Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively. Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent. These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.
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PMID:Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine. 822 55

Mg uptake was investigated with (28)Mg by a rapid filtration procedure in rat duodenal and jejunal brush border membrane (BBM) vesicles, prepared by CaCl(2)a or MgCl(2)b differential precipitation. At 1 mM Mg, 10 s uptakes were lower in jejunal vesicles (3.5(a) or 5.5(b) nmol/10 s per mg protein) than in duodenal vesicles (11.4(a) or 13.5(b) nmol/10 s per mg protein). The equilibrated 60 min uptakes were also lower in jejunum (11.0(a) or 26.6(b) nmol/60 min per mg protein) than in duodenum (l8.8(a) or 26.6(b) nmol/60 min per mg protein). The influence of medium osmolarity on 10s and 60 min uptakes of Mg indicated that Mg was 'transported' into osmotically active spaces. The effect of Mg concentration on the 10 s uptake suggested the existence of one single mechanism of transport in the duodenum, with an apparent K(T) of 1 mM, and of two mechanisms in the jejunum, with apparent K(T) values of 0.2 and 2-5 mM. Despite different amounts of calcium and magnesium in CaCl(2) and MgCl(2) precipitated vesicles, there were no large differences in magnesium uptakes depending on the mode of preparation of the vesicles. In contrast, calcium uptakes. measured with (45)Ca, were six to nine times higher in MgCl(2) prepared jejunal vesicles, and were always much higher than magnesium uptakes measured under the same conditions. At 0.1 mM calcium concentration, calcium uptake was depressed by 0.025 mM verapamil (50 percent) and by 0.1 mM ZnCl(2)(40-75 percent), while Mg uptakes were unaffected. L-leucine or L-phenylalanine (5 mM), two inhibitors of intestinal alkaline phosphatase, decreased Mg uptake by 30 to 40 percent at 1 mM Mg, but had no significant effect at 0.1 mM, and did not affect calcium uptakes at all. A possible involvement of alkaline phosphatase in magnesium uptake was ascertained in jejunal BBM vesicles treated with phosphatidylinositol-specific phospholipase C, which partially released alkaline phosphatase from the BBM. Calcium uptakes were unaffected by the treatment, while magnesium uptakes were significantly decreased at 1 mM Mg. These results confirm that magnesium and calcium are transported by distinct mechanisms in the jejunum.
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PMID:Uptake of (28)Mg by duodenal and jejunal brush border membrane vesicles in the rat. 886 Nov 32

An increase of alkaline phosphatase (ALP) activity has been observed in the bronchoalveolar lavage fluid (BALF) of patients affected by pulmonary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ agglutinin (WGA) precipitation, and an immunoassay specific for the bone-isoform of ALP. Only one anodic band representing a high-molecular-weight isoform of ALP (Mr approximately 2,000 kDa) was observed on electrophoresis of BALF. The inhibition assay results were consistent for a tissue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and resistant to L-phenylalanine and L-leucine. Less than 13% of ALP activity was heat-stable. After incubation of BALF specimens with glycosyl-phosphatidylinositol-phospholipase D plus Nonidet P-40, or with phosphatidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr approximately 220 kDa) appeared near the bone band of a standard serum. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less than 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.
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PMID:Identification of human pulmonary alkaline phosphatase isoenzymes. 910 92

Effects of leucine and related compounds on protein synthesis were studied in RLC-16 hepatocytes. The incorporation of [(3)H] tyrosine into cellular protein was measured as an indexof protein synthesis. In leucine-depleted RLC-16 cells, L-leucineand its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipase A(2) and C canceled stimulatory actions of L-leucine and KIC on protein synthesis, suggesting a possible involvement of either arachidonic acid metabolism by phospholipase A(2), cyclooxygenase or lipoxygenase, or phosphatidylinositol degradation by phospholipase C in the stimulatory actions of L-leucine and KIC.Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of protein kinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in RLC-16 cells via activation of phospholipase C and production of diacylglycerol and inositol triphosphate from phosphatidylinositol, which in turn activate protein kinase C.
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PMID:Possible involvement of phospholipase C and protein kinase C in stimulatory actions of L-leucine and its keto acid, alpha-ketoisocaproic acid, on protein synthesis in RLC-16 hepatocytes. 1900 15

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.
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PMID:Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes. 1900 13