Tetrameric alkaline phosphatase in human liver plasma membranes

Molecular weights of native membrane-bound alkaline phosphatase released by butanol and by nonionic detergents were more than twice that of the purified dimeric enzyme. Alkaline phosphatase released by phosphatidylinositol-specific phospholipase-C was of both high and low molecular weight: the former was a protomer of a single protein of the same molecular size as monomeric alkaline phosphatase. We conclude that the membrane-bound enzyme is probably a tetramer.     

ATPase and alkaline phosphatase activities of chick and rat small intestinal mucosa.

The alkaline phosphatase and (Ca2+ +Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of chick and rat small intestine have been investigated. The same pH optimum was found for membrane-bound and solubilized alkaline phosphatase, whereas those of the corresponding ATPases differed. The solubilised ATPases had inhibition and activation characteristics similar to those of alkaline phosphatase but markedly different from those of the membrane-bound ATPase. These results suggest that membrane-bound alkaline phosphatase and ATPase are not the same enzyme.     

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5''-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.     

Phospholipids of E. coli and activity of alkaline phosphatase]

The effects of liposomes prepared from the E. coli lipids on the activity of soluble alkaline phosphatase and on the complementation reaction between its subunits were studied. It was shown that the liposomes nonspecifically catalyze the dimerization of the enzyme subunits without changing the dimer activity. The effects of phospholipases A2 and C on the activity of membrane-bound alkaline phosphatase were studied. An interrelationship was found between the level of hydrolysis of membrane phosphatidyl glycerol (PG) by these enzymes and the changes in the activity of membrane-bound alkaline phosphatase. It was also shown that PG is less accessible to the effects of phospholipases in the cells with derepressed biosynthesis of alkaline phosphatase. It is assumed that the membrane PG interacts with the membrane-bound alkaline phosphatase during its translocation into the periplasm.     

Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.     

Characterization of brush border membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine

This study was conducted to characterize enterocyte apical membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine. Duodenal, jejunal, and distal ileal segments were isolated from three 26-kg pigs and enterocyte brush border membrane, enriched between 19- and 24-fold in sucrase specific activity, was prepared by Mg(2+) precipitation and differential centrifugation. With P-nitrophenyl phosphate as substrate, the optimum pH for porcine brush border membrane-bound alkaline phosphatase activity was defined to be 10.5 for all three segments. At the optimal pH, the kinetics of membrane-bound alkaline phosphatase were determined for the three intestinal segments. The affinity of this enzyme (K(m), mM) in the jejunum (0.64 +/- 0.07) was four times greater than that in the duodenum (2.75 +/- 0.59) and the distal ileum (2.71 +/- 1.14). These results indicate that different isomers of membrane-bound alkaline phosphatase might have been expressed in different segments of porcine small intestine. The maximal specific activity (V(max), micromol/mg protein . min) of this enzyme was highest in the duodenal (7.74 +/- 0.95), intermediate in the jejunal (4.31 +/- 0.18), and lowest in the distal ileal (3.53 +/- 0.84) brush border membrane. Therefore, the maximal specific activity of brush border membrane-bound alkaline phosphatase along the intestinal longitudinal axis in growing pigs decreases from the duodenum toward the distal ileum.     

Localization of alkaline phosphatase in cells of Bacillus intermedius

Alkaline phosphatase, an enzyme secreted by Bacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth of B. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na2HPO4 at a concentration of 0.01% diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon,Oncorhynchus keta

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stagespecific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a genetransfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were estblished. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 celllines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue nonspecific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.     

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.     

Hepatic alkaline phosphatase isoenzymes: isolation, characterization and differential alteration.

Although it is generally believed that hepatic alkaline phosphatase is localized to liver plasma membranes, 63% is present in the cytosol fraction after ultracentrifugation of rat liver homogenates. Divalent cation requirements, heat inactivation, pH optima, Km and chemical inhibition characteristics of partially purified alkaline phosphatase enzymes prepared from membrane and cytosol fractions suggested different structural forms. Furthermore, bile duct obstruction and ethinyl estradiol administration preferentially increased membrane-bound alkaline phosphatase activity, while cytosol activity was unaltered. In contrast, phenobarbital treatment decreased membrane-bound alkaline phosphatase and increased cytosol activity. These studies support the presence of two forms of hepatic alkaline phosphatase in rat liver which are regulated by different control mechanisms.     

Localization of alkaline phosphatase inBacillus intermedius cells

Alkaline phosphatase, an enzyme secreted byBacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth ofB. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na₂HPO₄ at a concentration of 0.01 % diminished the activity of membrane-bound and extracellular phosphatases. CoCl₂ at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

Immunological relatedness of cytoplasmic and membrane-bound acid phosphatases from Dioscorea cayenensis rotundata

Antibodies were raised against one cytoplasmic and two membrane-bound acid phosphatases purified from yam tubers (Dioscorea cayenensis rotundata). Experiments of immunoinactivation and immunoelectrophoresis revealed cross-immunological reactions between the cytoplasmic enzyme (acid phosphatase A) and one of two membrane-bound counterparts (acid phosphatase B) suggesting that these molecules share common antigenic determinants. The antibodies raised against the other membrane-bound enzyme (acid phosphatase C) only inhibited and precipitated this enzyme.     

Biochemical and cytochemical studies on enzymes that dephosphorylate inositol (1,4,5)-trisphosphate in neutrophils.

Guinea pig neutrophils contain membrane-bound and soluble phosphatases that catalyze the dephosphorylation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. The activities were 5.1 +/- 0.2 and 1.3 +/- 0.2 (SD; n = 5) nmoles phosphate (Pi) released/min/10(7) cell equivalents, respectively. The membrane-bound enzyme dephosphorylated many substrates (e.g., beta-glycerophosphate), exhibited alkaline pH optima, and was inhibited by levamisole. In contrast, the soluble phosphatase was specific for Ins(1,4,5)P3, exhibited a neutral pH optimum, and was insensitive to levamisole. A cerium-based ultrastructural cytochemical procedure was employed to identify the subcellular sites of the membrane-bound activity. Staining was observed on the exterior of the plasmalemma and in a population of granules. Staining in the granules was observed only in permeabilized cells. Treatment of neutrophils with p-diazobenzenesulfonate (DBSA) (4.0 mM) for 20 min at 37 degrees C blocked the cytochemical reaction on the cell surface using beta-glycerophosphate as the substrate, but did not affect the staining of the granules on subsequent permeabilization. In biochemical studies, this treatment with DBSA inhibited the membrane-bound activity by c. 50% but did not affect the soluble phosphatase. Therefore, the membrane-bound phosphatase is, in fact, an alkaline phosphatase that resides in locales not accessible to Ins(1,4,5)P3 generated during cell stimulation. Breakdown of Ins(1,4,5)P3 generated during cell stimulation, therefore, would be catalyzed by the soluble enzyme.     

Postnatal ontogeny of kinetics of porcine jejunal brush border membrane-bound alkaline phosphatase,aminopeptidase N and sucrase activities

Our objectives were to determine postnatal changes in the maximal enzyme activity (V(max)) and enzyme affinity (K(m)) of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg(2+)-precipitation and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V(max) of alkaline phosphatase was the lowest in the adult (4.27 micromol.mg(-1) protein.min(-1)), intermediate in the suckling (9.75 micromol.mg(-l) protein.min(-l)) and the highest in the weaning and post-weaning stage (12.83 and 10.40 micromol.mg(-l) protein.min(-l)). K(m) of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low in the adult (0.66 mM). V(max) of aminopeptidase N was low in the suckling (7.04 micromol.mg protein(-1).min(-1)) and high in the post-weaning stage (13.36 micromol.mg(-l) protein.min(-l)). K(m) of aminopeptidase N was the highest in the two weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V(max) of sucrase increased from the suckling to the adult (0.48-1.30 micromol.mg(-l) protein.min(-l)). K(m) of sucrase ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig.     

Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Prostatic acid phosphatase, a neglected ectonucleotidase

Two recent papers reveal that the soluble and secreted prostatic acid phosphatase, an enzyme that has long served as a diagnostic marker for prostate cancer, has a membrane-bound splice variant. This enzyme exhibits ecto-5′-nucleotidase activity, is widely distributed, and implicated in the formation of chronic pain. While prostatic acid phosphatase hydrolyzes phosphomonoesters other than 5′-nucleoside monophosphates these novel data suggest that, in addition to ecto-5′-nucleotidase and the alkaline phosphatases, prostatic acid phosphatase must be taken into account in future studies on extracellular adenosine production.     

Properties of membrane-bound and solubilized forms of alkaline phosphatase from human liver

To determine whether the properties of alkaline phosphatase in human liver are altered by releasing the enzyme from its native environment, we studied the membrane-bound and purified forms, and the enzyme released by applying phosphatidylinositol-specific phospholipase-C. The bound enzyme had the lowest affinities for eight substrates and the competitive inhibitor phenylphosphonate. The Ki for inorganic phosphate was lower with the bound enzyme than with the other forms, whereas the values for uncompetitive inhibitors were the same with all three. Phenylglyoxal reacted with essential residues of arginine at similar rates with the bound and purified enzymes, whereas essential cations were more readily removed and replaced in the bound and released forms. Arrhenius plots of the bound enzyme revealed two breaks, with activation energy above the second break similar to that of the purified enzyme. Activity of the bound enzyme increased when the membrane was perturbed by butanol and assayed below 30 degrees C. These experiments demonstrate that, even though binding of alkaline phosphatase to the plasma membrane is not essential for catalytic function, the properties of the enzyme in the membrane are different from those of the soluble form.     

Histochemical study of a number of hydrolases,including a new acid phosphatase,in various tissues of men and mice

Summary Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO₄ or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.