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)

We have treated bovine lung heparan sulfate with alkaline [3H]borohydride to end label the chains with [3H]xylitol. After subsequent periodate oxidation-alkaline elimination products were separated by gel permeation and ion exchange chromatography. The linkage region fragment expected to have 2 galactoses and 1 [3H]xylitol residue appeared in the tetra-/trisaccharide region after gel filtration and was bound to the anion exchange resin. A similar negatively charged fragment, expected to have 2 galactoses, 1 xylose and 1 serine, was isolated after periodate oxidation-alkaline elimination of unlabeled heparan sulfate. The negative charge was due to the presence of alkaline phosphatase-labile phosphate ester. The molar ratio of galactose:phosphate:xylose was 2.17:1.19:1.00. The phosphate ester was associated with the xylose/[3H] xylitol moiety as indicated by the formation of phosphoxylose/-xylitol by beta-galactosidase digestion of the phosphorylated trisaccharide. Furthermore, orcinol reactivity disappeared after periodate oxidation of the dephosphorylated trisaccharide. The phosphate ester must be located to C-2 of xylose/xylitol as the 1-3H radioactivity could be released by periodate oxidation when it was preceded by alkaline phosphatase treatment. It is estimated that almost every chain of heparan sulfate carries 2-phosphoxylose. It would be of interest to know if glycosaminoglycan chains that are artificially initiated onto exogeneous beta-D-xylosides also acquire the 2-phosphoxylose moiety.
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PMID:Structure of the heparan sulfate-protein linkage region. Demonstration of the sequence galactosyl-galactosyl-xylose-2-phosphate. 293 48

Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta subunits are derived from a single polypeptide precursor by one or more proteolytic cleavages. The predominant subunit configuration in the native insulin receptor is a disulfide-linked heterotetrameric structure containing two alpha and two beta subunits. The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. Other physiologically relevant substrates of the insulin receptor tyrosine kinase in target cells, if any, have not yet been identified. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. Tyrosine phosphorylation, catalyzed by either autophosphorylation or purified src kinase, of insulin receptor beta subunit in vitro activates the receptor kinase activity, whereas dephosphorylation with alkaline phosphatase deactivates the receptor kinase. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. The insulin receptor kinase is also inhibited in intact cells by phorbol esters that mediate serine/threonine phosphorylation of the insulin receptor, presumably via the Ca++-phospholipid-dependent protein kinase. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner.
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PMID:The nature and regulation of the insulin receptor: structure and function. 298 34

The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vmax values for the phosphorylation reaction were 0.4 microM topoisomerase II and 3.3 mumol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that post-translational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA.
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PMID:Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro. 298 12

Aging is associated with changes in the intestinal uptake of nutrients. This study was undertaken to determine whether the morphology, enzyme markers and the lipid content of the intestinal brush border membrane (BBM) was influenced by aging. There was an increase in the height of the jejunal villi and number of cells/villus, resulting in an age-related increase in the jejunal villus and mucosal surface area in young as compared with weanling rabbits. In mature 1-year-old animals, there was a decline in villus height, number of cells/villus, and mucosal surface area, so that the jejunal characteristics of the mature animals resembled those of the weanling rabbits. In the ileum, aging was associated with an increase (weanling vs. young), then a decrease (young vs. mature) in the height of the villi, and the number of cells/villus. Aging had no effect on the size of the villus cells. At all ages there was a direct positive relationship between the height of the villi and the mucosal surface area, and between villus surface area and sucrase activity. An established technique was used to purify rabbit BBM and to measure the BBM content of enzyme markers and lipids in weanling, young and mature animals. Both the BBM sucrase (S) and alkaline phosphatase (AP) increased in young as compared with weanling rabbits, and the ratio of AP/S remained unchanged. The S remained high in mature rabbits, but AP declined, so that AP/S fell. There was a positive linear correlation between S and villus surface area. In weanling rabbits, the total BBM phospholipid content and the ratio of total phospholipid/total cholesterol were lower in the ileum than in the jejunum. In the jejunal BBM of young animals, there was more total free fatty acids and cholesterol ester than in the weanling jejunum. The jejunal BBM total phospholipids and total cholesterol were higher in the mature than in the weanling animal jejunum when expressed as nmoles/mg protein, but the ratio of total phospholipid/total cholesterol was unaffected by aging. The greatest percentage of jejunal BBM phospholipid was comprised of lecithin and phosphatidyl ethanolamine. The increased BBM total phospholipid content in mature animals was associated with a higher amount and lower proportion of lecithin, but a higher proportion of sphingomyelin and phosphatidyl serine.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Intestinal morphology, marker enzymes and lipid content of brush border membranes from rabbit jejunum and ileum: effect of aging. 299 64

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of insulin receptor kinase by multisite phosphorylation. 300 Apr 58

The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.
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PMID:Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells. 300 31

The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Protein kinase activity of the insulin receptor. 301 97

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.
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PMID:Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor. 304 15

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.
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PMID:Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase. 307 19

A nerve growth factor (NGF)-sensitive S6 kinase was purified by alkaline lysis of PC12 cells. The activity in lysates from NGF-treated cells was 10-20-fold higher than that from controls. Half-maximal stimulation of the S6 kinase by NGF treatment occurred in approximately 5 min, and the activity returned almost to basal levels by 2 h. A rapid purification method was devised in which crude extract was applied directly to a PBE 94 column after buffer exchange on a PD-10 column (Sephadex G-25 M). The activated S6 kinase was purified at least 673-fold with a recovery of approximately 70%. The S6 kinase has an apparent molecular weight of 45,000 and is highly specific for S6. It is not inhibited by the specific inhibitor of cAMP-dependent protein kinases, or by chlorpromazine or sodium vanadate, nor is it activated by Ca2+/calmodulin. It was inhibited by EGTA, beta-glycerophosphate, or NaF. Phosphorylation occurred solely on serine residues. The S6 kinase activity from control cells and from NGF-treated cells eluted at pH 5.69 and 5.58, respectively, during PBE 94 column chromatography. Pretreatment of crude extract from NGF-stimulated cells with alkaline phosphatase resulted in an elution of the enzyme at the position of S6 kinase from control cells and a concomitant decrease in activity. These results indicate that phosphorylation is involved in the mechanism of S6 kinase activation.
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PMID:Purification and mechanism of activation of a nerve growth factor-sensitive S6 kinase from PC12 cells. 310 77


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