Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Homogeneous preparations of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.1.2.15] isolated as the enzyme-phosphoenolpyruvate complex from Escherichia coli are shown by atomic absorption analysis to contain approximately one mole of iron per mole of native enzyme. No cobalt was found, in contrast to suggestions of earlier workers. Pure enzyme preparations show a unique absorption maximum around 350 nm with an epsilon value of about 3500 M-1cm-1. The 350-nm band as well as the enzyme activity is lost when the enzyme is denatured with guanidine-hydrochloride, or when phosphoenolpyruvate, the first substrate to bind to the enzyme, is totally removed from the enzyme by incubation with an excess of erythrose 4-phosphate, the second substrate to bind to the enzyme. The iron remains bound to the enzyme when phosphoenolpyruvate is removed from the enzyme-phosphoenolpyruvate complex.
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PMID:Iron, an essential element for biosynthesis of aromatic compounds. 3 83

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.
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PMID:Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin. 12 64

Inactivation of apo-glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase(phosphorylating) (EC 1.2.1.12) from rat skeletal muscle at 4 degrees C in 0.15 M NaC1, 5 mM EDTA, 4 mM 2-mercaptoethanol pH 7.2 is a first-order reaction. The rate constant of inactivation depends on protein concentration. With one molecule of NAD bound per tetrameric enzyme, a 50 per cent loss in activity is observed and the rate constant of inactivation becomes independent of the protein concentration over a 30-fold range. Two moles of NAD bound per mole of enzyme fully protect it against inactivation. NADH affords a cooperative effect on enzyme structure similar to that of NAD. Inactivation of 7.8 S apoenzyme is reflected in its dissociation into 4.8-S dimers. In the case of enzyme-NAD1 complex, no direct relationship between the extent of inactivation and dissociation is observed, suggesting that these two processes do not occur simultaneously; we may say that dissociation is slower than inactivation. A mechanism in which the rate-limiting step for inactivation is a conformational change in the tetramer occurring prior to dissociation and affecting only the structure of the non-liganded dimer, is consistent with the experimental observations. Inorganic phosphate protects apoenzyme against inactivation. Its effect is shown to be due to the anion binding at specific sites on the protein with a dissociation constant of 2.6 plus or minus 0.4 mM. The NaC1-induced cold inactivation of glyceraldehyde-phosphate dehydrogenase is fully reversible at 25 degrees C in the presence of 20 mM dithiothreitol and 50 mM inorganic phosphate. The rate of reactivation is independent of protein concentration. Inactivated enzyme retains the ability to bind specific antibodies produced in rabbits, but diminishes its precipitating capability.
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PMID:Cold inactivation of glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle. 16 22

Phosphorylation of histone H1 occurs when spermatozoa of the sea urchin Strongylocentrotus purpuratus are treated with the macromolecular fraction of solubilized egg jelly. Phosphorylation is on serine residues in the N-terminal fragment of H1 bisected with N-bromosuccinimide. Phosphorylation is maximal by 4-8 min and dependent on Ca2+, but independent of Na+ or increased intracellular pH. Phosphorylation of H1 can be dissociated from the induction of the acrosome reaction. Only a fraction of the H1 molecules become phosphorylated upon treatment of sperm with egg jelly. The amount of phosphate per mole of H1 increases from 0.15 moles before jelly treatment to 0.46 moles after maximal phosphorylation. Phosphorylation of H1 occurs in a cAMP-dependent manner as indicated by the ability of the phosphodiesterase inhibitors IBMX and SQ20009 to induce H1 phosphorylation. This phosphorylation reaction can be blocked by digesting the sperm surface with Pronase, or preincubation of sperm in wheat germ agglutinin, showing that a ligand in egg jelly must interact with a sperm surface receptor to activate the kinase phosphorylating H1.
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PMID:Phosphorylation of sperm histone H1 is induced by the egg jelly layer in the sea urchin Strongylocentrotus purpuratus. 242 45

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16

Two proteins, termed P85gag-mos and P58gag, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85gag-mos is believed to be important for the transforming potential of ts110 MuSV and has been found to be associated with a thermolabile kinase activity that phosphorylates both P85gag-mos and P58gag in immune complexes. Modifications of the original kinase assay conditions are reported here that have allowed a 30-fold increase in the specific activity of P85gag-mos phosphorylated in vitro. The in vitro P85gag-mos-phosphorylating activity was found to be unresponsive to 10 microM-cAMP or 10 microM-cGMP. Addition of 1 mM-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85gag-mos and P58gag; the molar phosphate incorporation per mole of P85gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58gag increased 18-fold, from 0.013 to 0.234. pH curves of the in vitro kinase reaction further confirmed the presence of phosphatase activity; in the absence of pyrophosphate, a sharp optimum at pH 4 to 5 was observed, whereas it shifted broadly to pH 7.0 in the presence of pyrophosphate. Under the latter conditions, several experiments were performed in order to determine if the kinase was associated with either gag or mos sequences of P85gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62gag. An anti-mos serum generated against a synthetic peptide representing the predicted v-mos amino acid residues 37 to 55 recognizes P85gag-mos and allowed phosphorylation of P85gag-mos in vitro in the absence of P58gag. Peptide mapping of both phosphorylated P85gag-mos and P58gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v-mos. Phosphoamino acid analyses of P85gag-mos and P58gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically 32P-labelled P85gag-mos and P58gag were also found to contain phosphoserine and phosphothreonine. Based on these results, we conclude that a cAMP-independent, serine/threonine protein kinase activity is associated with the mos sequences of P85gag-mos.
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PMID:A cAMP-independent serine/threonine kinase activity is associated with the mos sequences of ts110 Moloney murine sarcoma virus-encoded P85gag-mos. 299 51

The balance of energy-rich compounds (ERC) was drawn up for the growth of Thiobacillus ferrooxidans in a medium with ferrous ions as an energy source. The balance items and the phosphorylating efficiency of oxidation (P/2e-) were calculated basing on the experimental yield values using the ERC balance equation. At a specific growth rate of 0.1 h-1, 55% of ferrous ions are used for the synthesis of cell biomass, 7.5% for maintainance, 4% of the ions are oxidized to reduce NAD+, and 34% are used to produce ERC necessary for the reduction. Here, 24% of ERC are used for the synthesis of monomers from CO2, 42% for the production of NADH, 24% for the biomass synthesis from monomers, and 10% for maintaining cell activity. The P/2e- for the oxidation of ferrous ions is 0.19 mole of ERC per 2e-. This is possible only when the [Fe3+]/[Fe2+] ratio in the cell periplasm is 1 X 10(3)-1 X 10(4).
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PMID:[Balance of macroergic compounds during the growth of Thiobacillus ferrooxidans]. 310 5

The fraction of secretory immunoglobulin A (sIgA) from the milk of healthy mothers was purified by sequential affinity chromatography on protein A-sepharose (in the presence of 1% triton x100), adsorbent Toyopearl HW-55 (gel-filtration), DEAE cellulose (separation of IgG and IgA antibodies), affinity sorbents with immobilized ATP and casein. The protein obtained corresponded to sIgA antibodies according to all known criteria and did not contain any protein contaminations. The ability of sIgA to phosphorylate selectively serine residues of casein (not histones) in the presence of [gamma-32P]ATP was shown. Purified kinase activity was stable at acid shock (pH 2.3), strongly interacted with immobilized antibodies against H-chain of sIgA and eluted from the sorbent with the peak corresponding to sIgA antibodies. The complex of sIgA and ATP was stable enough at the conditions of gel-filtration. Affinity modification of sIgA by chemically reactive analogs of ATP resulted in preferential modification of its light chain (2-3 mole reagent per mole of dimer form). In the condition of oligomer dissociation ATP-sepharose sorbed only the light chains of sIgA. sIgA have optimal conditions for phosphorylating activity different from those of known protein kinases. We suppose that sIgA antibodies with kinase activity are a first example of sIgA antibodies with catalytic activity. For the first time the possibility of existence of natural abzymes in a healthy human was shown. These abzymes catalyze the reaction of synthesis but not substrate degradation reaction.
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PMID:[Do catalytically active antibodies exist in healthy people? (Protein kinase activity of sIgA antibodies from human milk)]. 747 55

RS7-3G11 is a murine monoclonal antibody (MAb) raised against human non-small-cell lung carcinoma, and is under clinical evaluation. The epithelial/carcinoma antigen EGP-1, defined by RS7-3G11, was isolated and purified to homogeneity from a cervical carcinoma cell line, ME180. EGP-1 is a glycoprotein with an average molecular mass of 47.8 kDa. Metabolic labeling of the antigen with 32P-orthophosphate and subsequent immunoprecipitation with RS7-3G11 showed that it is a phosphoprotein. Phosphoamino acid analysis of the in vivo phosphorylated EGP-1 revealed that the phosphorylation is on serine. In vitro analysis with purified antigen demonstrated that protein kinase C, and not protein kinase A, is involved in phosphorylating the antigen in vitro. In vitro analysis indicated a stoichiometry of phosphorylation of 0.54 mole of phosphate per mole of EGP-1. Phosphoamino acid analysis and phosphopeptide mapping of the antigen phosphorylated in vitro by protein kinase C showed that phosphorylation occurred on a serine residue, specifically on serine 303, located in the cytoplasmic domain of EGP-1. Treatment of ME180 cells with phorbol ester increased the phosphorylation of EGP-1. The biological function of EGP-1 remains to be elucidated. In this report we elucidate an involvement of protein kinase C in phosphorylating EGP-1, which may signify a role for this antigen in signal transduction across the cell membrane.
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PMID:The epithelial/carcinoma antigen EGP-1, recognized by monoclonal antibody RS7-3G11, is phosphorylated on serine 303. 763 74

Streprococcus pyogenes gapN was cloned and expressed by functional complementation of the Escherichia gap mutant W3CG. The IPTG-induced NADP non-phosphorylating GAPDH (GAPN) has been purified about 75.4 fold from E. coli cells, using a procedure involving conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. The purified protein was characterised: it's an homotetrameric structure with a native molecular mass of 224 kDa, have an acid pI of 4.9 and optimum pH of 8.5. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 60 degrees C with activation energy of 51 KJ mole(-1). The apparent Km values for NADP and D-G3P or DL-G3P were estimated to be 0.385 +/- 0.05 and 0.666 +/- 0.1 mM, respectively and the Vmax of the purified protein was estimated to be 162.5 U mg(-1). The S. pyogenes GAPN was markedly inhibited by sulfydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfydryl groups in the catalytic activity.
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PMID:Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes expressed in E. coli. 1284 48


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