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PubMed1979

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium.

The alkaline phosphatase present on isolated brush border and basal lateral membranes of rat duodenal epithelium were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6--9.8, similar substrate km's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin of papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000--150,000, and both enzymes show an Rf value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The Vmax of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 mumoles/mg-h as opposed to 14 mumoles/mg-h. The differences in Vmax are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.

Alkaline Phosphatase↗

PubMed2000

Kinetic characteristics of ATP hydrolysis by a detergent-solubilized alkaline phosphatase from rat osseous plate.

Polidocanol-solubilized alkaline phosphatase was purified to homogeneity with a specific activity of 822.3 U/mg. In the absence of Mg2+ and Ca2+ ions and at pH 9.4, the enzyme hydrolyzed ATP in a manner that could be represented by biphasic curves with V = 94.3 U/mg, K0.5 = 17.2 microM, and n = 1.8 and V = 430.3 U/mg, K0.5 = 3.2 mM, and n = 3.2 for high- and low-affinity sites, respectively. In the presence of saturating concentrations of Mg2+ or Ca2+ ions, the hydrolysis of ATP also followed biphasic curves. However, the specific activity increased to as much as 1,000 U/mg, whereas the K0.5 and n values remained almost unchanged. In the presence of nonsaturating concentrations of metal ions, the hydrolysis of ATP was similar to that observed in the absence of these ions, but with a marked decrease in K0.5 values. At pH 7.5, the enzyme also hydrolyzed ATP with K0.5 = 8.1 microM and V = 719.8 U/mg. Apparently, alkaline phosphatase was able to hydrolyze ATP in vivo, either at pH 7.5 or pH 9.4. These data contribute to the knowledge of the biological properties of skeletal alkaline phosphatase and suggest that this enzyme may have a high-affinity binding site for ATP at alkaline pH.

Adenosine Triphosphatases↗

PubMed1975

Inhibition of serum alkaline phosphatase activity by phenylalanine and cholic acid.

Serum alkaline phosphatase activity was found to increase more markedly in patients with liver cirrhosis than in patients with peptic ulcer and this increase was found to be influenced by blood types. After testing several amino acids and bile acids, phenylalanine and cholic acid were chosen and their inhibitory effects upon serum alkaline phosphatase activity were studied in 66 patients with various liver diseases. It was found that the combination of both agents demonstrates different patterns of inhibition between the patients with liver cirrhosis and obstructive jaundice. This inhibitory effects were also variable among cases of different blood types. Basing upon the present observation, the possible source of the elevated alkaline phosphatase activity in liver cirrhosis was discussed.

Alkaline Phosphatase↗

PubMed1982

Plasma alkaline phosphatase activity: a screening test for rickets in preterm neonates.

Both rickets and raised plasma alkaline phosphatase activity are common in the preterm infant. Measurement of plasma alkaline phosphatase activity is valuable in screening for active disease and in diagnosis, but normal reference data are not available for preterm babies. In 30 consecutive preterm infants (birthweight 1580 +/- 410 g, gestational age 31 +/- 2.5 weeks) serial measurements of plasma alkaline phosphatase activity, plasma levels of calcium and inorganic phosphorus, and the pattern of alkaline phosphatase isoenzymes were made. Four patterns of changes in plasma alkaline phosphatase activity with time were seen. 4 of the 30 infants were shown to have rickets; these children and 14 of the 26 non-rachitic infants showed an increasing/peak/decreasing pattern with increasing age, but levels were much higher in the rachitic infants. The activity in all 30 was raised above the adult and childhood reference ranges at some point in time. The data suggest that an activity of five times the upper limit of the normal adult reference range is acceptable in the preterm infant but an activity higher than this may suggest rickets.

Alkaline Phosphatase↗

PubMed1981

Immunocytochemical demonstration of intracytoplasmic alkaline phosphatase in HeLa TCRC-1 cells.

The ultrastructural localization of alkaline phosphatase has been examined in cells of a HeLa subline (TCRC-1) that are monophenotypic for Regan isoenzyme expression. Enzyme activity was demonstrated at the cell surface plasma membrane and in certain lysosomes as revealed by the lead citrate method. The regular direct immunoperoxidase procedure utilizing antibodies in IgG or Fab' form showed the same distribution patterns of alkaline phosphatase. However, when the cell surface antigen was blocked in advance with specific unlabeled antibodies and direct immunocytochemistry performed in the presence of saponin, intracellular alkaline phosphatase antigen was observed in the perinuclear space, endoplasmic reticulum, and Golgi apparatus. The results appeared to be concordant with the current concept that membrane glycoproteins are formed in the endoplasmic reticulum, modified in the Golgi apparatus and then transported to the cell surface. Intracellular alkaline phosphatase was observed predominantly in some cell populations especially mitotic cells, suggesting that the enzyme protein was synthesized in and around the mitotic phase. Accordingly, this technique of differential membrane immunocytochemistry appears to provide an opportunity to follow ectopic gene expression as a function of cell cycle and enzyme induction.

Alkaline Phosphatase↗

PubMed1980

Anion-stimulated phosphohydrolase activity of intestinal alkaline phosphatase.

Phosphohydrolase activity of a highly enriched commercial preparation of calf intestinal alkaline phosphatase was stimulated in the presence of HCO3. SO4, Cl, SCN, and acetate did not stimulate hydrolysis, whereas SO3 exhibited a bimodal effect, stimulating at low (25mM) concentration but inhibiting at high (100 mM) concentration. The pH optimum of this stimulation by HCO3 or SO3 was 8.5--9.0 and was maximal at a Mg concentration of 0.5 mM. HCO3 increased the Vmax of the reaction without changing the Km for ATP. ATP, GTP, UTP, and xanthosine triphosphate were equally effective as substrates, whereas AMP and p-nitrophenyl phosphate were much less effective. Alkaline phosphatase activity was inhibited by L-cysteine and L-phenylalanine, compounds that also inhibited the HCO3-ATPase activity of the preparation. Passage of the commercial preparation through an anion-exchange column yielded a fraction with enriched alkaline phosphatase and HCO3-ATPase activities; this fraction proved to be a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on isoelectric focusing, and by immunologic techniques. These studies strongly suggest that alkaline phosphatase and anion-stimulated phosphohydrolase activities are properties of the same protein in small intestine. It is possible that alkaline phosphatase may function as a HCO3-ATPase involved in intestinal absorption and secretion.

Adenosine Triphosphatases↗

PubMedDate not supplied

[Alterations in acid and alkaline phosphatase activity in some steroid-producing organs of rats in autolysis].

Alterations in activity of acid and alkaline phosphatases as well as in concentration of inorganic phosphate were studied in tissue and homogenates of adrenal glands and testicles of rats within 48 hrs of the postmortal autolysis. During the autolysis acid phosphatase of homogenate and tissue of adrenal glands as well as alkaline phosphatase from the gland were more stable than acid and alkaline phosphatases from testicles. Activities of acid and alkaline phosphatases decreased gradually in homogenate and tissue of testicles during autolysis. Within the first day of autolysis distinct inhibition of alkaline phosphatase was also observed in the homogenate of testicles and adrenal glands. An increase in amount of inorganic phosphate in tissue and homogenate of adrenal gland and in tissue of testicles did not depend on alterations in activity of the phosphatases on autolysis. Prehomogenization of these tissues altered markedly the enzymatic activity both in adrenal glands and in testicles.

Acid Phosphatase↗

PubMed1988

Changes in the bone and liver isoenzymes of alkaline phosphatase in postmenopausal women being treated with norethisterone.

The effects of norethisterone therapy on alkaline phosphatase isoenzyme activities were studied in a group of postmenopausal women. There was a significant fall in total alkaline phosphatase activity after 8 wk which was still in evidence after 24 wk. Both bone and liver alkaline phosphatase isoenzyme activities were decreased during the first 16 wk on treatment, but after 24 wk only the bone phosphatase activity was significantly lower than the pretreatment level. The other biochemical indices of bone metabolism and liver function were also measured during the study. The results indicate that bone specific alkaline phosphatase activity is a more sensitive index of bone activity than total alkaline phosphatase and that monitoring of total activity may in some instances be misleading.

Alkaline Phosphatase↗

PubMed1987

Purification of bone alkaline phosphatase from human osteosarcoma.

Purification of human bone alkaline phosphatase, derived from human osteosarcoma tissue, has been carried to electrophoretic homogeneity. The purification procedure involved three major steps: (1) chromatography on hydroxylapatite; (2) ion exchange chromatography; and (3) gel filtration. The resultant purified enzyme is a glycoprotein, has a molecular weight of approximately 80,000 (consistent with previous reports for the bone isoenzyme), and is characteristically inhibited by modest heat (56 degrees C, 30 min) and L-homoarginine but not by L-phenylalanine. The isolation and purification procedure described can lead to the production of significant amounts of highly purified bone alkaline phosphatase. Purified ALP can be used for an analysis of minor structural differences that appear to exist between the bone, liver and kidney isoenzymes. Such information could lead to the development of a clinical diagnostic procedure specifically for bone alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1985

Detection of alkaline phosphatase activity after conventional isoelectric focusing by an indoxyl-tetrazolium salt technique.

Alkaline phosphatase was solubilized from human and rat tissues using papain in the presence of TRITON X-100 and subjected to isoelectric focusing (IEF) in polyacrylamide or agarose gels. Up till now, usually 1- and 2-naphthylphosphates have been used as substrates in order to specifically stain molecular forms of this enzyme by the azo-dye technique. In this paper, the use of another histochemical substrate, 5-bromo-4-chloro-3-indoxyl phosphate, in combination with tetrazolium salts [McGadey, J. (1970) Histochemie 23, 180-184] is presented. After hydrolysis, the released indoxyl moieties reduce tetrazolium salts to insoluble formazans at the zones of alkaline phosphatase activity. Zymogrammes showing molecular forms of alkaline phosphatase from 20 rat organs and the application of this staining technique for the detection of alkaline phosphatase activity in non-dialyzed human plasma after IEF are presented.

Acid Phosphatase↗

PubMed1988

Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity.

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.

Acid Phosphatase↗

PubMed1987

Comparative studies of acid and alkaline phosphatase activities in nonvascular smooth muscle membranes.

Acid and alkaline phosphosphatase activities of subcellular fractions isolated from rat gastric muscle and vas deferens by differential centrifugation, sucrose density gradient and cation-induced aggregation methods were studied using p-nitrophenyl phosphate as the substrate. Alkaline phosphatase and a large portion of acid phosphatase activities were found to be of plasmalemmal origin. Acid and alkaline phosphatase activities were different in the effect of Mg2+, fluoride, vanadate, EDTA and resistance to heat inactivation suggesting that these two phosphatase activities were not expressed by the same enzyme.

Acid Phosphatase↗

PubMed1974

Regulation of phosphate metabolism in Neurospora crassa: isolation of mutants deficient in ther repressible alkaline phosphatase.

Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pcon(c), and preg(c). Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.

Acid Phosphatase↗

PubMed2002

Bone mineralisation in premature infants cannot be predicted from serum alkaline phosphatase or serum phosphate.

BACKGROUND: The bone mineral content of premature infants at term is lower than in mature infants at the same postconceptional age. Serum alkaline phosphatase and serum phosphate are often used as indicators of bone mineralisation. OBJECTIVE: To analyse the association between bone mineral content and serum alkaline phosphatase and serum phosphate. METHODS: Serum alkaline phosphatase and phosphate were measured at weekly intervals during admission in 108 premature infants of gestational age below 32 weeks (mean (SD) gestational age 29 (2) weeks; mean (SD) birth weight 1129 (279) g). Bone mineral content was measured at term (mean gestational age 41 weeks) by dual energy x ray absorptiometry and corrected for body size. RESULTS: Serum alkaline phosphatase was significantly negatively associated with serum phosphate (p < 0.001). Bone mineral content was not associated with mean serum alkaline phosphatase (p = 0.8), peak serum alkaline phosphatase (p = 0.5), or mean serum phosphate (p = 0.2) at term. CONCLUSION: Routine measurements of serum alkaline phosphatase and serum phosphate are of no use in predicting bone mineralisation outcome in premature infants.

Absorptiometry, Photon↗

PubMedDate not supplied

Leukocyte and serum alkaline phosphatase after hip arthroplasty, synovectomy of the knee and fracture.

The alkaline phosphatase in leukocytes (LAP) was measured in patients who had undergone various types of total arthroplasty of the hip, in patients who had undergone synovectomy and in whom tibial fractures had been treated conservatively. The LAP-Score increased rapidly and reached a maximum within a few days and returned to original levels after 14 days both after soft tissue trauma and skeletal trauma. The size of the maximal increase appears to vary only with the severity of the trauma. LAP was not found to be correlated with the increase in alkaline phosphatase in the serum. The increase in serum alkaline phosphatase occurred much later and decreased much slower and occurred only after skeletal trauma. The increase in alkaline phosphatase in the serum appears to vary with severity of skeletal trauma. A significant difference in LAP (P less than 0.05) was found between patients operated upon with total arthroplasty with success and those with deep infection. The difference between these two groups was, however, larger (P less than 0.01) regarding ESR and CRP.

Adult↗

PubMed1989

Levamisole inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes.

1. The effect of levamisole (LMS) on alkaline phosphatase (EC 3.1.3.1) and 5'-nucleotidase (EC 3.1.3.5) activities of bovine milk fat globule membranes (MFGM) was examined. 2. LMS inhibited MFGM alkaline phosphatase activity in a concentration-dependent manner with 50% inhibition produced by 49 +/- 23 microM LMS. 3. 5'-Nucleotidase was resistant to LMS inhibition with 30.9% inhibition produced by 10 mM LMS, the highest concentration tested. 4. LMS was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 45 +/- 6 microM. 5. The extent of LMS inhibition of alkaline phosphatase was dependent on the substrate utilized in the assay. 6. The effect of LMS on bovine MFGM alkaline phosphatase was similar to LMS effects on other mammalian alkaline phosphatases of liver/kidney/bone/placental isoenzyme origin.

5'-Nucleotidase↗

PubMed1982

Electron histochemical study of alkaline phosphatase activity in the pecten oculi of the chick.

Alkaline phosphatase activity in the pecten of chicks was studied electron histochemically. Alkaline phosphatase activity was located in the plasma membrane of the cytoplasmic processes in the luminal and basal portions of the endothelial cells of the capillary, in the plasma membrane of cells with pigmented granules and in the plasma membrane of cells in the vitreous in contrast with the basement lamina of the cells with pigmented granules. The cytoplasmic processes of the luminal and basal portions of the capillary endothelium and the plasma membrane of the cells with pigmented granules may play a role in the exchange of metabolites between the pecten and the vitreous. In the pecten of dark adapted eye, alkaline phosphatase activity could not be demonstrated histochemically. Thus, darkness may make the exchange of metabolites between the pecten and the vitreous less active.

Alkaline Phosphatase↗

PubMed1982

[Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase].

The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E. coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared. For both phosphatases the type of interaction was found to be independent on the nature of substrate. For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively. In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction. The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.

Acid Phosphatase↗