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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoenolpyruvate carboxykinase (PEPCK) with a subunit molecular mass of 74 kDa has been purified 450-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.). This is the first purification of the native form of the enzyme from any plant tissue. Incubation of the purified enzyme with [gamma-32P]ATP and either phosphoenolpyruvate-carboxylase kinase or mammalian cAMP-dependent protein kinase led to labelling of the enzyme in a part of the molecule separate from the active site. This was reversed by incubation with protein phosphatase 2A. Cotyledons of cucumber seedlings were also supplied with 32Pi. Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by 32Pi in darkened cotyledons was reversed by illumination.
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PMID:Purification, and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons. 769 56

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. We have identified soluble rat brain phosphatases that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity were observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A, since they were 85-100% inhibited by 10 nM okadaic acid and contained a 36-kDa polypeptide recognized by a monoclonal antibody directed against the catalytic subunit of phosphatase 2A. Immunoblots performed using antibodies specific for isoforms of the B subunit of phosphatase 2A indicate that the two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and calmodulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcinerurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A or cyclosporin A to inhibit calcineurin increased apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites, as assayed by back phosphorylation. These results indicate that phosphatase 2A and calcineurin dephosphorylate sodium channels in brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity.
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PMID:Identification of soluble protein phosphatases that dephosphorylate voltage-sensitive sodium channels in rat brain. 770 24

A triacylglycerol lipase, presumably the first enzyme involved in the mobilization of lipid from the insect fat body, has been purified to homogeneity from the fat body of Manduca sexta. The purification procedure involved polyethyleneglycol precipitation, and chromatography on DEAE-cellulose, phenyl-Sepharose, Q-Sepharose and hydroxylapatite. The final product, a protein with an M(r) = 76,000 by SDS-PAGE, was purified nearly 8000-fold from the original homogenate in a yield of about 11%. The enzyme catalyzed the hydrolysis of tri-, di-, and mono-oleoylglycerols, but showed highest affinity for tri- or dioleoylglycerol. Thus, under initial reaction conditions, the end products of trioleoylglycerol hydrolysis were: free fatty acids (66%), sn-2-monooleoylglycerol (24%), sn-1,2(2,3)-dioleoylglycerol (7%), and glycerol (3%). The fat body lipase exhibited a preference for hydrolyzing the primary ester bonds of acylglycerols, and did not show stereoselectivity toward either the sn-1 or sn-3 position of trioleoylglycerol. The enzyme had a pH optimum of 7.9, and was inhibited by diisopropylfluorophosphate, ATP, ADP, Mg2+, and NaF. The enzyme showed a strong tendency to aggregate, but was stable in detergent solutions at high concentration of glycerol. The polypeptide was phosphorylated by the cAMP-dependent protein kinase from bovine heart; however, phosphorylation did not cause activation of the enzyme. It is suggested that this fat body lipase could be analogous to the "hormone-sensitive lipase" of vertebrate adipose tissue.
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PMID:Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta. 780 79

There is ample evidence that intracellular protein phosphorylation is a mandatory event in the process of macrophage activation by LPS, yet how this event is initiated and what roles the phosphorylated proteins are assigned to are poorly understood. We previously isolated a 65-kDa cytosolic protein (pp65) that was phosphorylated specifically in LPS-stimulated murine macrophages. In the present study, the complete primary structure of pp65 was determined on the basis of the cDNA containing an open reading frame of 1881 bases. The sequence of pp65 revealed that it is a murine homologue of human L-plastin, recently identified as a novel transformation-induced polypeptide of neoplastic human cells, and that it contains a unique series of Ca2+, calmodulin, and actin binding domains. A single phosphorylated peptide was isolated from the tryptic digest of pp65 by reverse-phase HPLC. From the amino acid sequence of the dodecapeptide Gly-Ser-Val-Ser-Asp-Glu-Glu-Met-Met-Glu-Leu-Arg, the phosphorylation site of pp65 was located at the N-terminal region adjacent to the first Ca2+ binding domain. This sequence contains a repeat of the casein kinase II motif Ser-Xxx-Xxx-Glu/Asp and, together with the preceeding Arg residue, constitutes the consensus sequence Arg-Xxx-Ser for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), but not mitogen-activated protein kinase (MAPK)-specific motif is found. These results, taken together with previous observations on the process of macrophage activation by LPS, demonstrate that pp65 is phosphorylated by an LPS-induced protein kinase other than MAPK and exerts its function on the cytoskeleton in a Ca2+/calmodulin-dependent manner.
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PMID:Complete primary structure and phosphorylation site of the 65-kDa macrophage protein phosphorylated by stimulation with bacterial lipopolysaccharide. 789 27

Two type II regulatory (R) subunits of cAMP-dependent protein kinase (PKA) of 50 and 47 kDa have been identified in Aplysia neurons by several criteria which include phosphorylation by the catalytic subunit of PKA and nanomolar affinity for a peptide fragment of the human thyroid protein Ht 31, properties that in mammals distinguish type II from type I R subunits. The neuronal type II R subunits are differentially localized within cells. For example, the 50-kDa polypeptide is enriched in taxol-stabilized microtubules. In addition, at least seven high molecular mass neuronal RII-binding proteins ranging in mass from 110 to 420 kDa have been demonstrated by a blot overlay technique, which uses 32P-labeled bovine RII alpha as a probe. The RII-binding proteins also exhibit discrete patterns of subcellular localization. For example, the 420 kDa species is enriched in taxol-stabilized microtubules and therefore may serve to anchor the 50-kDa RII subunit. The localization of PKA through the association of RII subunits with the binding proteins may anchor the multifunctional kinase close to key substrates and thereby contribute to the spatial organization required to mediate the orderly phosphorylation events that underly neuronal modulation.
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PMID:Type II regulatory subunits of cAMP-dependent protein kinase and their binding proteins in the nervous system of Aplysia californica. 790 81

Demembranated rat sperm flagellar polypeptides capable of binding the regulatory subunit (RII) of a type II cAMP-dependent protein kinase, having apparent subunit molecular masses of 120, 80 and 57 kDa were identified by an RII overlay procedure [Horowitz, J. A., Wasco, W., Leiser, M. & Orr, G. A. (1988) J. Biol. Chem. 263, 2098-2104]. In this study it is shown that all three polypeptides capable of binding RII on a solid-phase blot are tightly associated with the fibrous sheath. Purified fibrous sheath preparations were capable of binding (a) [3H]cAMP and (b) purified catalytic subunits of cAMP-dependent protein kinase forming a functional holoenzyme. The 57-kDa protein was identified as RII by photoaffinity labeling with 8-azido[32P]cAMP. This peptide was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. RII alpha was also shown to form tight, specific complexes with the fibrous sheath demonstrating the presence of functional RII alpha-binding sites. Truncated RII beta fusion proteins were used to identify the N-terminal amino acids at positions 1-50 as a primary determinant for RII-binding protein interaction. Differential extraction of adult testis with buffers containing Triton X-100, urea and sodium dodecyl sulfate revealed the presence of 80-kDa (major) and 120-kDa (minor) RII-binding proteins in particulate extracts. The 80-kDa polypeptide is only expressed at late stages of spermatogenesis, i.e. during spermiogenesis, suggesting a developmental role for RII anchoring to the sperm flagellar fibrous sheath.
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PMID:Association of the regulatory subunit of a type II cAMP-dependent protein kinase and its binding proteins with the fibrous sheath of rat sperm flagellum. 792 27

Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated.
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PMID:Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region. 806 27

Intact bovine adrenal medullary chromaffin cells were preincubated with 32PO4, and the multiple-site phosphorylation of tyrosine hydroxylase (TH) was studied. Up to eight 32P-labeled peptides were produced by tryptic hydrolysis of TH; however, all of the tryptic phosphopeptides were derived from four phosphorylation sites--Ser8, Ser19, Ser31 and Ser40. In situ regulation of 32P incorporation into the latter three sites was demonstrated with a diverse set of pharmacological agents. 32P incorporation into Ser19 was preferentially increased by brief exposures to depolarizing secretagogues. Longer treatments also increased Ser31 and Ser40 phosphorylation. Nicotine, muscarine and vasoactive intestinal polypeptide--reflecting cholinergic and non-cholinergic components of sympatho-adrenal transmission--each produced different patterns of multiple-site phosphorylation of TH. Nicotine, bradykinin and histamine increased 32P incorporation at each of the three sites whereas muscarine, angiotensin II, endothelin III, prostaglandin E1, GABA and ATP selectively increased Ser31 phosphorylation. Nerve growth factor did not influence TH phosphorylation in chromaffin cells from adult adrenal glands but selectively increased Ser31 phosphorylation in chromaffin cells isolated from calf adrenal glands. 32P incorporation into Ser40 was selectively increased by forskolin and other cAMP-acting agents whereas vasoactive intestinal polypeptide increased Ser31 and Ser40 phosphorylation. Thus, the phosphorylation of TH in bovine chromaffin cells appears to be regulated at three sites by three separate intracellular signaling pathways--Ser19 via Ca2+/calmodulin-dependent protein kinase II; Ser31 via ERK (MAP2 kinases); and Ser40 via cAMP-dependent protein kinase. These signaling pathways, as well as the extracellular signals that were effective in stimulating them, are similar to those previously described for TH in rat pheochromocytoma cells. However, several of the pharmacological agents produced different patterns of multiple-site TH phosphorylation in the bovine chromaffin cells. These differences between tissues could be accounted for by differences in the coupling/access between the extracellular signal transduction systems and the intracellular signaling pathways as opposed to differences in the intracellular signaling pathways per se.
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PMID:Multiple signaling pathways in bovine chromaffin cells regulate tyrosine hydroxylase phosphorylation at Ser19, Ser31, and Ser40. 809 28

To elucidate the mechanism causing the transient accumulation of intracellular cAMP in the FRTL-5 thyroid cell line, the short-term effect of thyroid-stimulating hormone (TSH) on phosphodiesterase (PDE) activity was studied. Together with an increase in cAMP levels, TSH produced a significant increase in total PDE activity as early as 3 min, with a maximal stimulation reached after 15 min. This short-term increase in PDE activity was dependent on the TSH concentration (ED50 = 4 x 10(-11) M TSH). Forskolin and dibutyryl cAMP produced an even larger stimulation than that produced by TSH, suggesting that the effect of TSH is mediated by cAMP. To determine the properties of the PDE forms activated by TSH, antibodies specific for the cAMP-PDEs were used to immunoprecipitate the PDEs present in control cells, and cells incubated for 15 min in the presence of 10 nM TSH. Comparison of the activity recovered in the immunoprecipitation pellets demonstrated that TSH produced more than a 2.5-fold increase in the cAMP-PDE form(s) recognized by this antibody. Conversely, the activity remaining in the supernatants was not affected by the TSH treatment. Most of the activity recovered in the immunoprecipitation pellets (90%) was inhibited by 10 microM Rolipram, an inhibitor specific for the high affinity cAMP-PDEs. No TSH stimulation of the Rolipram-insensitive PDE activity could be observed under these conditions. Western blot analyses with two different cAMP-PDE specific antibodies showed that a 15-min stimulation with TSH induced the appearance of a new band with electrophoretic mobility slower than the polypeptide present in unstimulated cells. The appearance of this band did not require ongoing protein synthesis because it occurred in the presence of cycloheximide. Metabolic [32P]orthophosphate labeling of intact FRTL-5 cells indicated that the TSH treatment caused an increased 32P incorporation into a polypeptide that co-purified with the stimulated PDE activity and had an electrophoretic mobility identical to that of the cAMP-PDE. Okadaic acid, a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A, elicited a potentiation of the TSH-stimulated PDE activity. The stimulating of a PDE with the same immunological properties and Rolipram sensitivity as the cAMP-PDE stimulated by TSH in the intact cells was reproduced, in a cell-free system, by incubating soluble extracts from FRTL-5 cells with the catalytic subunit of cAMP-dependent protein kinase. These data provide evidence that TSH produces a rapid activation of a cAMP-PDE in the FRTL-5 cells through a cAMP-dependent phosphorylation.
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PMID:The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation. 813 62

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. In this report, a simple and rapid purification as well as the properties of this enzyme from bovine spleen is described. Using combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow, phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Superose 12 (HR/30) gel filtration fast protein liquid chromatography, the enzyme was purified 1475-fold with a high yield. Under native conditions, the enzyme exhibited an apparent molecular mass of 58 kDa, whereas under denaturing conditions the enzyme represented an apparent molecular mass of 50 kDa, suggesting that spleen NMT is a monomeric protein. The NMT activity could be greatly activated to severalfold with the use of Tris-HCl buffer. Kinetic properties indicated that spleen NMT had an apparent low Km for pp60src and myristoylated alanine-rich C kinase substrate as compared with cAMP-dependent protein kinase and the M2 gene segment of reovirus type 3-derived peptides. Bovine spleen NMT was potently inhibited in a concentration-dependent manner by NIP71 (a bovine brain NMT inhibitory protein) with a half-maximal inhibition of 0.816 microgram/ml. Results of this study along with the existing knowledge on NMT indicate that the activity of enzyme resides in a single polypeptide chain of molecular mass between 50 and 68 kDa.
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PMID:Purification and properties of bovine spleen N-myristoyl-CoA protein:N-myristoyltransferase. 816 12


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