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)

A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.
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PMID:Alkaline phosphatase from rat osseous plates: purification and biochemical characterization of a soluble form. 206 78

Cell-associated alkaline phosphatase (ALPase) of Bacteroides gingivalis 381 was found in the outer part of the periplasmic space by using an ultracytochemical procedure. Cell-associated ALPase was solubilized by extraction with 1% Triton X-100, and the solubilized enzyme was purified 904-fold with 5.6% recovery by using affinity column chromatography for mammalian intestinal-form ALPase. The purified enzyme gave a single protein band that corresponded to the enzyme activity band on polyacrylamide gel electrophoresis preparations. A single protein band at a molecular weight of 61,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparations. The molecular weight of the native enzyme was estimated to be 130,000 by gel filtration with TSK-gel G3000SW. These findings indicate that B. gingivalis ALPase is a homodimer. The optimal pH of the enzyme was between 9.1 and 9.3 in the absence of divalent metal ions and was between 10.1 and 10.3 in the presence of manganese or zinc ions. The apparent km for p-nitrophenylphosphate was 0.037 +/- 0.003 mM (mean +/- standard deviation) at pH 9.2 in the absence of divalent metal ions and 0.22 +/- 0.02 mM at pH 10.2 in the presence of 1 mM manganese ions. Under both of the conditions described above, the purified enzyme was able to hydrolyze casein and O-phosphoserine, suggesting that B. gingivalis ALPase can act as a phosphoprotein phosphatase. ALPase that immunologically cross-reacted with the purified enzyme was found in the extracellular soluble fraction. This means that ALPase is released from the periplasmic space into the culture supernatant as a soluble form.
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PMID:Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381. 211 73

A vitamin and trace element supplement containing recommended dietary amounts or "safe and adequate" levels was given to ten healthy subjects for 12 to 35 weeks. Plasma levels of selenium and zinc, activity of glutathione peroxidase in plasma and platelets, whole blood manganese, activity of superoxide dismutase in hemolysate, activity of alkaline phosphatase in serum, iron status indices and urinary excretion of zinc and selenium were measured. A small but significant change in plasma selenium from 1.01 +/- 0.14 mumol/L to 1.08 +/- 0.10 mumol/L was observed after two weeks. However, at the end of the supplementation plasma selenium levels did not differ from the initial levels. Plasma glutathione peroxidase levels showed a similar trend and changes in glutathione peroxidase activity in platelets were also transient. A small increase in serum zinc values was observed after 30 weeks of supplementation. No significant changes were observed in the other blood and urine parameters studied. In seven of the subjects absorption of zinc, manganese and selenium was measured after 30-31 weeks of supplementation by a radionuclide technique. The absorption of selenium and manganese after long term supplementation was 30-50% lower than observed previously in non-supplemented subjects. In conclusion, present available indices of trace element status are only to a limited extent affected by 30 weeks of a doubling of the normal dietary intake.
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PMID:Effect of long-term trace element supplementation on blood trace element levels and absorption of (75Se), (54Mn) and (65Zn). 213 27

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.
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PMID:Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein. 215 97

1. Matrix-induced alkaline phosphatase prepared from rat osseous plate was solubilized with polidocanol and purified on a Sephacryl S-300 column. 2. Purified solubilized alkaline phosphatase has a molecular weight of ca 115,000 and bind one magnesium and two zinc ions. At least 110 detergent molecules are bound to each enzyme molecule. 3. Solubilization and purification procedures did not destroy the ability of the enzyme to hydrolyze adenosine-5'-triphosphate, p-nitrophenylphosphate, pyrophosphate and bis p-nitrophenylphosphate. 4. Magnesium, manganese and cobalt ions are stimulators of PNPPase activity of solubilized enzyme whereas calcium and zinc ions are inhibitors.
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PMID:Solubilization of membrane-bound matrix-induced alkaline phosphatase with polyoxyethylene 9-lauryl ether (polidocanol): purification and metalloenzyme properties. 215 26

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.
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PMID:Characterization of a nerve growth factor-stimulated protein kinase in PC12 cells which phosphorylates microtubule-associated protein 2 and pp250. 216 72

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.
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PMID:o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase. 247 84

The 33 kDa extrinsic polypeptide of photosystem II, also known as the manganese-stabilizing polypeptide (MSP), is located on the lumen side of the thylakoid and is involved in water oxidation. The gene for MSP, designated woxA, has been cloned from the nitrogen-fixing filamentous cyanobacterium Anabaena and sequenced. The woxA open reading frame was found to be 819 bp. The deduced amino acid sequence was 63% and 59% homologous with that of Synechococcus and Synechocystis, respectively, and 44% conserved when compared to the MSP of spinach or pea. Two cysteine residues at positions 48 and 73 were found to be conserved in cyanobacteria and plants. The first 29 amino acids are hydrophobic and may represent the transit peptide. woxA: :phoA translational fusion products, in which the body of Escherichia coli alkaline phosphatase was fused to the amino terminal portion of woxA between residues 35 and 130, yielded active alkaline phosphatase in E. coli. Thus the transit peptide of woxA functions in E. coli to transport phosphatase across the cytoplasmic membrane. S1 mapping and primer extension experiments showed that the woxA transcription initiation site is located 220 bp upstream from the translational start. The woxA promoter has some resemblance to the E. coli consensus and other known Anabaena vegetative cell promoters.
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PMID:Nucleotide sequence of the gene encoding the 33 kDa water oxidizing polypeptide in Anabaena sp. strain PCC 7120 and its expression in Escherichia coli. 251 33

Binding of epidermal growth factor (EGF) stimulates tyrosyl protein kinase activity of its receptor in the epidermis. This tyrosine residue phosphorylation is thought to be one mechanism by which EGF mediates its effects such as growth stimulation. To modulate a cellular response to EGF, an enzyme which dephosphorylates phosphotyrosyl residues should be present to oppose the effect of the tyrosyl kinase activity of the EGF receptor. We have identified an enzyme in the neonatal mouse epidermis which has the ability to dephosphorylate tyrosyl residues in vitro on EGF receptors. This phosphatase is a soluble protein with a molecular weight greater than 10,000 daltons and shows optimum activity at neutral pH. This epidermal tyrosyl protein phosphatase is not inhibited by tartrate, ATP, and micromolar levels of zinc, but is inhibited by millimolar levels of zinc, magnesium, manganese, and fluoride. Unlike other well-known phosphotyrosyl phosphatases, alkaline phosphatase, and calcineurin, this enzyme is not inhibited by EDTA. Thus, we have identified and partially characterized a possibly unique phosphotyrosyl phosphatase from the epidermis.
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PMID:Identification of a phosphotyrosyl-protein phosphatase in mouse epidermis. 253 66

In the presence of inhibitors for mitochondrial H+-ATPase, (Na+ + K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N.N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brush-border and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator. ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl-) or entry of a cation, e.g., Na+ via electrogenic Na+/D-glucose and Na+/L-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM- sensitive ATPases at the cytosolic side of the brush-border membrane.
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PMID:Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion. 253


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