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 small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.
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PMID:High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins. 170 75

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.
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PMID:Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C. 171 84

A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye-ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine-labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.
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PMID:Fetuin and alpha-2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes. 172 Oct 70

Monoclonal antibodies were produced against human pituitary gland 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes in the biosynthesis of tetrahydrobiopterin, by in vitro immunization with the antigen directly blotted from SDS-PAGE to polyvinylidene difluoride membranes. The antibodies produced show crossreactivity in the enzyme linked immunosorbent assay, not only with the human 6-pyruvoyl tetrahydropterin synthase but some also with the same enzyme isolated from salmon liver. 6-Pyruvoyl tetrahydropterin synthase was localized immuno-enzymatically in peripheral blood smears and in skin fibroblasts by the use of these monoclonal antibodies and the alkaline phosphatase monoclonal anti-alkaline phosphatase labeling technique.
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PMID:Production of monoclonal antibodies against human 6-pyruvoyl tetrahydropterin synthase and immunocytochemical localization of the enzyme. 173 83

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.
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PMID:Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells. 176 23

1. A monoclonal antibody APP, 1 against harp seal alkaline phosphatase has been prepared. It was found that the antibody was cross-reacted with the intestinal alkaline phosphatase of common seal Phoca vitulina larga. 2. Purified antibody was linked to Sepharose 4B and used for immunoaffinity chromatographic purification of alkaline phosphatase from the intestinal content of common seal. A spec. act. of the purified enzyme was 7300 units per mg of protein. 3. The enzyme in 7.5% polyacrylamide gel in the presence of 2-mercaptoethanol and SDS was migrated as a single band of Mr 67,000. The value of the apparent Km for common seal alkaline phosphatase was equal to 3.7 mM.
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PMID:Purification of alkaline phosphatase from the intestinal content of common seal (Phoca vitulina larga) by immunoaffinity chromatography. 176 1

Surface antigens of adult filarial parasite S. digitata was isolated by employing techniques from manual dissection to treatment with detergents. Among the surface antigen preparations (SAPs), the activities of marker enzymes such as alkaline phosphatase, adenosine triphosphatase and 5' nucleotidase were higher with that isolated by triton X-100 technique (SAP2). On SDS-PAGE, the SAP2 has three major proteins with molecular weights 17, 29 and 36 KD which were consistent with the PBS soluble cuticular proteins (SAP1). Besides these, few other minor protein bands were also observed with the other SAPs. All SAPs were antigenic and showed positive reaction against antiserum to SAP2, and the results confirmed the SAP2 as a better preparation. The release of 29 KD surface protein during in vitro culture of adult parasite and its cross-reactivity with antiserum to surface antigens revealed the possible natural shedding of surface molecules into the host system.
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PMID:Isolation and analysis of surface antigens of filarial nematode Setaria digitata. 176 14

p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos. We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment. In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively. In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.
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PMID:Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. 182 50

Some investigators have described the presence in Alzheimer's disease brain extracts of several abnormal forms of the microtubule-associated protein tau, based on their unusual mobility in SDS/PAGE. It has been proposed that these abnormal forms of tau may be the result of aberrant tau phosphorylation. In this study we show that tau in extracts of Alzheimer's disease brain can be separated into two fractions based upon its solubility (100,000 g x 1 h supernatant) in non-denaturing conditions (100 mM-Mes, pH 6.5, 0.5 mM-MgCl2, 1 mM-EGTA and 1 M-NaCl). The tau isoforms with decreased mobility in SDS/PAGE are predominantly in an insoluble fraction, whereas the soluble tau is indistinguishable by its mobility in SDS/PAGE from tau in soluble extracts of control brain. Insoluble tau displaying abnormal mobility on SDS/PAGE was only found in Alzheimer and adult Down's syndrome brains and was absent from the brains of age-matched controls and from foetal and infant Down's syndrome brains. There was a good correlation between the presence of insoluble tau in brain extracts and the abundance of neurofibrillary tangles and senile neuritic plaques. The monoclonal antibody Tau. 1 stained insoluble tau on Western blots only after treatment of the nitrocellulose transfers with alkaline phosphatase, implying that this insoluble tau is in a particular state of phosphorylation. We conclude that, in Alzheimer's disease, a fraction of tau has a modified phosphorylation state and a decreased solubility; these modifications may precede formation of the neurofibrillary tangles characteristic of Alzheimer's disease and Down's syndrome in adults.
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PMID:Tau in Alzheimer's disease and Down's syndrome is insoluble and abnormally phosphorylated. 182 35

Biotinylated granulocyte/macrophage colony-stimulating factor (GM-CSF) analogues with different linkage chemistries and levels of conjugated biotin were synthesized by reacting recombinant human GM-CSF with sulfosuccinimidyl 6-biotinamidohexanoate or biotin hydrazide/1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide. These chemically reactive forms of biotin produced derivatives biotinylated at amine or carboxyl groups, respectively. Amine-derivatized analogues of 1.2 and 3.8 mol of biotin/mol of protein (N1-bGM-CSF and N4-bGM-CSF) and a carboxyl-modified analogue of 4.6 mol of biotin/mol of protein (C5-bGM-CSF) were synthesized. These analogues were compared to determine the effect of biotinylation on biological activity and GM-CSF receptor binding characteristics. The biotinylated proteins migrated with the same molecular weight as the native, unmodified protein as determined by SDS-PAGE and could be detected by Western blotting with alkaline phosphatase conjugated streptavidin, thus demonstrating the biotin linkage. All three analogues retained full agonist activity relative to the native protein (EC50 = 10-15 pM) when assayed for the stimulation of human bone marrow progenitor cell growth. Cell surface GM-CSF receptor binding was characterized by the binding of the analogues to human neutrophils, with detection by fluorescein-conjugated avidin and fluorescence-activated cell sorting. The N-bGM-CSFs demonstrated GM-CSF receptor specific binding that was displaceable by excess underivatized protein, with the detected fluorescence signal decreasing with increasing biotin to protein molar ratio. In contrast, C5-bGM-CSF binding above background fluorescence could not be detected using this system, suggesting that this derivative could bind to and activate the receptor, but not simultaneously bind fluorescein-conjugated avidin. The amine-derivatized biotinylated GM-CSF analogues retained biological activity, could specifically label cell surface receptors, and may be useful nonradioactive probes with which to study GM-CSF receptor cytochemistry and receptor modulation by flow cytometry.
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PMID:Biotinylated granulocyte/macrophage colony-stimulating factor analogues: effect of linkage chemistry on activity and binding. 183 6


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