Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycosylphosphatidylinositol (GPI)-anchored proteins occur widely, perhaps universally, on the surface of animal cells, where they perform a variety of important functions. However, the existence of GPI-anchored proteins on plant cells has never been established. Evidence is presented in this communication for the occurrence of a 50 kDa GPI-anchored alkaline phosphatase (AP) induced in the duckweed Spirodela oligorrhiza by phosphate deprivation. Triton X-114 partitioning of the Spirodela proteins yielded two forms of AP activity. The detergent-associated form was labeled prominently by [3H]ethanolamine, [3H]myristic acid and [3H]palmitic acid. This amphiphilic form of AP, like authentic GPI-anchored AP from mammals, was clearly resolved from the remaining, water-soluble AP activity by two types of incompletely-denaturing polyacrylamide gel electrophoresis. Lipid covalently bound to the solvent-delipidated amphiphilic AP was resistant to cleavage by phosphatidylinositol-specific phospholipase C. Strong acid or alkaline hydrolysis of the 3H-fatty acid-labeled amphiphilic AP yielded radioactive fatty acids and a radioactive lipid tentatively identified as a long chain base. The more abundant water-soluble AP was also radioactive in plants incubated with [3H]ethanolamine and was labeled to a lesser extent by 3H-fatty acids. The water-soluble AP, unlike its amphiphilic counterpart, could be freed of all fatty acid radioactivity by mild alkaline hydrolysis, indicating the continued presence of an ester-linked fatty acid. All evidence supports the conclusion that Spirodela AP is synthesized as an amphiphilic protein with a ceramide-containing GPI anchor.
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PMID:Evidence for a glycosylinositolphospholipid-anchored alkaline phosphatase in the aquatic plant Spirodela oligorrhiza. 864 7

An electrophoretically homogeneous glycosylphosphatidylinositol- alkaline phosphatase fraction from calf intestine, obtained by hydrophobic chromatography, was used as "enzyme-labeled" substrate for testing phospholipase activity. The reaction products were separated by (i) hydrophobic chromatography in pipet tips and (ii) Triton X-114 phase partitioning. The chromatographic method presented permits high test frequencies, does not need temperature-controlled sample handling, and is only slightly disturbed by detergents, organic solvents, and proteins. The method was used to characterize phosphatidylinositol- specific phospholipase C from Bacillus cereus and phospholipase D from calf serum. Measurement of substrate hydrolysis by phospholipases is apparently linear to enzyme concentration and time. Relative activity of both enzymes is maximum at pH 6.5, corresponding to the optimal pH range found with other glycosylphosphatidylinositol substrates and phosphatidylinositol-specific phospholipases of other sources. Maximum activity of phospholipase C was found at 0.03% Triton X-100, 0.01% Brij 35, and 0.2% n-octylglucoside. The activity is not affected by Ca(2+), NaHCO(3), o-phenanthroline, or EDTA, increasingly inhibited by MgCl(2), MnCl(2), and ZnCl(2), and slightly activated by Na+ and K+. Calf serum phospholipase D shows maximum activity at 0.05% Triton X-100, 0.02% Brij 35, and 0.4% n-octylglucoside. The apparent Km values for phospholipase C (12.25 micron) and phospholipase D (4.94 micron) found with glycosylphosphatidylinositol-alkaline phosphatase are compared with values published for other glycosylphosphatidylinositol substrates.
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PMID:Glycosylphosphatidylinositol-alkaline phosphatase from calf intestine as substrate for glycosylphosphatidylinositol-specific phospholipases--microassay using hydrophobic chromatography in pipet tips. 867 26

Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.
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PMID:Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification. 875 Nov 58

The phosphatidylinositol-specific phospholipase C, presumably a Zn-metalloenzyme, catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate to inositol-1,4,5-trisphosphate (IP3) and s,n-1,2-diacylglycerol (DAG). The activity of phosphatidylinositol-specific phospholipase C was measured indirectly by determination of the metabolites IP3 and DAG in Zn deficiency. For this purpose 24 male Sprague-Dawley rats with an average live mass of 117 g were divided into 2 groups of 12 animals each. The Zn-deficient and the control group received a semisynthetic casein diet with a Zn content of 1.6 ppm and 115 ppm, respectively. In order to prevent the reduced feed intake that occurs in Zn deficiency and the associated energy and protein depletion from interfering with the experimental parameters, all animals were fed four times daily by gastric tube. This made it possible to supply all animals with adequate nutrients and to synchronize the feed intake exactly. After 12 d, the depleted rats were in a severe state of Zn deficiency, as demonstrated by the reduction of Zn in the serum and the femur by 74% and 43%, respectively, and the 28% lower serum activity of alkaline phosphatase. The radioimmunologically determined concentrations of IP3 were reduced by a significant 53% in the testes of the Zn-deficient rats (0.24 nmol IP3/g wet wt) compared to the control animals (0.51 nmol IP3/g wet wt), while the IP3 concentration in the brain was not affected by the alimentary Zn supply (1.7 and 1.6 nmol IP3/g wet wt, respectively). The DAG concentrations in the testes (474 vs 471 nmol DAG/g wet wt) and the brain (594 vs 640 nmol DAG/g wet wt), which were determined by radioenzymatic methods, showed no significant differences in relation to the alimentary Zn supply. The fact that the Zn concentration in the Zn-deficient rats was reduced only in the testes and not in the brain and that high concentrations of DAG may also result from other metabolic processes suggests that the phosphatidylinositol-specific phospholipase C in the mammalian organism is a Zn-metalloenzyme whose activity is reduced in alimentary Zn deficiency in tissues suffering Zn loss.
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PMID:Influence of alimentary zinc deficiency on the concentration of the second messengers D-myo-inositol-1,4,5-trisphosphate (IP3) and s,n-1,2-diacylglycerol (DAG) in testes and brain of force-fed rats. 881 Dec 85

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

The purpose of the present study was to investigate whether alimentary zinc (Zn) deficiency affects the activities of the Zn metalloenzymes protein kinase C (pKC) and the phosphatidylinositol-specific phospholipase C (PLC) in force-fed Zn-deficient rats. The in vivo activity of pKC was determined by measuring the subcellular distribution of the enzyme between the cytosolic and the particulate fraction of erythrocytes, whereas the activity of PLC was measured indirectly through the concentration of its metabolite inositol-1,4, 5-trisphosphate (IP3) in platelets and monocytes. For this purpose, 24 male Sprague-Dawley rats with an average live mass of 126 g were divided into 2 groups of 12 animals each. The Zn-deficient and the control rats received a semisynthetic casein diet with a Zn content of 1.2 and 24.1 ppm, respectively. All animals were fed the same amount of the diet (10.8 g dry matter [DM]/d and rat) four times daily by gastric tube. After 12 d, the depleted rats were in a state of severe Zn deficiency, as demonstrated by a 70% lower Zn concentration and a 66% reduction in the serum activity of alkaline phosphatase. The radio-immunologically determined concentration of IP3 was reduced by a significant 55% in the platelets of the Zn-deficient rats (8.4 pmol IP3/ 5 x 10(8)) as compared with the control rats (18.8 pmol IP3/5 x 10(8)), whereas the IP3 concentration in the monocytes was not affected by the alimentary Zn supply (1.4 vs 1.2 pmol IP3/10(6)), nor was there any difference between the Zn-deficient and the control rats with regard to the radioenzymatically determined specific activity of pKC, either in the cytosolic fraction (32.7 vs 32.5 pmol P/min/mg protein) or in the particulate fraction (38.1 vs 36.5 pmol P/min/mg protein) of the erythrocytes.
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PMID:Subcellular distribution of protein kinase C (pKC) in erythrocytes and concentration of D-myo-inositol-1,4,5-trisphosphate (IP3) in platelets and monocytes of force-fed zinc-deficient rats. 886 51

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5'-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca(2+)-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the mental chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.
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PMID:Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes. 892 Sep 95

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.
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PMID:Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1. 901 Feb 30

This study investigated ectoenzyme release from small intestine brush border membranes (duodenum and jejunum, Preparation A; ileum, Preparation B) of mice by the action of phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D. Most of the alkaline phosphatase was solubilized from Preparation A, but about 60% was released from Preparation B. As for alkaline phosphodiesterase I activity, 15 and 10% were released from Preparations A and B, respectively. With Preparation B, octylglucoside treatment followed by phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D completely solubilized the alkaline phosphatase activity. However, this treatment did not change the ratio of release of alkaline phosphodiesterase I from Preparation A or B. These results indicate that the resistance to alkaline phosphatase found in Preparation B is due to hindered accessibility of the bonding splitting by phosphatidylinositol-specific phospholipase C and not to a modified glycosyl-phosphatidylinositolanchor.
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PMID:Release of ectoenzymes from small intestine brush border membranes of mice by phospholipases. 905 73

Treatment with phosphatidylinositol-specific phospholipase C of rat osseous plate membranes released up to 90-95% of alkaline phosphatase, but a specific ATPase activity (optimum pH = 7.5) remained bound to the membrane. The hydrolysis of ATP by this ATPase was negligible in the absence of magnesium or calcium ions. However, at millimolar concentrations of magnesium and calcium ions, the membrane-specific ATPase activity increased to about 560-600 U/mg, exhibiting two classes of ATP-hydrolysing sites, and site-site interactions. GTP, UTP, ITP, and CTP were also hydrolyzed by the membrane-specific ATPase. Oligomycin, ouabain, bafilomycin A1, thapsigargin, omeprazole, ethacrynic acid and EDTA slightly affected membrane-specific ATPase activity, while vanadate produced a 18% inhibition. The membrane-specific ATPase activity was insensitive to theophylline, but was inhibited 40% by levamisole. These data suggested that the membrane-specific ATPase activity present in osseous plate membranes, and alkaline phosphatase, were different proteins.
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PMID:Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochodral ossification. 945 89


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