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)

Treatment of the U-2 OS human osteosarcoma cell line with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) dramatically decreased the rate of DNA synthesis. This decrease in proliferation as well as the change in morphology of the TPA-treated cells can be blocked by the protein kinase C inhibitor GF 109203X. The U-2 OS cells are known to express the c-sis oncogene [platelet-derived growth factor (PDGF) B-chain], PDGF-A, and receptors for PDGF, thus providing a potential autocrine loop of growth stimulation. TPA was found to induce the expression of both the PDGF-A and the PDGF-B chains. However, the levels of the PDGF receptor beta subunits and of the PDGF-BB inducable tyrosine phosphorylation of the PDGF receptor were markedly reduced. The TPA treatment of the U-2 OS cells also induced changes typical for maturing bone cells, such as increased expression levels of alkaline phosphatase and osteopontin. The expression levels of type I collagen and bone sialoprotein were reduced. The results show a TPA-dependent down-regulation of the PDGF receptor beta subunits that correlates with an increased expression of osteoblast phenotypic markers.
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PMID:Phenotypic modification of human osteosarcoma cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. 779 13

The transforming gene v-crk found in CT10 and ASV-1 avian sarcoma viruses induces marked phosphorylation of several proteins in cells expressing p47v-crk (v-Crk). In this work, the main tyrosine-phosphorylated proteins in ASV-1-infected chicken cells and v-crk-transfected rat cells were characterized biochemically. Both these proteins have a molecular mass of about 130 kDa and are tightly associated with v-Crk in vivo. Two-dimensional gel electrophoresis revealed that they are both essentially single proteins (p130) with modifications that result in a broad spot in an acidic region. The broad band of semi-purified p130 became sharp at an elevated position in the gel upon treatment with orthovanadate in vivo or with c-Src kinase produced using a baculovirus vector in vitro, whereas it shifted at a lower position upon treatment with alkaline phosphatase in vitro. These results suggest multiple phosphorylation states of p130, which result in a broad band of p130. Two procedures of immunoaffinity purification were used to purify p130 from 3Y1 cells transfected with v-crk. Approximately 30 pmol of purified p130 was obtained in an immobilized form on a filter starting from 3 x 10(10) cells. Peptide mapping of p130 digested in situ by peptidase revealed that the purity and quantity of the final material were enough for peptide sequencing. Several stretches of partial amino acid sequences were determined, and they indicated that p130 is a novel protein.
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PMID:Characterization, partial purification, and peptide sequencing of p130,the main phosphoprotein associated with v-Crk oncoprotein. 780 94

Neu differentiation factor (NDF or heregulin) elevates tyrosine phosphorylation of the ErbB-2 receptor tyrosine kinase, and it was, therefore, thought to function as a ligand of this receptor. However, several lines of evidence raised the possibility that the interaction between NDF and ErbB-2 involves another molecule, which belongs to the family of epidermal growth factor receptors. To address this question we constructed soluble chimeric proteins between alkaline phosphatase and the extracellular domains of ErbB-2 and either ErbB-3 or ErbB-4, two newly recognized members of the epidermal growth factor receptor family. Using the soluble proteins we found that beta isoforms of NDF specifically bind to the ErbB-3 and ErbB-4 receptors but not to the soluble ErbB-2 protein. When ectopically expressed in monkey fibroblasts, the full-length ErbB-3 and ErbB-4 receptors conferred specific binding to NDF. In these cells ErbB-3 displayed lower ligand binding affinity than ErbB-4, but like the latter receptor it preferred to bind the beta isoform over the alpha class of NDFs. These results indicate that both ErbB-3 and ErbB-4 function as physiological receptors of all NDF isoforms and suggest that a still unknown ligand of ErbB-2 exists.
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PMID:ErbB-3 and ErbB-4 function as the respective low and high affinity receptors of all Neu differentiation factor/heregulin isoforms. 792 12

The direct effect of different vanadium compounds upon alkaline phosphatase (ALP) activity was investigated. Vanadate and vanadyl inhibited both the soluble and particulate ALP activity from UMR.106 cells and from bovine intestinal ALP. We have also shown the inhibition of ALP activity in the soluble fraction of osteoblasts by peroxo and hydroperoxo vanadium compounds. ALP activity in the particulate fraction was not inhibited by these species; nor was the bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), the soluble ALP was partially characterized as a PTPase. The major activity in the particulate fraction represents the bone-specific ALP-activity. This study demonstrates that different forms of vanadium are direct inhibitors of ALP activity. This effect is dependent on the enzymatic activity investigated and on the origin of the ALP.
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PMID:Vanadium compounds. Their action on alkaline phosphatase activity. 794 23

Various sensor technologies require a monolayer of tightly bound proteins. Five methods for protein immobilization were evaluated, comprising two methods of linker attachment to a silicon nitride surface (CNBr, silanization) and three methods of linker attachment to the protein (via primary amines, carboxyl groups, and the aromatic rings of tyrosine and histidine). These covalent binding methods were evaluated by immobilization of the enzyme alkaline phosphatase and with monoclonal antibodies tagged with 125I. It was determined that approximately 75% of the protein on the "covalently" immobilized samples was simply adsorbed. Adsorption was reduced by varying the pH and ionic strength of the wash buffers as well as by adding chaotropes and competitors to the wash buffer. A more efficient method of reducing adsorption was to perform the immobilization in the presence of a detergent; 0.5% Tween 60 was the most effective detergent studied. The optimized procedure gives a surface loading of 1.20 x 10(12) MAb/cm2. of which approximately 10% was adsorbed protein. This surface density is consistent with a monolayer of immobilized protein. This protein was shown to be active by challenging immobilized antibodies with 35S-labelled antigen; only the antibody/antigen pair released radioactive material upon elution.
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PMID:Covalent immobilization of protein monolayers for biosensor applications. 801 17

A synthetic tris-sulfotyrosyl dodecapeptide (TRDIY(S)ETDY(S)Y(S)RK-amide), whose primary sequence is identical to the 1142-1153 sequence of the insulin proreceptor, inhibited insulin receptor dephosphorylation in solubilized membranes, and digitonin-permeabilized cells derived from Chinese hamster ovary (CHO) cells expressing high levels of human insulin receptors (CHO/HIRc). It also inhibited the dephosphorylation of a synthetic tyrosine phosphorylated substrate by recombinant PTP-1B, a protein tyrosine phosphatase (PTPase), indicating that it acted via interaction with PTPase(s). A N-stearyl derivative of the peptide caused an approximately 4.5-fold increase in insulin-stimulated receptor autophosphorylaction in intact CHO/HIRc cells. The peptide displayed specificity toward tyrosine-class phosphatases only, as it had no effect on the activities of the serine/threonine phosphatases PP-1 and PP-2A, or alkaline phosphatase. The tyrosine sulfate ester bonds of the peptide were stable when incubated with PTP-1B (1 h, 30 degrees C). These data suggest that the sulfotyrosyl peptide functions as a nonhydrolyzable phosphotyrosyl peptide analogue capable of direct interaction with PTPase catalytic domain.
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PMID:A synthetic tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain inhibits tyrosine dephosphorylation of the insulin receptor in situ. 808 98

Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.
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PMID:[Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of directed mutations into the alkaline phosphatase gene]. 814 43

The ligand-stimulated tyrosine kinase activity of the normal human epidermal growth factor (EGF) receptor and a truncated EGF receptor lacking 164 carboxy-terminal (C-terminal) amino acids was examined in intact cells and after Triton X-100 extraction into Triton-soluble and -insoluble (cytoskeletal) preparations. Detergent extraction of the intact and truncated receptors appeared complete using 0.3% Triton as demonstrated by anti-EGF receptor immunoblots, tyrosine kinase assays, and marker enzyme (alkaline phosphatase) solubilization. Higher Triton concentrations yielded no additional EGF receptor extraction and began to inhibit EGF-stimulated kinase activity toward angiotensin II (AII). Furthermore, the tyrosine kinase activity of the truncated EGF receptor exhibited increased sensitivity to Triton extraction, suggesting a lower affinity or a more labile association of this receptor with the cytoskeleton. However, both EGF receptor forms had altered catalytic activity when associated with the cytoskeletal fraction, as evidenced by the increased phosphorylation of the exogenous substrates: AII, src-peptide, and [Val5]AII. Kinetic analyses of both receptor types revealed that the cytoskeletal fractions obtained using 0.3% Triton contain EGF receptor activity that exhibits a Michaelis-Menten constant (Km) for AII that is 2- to 3-fold more favorable than that calculated for the soluble receptor forms. EGF treatment of intact cells containing either the intact or truncated receptor revealed similar phosphorylated proteins in the soluble fraction of both cell types, although there was evidence for the enhanced phosphorylation of certain proteins (e.g. 115 and 50 kilodalton proteins) in cells containing the truncated receptor. There was also a greater number of tyrosine-phosphorylated proteins in the Triton-insoluble fraction of cells containing the truncated receptor, suggesting an altered specificity of this receptor toward selected cytoskeletal proteins. This work indicates that EGF receptor-cytoskeletal interaction may be an important consideration in the control of receptor-kinase activity and has examined the detergent sensitivity of this association. These studies also suggest that the C-terminal domain of the EGF receptor may affect cytoskeletal interaction in addition to influencing the receptor's catalytic capacity.
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PMID:Evidence for the potentiation of epidermal growth factor receptor tyrosine kinase activity by association with the detergent-insoluble cellular cytoskeleton: analysis of intact and carboxy-terminally truncated receptors. 824 11

Nerve growth factor (NGF) treatment of PC12 cells led to the rapid phosphorylation of a calmodulin-binding protein of 100 kDa (CaM-BP100) identified on blot overlays with 125I-labeled CaM. The effect was detected as a retardation in the mobility of the protein by an apparent 10 kDa on SDS gels. The mobility shift was complete within 5 min and was maintained for 24 h in the continued presence of NGF. The protein was present in both the soluble and crude particulate fractions, and the gel mobility shift occurred in both fractions. Epidermal growth factor elicited a similar response, but the mobility shift was reversed within 12 h. The gel retardation was due to phosphorylation of CaM-BP100, as it could be reversed if cytoplasmic extracts were held under dephosphorylating conditions at 37 degrees C for 10 min prior to electrophoresis; dephosphorylation was inhibited by okadaic acid but not vanadate, suggesting the participation of a Ser/Thr phosphatase. Treatment with either acid or alkaline phosphatase also reversed the mobility shift. CaM-BP100 phosphorylation was stimulated by 12-O-tetradecanoylphorbol-13-acetate in intact cells, but the effect of NGF did not involve a protein kinase C-dependent process, because it occurred in PC12 cells depleted of protein kinase C. The phosphorylation event appeared to be due to an NGF-stimulated protein kinase, as mixing extracts from NGF-treated cells with extracts from control cells in the presence of ATP and Mg2+ reconstituted the mobility shift in vitro. CaM-BP100 appears to be a minor cellular phosphoprotein, as 32P labeling of the protein could not be detected in crude cell extracts. These results suggest that receptor tyrosine kinases communicate with at least one component of the Ca2+/calmodulin-signaling pathway early in signal transduction.
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PMID:Rapid and sustained phosphorylation of a calmodulin-binding protein (CaM-BP100) in NGF-treated PC12 cells. 839 55

Microtubule-associated protein tau is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of tau to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated tau could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of tau might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used 32P-labeled phosphorylase kinase and poly(Glu, Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of tau in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of beta-amyloid by augmenting the amyloidogenic pathway processing of beta-amyloid precursor protein.
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PMID:Phosphoprotein phosphatase activities in Alzheimer disease brain. 839 66


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