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PubMed1984

Quantitation of human intestinal and liver/bone alkaline phosphatase in serum by rocket electroimmunoassay.

The separation and quantitation of the various human serum alkaline phosphatases has been largely by indirect and/or imprecise methods. A rocket electroimmunoassay method has been developed for the quantitation of the alkaline phosphatases, which uses enzymatic activity to detect the rocket and bromochloroindolylphosphate as substrate. The assay for intestinal phosphatase requires only one-dimensional electrophoresis, since the antibody to the intestinal enzyme does not cross-react with the other phosphatases. Both liver and bone enzymes give a line of identity when tested against antibody directed against the liver phosphatases, and these phosphatases require separation first by acrylamide gel electrophoresis before quantitation by crossed immunoelectrophoresis. Liver/bone enzyme levels in serum are easily detected within the linear range of the assay without concentration of the serum. The assay is 10-fold more sensitive than quantitation by acrylamide gel electrophoresis. The inability to detect circulating intestinal alkaline phosphatase in the serum of most fasting hospitalized patients has been documented by this sensitive assay, and the predominance of the liver isoenzyme has been confirmed. The assay should prove useful for determining the tissue of origin of serum alkaline phosphatases, and in providing quantitative data for physiological studies. It is not adaptable for automation and will not prove useful in the routine clinical laboratory.

Alkaline Phosphatase↗
PubMed1983

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1.

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.

Animals↗
PubMed1984

Oviduct dolichyl phosphate phosphatase: estrogen effects and a possible biosynthetic role.

Estrogen-induced chick oviduct differentiation is accompanied by an increased capacity for protein glycosylation. A portion of this increase has been attributed to increased levels of dolichyl phosphate (Dol-P). Hormone withdrawal leads to an apparent decrease in Dol-P. Dol-P metabolism in the oviduct has been studied, and one of the enzymes having a direct effect on Dol-P, Dol-P phosphatase is herein described. Dol-P phosphatase has a pH optimum of 6.0, does not require a metal ion, and is inhibited by Mn2+ at concentrations greater than 5 mM. Inhibitor studies indicate that Dol-P hydrolysis is inhibited by polyprenyl phosphates having both saturated and unsaturated alpha-isoprene residues, but not by the corresponding alcohols. The enzyme is also inhibited by phosphatidic acid unless 2 mM Mn2+ is included in the incubations. Under these conditions Dol-P hydrolysis is only slightly inhibited (less than 10%), but phosphatidate inhibition is totally eliminated. Oviduct membranes also possess phosphatidate phosphatase, but this enzyme is distinct from Dol-P phosphatase based on thermolability, metal ion sensitivity, and sulfhydryl reagent sensitivity. Studies of enzyme activity in response to estrogen treatment reveal that both Dol-P phosphatase and phosphatidate phosphatase have maximal specific activity early in the differentiation process (peaking after 3 days of treatment), and low specific activity in fully differentiated oviducts, including laying hen oviduct. Hormone withdrawal elicits a small increase in specific activity of both phosphatases. The hormone effects suggest that Dol-P phosphatase may be a biosynthetic enzyme.

Animals↗
PubMed1992

The phospholamban phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme.

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic protein phosphatase activity, which can dephosphorylate phospholamban and regulate calcium transport. This phosphatase has been suggested to be a mixture of both type 1 and type 2 enzymes (E. G. Kranias and J. Di Salvo, 1986, J. Biol. Chem. 261, 10,029-10,032). In the present study the sarcoplasmic reticulum phosphatase activity was solubilized with n-octyl-beta-D-glucopyranoside and purified by sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, and DEAE-Sephadex. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. The partially purified phosphatase could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase(s). Enzymatic activity was inhibited by inhibitor-2 and by okadaic acid (I50 = 10-20 nM), using either phosphorylase a or phospholamban as substrates. The sensitivity of the phosphatase to inhibitor-2 or okadaic acid was similar for the two sites on phospholamban, phosphorylated by the cAMP-dependent and the calcium-calmodulin-dependent protein kinases. Phospholamban phosphatase activity was enhanced (40%) by Mg2+ or Mn2+ (3 mM) while Ca2+ (0.1-10 microM) had no effect. These characteristics suggest that the phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme, and this activity may participate in the regulation of Ca2+ transport through dephosphorylation of phospholamban in cardiac muscle.

Animals↗
PubMed1992

Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.

The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.

Animals↗
PubMed1976

Presence and androgen control of an alkaline phosphatase in the nucleus of rat ventral prostate.

The presence of alkaline phosphatase (EC 3.1.3.1) activity has been demonstrated in nuclei of rat ventral prostate. This enzyme activity remained after washing of isolated nuclei with 0.5% Triton X-100; an acid phosphatase initially present with the nuclear fraction was removed by this treatment. The nuclear alkaline phosphatase, examined by utilizing p-nitrophenyl phosphate as substrate, had a pH optimum of 9.5-10.3, and a broad substrate specificity: p-nitrophenyl phosphate greater than phosphothreonine greater than beta-glycerophosphate greater than phosphoserine. The nuclear phosphatase was sensitive to denaturation by heat or urea treatments and was also inhibited by Pi, L-phenylalanine, homoarginine, dithiothreitol, and EDTA. The EDTA-inhibited enzyme was maximally reactivated by Zn2+, although Mg2+, or Ca2+ were also effective at somewhat higher concentrations. Orchiectomy of adult rats resulted in an increase in the nuclear alkaline phosphatase activity (2-3-fold at 24 or 48 h postorchiectomy). A decline in the protein: DNA ratio also occurred following orchiectomy, but the increase in phosphatase specific activity was evident whether expressed per unit of protein or per unit of DNA. Testosterone replacement following orchiectomy abolished the increase in nuclear phosphatase activity. The results suggest that the prostatic nuclear alkaline phosphatase may be involved in events related to inactivation of the prostate nucleus following androgen deprivation.

Alkaline Phosphatase↗
PubMed1977

Calf thymus alkaline phosphatase. II. Interaction with detergents.

1. A number of detergents were used to dissolve calf thymus plasma membranes rich in alkaline phosphatase (orthophosporic-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activity. 2. The Stokes' radius (r) of alkaline phosphatase in each detergent was measured by gel filtraton. The size of the solubilized enzyme varied from r = 6.2 nm in sodium cholate to r = 8.3 nm in Berol EMU-043. With N-alkylsulphates, the apparent size increased with alkyl chain length, with r = 6.4 nm (C9) and r = 7.3 nm (C12). Tween 20 failed to solubilise the enzyme. 3. The effect of each detergent on the catalytic activity of alkaline phosphatase was determined. The non-ionic detergents Triton X-100, Nonidet P-40, Berol EMU-043, Tween 20 and the zwitterionic detergent Empigen BB increased V by 10--50% without substantially altering the Km for p-nitrophenylphosphate. The bile salts sodium deoxycholate and sodium cholate decreased V and increased the apparent affinity of the enzyme for nitrophenylphosphate. Inhibition was concentration-dependent up to the critical micellar concentration, above which it remained constant (deoxycholate, 33% cholate, 76%). Alkylsulphates (C8-12) had no significant inhibitory effect during 24 h at 23 degrees C. 4. Exchanging one detergent for another altered alkaline phosphatase activity to a state characteristic for the second detergent, e.g. the activity of cholate-inhibited alkaline phosphatase was restored to normal levels by excess of Triton X-100 and vice versa. The inhibitory effect of deoxycholate and cholate therefore result primarily from interactions between detergent and alkaline phosphate, rather than from selective removal of lipids from the enzyme. 5. Pure lecithin, lysolecithin and an ether-deoxylysolecithin each reactivated cholate-inhibited alkaline phosphatase in a concentration-dependent fashion. Cholesterol had no effect. 6. The half-life (t1/2) of membrane-bound alkaline phosphatase at 55 degrees C was 64 min. With the exception of Berol, solubilisation in non-ionic detergents caused no marked change in this sensitivity. The enzyme became more labile in deoxycholate (t1/2) = 31 min), but less labile in cholate (t1/2 = 99 min). Alkylsulphates, which are strong denaturants, markedly increased the sensitivity of the enzyme to heat-inactivation (C8, t1/2 = 13 min; C9--12, t1/2 less than 2 min). 7. It is concluded that membrane-bound alkaline phosphatase is separated from most if not all of its neighbouring lipid moieties by these detergents, which bind to the solubilised enzyme. The number and character of molecules binding to the enzyme influence its size and shape, its susceptibility to inactivation and its catalytic activity.

Alkaline Phosphatase↗
PubMed1977

Determination of serum acid phosphatase in Gaucher's disease using 4-methylumbelliferyl phosphate.

We describe a new assay that is useful for identifying individuals who may be affected with Gaucher's disease. The assay involves the determination of serum acid phosphatase activity using the fluorogenic substrate 4-methylumbelliferyl phosphate. The assay measures acid phosphatase activity at pH 6.0 in the presence of 3.0 M 2-mercaptoethanol and requires a 5 microliter serum sample and a 15-min incubation period. Under these conditions, 2-mercaptoethanol preferentially inhibited the acid phosphatase activity in control serum but did not inhibit the elevated acid phosphatase present in the serum of patients with Gaucher's disease. Using this assay, we observed a 5-50-fold elevation in serum acid phosphatase activity in 8 patients with the adult, non-neuropathic form of Gaucher's disease when compared to control serum assayed under the same conditions. Serum from several heterozygotes free from pathology exhibited normal acid phosphatase activity when assayed at pH 6.0 in the presence of 2-mercaptoethanol. Acid phosphatase activity in serum from patients with prostatic cancer can be distinguished from that in Gaucher serum on the basis of the well-documented sensitivity of the former to inhibition by sodium tartrate. A serum sample from a patient with Niemann-Pick disease exhibited a mild elevation in tartrate-resistant acid phosphatase activity so that conclusive diagnosis of Gaucher's disease requires assaying leukocytes or fibroblasts from suspected patients for glucocerebroside:beta-glucosidase activity.

Acid Phosphatase↗
PubMed1978

Further observations on the differential precipitation of alkaline phosphatase isoenzymes by ethanol.

The proportions of the total activities of different isoenzymes of human alkaline phosphatase precipitated from serum by ethanol (20% v/v) were: liver phosphatase, 37%; placental phosphatase, 23%; bone phosphatase, 8.0%, and small-intestinal phosphatase, 3.7%. Treatment of the isoenzymes with neuraminidase reduced the percentages of non-intestinal phosphatases precipitated by ethanol to below 10%. Precipitation of intestinal alkaline phosphatase was unaffected by this treatment. The degree of solubility in ethanol therefore appears to be largely determined by the content of terminal sialic acid residues in the alkaline phosphatase molecules. In contrast the stabilities of the isoenzymes to heating at 56 degrees C were not significantly altered by neuraminidase digestion.

Alkaline Phosphatase↗
PubMed1980

Mechanism of elevation of serum alkaline phosphatase activity in biliary obstruction: an experimental study.

Bile duct ligation in rats increased alkaline phosphatase activity in serum and liver. In the serum, the activity reached a peak 24 h after bile duct ligation, earlier than in the liver. This finding indicates that the elevation of serum alkaline phosphatase activity is not due to simple overspill of this enzyme from the liver into the circulation. An electrophoretic study, employing polyacrylamide gel with Triton X-100, and a gel filtration study disclosed that 24 h after bile duct ligation the serum contained a high molecular weight form of alkaline phosphatase in addition to the hepatic and intestinal isoenzymes. The high molecular weight form was also found in bile, indicating that regurgitation of bile contributed to the increase in alkaline phosphatase activity in the serum. The absence of the high molecular weight alkaline phosphatase in the sera of rats with intrahepatic cholestasis induced by alpha-naphthylisothiocyanate suggests that, in this type of cholestasis, regurgitation of bile alkaline phosphatase does not play an important role in the elevation of serum alkaline phosphatase activity. These findings indicate that the high molecular weight alkaline phosphatase in serum is a useful diagnostic marker of biliary obstruction.

1-Naphthylisothiocyanate↗
PubMed1987

A novel alkaline phosphatase isozyme in human adipose tissue.

A novel alkaline phosphatase (AP) isozyme was found in human adipose tissue. Adipose tissue alkaline phosphatase differed in enzymatic properties from liver, placental and intestinal alkaline phosphatases. On electrophoresis it showed the same mobility as intestinal alkaline phosphatase, but after treatment with neuraminidase its mobility was decreased to the same as or slightly less than that of neuraminidase-treated liver alkaline phosphatase. Its inhibition by amino acids, inactivation by urea and activation by Mg2+ were almost the same to those of liver alkaline phosphatase. However, at 56 and 65 degrees C it was more stable than liver alkaline phosphatase. Alkaline phosphatase activity was demonstrated histochemically in adipose tissue with naphthol AS-MX phosphate as substrate. It was localized in the wall of blood capillaries, but not present in adipocytes.

Adipose Tissue↗
PubMed1985

Chronic ethanol consumption alters rat liver plasma membranes and potentiates release of alkaline phosphatase.

The aim of this study was to investigate possible mechanisms involved in the elevation of serum alkaline phosphatase activity in alcoholics. Male Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4-5 wk. Serum alkaline phosphatase activity was increased moderately but significantly. Hepatocytes isolated from ethanol-fed animals exhibited pronounced morphologic alterations of their plasma membranes by scanning electron microscopy and a reduced content of alkaline phosphatase despite an increase in total liver alkaline phosphatase content. Chronic ethanol feeding also potentiated the release of alkaline phosphatase from the cells during incubation with 50 mM ethanol. Furthermore, chronic ethanol feeding resulted in reduced recovery of alkaline phosphatase in hepatic plasma membranes isolated by sucrose gradient centrifugation but did not affect the recoveries of other plasma membrane markers (5'-nucleotidase and Na+,K+-adenosine triphosphatase) nor the subcellular distribution of alkaline phosphatase in the nuclear, mitochondrial, microsomal, and cytosolic fractions. These findings suggest that the increased serum alkaline phosphatase levels observed in response to chronic ethanol feeding may be due, at least in part, to increased lability of this plasma membrane enzyme.

Alkaline Phosphatase↗
PubMed1981

Alkaline phosphatase activity in chronic streptozotocin-induced insulin deficiency in the rat: effect of insulin replacement.

Alterations in circulating alkaline phosphatase have been described in both man and the experimental animal with chronic insulin deficiency. We evaluated plasma and tissue alkaline phosphatase levels in freely-fed control, streptozotocin-induced diabetic and insulin-treated diabetic rats, seven weeks after the induction of diabetes. Circulating alkaline phosphatase activity was markedly elevated in the insulin deficient animal (p less than 0.001) and completely normalized following insulin administration. The elevated plasma alkaline phosphatase activity observed in the insulin deficient animals was heat-resistant and phenylalanine-sensitive, a pattern typical of the intestinal isoenzyme. Small intestinal alkaline phosphatase activity was significantly higher (p less than 0.01) in the diabetic animals, but comparable in the insulin-replaced and control rats. The intestinal isoenzyme activity was found to be strikingly insulin-sensitive; withholding insulin therapy for 36 hr prior to sacrifice resulted in an abrupt rise in both plasma and intestinal alkaline phosphatase values comparable to those observed in the insulin-deficient state. In contrast to these observations, skeletal alkaline phosphatase activity was decreased in the insulin deficient animal (p less than 0.01) and this abnormality was corrected by insulin replacement. Neither insulin deficiency nor insulin replacement resulted in any significant changes in the hepatic alkaline phosphatase isoenzyme.

Alkaline Phosphatase↗
PubMed1994

Effect of alkaline phosphatase on thromboxane mimetic induced platelet activation.

Recently it has been reported that alkaline phosphatase selectively inhibited thromboxane mimetic induced platelet aggregation and secretion suggesting that the phosphorylation state on the platelet surface may be important for thromboxane induced platelet activation. We report here studies attempting to elucidate the mechanism of action of alkaline phosphatase. Washed human platelet aggregation induced by the thromboxane mimetic IBOP was completely abolished when incubated with alkaline phosphatase (1 unit/ml) for 5 min. The effect was inhibited by co-incubation with 5mM phosphate. Binding studies using [125I]BOP showed that neither the affinity of IBOP for the receptor (control: 9.2 +/- 2.1 nM, alkaline phosphatase: 7.9 +/- 1.8 nM) nor the Bmax (control: 1780 +/- 320 sites/plt. alkaline phosphatase: 1920 +/- 290 sites/plt) were effected by alkaline phosphatase treatment. GTPase activity was measured in platelet membranes treated with and without alkaline phosphatase as measured by IBOP induced hydrolysis of [gamma-32P]GTP. The EC50 values for IBOP induced GTPase were similar whereas the maximum amount of released Pi in the control membranes was more than two fold greater than in alkaline phosphatase treated membranes. These studies suggest that thromboxane induced platelet activation may be dependent upon the phosphorylation state of the thromboxane receptor and/or closely associated protein.

Alkaline Phosphatase↗
PubMed1992

Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.

Animals↗
PubMed1994

Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation.

In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.

Animals↗
PubMed1992

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

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

Alkaline Phosphatase↗
PubMed2006

Studies of small molecule interactions with protein phosphatases using biosensor technology.

Reversible protein phosphorylation of serine, threonine, and tyrosine residues by protein kinases and phosphatases is important for the regulation of cellular signal transduction and controls many cellular functions. Disturbances in this regulation have been implicated in a growing number of diseases, making kinases and phosphatases useful targets for therapeutic intervention. The suitability of surface plasmon resonance (SPR) technology has been widely demonstrated in many drug discovery applications. A novel and straightforward methodology is presented for analyzing small molecule binding to two serine/threonine phosphatases, PP1 and PP2B (calcineurin), and to the prototypic tyrosine phosphatase, PTP1B. Emphasis was placed on investigating the immobilization conditions of the phosphatases by using reducing conditions, inhibitors and metal ions. A comparison of inhibitor binding, either to phosphatase (PP2B) alone or in complex with the regulatory protein subunit calmodulin, revealed different kinetics. The methodology was also used to test inhibitor specificity toward different phosphatases. Inhibition of regulatory protein PP-inhibitor-2 binding to PP1 by a small molecule inhibitor was demonstrated. This type of information, together with data on compound binding that is independent of enzyme activity and in which affinities are resolved into kinetic rate constants, may be of great significance for the development of highly specific and high-affinity phosphatase inhibitors.

Binding Sites↗