Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole.
(1/17)
Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [(32)P]PO(4) release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)-p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate. (+info)
Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.
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BACKGROUND: In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. RESULTS: To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. CONCLUSION: Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. (+info)
Necessity of enzymatic activity of alkaline phosphatase for mineralization of osteoblastic cells.
(3/17)
Alkaline phosphatase (ALP) is supposed to be important for bone formation; however, its role is not clear. In this study, we examined the importance of enzymatic activity of ALP and anchoring of ALP protein to the cells for mineralization of an osteoblastic cell line, MC3T3-E1. While we cultured the cells in the presence of tetramisole, an inhibitor of ALP activity, ALP protein was expressed at a similar level to that in the control. Although tetramisole showed no effect on cell growth and increased hydroxyproline accumulation, it decreased the osteocalcin production and the accumulation of calcium and phosphate in the matrices. Tetramisole also inhibited mineralized nodule formation, which was observed by optical microscopy and detected by Von Kossa staining. On the other hand, when ALP protein was released from the cell membranes with the use of phosphatidylinositol-specific phospholipase C, no marked changes were detected in hydroxyproline, calcium and phosphate accumulations in the matrices at late calcification stage, which was consistent with the morphological findings. These results clearly show that enzymatic activity of ALP is necessary for mineralization of MC3T3-E1 cells, but not the presence of ALP protein or anchoring of ALP to the cells. (+info)
L-tetramisole. Enhancement of human lymphocyte response to antigen.
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The anthelminthic drug L-Tetramisole enhanced in vitro lymphocyte transformation of normal young adults in the presence of PPD, Candida or measles antigen. This enhancement occurred regardless of evident pre-existing immunity to the antigen used. The increase in stimulation obtained by the drug and antigen combined, beyond that by antigen alone, varied between 1-25 and 5-99 for all three antigens in fourteen cell donors. Lymphocyte transformation was induced in seven individuals by the drug alone and suppressed in two. In levamisole dose--response tests, the optimum dose for this in vitro effect in six donors was similar for the three antigens. (+info)
Benzotetramisole-catalyzed dynamic kinetic resolution of azlactones.
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Measurement of alkaline phosphatase of intestinal origin in plasma by p-bromotetramisole inhibition.
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L-p-bromotetramisole was used to inhibit non-intestinal alkaline phosphatase (of liver or bone origin) (EC 3.1.3.1; ALP) in plasma, and intestinal ALP was measured from the uninhibited activity. The method of determination is convenient and correlated well with measurement by immunocapture assay. If carried out in parallel with wheat-germ lectin precipitation of bone ALP, subtraction of intestinal ALP activity from that of non-bone ALP in the supernatant can be used to measure the ALP that originates from the liver in men and non-pregnant women. (+info)
Determination of absolute configuration of secondary alcohols using thin-layer chromatography.
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Quantitative method for determining serum alkaline phosphatase isoenzyme activity: estimation of intestinal component.
(8/17)
Intestinal alkaline phosphatase activity was measured using levamisole inhibition, and results were compared with a previously reported method using L-phenylalanine. Sixty two per cent intestinal, 39% placental, and 1.3% of either bone or liver alkaline phosphatase activity remained when alkaline phosphatase activity was inhibited in a 2-amino-2-methyl-1-propanol (AMP) buffer reagent system with 10 mmol/l levamisole (final assay concentration 8.1 mmol/l). The assay imprecision (SD) was 0.6 U/l compared with 3.9 U/l using L-phenylalanine for specimens with total alkaline phosphatase activity less than 250 U/l (reference range 30-120 U/l). In serum pools with raised total alkaline phosphatase activity errors in recovered intestinal activity were small (usually less than 3 U/l) when intestinal alkaline phosphatase was added. Much larger errors and many underestimated results were found using L-phenylalanine. For non-haemolysed specimens it is concluded that an assay based on levamisole inhibition provides a better measure of intestinal alkaline phosphatase activity than L-phenylalanine. (+info)