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)

The activities of hybrid dimers of alkaline phosphatase containing two chemically modified subunits have been investigated. One hybrid species was prepared by dissociation and reconstitution of a mixture of two variants produced by chemical modification of the native enzyme with succinic anhydride and tetranitromethane, respectively. The succinyl-nitrotyrosyl hybrid was separated from the other members of the hybrid set by DEAE-Sephadex chromatography and then converted to a succinyl-aminotyrosyl hybrid by reduction of the modified tyrosine residues with sodium dithionite. A comparison of the activities of these two hybrids with the activities of the succinyl, nitrotyrosyl and aminotyrosyl derivatives has shown that either the subunits of alkaline phosphatase function independently or if the subunits turnover alternately in a reciprocating mechanism, then the intrinsic activity of each subunit must be strongly dependent on its partner subunit.
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PMID:Hybrids of chemical derivatives of Escherichia coli alkaline phosphatase. 0 86

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.
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PMID:Purification and characterization of alkaline phosphatase from rat kidney. 0 27

A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.
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PMID:Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. 1 Sep 86

Calf pancreas microsomes incorporated radioactive D-mannose from GDP-D-[14C]mannose into lipid-bound oligosaccharides extracted with chloroform/methanol/water (10/10/2.5, v/v). Several products, which probably differed in the size of the oligosaccharide moiety, were labeled. These could be partially resolved by thin layer chromatography and DEAE-cellulose chromatography. The labeled lipid-bound oligosaccharides were retained on DEAE-cellulose more strongly than synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a neutral 14C-labeled oligosaccharide fraction which was estimated by gel filtration to have a minimum of 8 monosaccharide residues. Hot alkali treatment yielded a mixture of neutral and acidic 14C-labeled oligosaccharides which could be transformed into neutral products by alkaline phosphatase. The D-[14C]mannose residues were alpha-linked at the nonreducing terminus of the oligosaccharides since they could be removed completely with alpha-mannosidase. Most of the D-[14C]mannose-labeled oligosaccharides were retained on concanavalin A Sepharose and eluted with methyl alpha-D-mannopyranoside. Pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate incubated with calf pancreas microsomes in the presence of sodium taurocholate was efficiently utilized as donor of alpha-D-mannosyl residues in lipid-bound oligosaccharides. The products formed from dolichyl beta-D-[14C]mannopyranosyl phosphate were identical with those formed from GDP-D-[14C]mannose, and evidence was obtained to show that the dolichyl beta-D-[14C]mannopyranosyl phosphate was serving as donor without prior conversion to GDP-D-[14C]mannose. Transfer of mannose from dolichyl beta-D-[14C]mannopyranosyl phosphate to lipid-bound oligosaccharides took place at a pH optimum of 7.3, whereas transfer to the precipitate containing glycoproteins was greatest at pH 6.0 in Tris/maleate buffer. The addition of divalent cation was not required, but low concentrations of EDTA were extremely inhibitory. The carbohydrate composition of the lipid-bound oligosaccharides of microsomal membranes was investigated by gas-liquid chromatography and by reduction with sodium borotritide. A heterogeneous mixture of oligosaccharides containing N-acetyl-D-glucosamine, D-mannose, and D-glucose varying in proportions from approximately 1/2.5/0.5 to 1/5/1.5 was obtained with glucosamine at the reducing end. Acid treatment of the lipid-bound oligosaccharide fraction yielded dolichyl pyrophosphate, suggesting that at least some of the oligosaccharides were linked to dolichol through a pyrophosphate group.
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PMID:Mannosyltransferase activity in calf pancreas microsomes. Formation of 14C-labeled lipid-linked oligosaccharides from GDP-D-[14C]mannose and pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate. 1 65

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.
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PMID:Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain. 1 41

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in guinea pig thymus was extracted optimally in 10 mM Tris - HCl buffer at pH 8.0 containing 5 g/l Triton X-100. 2. alpha-Glycerophosphate, beta-glycerophosphate and phenolphthalein monophosphate were hydrolyzed by thymus extract with a pH optimum at 9.8-10.0, whereas p-nitrophenylphosphate and alpha-naphthylphosphate were hydrolyzed with pH optima at 10.7-10.8 and beta-naphthylphosphate at pH 11.2. P-Nitrophenylphosphate and phenolphthalein monophosphate proved to be the most suitable substrates. 3. Alkaline phosphatase was effectively inhibited by EDTA, Zn2+, histidine and urea therefore resembling the inhibition characteristics of alkaline phosphatase in the placenta and kidney, but not that in the liver and intestine, which differed markedly. 4. DEAE-cellulose chromatography and polyacrylamide disc electrophoresis revealed three enzyme peaks which did not differ in their substrate specificities and modifier characteristics. 5. Polyacrylamide disc electrophoresis of thymus, serum, placenta, kidney, liver, bone and intestine revealed no alkaline phosphatase bands definitely unique to thymus.
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PMID:Biochemical characterization of alkaline phosphatase in guinea pig thymus. 1 86

Vicia faba root cells contain several nucleolytic activities: phosphomonoesterase and phosphodiesterase (which however were not studied in details), one nuclease and four ribonucleases. These results were obtained by separating the extracted proteins into anionic and cationic species by chromatography on CM-cellulose at pH 5.5 and analysing each kind of proteins. Anionic species were subjected to chromatography on DEAE-cellulose which lead to isolation of one nuclease (A1) and two RNAases (A2, A3), the properties of which were studied. It was shown that the RNAases pH optima are near 6; A2 is more thermolabile than A3; both are endonucleases unable to attack double-stranded structure; studies with homopolymers, i.e. poly(A), poly(I), poly(C), poly(U), showed that their base specificities were analogous to that of already known plant RNAases. The cationic proteins, analysed with CM-cellulose, contain two RNAases (C1, C2). The pH optima were near 6 and 7, respectively; C1 is much more thermolabile than C2; both were endonucleases inactive on double-stranded structures. C1 and C2 hydrolysed poly(C) and poly(U) but not poly(A) and poly(U).
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PMID:Study on plant RNAases. Isolation and properties of several activities from Vicia faba root cells. 2 32

Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.
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PMID:Purification and characterization of alkaline phosphatase in cultured rat liver cells. 2 75

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.
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PMID:Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography. 2 75

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0.2% (v/v) Triton X-100 and extracted wtih 20% (v/v) n-butanol. The procedure, which resulted in 182-fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex G200 gel filtration. Solubilization with Triton X-100 was an important step in the procedure since extraction with n-butanol alone only partially solubilized the enzyme and gave low extraction yields, much of the enzyme activity remaining in association with negatively charged residues. However, butanol extraction of Triton X-100-treated homogenates gave high yields of enzyme and eliminated p-nitrophenyl phosphatases which displayed activity in the pH range 3.0--7.5, together with a large proportion of inactive protein. The activity of the purified enzyme preparations was electrophoretically homogeneous on cellulose acetate membranes, suggesting that the alkaline phosphatase in the mouse uterus exists in a single isozymic form. Polyacrylamide-gel electrophoresis revealed that the purified preparations contained at least one protein as an impurity. Attempts to further purify the alkaline phosphatase by isoelectric focusing were unsuccessful since the enzyme was found to have an isoelectric point of about 5.0 and at this pH it was rapidly inactivated.
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PMID:Extraction and partial purification of mouse uterine alkaline phosphatase. 4 May 39


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