Studies of reversible inhibition,irreversible inhibition,and activation of alkaline phosphatase by capillary electrophoresis

Reversible inhibition, irreversible inhibition, and activation of calf intestinal alkaline phosphatase (EC 3.1.3.1) have been studied by capillary electrophoresis. The capillary electrophoretic enzyme-inhibitor assays were based on electrophoretic mixing of inhibitor and enzyme zones in a substrate-filled capillary. Enzyme inhibition was indicated by a decrease in product formation detected in the capillary by laser-induced fluorescence. Reversible enzyme inhibitors could be quantified by Michaelis-Menten treatment of the electrophoretic data. Reversible, competitive inhibition of alkaline phosphatase by sodium vanadate and sodium arsenate has been examined, and reversible, noncompetitive inhibition by theophylline has been studied. The K(i) values determined for these reversible inhibitors using capillary electrophoresis are within the range of values reported in the literature for the same enzyme-inhibitor combinations. Irreversible inhibition of alkaline phosphatase by EDTA at concentrations of 1.0mM and above has been observed. Activation of alkaline phosphatase has also been observed for EDTA at concentrations from 20 to 400 microM.     

A major lienal phosphotyrosine phosphatase is inhibited by phospholipids and inositol trisphosphate.

A major "non-receptor" phosphotyrosine-specific protein phosphatase isolated from the 30,000g pellet fraction of porcine spleen is related to the human T-cell tyrosine phosphatase (Cool et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5257-5261) and is strongly inhibited by micromolar concentrations of phosphatidyl inositol (IC50 6 microM) and phosphatidyl serine (IC50 3.7 microM). In addition, the enzyme is inhibited by myo-inositol 1,4,5-trisphosphate (IC50 ca. 2 microM) in a non-competitive manner but not by myo-inositol hexaphosphate. Since the overall cellular tyrosine phosphatase activity greatly exceeds tyrosine kinase activity, inhibition of the phosphatase may be of importance for the regulation of the extent of tyrosine phosphorylation of cellular proteins.     

Inhibitory effect of the regulatory subunit of type I cAMP-dependent protein kinase on phosphoprotein phosphatase

The regulatory subunit of type I cAMP-dependent protein kinase (RI) from rabbit skeletal muscle inhibited the activity of a low molecular weight phosphoprotein phosphatase. The inhibition was concentration and time dependent. A maximum inhibition, about 70%, was observed at 2 microM of RI with an apparent Ki of 0.8 microM. Inhibition was associated with a decrease in Vmax with no change in Km for substrate, phosphorylase a. On the other hand, cAMP-dependent protein kinase holoenzyme or its catalytic subunit was without any effect. The inhibition of phosphoprotein phosphatase by RI may be of physiological significance since the dissociation of cAMP-dependent protein kinase by cAMP would result in a simultaneous increase in the phosphorylation and decrease in the dephosphorylation rates of target proteins.     

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells.     

Alkaline phosphatase isozymes as possible markers of differentiation in human testicular teratocarcinoma cell lines

The alkaline phosphatase (ALP) activity in eight independent cell lines derived from human testicular germ cell tumors was characterized. Seven out of eight of the lines had high ALP levels, and most of the activity in each case was of the liver/bone/kidney ALP type, as judged by thermostability, inhibition, and electrophoretic studies. Low levels of a heat stable, placental-like ALP were also present in these seven lines; only a small subpopulation of the cells of each line reacted strongly with an anti-placental ALP monoclonal antibody. The heat-stable, placental-like isozyme characteristic of these lines differed from the normal placental ALP in its inhibition profile. Thus it is possible that a subpopulation of the cells in these lines expresses a new embryonic ALP form.     

Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression

Non-transformed, rat intestinal epithelial cells (IEC-6), and human intestinal colonic carcinoma cells (CACO-2) have both been used to study processes of epithelial cell differentiation. However, only CACO-2 cells have been described as spontaneously expressing phenotypic changes of differentiation in culture. We report here that when IEC-6 cells are grown in post-confluent culture, they develop structural changes similar to those seen in cells induced to differentiate by culture on Englebreth-Holm-Swarm (EHS) extracellular matrix proteins. Correlated with this morphological change is loss of nuclear localization of c-myc protein and development of cell surface alkaline phosphatase (ALP) enzymatic activity. Messenger RNAs for liver and intestinal isoforms of ALP were expressed in both pre- and post-confluent cells. Inhibition of ALP activity in post-confluent cells by levamisole indicated the expressed ALP activity to be of the liver isoform. We suggest the expression of ALP activity, which occurs concomitantly with morphological alterations in post-confluent IEC-6 cells, represents increased expression and localization to the cell surface of the liver isoform of ALP. Cultured IEC-6 cells may provide a non-transformed, in vitro alternative to CACO-2 cells for study of epithelial cell differentiation.     

Comparative biochemical studies of placental alkaline phosphatase in animals (author's transl)]

Biochemical properties of alkaline phosphatase (ALP) from placenta were compared between man, dog, cat, rabbit and cattle. 1) Optimum pH of the enzyme was essentially identical through the species of the animals but the inhibition of L-phenylalanine was clearly demonstrable with human ALP but little with that of other animals. 2) ALP of human placenta was not inactivated by heating at 65 degrees C for 15 min. but one of the other animals was thermolabile. Such thermostability of human placental ALP almost disappeared after treatment with EDTA, suggesting that the divalent metal ions were required for the thermostability. 3) Activities of placental ALP were inhibited by cationsurfactants in human and rat but not in the other animals, while the inhibition by DOC-Na, an anion-surfactant, was seen only in human. 4) The affinity to beta-glycerophosphae of placental ALP was seen only in human.     

Okadaic acid, a phosphatase inhibitor, induces activation and phosphorylation of the Na+/H+ antiport.

We determined the effect of okadaic acid (OA), a potent phosphoprotein phosphatase inhibitor, on the intracellular pH (pHi) of rat thymic lymphocytes and human bladder carcinoma cells. OA induced a rapid and sustained cytosolic alkalinization. This pHi increase was Na(+)-dependent and was inhibited by 5,N-disubstituted analogs of amiloride, indicating mediation by the Na+/H+ antiport. As described for other stimulants, such as mitogens and hypertonic challenge, activation of the antiport by OA is attributable to an upward shift in its pHi dependence. Accordingly, the alkalinization produced by the phosphatase inhibitor was not additive with that induced osmotically. Activation of the antiport by OA was accompanied by a marked increase in phosphoprotein accumulation, revealing the presence of active protein kinases in otherwise unstimulated cells. We considered the possibility that phosphorylation of the antiport itself or of an ancillary protein is responsible for activation of Na+/H+ exchange. Consistent with this notion, the alkalinization induced by OA was absent in ATP depleted cells. More importantly, immunoprecipitation experiments demonstrated increased phosphorylation of the antiport following treatment with OA. We conclude that, upon inhibition of phosphoprotein phosphatase activity, constitutively active kinases induce the activation of Na+/H+ exchange, possibly by direct phosphorylation of the antiport.     

Calpain controls the balance between protein tyrosine kinase and tyrosine phosphatase activities during platelet activation.

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]i< or = 1 microM, proteins were phosphorylated. At ionized [Ca2+]i > or = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]i < or =1 microM, whereas at ionized [Ca2+]i > or =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.     

Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells

Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane. ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5'-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility. Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity. The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (microM) of PEA and PLP. TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) greater than 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C. Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH.     

Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV)

Vanadate has been recognized as a specific and potent phosphatase inhibitor since its structure is similar to that of phosphate. In this study, we measured the inhibition of glutathione S-transferase-tagged protein tyrosine phosphatase 1B (GST-PTP1B) and alkaline phosphatase (ALP) by the insulin enhancing compounds, bis(maltolato)oxovanadium(IV) (BMOV). The results showed that the activity of GST-PTP1B was reversibly inhibited by solutions of BMOV with an IC₅₀ value of 0.86 ± 0.02 μM. Steady state kinetic studies showed that inhibition of GST-PTP1B by BMOV was of a mixed competitive and noncompetitive type. In addition, incubation of GST-PTP1B with BMOV showed a time-dependent biphasic inactivation of the protein. On the other hand, the inhibitory behavior of BMOV on ALP activity was reversible and competitive with an IC₅₀ value of 32.1 ± 0.6 μM. Incubation with BMOV did not show biphasic inactivation of ALP. The reversible inhibition of GST-PTP1B by BMOV is more potent than that of ALP, but solutions of BMOV inhibited both enzymes. This data support the suggestion that mechanisms for the inhibitory effects of BMOV on GST-PTP1B and ALP are very different.     

Protein tyrosine phosphatase activity modulation by endothelin-1 in rabbit platelets

Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin-1 stimulation of rabbit platelets resulted in a dose- and time-dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70–100 and 100–200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose-dependent inhibition of the endothelin-stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6-fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti-protein tyrosine phosphatase-1C from endothelin-stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase-1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin-1 in rabbit platelets can be proposed through a cAMP-dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.     

Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors.

Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.     

Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Phosphorylable proteins and alkaline phosphatase of brush border membranes from different parts of the rat small intestine

1. The distribution along the small intestine of phosphorylable proteins from the brush border has been studied by gel electrophoresis. 2. Four proteins, with apparent Mr of 190, 160, 140 and 120 kDa were distributed unequally along the gut, which incorporated 32P from gamma 32P (ATP) to different degrees. 3. Alkaline phosphatase activity has been shown to follow the same distribution. 4. Under denaturing conditions 90, 85 and 65 kDa proteins were observed, whilst the proteins of 190, 160, 140 and 120 kDa had disappeared. 5. All these proteins, with the exception of the 190 kDa protein, had also been labelled with 32Pi. Furthermore, a difference in the phosphorylation of the 65 kDa and the 90-85 kDa proteins was observed. 6. The 65 kDa protein like commercial calf alkaline phosphatase had a ratio of phosphorylation from ATP to phosphorylation from Pi less than the 90 and 85 kDa proteins. 7. Mg2+ (2.5-10 mM) decreased phosphorylation of only the 65 kDa protein whilst beta-glycerophosphate inhibited phosphorylation of all forms of alkaline phosphatase. 8. Incorporation of gamma 32P (ATP) into the proteins was enhanced in the presence of 5 mM theophylline or EDTA. 9. The nature of the phosphorylation of these different proteins is discussed.     

Inhibition of PP-2A upregulates CaMKII in rat forebrain and induces hyperphosphorylation of tau at Ser 262/356

The regulation of the activity of CaMKII by PP-1 and PP-2A, as well as the role of this protein kinase in the phosphorylation of tau protein in forebrain were investigated. The treatment of metabolically active rat brain slices with 1.0 microM okadaic acid (OA) inhibited approximately 65% of PP-2A and had no significant effect on PP-1 in the 16000xg tissue extract. Calyculin A (CL-A), 0.1 microM under the same conditions, inhibited approximately 50% of PP-1 and approximately 20% of PP-2A activities. In contrast, a mixture of OA and CL-A practically completely inhibited both PP-2A and PP-1 activities. The inhibition of the two phosphatase activities or PP-2A alone resulted in an approximately 2-fold increase in CaMKII activity and an approximately 8-fold increase in the phosphorylation of tau at Ser 262/356 in 60 min. Treatment of the brain slices with KN-62, an inhibitor of the autophosphorylation of CaMKII at Thr 286/287, produced approximately 60% inhibition in CaMKII activity and no significant effect on tau phosphorylation at Ser 262/356. The KN-62-treated brain slices when further treated with OA and CL-A did not show any change in CaMKII activity. In vitro, both PP-2A and PP-1 dephosphorylated tau at Ser 262/356 that was phosphorylated with purified CaMKII. These studies suggest (i) that in mammalian forebrain the cytosolic CaMKII activity is regulated mainly by PP-2A, (ii) that CaMKII is the major tau Ser 262/356 kinase in brain, and (iii) that a decrease in PP-2A/PP-1 activities in the brain leads to hyperphosphorylation of tau not only by inhibition of its dephosphorylation but also by promoting the CaMKII activity.