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 treatment of human platelets with the dibutyryl cyclic AMP (dbcAMP) revealed the presence of a 250 kDa protein which enhanced its GTP-binding activity. This protein was purified from platelet membranes by successive chromatographies on DEAE-cellulose, Ultrogel AcA34, Mono Q, HCA-hydroxyapatite, and TSK-3000SW columns. The positive cross-reaction of the 250 kDa protein with the anti-filamin antibody indicated that this protein is filamin or very close to it. The GTP gamma S-binding activity of this protein, when phosphorylated with cyclic AMP-dependent protein kinase (A-kinase), showed an over tenfold increase, with the specific activity being 3.6 nmol/mg protein. Dephosphorylation of the phosphorylated protein with alkaline phosphatase reduced the GTP gamma S-binding activity to the control untreated level.
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PMID:Enhancement of GTP gamma S-binding activity by cAMP-dependent phosphorylation of a filamin-like 250 kDa membrane protein in human platelets. 217 20

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.
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PMID:The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays. 230 58

Properties of human ileal and duodenal alkaline phosphatase (ALP) were compared. The pH optimum, Km values, heat stability, inhibition of activity by amino acids, and antigenicity of ileal and duodenal ALPs were similar. Affinity for DEAE and Tyraminyl derivatives/Sepharose chromatographies, substrate specificity, molecular mass, isoelectric point, and sugar chain structure differed, suggesting two forms of intestinal enzyme. The N-terminal amino acid sequence, or peptide mapping or both suggest that the two major intestinal ALPs are identical, but the minor ALP may be differed from the sequence of major one.
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PMID:Partial characterization of human ileal alkaline phosphatase: differences between human ileal and duodenal enzymes. 243 69

Seven Tyr-protein phosphatase activities were isolated from bovine brain using phosphotyrosyl-casein as a model substrate. The activities were resolved from the cytosolic fraction by a three-step procedure employing successive DEAE-cellulose, phosphocellulose, and gel permeation chromatography steps. The seven activities accounted for 70% of the Tyr-protein phosphatase activity in bovine brain extracts and were distinct from type 1 and type 2 Ser/Thr-protein phosphatases and from the major alkaline phosphatase activities. Apparent molecular weights of the activities by gel permeation chromatography were: phosphotyrosyl-protein phosphatase (PTP)-1A (Mr 86,000), PTP-1B (Mr 24,000), PTP-2 (Mr 88,000), PTP-3 (Mr 90,000), PTP-4 (Mr 80,000), PTP-5 (Mr 48,000), and PTP-6 (Mr 104,000). PTP-5 was the major activity accounting for 26% of total while the remaining activity was divided rather evenly among the other six activities. PTP-5 was further purified to near homogeneity by additional chromatographies on Affi-Gel Blue, heparin-agarose, and Mono S giving an overall purification of 50,000-fold and a yield of 5.8%. One of two major polypeptides (Mr 46,000) in the preparation was identified as PTP-5 since it alone expressed protein phosphatase activity when protein-staining bands were eluted from sodium dodecyl sulfate-polyacrylamide gels and renatured. PTP-5 had a neutral pH optimum, and using phosphotyrosyl-casein as substrate it had a Km of 130 nM and a Vmax of 10 mumol Pi released.min-1.mg protein-1. These kinetic parameters are well within the range of values obtained for other pure protein phosphatases. PTP-5 also dephosphorylated pp60v-src (autophosphorylated at Tyr-416) at 10% of the rate observed with phosphotyrosyl-casein. Additionally the ratio of phosphotyrosyl-casein/pp60v-src phosphatase activity was relatively constant throughout the PTP-5 purification procedure. These results indicate that PTP-5 is able to bind and efficiently dephosphorylate phosphotyrosyl-proteins and suggest that it is a physiologically relevant Tyr-protein phosphatase.
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PMID:Phosphotyrosyl-protein phosphatases. I. Separation of multiple forms from bovine brain and purification of the major form to near homogeneity. 246 73

The end-stage maturation of neutrophilic granulocyte precursor cells isolated from normal human bone marrow by Ficoll density centrifugation was studied in a liquid culture assay system used previously to study the maturation of guinea pig granulocyte precursors. Dialyzed normal human serum induced end-stage morphological maturation of human granulocyte precursors and this induction was proportional to a serum level of up to 5.0% in the assay medium. At serum concentrations greater than 5.0% a pronounced inhibition of maturation was observed. Passage of serum through a DEAE-Fractogel 650S column equilibrated with 0.01 M phosphate buffer (pH 7.0) resulted in the binding of the end-stage granulocyte maturation factor to the column. The activity eluted from the column in a fraction containing 17% of the starting serum protein that was inhibitor-free and was also capable of inducing the appearance of granulocyte alkaline phosphatase, a specific biochemical marker for granulocyte end-stage maturation. GMF is most likely a protein since it was destroyed by protease digestion. The data also indicate that neither purified human transferrin nor human recombinant granulocyte colony-stimulating factor can substitute for human serum GMF as a granulocyte end-stage maturation factor in this assay system. It was observed, however, that purified human transferrin greatly potentiated the effect of GMF suggesting that transferrin plays a supporting role in the end-stage maturation of human granulocytes in vitro. To our knowledge the evidence presented here indicates for the first time the existence of a neutrophilic granulocyte end-stage maturation factor in normal human serum.
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PMID:Evidence for a factor in normal human serum that induces human neutrophilic granulocyte end-stage maturation in vitro. 247 45

An improved procedure for isolating lambda DNA and screening lambda gt10 or lambda gt11 libraries is described. Recombinant lambda gt11 bacteriophage particles (150,000) were amplified on three agarose plates (50,000 per plate) with Escherichia coli Y1090 as plating bacteria. After confluent lysis, recombinant bacteriophage was extracted with SM buffer. Bacterial debris was removed by centrifugation. A small aliquot of amplified lambda gt11 bacteriophage was kept to rescreen the bacteriophage, should a large or full-length clone be found to be present, after analysis of the size of the cDNA inserts. The major portion of the bacteriophage particles was purified by treatment with equilibrated DEAE-cellulose, pH 7.5. Purified phage particles were precipitated with polyethylene glycol from the DEAE supernatant and extracted with phenol, phenol-chloroform, and chloroform. Such lambda gt11 DNA was readily digested with EcoRI. Liberated insert cDNA was separated on 1.2% agarose gels, transferred onto a nylon membrane, and hybridized with an alkaline phosphatase cDNA probe in an iterative procedure that allows isolation of the largest cDNA clones present in the library. We have used this procedure to isolate a full-length alkaline phosphatase cDNA. The method is quick, reliable, and less costly than conventional procedures for the isolation of full-length cDNAs.
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PMID:A simple purification procedure for lambda gt bacteriophage DNA with hybridization size screening for isolation of longest length cDNA clones. 253 67

A method for determination of levels of incorporation of nonradiolabeled 5-fluorouracil (FUra) into RNA (F-RNA) in tissue samples is shown to be applicable to tissues in vivo. BDF1 mice bearing L1210 ascites cells were injected intraperitoneally with [14C]FUra, 100 mg/kg. The time course of F-RNA levels in L1210 cells was determined by following the radiolabeled drug, and by NaB3H4 labeling of isolated and derivatized nucleoside. RNA ribonucleotides were obtained by KOH hydrolysis of perchloric acid precipitates of cell sonicates. FUMP nucleotides were separated from remaining nucleotides by DEAE-cellulose chromatography. FUMP fractions were treated with alkaline phosphatase, and FUrd was separated from non-FUrd nucleoside contaminants by additional DEAE-cellulose chromatography. FUrd was quantitated by periodate oxidation of ribose and NaB3H4 reduction of the resulting nucleoside dialdehydes. Isolation of tritiated FUrd-trialcohol from remaining tissue contaminants and background radioactivity was done by silica gel thin layer chromatography. Comparison of results obtained by isolation of [14C]FUrd with results of NaB3H4 labeling of the same samples showed parallel results with comparable biological standard deviations, although the tritium method consistently gave slightly lower values. The peak level of F-RNA at 3 hr was 1 base substitution per 174 normal nucleotides. The level of F-RNA after 3 hr declined slowly, so that at 96 hr there still remained 1 FUra base per 597 normal nucleotides. Serial determinations of RNA content showed marked decreases, on the basis of either DNA or protein level, that continued up to 96 hr after FUra administration. These biochemical effects are among the most prolonged reported for FUra, suggesting the possibility that F-RNA represents a storage compartment for release of toxic metabolites and emphasize the need for additional study of RNA effects at long time points. Our method for assay of F-RNA appears to be suitable for study of biopsy specimens of tumors and normal tissues following nonradiolabeled-FUra administration.
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PMID:Assay and time course of 5-fluorouracil incorporation into RNA of L1210/0 ascites cells in vivo. 257 5

Alkaline phosphatase from calvaria of 8 to 12-day-old Wistar rats was purified to electrophoretic homogeneity by a simple procedure (homogenisation, solubilisation by Triton X-100, DEAE-Sephacel ion exchange chromatography). For the holoenzyme, a Mr of about 160 kDa was determined, and it seems to consist of two identical subunits. The pH optima for the hydrolysis of phosphoethanolamine and p-nitrophenylphosphate are 10.0 and pH 9.0-10.5, respectively. The rate constants for the hydrolysis of phosphoethanolamine, p-nitrophenylphosphate and other phosphomonoesters at pH 10.0 are comparable, but the Km values differ by one to two orders of magnitude. At physiological pH (7.5) the maximum hydrolysis rate of the substrates phosphoethanolamine and p-nitrophenylphosphate was only 8% and 5%, respectively, of that determined at the pH optimum. On the basis of the kinetic data an in vivo function of alkaline phosphatase in bones as a monophosphate ester hydrolyzing enzyme seems unlikely.
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PMID:[Phosphoethanolamine--a substrate of alkaline phosphatase isolated from rat calvaria]. 261 22

Some enzymatic activities and toxic properties of four samples of Ophiophagus hannah (king cobra) venom were investigated. There is little intraspecific variation in enzyme contents, protein composition and toxic properties of the venom. The venom does not exhibit hemolytic or edema-inducing activity but is characterized by an exceptionally high alkaline phosphomonoesterase activity. DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography of the venom indicate that the major lethal toxins are the low mol.wt, non-enzymatic basic proteins. Venom fractions exhibiting high enzymatic activities apparently do not play an important role in the lethality in mice of Ophiophagus hannah venom.
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PMID:Enzymatic and toxic properties of Ophiophagus hannah (king cobra) venom and venom fractions. 274 65

Four forms of nephrocalcin have been routinely isolated from mammalian kidney tissues and urine using DEAE-cellulose column chromatography with a linear NaCl gradient. We have demonstrated that these four forms of nephrocalcin, isolated from bovine kidneys, contain different amounts of phosphate residues, and that alkaline phosphatase digestion converts these to only one form of nephrocalcin. The changes in the nephrocalcin before and after removal of phosphate residues were measured by 31P-NMR spectrometer. Loss of phosphate residues decreased the dissociation constant of nephrocalcin 10-fold toward calcium oxalate monohydrate crystals, suggesting the phosphate residues appear to be important in the inhibitory effects of calcium oxalate monohydrate crystal growth.
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PMID:Elucidation of multiple forms of nephrocalcin by 31P-NMR spectrometer. 275 26


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