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 investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.
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PMID:Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum. 311 Jun 3

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.
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PMID:Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. 312 81

Two enzymes, alkaline phosphatase and acetylcholinesterase (AChE), have been shown previously to be components of the surface of the trematode parasite Schistosoma mansoni. In this study we report that both these enzymes and other serine hydrolases are susceptible to release from the S. mansoni tegumental membrane by a phosphatidylinositol-specific phospholipase C (PIPLC) of bacterial origin. These data suggest that AChE and alkaline phosphatase of S. mansoni, as in higher organisms, are anchored to the membrane via covalently attached phosphatidylinositol. The release of AChE from the vesicular fraction of the parasite with PIPLC occurs in a concentration-dependent manner. Sucrose gradient centrifugation of the PIPLC-released AChE showed a single 8.3 S molecular form, similar to that observed for AChE solubilized by Triton X-100. PIPLC removed large amounts of AChE from the surface of intact schistosomula in culture, with no impairment of the viability of the parasite. In this case, an increase in the overall levels of AChE in the intact parasite was observed after addition of PIPLC.
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PMID:Acetylcholinesterase in Schistosoma mansoni is anchored to the membrane via covalently attached phosphatidylinositol. 313 66

The T cell antigen receptor is composed of at least seven chains derived from six different gene products. Upon stimulation, several chains can be phosphorylated. Two of these, CD3-gamma and CD3-epsilon are phosphorylated on serine residues. In addition, a 21-kDa nonglycosylated receptor component is phosphorylated, upon activation, on tyrosine residues. We have referred to this phosphoprotein as p21 because we have previously not been able to assign the tyrosine phosphorylation to any of the described receptor subunits (Samelson, L. E., Patel, M. D., Weissman, A. M., Harford, J. B., and Klausner, R. D. (1986) Cell 46, 1083-1090). In this paper, we demonstrate that it is the 16-kDa zeta chain which is the tyrosine phosphorylated subunit, and thus the p21 nomenclature can be replaced. This phosphorylation results in a shift of the apparent Mr of zeta to 21 kDa. Proof that p21 is tyrosine phosphorylated zeta was afforded by a number of approaches. Specific anti-zeta antibodies directly precipitated phospho-p21. Metabolically labeled protein corresponding to p21 could only be observed after activation. When this 21-kDa band was isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with alkaline phosphatase, its migration was identical with that of zeta. Furthermore, peptide mapping of metabolically labeled p21 (after gel isolation and dephosphorylation) showed it to be indistinguishable from p21. Thus, one of the early events of T cell activation is the tyrosine phosphorylation of the zeta chain of the T cell antigen receptor.
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PMID:The T cell antigen receptor zeta chain is tyrosine phosphorylated upon activation. 314 73

Three peptides have been formed by proteolytic digestion of individual casein proteins and their secondary structures characterised by far-UV circular dichroism (CD). Peptide alpha s1(1-23), residues 1-23 of alpha s1-casein, was generated by treatment of the parent protein with chymosin. Peptides beta(1-28) and beta(1-52), residues 1-28 and 1-52 of beta-casein, were plasmin- and chymotrypsin-generated fragments, respectively. Analysis of the CD spectra revealed that in aqueous solution all three peptides have secondary structures composed exclusively of beta-sheet and random coil. A limited amount of alpha-helix was formed in two of the three peptides upon treatment with high concentrations (greater than 40% (v/v] of 2,2,2-trifluoroethanol. Partial dephosphorylation (60%) of beta(1-28) and beta(1-52) by treatment with alkaline phosphatase resulted in homogeneous preparations, as judged by polyacrylamide gel electrophoresis, which exhibited increased hydrophobicity. This reduction in the level of phosphorylation of serine residues 15, 17, 18 and 19 led to increased propensity for helix formation in the peptides in the presence of 2,2,2-trifluoroethanol, but no alpha-helical structures were detected in the dephosphorylated peptides in the absence of 2,2,2-trifluoroethanol.
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PMID:The secondary structure of peptides derived from caseins: a circular dichroism study. 316 68

Enzyme profiles of oral Treponema species were determined by using RapID-ANA (Innovative Diagnostic System, Atlanta, Ga.), a 4-h test system which detects 18 enzymatic reactions, including aminopeptidases and glycosidases. Seventy-two clinical isolates of Treponema denticola, four reference strains of T. denticola (ATCC 35404, ATCC 35405, ATCC 35520, and ATCC 33521), one strain of T. vincentii (ATCC 35580), and two strains of T. socranskii subspecies (T. socranskii subsp. buccale ATCC 35534 and T. socranskii subsp. socranskii ATCC 35536) were used in this study. All T. denticola strains produced indole and a variety of aminopeptidases and glycosidases. These organisms could be differentiated into two groups on the basis of tetrazolium reductase and serine, phenylalanine, and glycine aminopeptidase activities. T. vincentii produced N-acetylglucosaminidase and arginine aminopeptidase, which facilitated the differentiation of this organism from T. socranskii subspecies and the T. denticola group. T. socranskii subspecies gave positive reactions for alkaline phosphatase only. These findings suggest that the RapID-ANA system is useful for enzymatic characterization and differentiation of oral spirochetes.
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PMID:Enzyme profiles of oral spirochetes in RapID-ANA system. 318 13

Glucokinase, purified from rat liver, was phosphorylated to an extent of 1 mol [32P]-phosphate/mol of enzyme when incubated with [32P]ATP and protein kinase A from pig or rabbit muscle. The phosphate was bound to serine residues. K0.5 increased and Vmax decreased upon phosphorylation. The phosphate group was removed during incubation of the phosphorylated glucokinase with alkaline phosphatase. Enzymatically inactive glucokinase was not phosphorylated by the protein kinase.
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PMID:Phosphorylation of glucokinase from rat liver in vitro by protein kinase A with a concomitant decrease of its activity. 335 51

Phosphoenolpyruvate carboxylase (PEPCase) from light- and dark-adapted maize leaves was rapidly purified in the presence of L-malate and glycerol to apparent electrophoretic homogeneity by ammonium sulfate fractionation, hydroxylapatite chromatography, and fast-protein liquid chromatography on Mono Q. The resulting preparations were totally devoid of pyruvate, orthophosphate dikinase protein based on immunoblot analysis. Throughout the purification, both forms of PEPCase retained their different enzymatic properties. The specific activity of the light enzyme was consistently about twice that of the dark form when assayed at suboptimal (but physiological) pH (pH 7.0-7.3), and the former was also less sensitive to feedback inhibition by L-malate than that from darkened leaves under various conditions. Covalently bound phosphate and high-performance liquid chromatography-based phosphoamino acid analyses showed that both forms of purified PEPCase were phosphorylated exclusively on serine residues, but the degree of phosphorylation was about 50% greater in the light enzyme. Notably, incubation of purified PEPCase in vitro with exogenous alkaline phosphatase led to an increase in malate sensitivity and a decrease in specific activity of the light form enzyme to levels observed with the dark form, which was essentially not affected by phosphatase treatment. These results with the purified enzyme from light- and dark-adapted maize leaves indicate that the light-induced changes in activity and malate sensitivity of C4 PEPCase are related, at least in part, to the degree of covalent seryl phosphorylation of the protein in vivo.
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PMID:Light/dark regulation of maize leaf phosphoenolpyruvate carboxylase by in vivo phosphorylation. 335 58

The catalytically essential amino acid in the active site of bacterial alkaline phosphatase (Ser-102) has been replaced with a cysteine by site-directed mutagenesis. The resulting thiol enzyme catalyzes the hydrolysis of a variety of phosphate monoesters. The rate-determining step of hydrolysis, however, is no longer the same for catalysis when the active protein nucleophile is changed from the hydroxyl of serine to the thiol of cysteine. Unlike the steady-state kinetics of native alkaline phosphatase, those of the mutant show sensitivity to the leaving group of the phosphate ester.
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PMID:Modification of the active site of alkaline phosphatase by site-directed mutagenesis. 351 Apr 54

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.
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PMID:Molecular cloning and sequence analysis of human placental alkaline phosphatase. 351 48


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