liver/Kidney/Bone (L/K/B) human alkaline phosphatase was investigated. For this reason Ala-161 in the E. coli enzyme was substituted with a threonine residue via site-specific mutagenesis. The homologous amino acid in the L/K/B alkaline phosphatase sequence has been shown to cause inactivation of the enzyme. In E. coli alkaline phosphatase the Ala-161-->Thr substitution results in a mutant enzyme with virtually unchanged catalytic properties when compared to the wild-type enzyme. Our results show that Ala-161 in the E. coli alkaline phosphatase does not have an important catalytic role. The results suggest that the three-dimensional topology of the L/K/B alkaline phosphatase may be different from that observed for the enzyme from E. coli.

Alanine↗

Alpha 1 isoenzyme of alkaline phosphatases. Clinical importance and value for the detection of liver metastases.

Measurement of the alpha 1 (fast liver) fraction of alkaline phosphatases in the serum for 217 cancer patients, 92 patients with nonmalignant hepatic affections and 131 controls, revealed that the alpha 1 fraction offers better global value (94%), sensitivity (96%), and specificity (93%) than gamma GT or total alkaline phosphatase determinations for the detection of liver metastases during cancer. Initial data from study of the time of appearance of the alpha 1 fraction reveals that this fraction shows up earlier than rises in the gamma GT or total alkaline phosphatases. Results of a multiparametric study conducted on the alpha 1 fraction and various hepatic enzymatic tests (SGOT, SGPT, GLDH, ALP, gamma GT) indicate that the alpha 1 fraction used alone is better than any other test or combination of tests for biological detection of liver metastases. As concerns the influence of chemotherapy on the appearance of the alpha 1 fraction, the majority of the drugs used for anticancer chemotherapy do not seem to affect measurement of the alpha 1 ALP fraction. The alpha 1 fast liver fraction of alkaline phosphatases, detected by electrophoresis on cellulose acetate, can be considered one of the best known tests for the detection of liver metastases.

Alkaline Phosphatase↗

Another Kasahara-variant alkaline phosphatase in renal cell carcinomas.

Another Kasahara-variant alkaline phosphatase isoenzyme was found in 2 out of 25 human renal cell carcinoma tissues. This enzyme electrophoresed in a single diffuse band which is cathodal to but continuous with the liver alkaline phosphatase. After neuraminidase treatment, this enzyme electrophoresed in the same position as that of neuraminidase-treated Kasahara isoenzyme. The enzymic properties of another neuraminidase-treated Kasahara-variant enzyme such as inhibitions by L-phenylalanine, L-homoarginine, L-tryptophan, and L-leucine, effects of inorganic phosphate, urea, and sodium dodecyl sulfate, heat stability, and the reactivity with concanavalin-A are consistent with those of Kasahara isoenzyme. On Ouchterlony's double diffusion, the precipitin lines of Kasahara and the new variant enzyme produced by antibody to Kasahara isoenzyme fused completely. These facts may indicate the occurrence of another Kasahara-variant isoenzyme.

Adenocarcinoma↗

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum.

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl(2), BaCl(2), CuCl(2), and ZnCl(2). All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but beta-glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70-75 degrees C for both activities. The enzymes were fully stable up to 1 h at 60 degrees C. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications.

Alkaline Phosphatase↗

On the value of placental alkaline phosphatase as a marker for gynecological malignancy.

A sensitive catalytic assay for placental alkaline phosphatase (PLAP) was used to quantify enzyme levels in sera from women with malignant gynecological tumors, i.e. cervical carcinoma, ovarian carcinoma and carcinoma of the breast. Values were compared with those of a reference group of 155 healthy individuals. In this group, basic mean levels of the enzyme amounted to 0.06 +/- 0.08 mumoles X min-1 X 1(-1) (mean +/- SD), which corresponds to 0.3 +/- 0.4 ng/ml (mean +/- SD) purified placental alkaline phosphatase. Enzyme levels were not significantly affected by age, whereas sex gave slightly higher values for women in all age groups. This difference was statistically significant (p less than 0.001). The discriminating capacity of PLAP as a tumor marker was analysed with this assay and compared in sensitivity with a radio-immunoassay. A cut-off level of 0.2 mumoles X min-1 X 1(-1), corresponding to 1 ng/ml enzyme protein as measured by the radio-immunoassay, detected 23-68% of the tumor patients, giving false-positive results from the control group in the order of 5%. Corresponding values at a cut-off level of 0.3 mumoles X min-1 X 1(-1) (1.5 ng/ml) gave values 20-44% and 2%, respectively. The present investigation emphasizes that sensitivity in assays of placental alkaline phosphatase, whether catalytic or immunological, must not exceed ng-level.

Adolescent↗

Familial increased serum intestinal alkaline phosphatase: a new variant associated with Gilbert's syndrome.

Investigation of mild, inherited increased serum alkaline phosphatase activity partially combined with Gilbert's syndrome in one family showed, apart from a normal liver fraction, an intestinal isoenzyme pattern and an extra band in the agar electrophoresis. Analysis by agarose electrophoresis before and after incubation of neuraminidase showed that the extra fraction was an intestinal variant isoenzyme. The precise genetic background of the two disorders in this family could not be determined from the available data. Abnormal activities of (regular) intestinal alkaline phosphatase isoenzyme caused the increase in serum alkaline phosphatase in the absence of disease.

Adult↗

Specific gel electrophoresis method detects two isoforms of human intestinal alkaline phosphatase.

We have demonstrated that the 6.0% polyacrylamide disc gel electrophoresis (PAGE) method in the presence of 1% Triton X-100 clearly separated both normal molecular mass intestinal alkaline phosphatase (NIAP) and bone alkaline phosphatase (BAP) in serum regardless of the ABO blood group and the secretor status of the subjects. From the results under the usual 7.5% PAGE condition, overlapping mobilities of NIAP and BAP were found in particular in nonsecretor subjects after a high-fat meal. Under the above conditions, the apparent BAP percentage three hours after a meal was higher in nonsecretors than in subjects under fasting conditions, because NIAP activity in serum rose sharply following a high-fat meal. In contrast, under our 6.0% PAGE method, the NIAP and BAP were clearly separated from each other regardless of whether the subjects were fasting or had ingested a high-fat meal. In addition, an elevated level of the circulating NIAP can be another marker for patients with liver cirrhosis. Considering all these factors, the 6.0% PAGE method proposed by us is not only a useful method for the separation of intestinal alkaline phosphatase (IAP) isoforms, but can also be useful for the analysis of other usual AP isozymes.

Adolescent↗

Placental type alkaline phosphatase in possibly premalignant alterations of human gastric mucosa.

The occurrence of placental type alkaline phosphatase (Regan enzyme) was studied in various pathological conditions of the human stomach using polyacrylamide gel electrophoresis and various inhibition techniques. Placental type alkaline phosphatase could be demonstrated in 16 out of 22 gastric carcinomas, in 3 out of 11 moderate and 6 out of 6 severe dysplasias, we failed however to demonstrate it in any other pathological conditions of the stomach, i.e. in intestinal metaplasia. There was no correlation between the occurrence of Regan enzyme and histological type of gastric carcinomas. The appearance of placental type alkaline phosphatase in gastric dysplasias may be suggestive of malignant transformation and its determination in repeated biopsy samples may be a help in the follow-up of patients with abnormal gastroscopic findings.

Adenocarcinoma↗

The Preterm Prediction Study: the value of serum alkaline phosphatase, alpha-fetoprotein, plasma corticotropin-releasing hormone, and other serum markers for the prediction of spontaneous preterm birth.

OBJECTIVE: High levels of a number of analytes are found in maternal blood; alkaline phosphatase,alpha-fetoprotein, and corticotropin-releasing hormone have been associated with spontaneous preterm birth. We investigated the relationship between 8 potential blood markers and subsequent spontaneous preterm birth in asymptomatic pregnant women. STUDY DESIGN: We performed a nested case control study that involved 127 women who were enrolled in the preterm prediction study and who had a singleton spontaneous preterm birth at <35 weeks and 127 women who had a term birth and served as matched (age, parity, center) controls. Serum that was collected at 24 and 28 weeks was analyzed for alkaline phosphatase, alpha-fetoprotein, corticotropin-releasing hormone, and 5 other analytes. RESULTS: Alkaline phosphatase, alpha-fetoprotein, and corticotropin-releasing hormone, but not other analytes, were significantly elevated in pregnancies that ended in spontaneous preterm birth. For alkaline phosphatase at 24 weeks, the odds ratio for spontaneous preterm birth at <32 weeks was 6.8 (range, 1.4-32.8) and for spontaneous preterm birth at <35 weeks 5.1 (range, 1.7-15.6). Similar results were found at 28 weeks. For alpha-fetoprotein at 24 weeks, the odds ratio for spontaneous preterm birth at <32 weeks was 8.3 (range,2.2-30.9) and for spontaneous preterm birth at <35 weeks was 3.5 (range, 1.8-6.7). The levels at 28 weeks were still predictive but less so than at 24 weeks. Corticotropin-releasing hormone, at 28 weeks but not at 24 weeks, was predictive for spontaneous preterm birth at <35 weeks, with an odds ratio 3.4 (range, 1.0-10.9). CONCLUSION: Elevated alkaline phosphatase and alpha-fetoprotein are associated with subsequent spontaneous preterm birth in asymptomatic pregnant women at 24 and 28 weeks. Elevated corticotropin-releasing hormone levels at 28 weeks are associated with spontaneous preterm birth at <35 weeks.

Alkaline Phosphatase↗

Separation of pig bone alkaline phosphatase activities.

A simple method for the separation of alkaline phosphatase and pyrophosphatase activities of pig bone ribs is described. Using anionic exchange chromatography (DEAE-cellulose) and affinity chromatography on Concanavalin A sepharose (Con A) eluted by a step pH gradient and Na4P2O7, several activities were obtained. A pyrophosphatase containing very little alkaline phosphatase activity was isolated from Con A sepharose by elution with pyrophosphatase. Our data are consistent, with the hypothesis that cortical alcaline phosphatase and pyrophosphatase activities are not due to a single enzyme protein. The method was used on whole bone, on bone marrow and on cortical bone.

Alkaline Phosphatase↗

[Activity of the alkaline phosphatase in the serum and bones of zinc-deficient and zinc-replenished cows].

In a study with five lactating dairy cows fed a semisynthetic diet, the changes in the activity of the alkaline phosphatase in the serum, bones and milk were investigated in response to dietary depletion and repletion of zinc. During a six-week depletion period (6 mg Zn/kg dry matter) the alkaline phosphatase in the serum decreased to only 82% of its initial level which, however, was reduced already due to a low zinc intake before the start of the study. After 19 weeks of depletion the activity had decreased to 43% of its initial level. Additions of zinc during the repletion increased the activity very clearly and to much higher values than the ones at the start of the study. Thus this effect of repletion was as large as that for the zinc content in serum and milk. The serum alkaline phosphatase indicated a dependence on the performance and represents a sensitive indicator of the zinc status. The activity of the alkaline phosphatase in the bones (Tuber coxae) decreased to 68% of its initial level after six weeks of zinc depletion. The alkaline phosphatase in the milk was not influenced by the zinc supply.

Alkaline Phosphatase↗