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)

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.
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PMID:Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. 285 69

Distal urinary acidification is thought to be mediated by a proton ATPase (H+-ATPase). We isolated a plasma membrane fraction from human kidney cortex and medulla which contained H+-ATPase activity. In both the cortex and medulla the plasma membrane fraction was enriched in alkaline phosphatase, maltase, Na+,K+-ATPase and devoid of mitochondrial and lysosomal contamination. In the presence of oligomycin (to inhibit mitochondrial ATPase) in the presence of ouabain (to inhibit Na+,K+-ATPase) and in the absence of Ca (to inhibit Ca2+-ATPase) this plasma membrane fraction showed ATPase activity which was sensitive to dicyclohexylcarbodiimide and N-ethylmaleimide. This ATPase activity was also inhibited by vanadate, 4,4'-diisothiocyano-2,2'-disulfonic stilbene and ZnSO4. In the presence of ATP, but not GTP or UTP, the plasma membrane fraction of both cortex and medulla was capable of quenching of acridine orange fluorescence, which could be dissipated by nigericin indicating acidification of the interior of the vesicles. The acidification was not affected by presence of oligomycin or ouabain indicating that it was not due to mitochondrial ATPase or Na+,K+-ATPase, respectively. Dicyclohexylcarbodiimide and N-ethylmaleimide completely abolished the acidification by this plasma membrane fraction. In the presence of valinomycin and an outward-directed K gradient, there was increased quenching of acridine orange, indicating that the H+-ATPase is electrogenic. Acidification was not altered by replacement of Na by K, but was critically dependent on the presence of chloride. In summary, the plasma membrane fraction of the human kidney cortex and medulla contains a H+-ATPase, which is similar to the H+-ATPase described in other species, and we postulate that this H+-ATPase may be involved in urinary acidification.
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PMID:Plasma membrane proton ATPase from human kidney. 287 34

The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide. The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding. Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture. It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding. A similar enhancement is observed upon interaction with TNP-ADP. Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment. Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement. This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain. This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.
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PMID:Mitochondrial ATP synthase. Overexpression in Escherichia coli of a rat liver beta subunit peptide and its interaction with adenine nucleotides. 290 92

This communication presents the results obtained in tubular aggregates of 24 enzyme histochemical techniques for demonstrating activity of oxidoreductases, transferases, hydrolases and isomerases. The activity characteristics of the tubular aggregates in m. gluteus medius of 18 patients with diseases of the neuromuscular system were almost identical. A high activity of the mitochondrial enzymes, NADPH: tetrazolium oxidoreductase, NADH:tetrazolium oxidoreductase and cytochrome c oxidase, could be shown in the pathological structures, whereas the activity of the mitochondrial enzymes, glycerol-3-phosphate:menadione oxidoreductase, succinate:PMS oxidoreductase, malate:NAD+ oxidoreductase and isocitrate:NAD+ oxidoreductase, and the partial mitochondrial enzymes, malate:NADP+ oxidoreductase and isocitrate:NADP+ oxidoreductase, was very slight or even absent. There was a moderate to strong activity of the glycolytic enzymes lactate:NAD+ oxidoreductase, glyceraldehyde-3-phosphate:NAD+ oxidoreductase, phosphofructokinase, phosphoglucomutase and glucose phosphate isomerase. In contrast, the activity of alpha-glucan phosphorylase was slight. The activity of phosphogluconate:NADP+ oxidoreductase, glucose-6-phosphate:NADP+ oxidoreductase and 5'-nucleotidase was slight, whereas there was no activity of myosin ATPase and mitochondrial ATPase, acid phosphatase or alkaline phosphatase. The high activity of AMP-deaminase was very striking. The activity of peroxidase was moderate. Results obtained with adsorption studies point to adsorption of some of the enzymes studied to the tubular aggregates in vivo and this phenomenon very probably determined the histochemical characteristics of these structures.
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PMID:Histochemical features of tubular aggregates in diseased human skeletal muscle fibres. 317 98

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.
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PMID:4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase. 644 30

In late infantile and juvenile forms of neuronal ceroid lipofuscinosis, commonly known as Batten disease (BD). ATP synthase subunit c accumulates in the lysosomes of neural cells. By using polyclonal antibodies, raised against bovine liver subunit c and an image analysis system for the quantification of antibody-linked alkaline phosphatase reaction, we have demonstrated that polymorphonucleocytes (PMN) from a late infantile and a juvenile BD patient stored several-fold more subunit c as compared to normal PMN.
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PMID:ATP synthase subunit C storage in the polymorphonucleocytes of late infantile and juvenile batten patients. 748 16

Purified chloroplast ATP synthase (CF1) contains 1.2-2 mol of tightly bound ADP/mol of enzyme that resists removal by gel filtration or dialysis. CF1 was depleted of its endogenous nucleotide by treatment with alkaline phosphatase. Tightly bound nucleotide was demonstrated not to have an essential structural role. CF1 depleted of endogenous nucleotide retains its ability to catalyze Ca2+- and Mg2+-dependent ATPase activity and is not more sensitive to cold inactivation than untreated CF1. 2'(3')-O-Trinitrophenyladenosine 5'-diphosphate (TNP-ADP) binds tightly to two sites on nucleotide-depleted CF1, binding to either site at a faster rate than that of exchange of bound nucleotide for medium nucleotide. The nucleotide-depleted enzyme binds about one additional mol of TNP-ADP/mol of CF1, indicating that there is a tight TNP-ADP binding site that does not exchange readily with medium nucleotide. It is MgADP in this nonexchanging site, not the easily exchanging ADP binding site, that is responsible for the MgADP-induced inhibition of the ATPase activity. The rate of exchange of tightly bound ADP from CF1 matches the rate at which the Mg2+ATPase activity of CF1 is activated but is not itself responsible for the activation.
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PMID:Differences between two tight ADP binding sites of the chloroplast coupling factor 1 and their effects on ATPase activity. 870 14

The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag). None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium. Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell. Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography. The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase. Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131. On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.
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PMID:Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues. 963 82

Using the cultured chicken embryonic chondrocytes as a model, the effects of simulated microgravity on the microtubular system of the cellular skeleton, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration and mitochondrial ATP synthase activity with its oligomycin inhibition rate were studied with a clinostat. The microtubular content was measured by a flow cytometer. The decrease of microtubular content showed the impairment of the cellular skeleton system. Observation on the extracellular matrix by the scanning electron microscopy showed that it decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly than that of the control group. It can be concluded that the simulated microgravity can affect the secreting and assembly of the extracellular matrix. In contrast to the control, there was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. These results indicate that simulated microgravity can suppress matrix calcification of cultured chondrocytes, and intracellular free calcium may be involved in the regulation of matrix calcification as the second signal transmitter. No significant changes happened in the mitochondrial ATP synthase activity and its oligomycin inhibition rate. Perhaps the energy metabolism wasn't affected by the simulated microgravity. The possible mechanisms about them were discussed.
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PMID:The effects of simulated microgravity on cultured chicken embryonic chondrocytes. 1500 13

Proteins such as aminopeptidases and alkaline phosphatases, both glycosyl-phosphatidyl-inositol (GPI) anchored proteins, were previously identified as Cry1Ac binding proteins in the Heliothis virescens midgut. To identify additional toxin binding proteins, brush border membrane vesicles from H. virescens larvae were treated with phosphatidyl inositol phospholipase C, and released proteins were resolved by two-dimensional electrophoresis. Protein spots selected by their ability to bind Cry1Ac were identified by MALDI-TOF mass spectrometry coupled to peptide mass fingerprinting (PMF) and database searching. As in previous studies, H. virescens alkaline phosphatase was identified as a Cry1Ac binding protein. V-ATP synthase subunit A and actin were identified as novel Cry1Ac binding proteins in H. virescens. Additional toxin-binding proteins were predicted based on MS/MS fragmentation and de novo sequencing, providing amino acid sequences that were used in database searches to identify a phosphatase and a putative protein of the cadherin superfamily as additional Cry1Ac binding proteins.
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PMID:Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses. 1729 94


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