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 ATPase activity of rabbit-kidney brush border can be activated almost equally well by Ca2+ and Mg2+ and, therefore, should be called (Ca2+ or Mg2+)-ATPase. This enzyme was solubilized and enriched 14-fold by the following steps: pretreatment with papain removed 69% of alkaline phosphatase without attacking a significant portion of the ATPase activity. Addition of 1% cholate removed 65% of the protein but no ATPase activity. The combination of cholate (0.5%) and deoxycholate (0.4%) solubilized most of the ATPase activity and most of the remaining protein. A column chromatography of the extract on Sepharose CL-2B resulted in an 6.5-fold increase of specific ATPase activity. A precipitation by ammonium sulfate (40% saturation) produced an additional 1.9-fold increase. The yield of this partial purification was 16%. Towards the nucleotides UTP and GTP the enzyme showed an activity slightly higher, and towards ITP and CTP an activity slightly lower than that with ATP. ADP was split about half as fast as ATP. AMP was not accepted by the enzyme. Replacing MgCl2 by CaCl2 resulted in an ATPase activity of 92% of that with MgCl2. Using calcium- and magnesium-ATP as substrates, apparent Km values of 0.22 and 0.33 mM, respectively, were obtained. The gel electrophoresis revealed the enrichment of a protein with an apparent Mr of 95000 and also that of microvillus actin.
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PMID:Partial purification and characterization of rabbit-kidney brush-border (Ca2+ or Mg2+)-dependent adenosine triphosphatase. 614 3

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.
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PMID:Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C. 618 49

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.
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PMID:Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. 633 62

Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the alkaline phosphatase (ALKP) of the mosquito, Culex tarsalis, are presented. With p-nitrophenylphosphate (Pnp) as substrate, ALKP was optimally active at 37 degrees C, pH 8.0, 30 mM MgCl2, Vmax was 35.8 mumoles/10 min and the Km was 5.7 mM, with no demonstrable requirement for Zn2+. The spectrophotometric enzyme(s) was stimulated by dithiothreitol, 2-mercaptoethanol, and poly-vinylpyrollidone (PVP); inhibited by NaF, several alternative cations (Ca2+, Ba2+, Fe2+, Cu2+), and EDTA. ALKP activity was cyclic during the 15 day post-adult emergence period of the study. No significant differences were noted between the specific activities of males and females. IEF electrophoresis revealed 6 ALKP isozymes detected with alpha-naphthylphosphate within the pH range 4.0-5.5, with a second group of 3 rather indistinct species in the pH 6.0-7.0 range. IEF ALKP isozymes were stimulated by Mg2+ and PVP and inhibited by EDTA (except ALKP5.0) and cysteine; partial inhibition with phenylalanine. IEF detection of ALKP activity with Pnp indicated that the majority of the activity was localized in the pH 4.0-5.5 range, in close agreement with the alpha-naphthylphosphate results.
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PMID:Alkaline phosphatases of the mosquito, Culex tarsalis Coquillett. 646 96

Boar sperm plasma membranes were purified by differential and sucrose density equilibrium centrifugation and were found to yield a single band at a density of 1.14 g/cm3. Both alkaline and acid phosphatase activities were enriched in this fraction. The alkaline phosphatase activity was optimal in 100 mM tris (hydroxymethyl) methylamine (Tris)-NaHCO3 at pH 9.9 with 0.05% Triton X-100 and 1 mM MgCl2. This activity was inhibited by ethylenediaminetetraacetic acid (EDTA), cadmium, zinc or heating at 60 degrees C for 30 min. Also, L-homoarginine caused approximately 70% inhibition and L-phenylalanine or L-leucine caused about 10 to 20% inhibition. Acid phosphatase activity was optimal in 100 mM sodium acetate at pH 5.1 with 0.05% Triton. Sodium dodecyl sulfate, potassium fluoride (KF) or sulfhydryl reagents inhibited the activity, while EDTA or heating at 60 degrees C had no effect. These data for enzymes from boar sperm plasma membranes can be used for future work on the quantitation of the enzymes, distinguishing these two phosphatases from other phosphohydrolases, purification of the enzymes and for comparison to phosphatases in other tissues.
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PMID:Some properties of acid and alkaline phosphates from boar sperm plasma membranes. 650 37

The objective of this investigation was to examine the in vivo characteristics of binding sites for alkaline phosphatase in Bacillus licheniformis cell surface. An attempt was made to correlate the results from several experimental approaches, namely (i) cell fractionation; (ii) ultrastructural cytochemistry; (iii) MgCl2 extraction and sodium dodecyl sulphate--polyacrylamide electrophoresis of the extracted material; (iv) labelling with 125I-labelled diazonium salt to determine the subcellular origin of MgCl2-extracted material. Results show that 40% of the alkaline phosphatase was bound to the plasma membrane, 35% to the cell wall, and 15% was free in the cytosol. The enzyme was present as aggregates in a few discrete sites in the membrane, wall, and cytoplasm. The membrane enzyme was associated with the inside surface. A few aggregates were enclosed in single-layered vesicles which appeared to protrude through the cell wall. The material extracted with magnesium salt consisted of 8-10 proteins including alkaline phosphatase. The majority of the proteins extracted by MgCl2 originated from the outside half of the plasma membrane, whereas, only a few, including alkaline phosphatase, came from the inside half of the plasma membrane. All of these proteins may have formed a complex which was removed by MgCl2 extraction. Patch formation in the membrane indicated specific aggregation of intramembrane proteins after MgCl2 treatment.
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PMID:Specificity of subcellular distribution of alkaline phosphatase in Bacillus licheniformis 749/C. 671 99

Xanthomonas oryzae was shown to contain a constitutive alkaline phosphatase (EC 3.1.3.1.). The enzyme was detectable in intact cells and releasable by osmotic shock or spheroplast formation indicating its periplasmic location in the cell. Sonication released about 85% of the total enzyme, and the releasable amount was increased to 97% when lysozyme was added to the sonicated cells prior to centrifugation. These changes suggest an association of the enzyme with peptidoglycan; the enzyme is not released unless the polymer is digested to small units. Adapted usual method of spheroplast formation released 85%, while modified osmotic shock procedure released about 75% of the enzyme. These procedures released decreased amounts of the enzyme following cell growth reflecting that some changes were taking place toward a tighter association between the enzyme and the cell envelope during aging of the culture. During osmotic shock, the major portion of the released enzyme distributed in supernatant of the first stage (S1) hypertonic solution. The enzyme was inhibited by EDTA, whereas the inhibition was partially removed by dialysis and completely reversed by addition of MgCl2. Data obtained also indicated that X. oryzae seems to have relatively high content of periplasmic protein.
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PMID:Release of alkaline phosphatase from cells of Xanthomonas oryzae by manipulation of surface permeability. 679 31

A simple method is described for the enzymatic determination of sphingomyelin in the apolipoprotein B-free supernatants prepared by precipitation of blood sera with phosphotungstate/MgCl2. The analysis is based on the enzymatic hydrolysis of sphingomyelin, by sphingomyelinase from B. cereus, into phosphorylcholine and N-acylsphingosine, and subsequent hydrolysis of phosphorylcholine by alkaline phosphatase. The choline formed is determined by choline kinase in an optical test. The results from this method were in good agreement with those obtained by the conventional chemical sphingomyelin determination. Furthermore, there was a good correlation between the sphingomyelin concentrations obtained from the HDL fractions isolated by ultracentrifugation (1.063-1.21 kg/l) and those obtained from the apolipoprotein B free supernatants after phosphotungstate/mgCl2 precipitation of sera.
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PMID:[The enzymatic analysis of sphingomyelin in HDL (author's transl)]. 680 83

Acid phosphatase activity was studied biochemically in homogenates of secretory enamel organs from the rat. Incubations with crude homogenate failed to show distinct pH optima or kinetics characteristic for single enzymes. Crude homogenate activity was strongly inhibited by concentrations higher than 1 mM of NaF and Na-tartrate, and higher than 10 mM of ZnSO4 and para-bromotetramisole oxalate. 10 mM MgCl2 gave a slight stimulation. CaCl2, KCl and EDTA were uneffective. Electrophoretic separation of the crude homogenate acid phosphatase on Triton X-100 containing polyacrylamide gel demonstrated the presence of at least three multiple forms of the enzyme. Two of them showed distinct pH optima at pH 4.4. The third one showed a broad pH plateau in the acid pH range. Kinetic studies of the three forms indicated single enzyme reactions. Two forms had electrophoretic mobilities similar to alkaline phosphatase. One form could be solubilized only after Triton X-100 treatment. All forms were strongly sensitive to 10 mM NaF when added to the reaction mixture. The sensibility to 10 mM ZnSO4, CuSO4, Na-tartrate and para-bromotetramisole oxalate differed between the different forms.
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PMID:Multiple forms of acid phosphatase in rat secretory enamel organ. 695 66

Many researches have shown the role of some bivalent ions on the structure and function of alkaline phosphatase. For this reason we considered interesting to assay the effect of Zn++ and Mg++, at various concentrations, on the activity of alkaline phosphatase (APase) from different sources. The isoenzymes of alkaline phosphatase used for the experiments were from rat kidney and bone, from calf intestinal mucosa and from Escherichia coli. In order to investigate the effect of Zn++ and Mg++ on the enzyme activity, the two ions were removed using EDTA as chelating agent. The residual enzymatic activity was measured after having preincubated for 15 min the enzyme with EDTA at a final concentration of 0.05 mM, 0.1 mM, 0.5 mM, 5 mM, 25 mM. The reactivation of the enzyme was studied using as reference a sample, in which the final concentration of EDTA was 5 mM. In these series of experiments the enzymatic activity was assayed after a preincubation of the reaction mixture with ZnCl2 10 mM, MgCl2 10 mM and ZnCl2 +MgCl2 10 mM. The inactivation in the time of the enzyme by 5 mM EDTA was also studied. The results obtained show that APase from intestinal mucosa maintained, at the lower concentrations of EDTA (0.05, 0.1 and 0.5 mM), a residual activity higher than that of the enzymes of other source. Moreover, whilst the activity of the mucosal enzyme was completely restored by the addition of Zn++, the complete reactivation of the other enzyme activities was obtained only by the addition of Zn++ and Mg++ together. Concerning the inactivation by EDTA during the time, it was shown that APase from calf intestinal mucosa was inactivated after 60 min of incubation, while the enzymes from other sources lost completely their activity after 10 min.
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PMID:[Influence of Zn++ and of Mg++ on alkaline phosphatase activity of different origins]. 700 69


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