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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microtubules purified from brain tissue contain endogenous cyclic AMP (cAMP)-dependent protein kinase activity, and microtubule-associated protein 2 (MAP2) is the major substrate. Beef brain microtubules were prepared and used as a model system to study the differential effects of rationally selected cyclic nucleotide analogues on microtubule receptor protein kinase. Data are presented to indicate that the following molecular interactions are essential for activation of the phosphorylation of MAP2: (a) hydrogen bond formation toward the 2', 3', or 5' position, (b) interaction with phosphorus, and (c) no hydrogen bonds but hydrophobic interactions at the base moiety. Thus, the activation mechanism of the type II protein kinase associated with brain microtubules resembles the mechanism found in protein kinases of other systems. In addition, we have studied the effect of the two diastereomers of adenosine 3',5'-monophosphorothioate (cAMPS). The (Sp)-cAMPS isomer was found to activate MAP2 protein kinase, whereas the (Rp)-cAMPS isomer had no activating effect. In contrast, this compound was able to inhibit cAMP-stimulated MAP2 phosphorylation and thus acts as an antagonist of the Sp diastereomer and cAMP. Hence, this analogue provides a useful means to clarify further the effect of cAMP-dependent phosphorylation on functional properties in microtubules in general.
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PMID:In vitro phosphorylation of microtubule-associated protein 2: differential effects of cyclic AMP analogues. 299 22

A 107 kDa (pp107) casein kinase G (ck-G) substrate has been purified from mouse and beef thyroid cytosol; ck-G was purified from beef thyroid cytosol. Ck-G and pp107 were found to co-elute on DEAE cellulose chromatography at approximately 300 mM NaCl. Ck-G and pp107 were separated by spermine-agarose affinity chromatography; pp107 is eluted with a stepped gradient at 250 mM NaCl and ck-G is eluted at 500 mM NaCl. Ck-G was subsequently purified by casein-agarose and GTP-agarose affinity chromatography. The 107 kDa protein was purified using heparin-agarose affinity chromatography. Phosphorylation of purified pp107 by ck-G was stimulated by spermine (ED50 = 0.2 mM) and inhibited by low concentrations of heparin (0.1-5 micrograms/ml). The Km and Vmax for the reaction were 1.46 microM and 32.2 nmoles P transferred/20 min/mg protein, respectively; 1 mole pp107 incorporated 0.81 mole phosphorus. pp107 was found to be an acidic substrate with a pI of 3.87 and was absorbed to wheat-germ agglutinin-agarose. The specificity of pp107 phosphorylation was studied using diacylglycerol-activated calcium/phospholipid-dependent protein kinase C, calcium-activated calmodulin-dependent protein kinase, and the catalytic subunit of cAMP-dependent protein kinase A. Phosphorylation of pp107 by the other protein kinases tested never exceeded 4% of that of ck-G. Our data show that pp107 is an acidic glycoprotein which may serve as a high-affinity and specific substrate for ck-G.
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PMID:Purification of a 107 kilodalton (kDa) casein kinase G substrate from thyroid cytosol. 320 Feb 52

Phosphorus-31 (31P) NMR is proving to be a powerful analytical method for investigating molecular/metabolic issues in neural tissues. Recent studies have demonstrated high levels of phosphomonoesters and phosphodiesters in mammalian brain, and revealed the influence of brain maturation, development, and aging on these levels. Preliminary studies in Alzheimer's disease have demonstrated elevated levels of phosphomonoesters and phosphodiesters in the areas of Alzheimer's brain which exhibit neuropathological changes. Moreover, phosphomonoester levels were also elevated in areas of Alzheimer's brain that were devoid of neuropathological changes. These findings suggest that the phosphomonoester elevations in Alzheimer's brain antedate changes in cellular morphology and structure. Abnormalities in protein kinase function could potentially explain these findings, as well as the reported hyperphosphorylation of tau protein in Alzheimer's brain. Recent studies from this laboratory suggest that aluminum could also be involved in the changes in phosphomonoesters and phosphodiesters.
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PMID:31P nuclear magnetic resonance (NMR) spectroscopy of brain in aging and Alzheimer's disease. 331 99

The specific activity of the gamma-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717-720). The HPLC method also allowed the incorporation of 32P into the (alpha + beta)-positions of ATP to be determined. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [gamma-32P]ATP attained a steady-state value after 1-2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the beta-agonist isoprenaline increased this value by 5-10% within 15 min. Under these conditions the steady-state specific activity of [gamma-32P]ATP was 30-40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the gamma-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.
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PMID:Studies on the specific activity of [gamma-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate. 351 72

O5-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography. Clean separation of phosphohydroxylysine from the other phospho amino acids, phosphoethanolamine, and phosphocholine was achieved. Conditions were also determined to permit hydrolysis of proteins in 2 M HCl without loss of the phosphono group of phosphohydroxylysine residues. Experiments were then performed showing that 32P was incorporated into the hydroxylysine residues of cell-associated collagens when cultured calf aorta medial smooth muscle cells were incubated with [32P]orthophosphate. In other experiments, the cells incorporated [3H]lysine into hydroxylysine residues of cell-associated collagen and then 32P into phosphohydroxylysine residues. The doubly labeled phosphohydroxylysine subsequently isolated showed nearly 1:1 stoichiometry with respect to incorporation of precursor lysine and phosphorus. Finally, in preliminary experiments done with a cell-free extract of the smooth muscle cells, 32P was transferred from [gamma-32P]ATP to hydroxylysine residues in several kinds of collagenous substrates. Thus, this work shows that smooth muscle cells have the capacity to phosphorylate hydroxylysine residues in their cell-associated collagens and provides preliminary evidence that a protein kinase is involved.
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PMID:Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells. 385 6

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS and the Rp and Sp isomers adenosine 3', 5'-monophosphodimethylamidate. cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)-cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.
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PMID:Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases. 608 45

The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and RP-cAMPN(CH3)2. We have investigated the agonistic and antagonistic activities of these compounds in four cAMP-dependent reactions: activation of the cellular slime mold Dictyostelium discoideum via its cell surface cAMP receptor, and phosphorylation by cAMP-dependent protein kinases type I, type II (both mammalian enzymes), and type D (derived from D. discoideum). The results show that 1) the compounds (SP)-cAMPS and (SP)-cAMPN(CH3)2 are (mostly full) agonists for the four proteins. Half-maximal activation is at micromolar concentrations (0.8-7 microM). 2) (RP)-cAMPS is a full antagonist for the cell surface receptor and protein kinases type I and II, with apparent inhibition constants between 0.8 and 8 microM. This compound is a partial agonist for protein kinase type D, where it induces maximally 50% activation of the enzyme if compared with cAMP. 3) (RP)-cAMPN(CH3)2 is a full antagonist for the cell surface receptor, and for protein kinase type II. This compound is a partial agonist for protein kinase type I (at least 50% activation if compared with cAMP), and inactive for protein kinase type D. This derivative is at least 25-fold less active as an antagonist than (RP)-cAMPS. 4) The activity of mixtures of different concentrations of the antagonist (RP)-cAMPS with different concentrations of cAMP reveals that the compound is a competitive antagonist of cAMP at micromolar concentrations.
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PMID:Competitive cAMP antagonists for cAMP-receptor proteins. 608 78

Tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2](TH) was purified from bovine corpus striatum. The purification involved sequential DEAE cellulose, hydroxylapatite and CM Sephadex C-50 chromatography, followed by glycerol density gradient centrifugation. Final preparations appeared to be 90 to 100% pure as judged by polyacrylamide gel electrophoresis under denaturing conditions in acetic acid-urea. The enzyme was estimated to have a minimum molecular weight of approximately 60,000 daltons. Purified TH could be activated in vitro by incubation with magnesium adenosine triphosphate and the catalytic subunit of cyclic AMP-dependent protein kinase (ATP/protein phosphotransferase; EC 2.7.1.37). When the final purified preparation of TH was incubated under these conditions utilizing [gamma-32P]ATP, it was found to incorporate 0.7 to 0.9 mol of phosphorus/mol of protein. These results suggest that the activation of TH in the presence of phosphorylating conditions is due to its phosphorylation by cyclic AMP-dependent protein kinase.
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PMID:Tyrosine hydroxylase: studies on the phosphorylation of a purified preparation of the brain enzyme by the cyclic AMP-dependent protein kinase. 611 Jul 71

The phosphoryl transferring enzymes pyruvate kinase, cAMP-dependent protein kinase and the pyrophosphoryl transferring enzyme PP-Rib-P synthetase utilize the beta, gamma bidentate metal--ATP chelate (delta-isomer) as substrate, as determined with substitution-insert CrIIIATP or CoIII(NH3)4ATP complexes. In addition, these enzymes bind a second divalent cation, which is an essential activator for pyruvate kinase and PP-Rib-P synthetase and an inhibitor of protein kinase. The enzyme-bound metal has been used as a paramagnetic reference point in T1 measurements to determine distances to the protons and phosphorus atoms of the bound nucleotide and acceptor substrates. These distances have been used to construct models of the conformations of the bound substrates. The activating metal forms a second sphere complex of the metal-nucleotide substrate on pyruvate kinase and PP-Rib-P synthetase while the inhibitory metal directly coordinates the polyphosphate chain of the metal-nucleotide substrate on protein kinase. Essentially no change is found in the dihedral angle at the glycosidic bond of ATP upon binding to pyruvate kinase (chi = 30 degrees), an enzyme of low base specificity, but significant changes in the torsional angle of ATP occur on binding to protein kinase (chi = 84 degrees) and PP-Rib-P synthetase (chi = 62 degrees), enzymes with high adenine-base specificity. Intersubstrate distances, measured with tridentate CrATP or beta, gamma bidentate CrAMPPCP as paramagnetic reference points, have been used to deduce the distance along the reaction coordinate on each enzyme. The reaction coordinate distances on pyruvate kinase (# +/- 1 A) and PP-Rib-P synthetase (not less than 3.8 A) are consistent with associative mechanisms, while that on protein kinase (5 +/- 0.7 A) allows room for a dissociative mechanism.
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PMID:Conformations and arrangement of substrates at active sites of ATP-utilizing enzymes. 611 25

The effect of cAMP-dependent protein kinases from rabbit skeletal muscles on Ca2+ uptake by fragments of skeletal muscle sarcoplasmic reticulum was studied. It was shown that incubation of the sarcoplasmic reticulum fragments with protein kinase increases the rate of Ca2+ uptake without changing the activity of Ca2+-dependent ATPase. This phenomenon is not accompanied by phosphorus incorporation into the protein components of the reticulum membranes. The protein kinase preparation subjected to "self-phosphorylation" is also capable to increase the rate of Ca2+ uptake. Using (14C) -oleic acid, it was shown that the increase of the rate of Ca2+ transport under effects of the "self-phosphorylated" protein kinase occurs due to the binding of free fatty acids present in the sarcoplasmic reticulum membranes. It was found that the effect observed is due to phosphofructokinase (ATP : D-fructose-6-phosphate-1-phosphotransferase) present in the protein kinase preparation.
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PMID:[Cause of increase in the efficiency of Ca2+ transport by fragments of sarcoplasmic reticulum from fast skeletal muscles induced by protein kinase]. 624 73


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