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)

Previous studies suggested that insulin receptor tyrosine kinase (IRTK) is the sole tyrosine kinase in rat adipocytes. We now report that this cell type also contains a cytosolic soluble protein tyrosine kinase (CytPTK) which is not related to IRTK. The enzyme phosphorylated PolyGlu4Tyr with high efficiency at a rate of 20 +/- 2 pmol PTyr/20 micrograms PolyGlu4Tyr/20 min/micrograms cytosolic protein. Upon gel filtration chromatography the enzyme activity was eluted as a single peak corresponding to a molecular mass of 53 +/- 3 kDa. Unlike IRTK, CytPTK activity was supported by Co2+ rather than by Mn2+, and it was not inactivated by N-ethylmaleimide. The enzyme was extremely sensitive to inhibition by staurosporine (ID50 = 3 nM) as opposed to IRTK (ID50 = 8 microM). In addition, CytPTK (but not IRTK) was largely activated by vanadate ions. Agents which affect the serine/threonine phosphorylation state of cell proteins did not alter CytPTK activity when subjected to intact adepocytes. In a cell-free system CytPTK activity was largely reduced by pretreatment with immobilized alkaline phosphatase at physiological pH. The possibility that CytPTK participates in insulin-independent regulation of glucose metabolism is suggested.
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PMID:A cytosolic protein tyrosine kinase in rat adipocytes. 154 96

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.
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PMID:The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases. 157 91

In this report we describe the purification of the murine interleukin 3 receptor (mIL-3R) to apparent homogeneity using a two-step procedure involving biotinylated mIL-3 (B-mIL-3) and affinity binding to immobilized antiphosphotyrosine and streptavidin agarose (SA). Purification was monitored using an assay for detergent solubilized-mIL-3Rs that utilized unglycosylated 125I-mIL-3 and concanavalin A (ConA)-Sepharose beads. The final material consisted of a 140-kDa tyrosine and serine phosphorylated protein that was greater than 98% pure as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of either [35S]methionine-labeled, silver-stained, or radioiodinated preparations. Characterization of the purified receptor revealed that it migrated identically under reducing and nonreducing conditions in SDS gels, possessed 10 kDa of N-linked carbohydrate, and was cleaved upon storage at 4 degrees C to a 70-kDa form. These properties suggested that the purified mIL-3R was identical to that identified by cross-linking studies. The KD of the purified receptor was 1-5 nM, similar to estimates obtained using intact normal mouse bone marrow cells and mIL-3-dependent cell lines. The two-step purification procedure also isolated a 120-kDa serine phosphorylated but nontyrosine phosphorylated mIL-3R species. Apart from phosphorylation differences, the 140- and 120-kDa species were apparently identical, yielding, after alkaline phosphatase treatment, the same molecular mass on SDS gels and similar chymotryptic peptide maps. Amino acid sequences and composition data obtained from the more abundant and more stable serine phosphorylated 120-kDa mIL-3R, further purified by SDS-polyacrylamide gel electrophoresis, suggested that the purified mIL-3R may be identical to the predicted sequence of the recently isolated cDNA clone AIC2A. This was further suggested by comparing chymotryptic maps of the 120-kDa mIL-3R with the Aic2A protein and using antibodies corresponding to the amino and carboxyl termini of the AIC2A cDNA product. However, the Aic2A protein, when expressed on the surface of COS or 3T3 cells or following detergent solubilization and partial purification with biotinylated mIL-3 and SA, displayed a substantially lower affinity for mIL-3.
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PMID:Purification of the murine interleukin 3 receptor. 164 33

The insulin-like growth factor-binding protein IGF-BP1 is a major secretory protein of human endometrial stromal cells decidualized in culture. Anion exchange chromatography and nondenaturing gel electrophoresis showed IGF-BP1 to exist in five electrophoretically and chromatographically distinct isoforms. IGF-BP1 variants migrated as a quintet on nondenaturing polyacrylamide gels and as a single band (28 kDa) on sodium dodecyl sulfate-polyacrylamide gels. Alkaline phosphatase treatment reduced the IGF-BP1 variants to a single band. Cells incubated with [32P]orthophosphate for 12 h secreted four 32P-labeled IGF-BP1 phosphovariants, and their migration coincided with those bands that were eliminated by alkaline phosphatase treatment. In cells treated with medroxyprogesterone acetate and relaxin, the concentration of phosphorylated IGF-BP1 was increased dramatically as compared with controls. All the phosphovariants were confirmed to be IGF-BP1 by their ability to be supershifted on nondenaturing polyacrylamide gels after binding a monoclonal antibody to IGF-BP1. Thin layer electrophoresis of IGF-BP1 acid hydrolysates showed IGF-BP1 to be phosphorylated exclusively on serine. Non-phosphorylated IGF-BP1 was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and casein kinase II in vitro. This suggests that IGF-BP1 may be a substrate of multiple protein kinases in vivo.
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PMID:Insulin-like growth factor-binding protein-1 is phosphorylated by cultured human endometrial stromal cells and multiple protein kinases in vitro. 165 36

We investigated whether phosphorylation of the essential components involved in the 3' end processing of mRNAs was required for mRNA polyadenylation. The proteins in HeLa nuclear extract were dephosphorylated with alkaline phosphatase, which is known to remove the phosphate moieties from serine and tyrosine. The dephosphorylated extract was used for analyzing cleavage-dependent polyadenylation of SV40 late pre-mRNA. The phosphatase treatment of the extract completely blocked the polyadenylation reaction, whereas dephosphorylation of the extract did not inhibit the cleavage reaction. Since the cleavage depends upon functional integrity of the specificity factor, it is unlikely that the phosphorylated state of the latter factor is required for the 3' end processing. Sodium vanadate, a potent inhibitor of alkaline phosphatase, markedly reduced the inhibitory effect of the phosphatase on the polyadenylation reaction. Dephosphorylation of the extract also prevented formation of the polyadenylation-specific complex with pre-mRNA, whereas the cleavage-specific complexes were formed under this condition. The Mn-dependent polyadenylation, which is largely poly(A) extension reaction, was relatively resistant to the phosphatase treatment. These data indicate that phosphorylation of a key factor is essential for the 3' end processing of pre-mRNA, and suggest that the factor may be poly(A) polymerase.
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PMID:Polyadenylation of SV40 late pre-mRNA is dependent on phosphorylation of an essential component associated with the 3' end processing machinery. 166 46

Alz-50 is a monoclonal antibody that detects antigens enriched in the brain tissue of Alzheimer's disease (AD) patients. Although Alz-50 recognizes tau, an identified integral constituent of the AD paired helical filament (PHF), the exact nature of the antigenic site is unknown. An immunoblot analysis demonstrated that the antigenic sites to Alz-50 are diminished by acid phosphatase treatment. Consistent with this finding, Alz-50 antigens were more concentrated in brain homogenates prepared with phosphatase inhibitors. The epitope in tau with which Alz-50 reacts is located in the carboxy terminus within a 14-amino acid region from just beyond the microtubule-binding repeats to the carboxy terminus. An isolated carboxy-terminal chymotryptic peptide from bovine brain tau reactive with Alz-50 was analyzed by fast-atom-bombardment mass spectroscopy (FAB-MS) and was found to be present as both a monophosphopeptide and a nonphosphorylated peptide. The immunohistological analysis has demonstrated that Alz-50 staining of neurofibrillary tangles (NFTs) is sensitive to acid phosphatase but not to alkaline phosphatase. Furthermore, Alz-50 staining of NFTs was effectively adsorbed by a high concentration of phosphoserine but not by serine or phosphothreonine. These results strongly suggest that Alz-50 recognizes a phosphorylated epitope in the carboxy terminus of tau which has not been previously detected by using alkaline phosphatase. The strong Alz-50 staining in AD samples may represent another association between a phosphorylation state and neurofibrillary lesions. As a marker of the inchoate tangle-bearing neuron, the characterization of the Alz-50 epitope in tau offers a partial molecular basis for the modifications that contribute to the assembly of PHFs.
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PMID:Alz-50 recognizes a phosphorylated epitope of tau protein. 169 46

Nascent precursors of phosphatidylinositol-glycan (PI-G)-linked membrane proteins contain a hydrophobic COOH-terminal sequence of 15-30 residues that is eliminated during processing to yield a newly exposed COOH terminus to which the PI-G moiety is added. There is no consensus as to the primary structure of the terminal peptide but there is a specific requirement for the amino acid destined to become the COOH terminus. In nascent human placental alkaline phosphatase (PLAP), the PI-G tail is attached to Asp-484. Site-directed mutants with glycine, alanine, cysteine, serine, or asparagine (category I) at residue 484 become PI-G tailed, appear in the plasma membrane, and are enzymatically active when expressed in COS cells. Although mutants with glutamic acid, glutamine, proline, tryptophan, leucine, valine, phenylalanine, threonine, methionine, and tyrosine (category II) are expressed equally well, only small amounts appear on the plasma membrane. Furthermore, they are not PI-G tailed and have little alkaline phosphatase activity. Studies with truncated PLAP-489 rule out nonspecific conformational changes in category II mutant proteins as a reason for their failure to be processed in COS cells and point to a specific COOH-terminal processing enzyme. Direct evidence that the selectivity for category I amino acids is enzymatically determined was obtained in a cell-free translation/processing system by using rabbit reticulocyte lysate and CHO cell rough microsomal membranes. In this in vitro system, both category I and category II mutants of PLAP-513 were translated, glycosylated, and cleaved by NH2-terminal signal peptidase. However, an additional and selective cleavage at residue 484 was observed only with category I mutants.
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PMID:Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined. 170 Apr 20

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

The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.
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PMID:Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat. 179 2

The insulin resistance seen in diabetes mellitus has been attributed partly to impaired autophosphorylation of the insulin receptor. It has been suggested that the phosphorylation of serine and/or threonine residues of the insulin receptor may reduce tyrosine autophosphorylation in streptozotocin-induced diabetic rats (STZ-D rats). To elucidate the mechanisms of decreased autophosphorylation of the insulin receptor in diabetic rats, we have investigated the effect of dephosphorylation of the insulin receptor by alkaline phosphatase on the insulin- and protein kinase-stimulating incorporation of 32P into the receptor of the liver from STZ-D rats. Both basal and insulin-stimulated autophosphorylations of the insulin receptor from STZ-D rats were significantly impaired to those from normal rats. Dephosphorylation of the insulin receptor by alkaline phosphatase resulted in an increase in insulin-stimulated autophosphorylation of the insulin receptor from STZ-D rats (43 +/- 13% to 66 +/- 14%, P less than 0.05), but not from normal rats (100% to 109 +/- 12%, NS). Although maximal autophosphorylation of the dephosphorylated insulin receptor was still lower in STZ-D rats than in normal rats, the increase in insulin-stimulated autophosphorylation of the insulin receptor from STZ-D rats by dephosphorylation was higher than that from normal (159.2 +/- 27.2% vs 108.0 +/- 12.4%, p less than 0.01), supporting the idea that the residues of the insulin receptor of STZ-D rats was highly phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dephosphorylation of the insulin receptor partially restores the decreased autophosphorylation in streptozotocin induced diabetic rats. 181 77


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