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)

Plasma membranes of porcine liver, highly purified by aqueous two-phase partition, oxidized NADH in the absence of added external acceptors. The oxidation was resistant to cyanide and responded to nanomolar concentrations of ATP alone or ATP in the presence of cyclic AMP. Both the Km for NADH and the long-term activity of the oxidase were affected. Upon incubation at 37 degrees C with cyclic AMP (0.1-10 nM) and ATP (1-100 nM), the NADH oxidase activity was inhibited. The inhibition was complex and due to an approx. 5-fold increase in the Km for NADH compared to the NADH oxidase of membranes incubated in the absence of cyclic AMP + ATP. The response to cAMP + ATP was rapid and occurred within seconds of ATP addition. The response was inhibited by the selective inhibitor of cyclic AMP-dependent protein kinase, H-89. Neither cyclic AMP alone nor ATP alone at nanomolar concentrations elicited a rapid response. However, 10 nM ATP alone did result in similar alteration of Km and Vmax as did ATP + 0.1 nM cyclic AMP. The response to ATP alone or in preparations depleted of cyclic AMP required higher ATP concentrations than with cAMP present or occurred more slowly with a lag of 1-2 min. The NADH oxidase activity of porcine plasma membranes after cyclic AMP + ATP treatment retained high activity with storage at 4 degrees C, whereas that of unincubated or sham-incubated plasma membranes was reduced with time of storage at 4 degrees C. In some but not all instances, NADH oxidase activity inactivated by incubation with NADH at 37 degrees C or after storage at 4 degrees C could be reactivated by incubation with cyclic AMP plus ATP. As with the alteration in Km, cyclic AMP alone was without effect and ATP alone was much less effective than the combination. The results demonstrate ATP-dependent modulation of the NADH oxidase activity of isolated plasma membranes at physiological concentrations of ATP. This modulation may have functional significance in mediating the hormone and growth factor responsiveness of the plasma membrane NADH oxidase activity.
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PMID:Cyclic AMP-plus ATP-dependent modulation of the NADH oxidase activity of porcine liver plasma membranes. 780 17

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.
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PMID:GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. 819 51

Previously, our laboratory reported that lactosylceramide (LacCer) stimulated human aortic smooth muscle cell proliferation via specific activation of p44 mitogen-activated protein kinase (MAPK) in the p21(ras)/Raf-1/MEK2 pathway and induced expression of the transcription factor c-fos downstream to the p44 MAPK signaling cascade (Bhunia A. K., Han, H., Snowden, A., and Chatterjee S. (1996) J. Biol. Chem. 271, 10660-10666). In the present study, we explored the role of free oxygen radicals in LacCer-mediated induction of cell proliferation. Superoxide levels were measured by the lucigenin chemiluminescence method, MAPK activity was measured by immunocomplex kinase assays, and Western blot analysis and c-fos expression were measured by Northern blot assay. We found that LacCer (10 microM) stimulates endogenous superoxide production (7-fold compared with control) in human aortic smooth muscle cells specifically by activating membrane-associated NADPH oxidase, but not NADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants, N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate. NAC and DPI both abrogated individual steps in the signaling pathway leading to cell proliferation. For example, the p21(ras).GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduced glutathione (GSH). In contrast, depletion of GSH by L-buthionine (S, R)-sulfoximine up-regulated the above described signaling cascade. In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxide (a redox stress signaling molecule), which mediates cell proliferation via activation of the kinase cascade. Our findings may explain the potential role of LacCer in the pathogenesis of atherosclerosis involving the proliferation of aortic smooth muscle cells.
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PMID:Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. 918 53

NADH oxidase activity of rat liver plasma membranes was inhibited by low concentrations (1-100 nM) of ATP. The inhibition was amplified by addition of nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition was complex and related to a marked increase in the Km for NADH at high NADH concentrations together with a concomitant decrease in the Vmax. In the absence of added or residual ATP, cyclic AMP was without effect. The response of cyclic AMP + ATP was inhibited by low concentrations of the selective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not by staurosporin. The Vmax but not the Km was modified by treating the plasma membranes with a mild oxidizing agent, N-chlorosuccinamide, or with the reducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmax was reduced by cyclic AMP + ATP. In contrast, in the presence of N-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative to cyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmax could be either an increase or a decrease depending on whether the membranes were oxidized or reduced. The results demonstrate regulation of NADH oxidase activity of rat liver plasma membranes through cyclic AMP-mediated phosphorylation by membrane-located protein kinase activities where the final response is dependent on the oxidation-reduction status of the plasma membranes.
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PMID:Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP. 927 56

17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a compound that is proposed for clinical development, shares the ability of geldanamycin to bind to heat shock protein 90 and GRP94, thereby depleting cells of p185erbB2, mutant p53, and Raf-1. Urine and plasma from mice treated i.v. with 17AAG contained six materials with absorption spectra similar to that of 17AAG. Therefore, in vitro metabolism of 17AAG by mouse and human hepatic preparations was studied to characterize: (a) the enzymes responsible for 17AAG metabolism; and (b) the structures of the metabolites produced. These materials had retention times on high-performance liquid chromatography of approximately 2, 4, 5, 6, 7, and 9 min. When incubated in an aerobic environment with 17AAG, murine hepatic supernatant (9000 x g) produced each of these compounds; the 4-min metabolite was the major product. This metabolism required an electron donor, and NADPH was favored over NADH. Metabolic activity resided predominantly in the microsomal fraction. Metabolism was decreased by approximately 80% in anaerobic conditions and was essentially ablated by CO. Microsomes prepared from human livers produced essentially the same metabolites as produced by murine hepatic microsomes, but the 2-min metabolite was the major product, and the 4-min metabolite was next largest. There was no metabolism of 17AAG by human liver cytosol. Metabolism of 17AAG by human liver microsomes also required an electron donor, with NADPH being preferred over NADH, was inhibited by approximately 80% under anaerobic conditions, and was essentially ablated by CO. Liquid chromatography/mass spectrometry analysis of human and mouse in vitro reaction mixtures indicated the presence of materials with molecular weights of 545, 601, and 619, compatible with 17-(amino)-17-demethoxygeldanamycin (17AG), an epoxide, and a diol, respectively. The metabolite with retention time of 4 min was identified as 17AG by cochromatography and mass spectral concordance with authentic standard. Human microsomal metabolism of 17AAG was inhibited by ketoconazole, implying 3A4 as the responsible cytochrome P450 isoform. Incubation of 17AAG with cloned CYP3A4 produced metabolites 4 and 6. Incubation of 17AAG with cloned CYP3A4 and cloned microsomal epoxide hydrolase produced metabolites 2 and 4, with greatly decreased amounts of metabolite 6. Incubation of 17AAG with human hepatic microsomes and cyclohexene oxide, a known inhibitor of microsomal epoxide hydrolase, did not affect the production of metabolite 4 but decreased the production of metabolite 2 while increasing the production of metabolite 6. These data imply that metabolite 2 is a diol and metabolite 6 is an epoxide. Mass spectral fragmentation patterns and the fact that 17AG is not metabolized argue for the epoxide and diol being formed on the 17-allylamino portion of 17AAG and not on its ansamycin ring. These data have implications with regard to preclinical toxicology and activity testing of 17AAG as well as its proposed clinical development because: (a) production of 17AG requires concomitant production of acrolein from the cleaved allyl moiety; and (b) 17AG, which was not metabolized by microsomes, has been described as being as active as 17AAG in decreasing cellular p185erbB2.
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PMID:Metabolism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and human hepatic preparations. 962 79

Plant 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR; EC 1.1.1.34) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) and synthetic peptides designed from the known phosphorylation sites of plant HMGR (SAMS*: KSHMKYNRSTKDVK), rat acetyl-CoA carboxylase (SAMS: HMRSAMSGLHLVKRR), spinach SPS (SP2: GRRJRRISSVEJJDKK), and spinach NADH:nitrate reductase (NR6: GPTLKRTASTPFJNTTSK) were used to characterize kinase activities from cauliflower (Brassica oleracea L. ) inflorescences. The three major peaks of protein kinase activity resolved by anion-exchange FPLC are homologs of those observed previously in spinach leaves and thus are designated PKI, PKIV, and PKIII, listed in order of elution. PKIV was the most active in terms of phosphorylation and inactivation of recombinant Nicotiana HMGR and was also strictly Ca2+ dependent. The novel aspects are that PKIII has not been detected in previous cauliflower studies, that SAMS* is a more specific peptide substrate to identify potential HMGR kinases, and that the major HMGR kinase in cauliflower is Ca2+ dependent. Of the three major kinases that phosphorylated the SP2 peptide only PKI (partially Ca2+ sensitive) and PKIII (Ca2+ insensitive) inactivated native spinach leaf SPS. Cauliflower extracts contained endogenous SPS that was inactivated by endogenous kinase(s) in an ATP-dependent manner and this may be one of the substrate target proteins for PKI and/or PKIII. The substrate specificity of the three kinase peaks was studied using synthetic peptide variants of the SP2 sequence. All three kinases had a strong preference for peptides with a basic residue at P-6 (as in SP2 and SAMS*; SAMS has a free amino terminus at this position) or a Pro at P-7 (as in NR6). This requirement for certain residues at P-6 or P-7 was not recognized in earlier studies but appears to be a general requirement. In plant HMGR, a conserved His residue at P-6 is involved directly in catalysis and this may explain why substrates reduced HMGR phosphorylation in vitro.
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PMID:3-Hydroxy-3-methylglutaryl-coenzyme A reductase kinase and sucrose-phosphate synthase kinase activities in cauliflower florets: Ca2+ dependence and substrate specificities. 967 40

In order to characterize the hepatitis B virus (HBV) hepatocellular receptor, several proteins have previously been identified in HepG2 hepatoma cells and in primary cultured normal human hepatocytes (PCHs) that reacted with an anti-idiotypic antibody against a preS1(21-47)-specific MAb (F35.25). Here, we report the identification of one of these preS1-binding proteins, a 35 kDa protein (preS1-BP35), as glyceraldehyde-3-phosphate dehydrogenase (GAPD). GAPD is well-known as a key enzyme involved in glycolysis and gluconeogenesis. Nevertheless, GAPD has also been shown to have many other functions such as protein kinase activity (GAPD-PK). HBV core particles derived from infected hepatocytes possess an associated kinase activity that phosphorylates HBcAg, and the nucleocapsid may acquire sequential functions through selective phosphorylation. Therefore, we have investigated the potential role of GAPD-PK in HBV replication. In this study, we found that the endogenous PK associated with human liver-derived HBV core particles (hL-HBcAg) and GAPD-PK were sensitive to the same types of inhibitors. Interestingly, capsid protein phosphorylation decreased in a concentration-dependent manner (at concentrations of 5-30 mM) in the presence of specific inhibitors for GAPD-PK (NADH and GAP). Furthermore, we demonstrated in vitro that GAPD-PK could phosphorylate the major core protein P22 in hL-HBcAg particles. The data suggest that GAPD is an additional cellular kinase which might interfere in the life-cycle of HBV.
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PMID:Phosphorylation of the hepatitis B virus core protein by glyceraldehyde-3-phosphate dehydrogenase protein kinase activity. 968 Jan 29

It is still a matter of debate, whether decreased protein expression of SERCA 2a and phospholamban (PLB), or alterations in the phosphorylation state of PLB are responsible for the reduced SERCA 2a function in failing human myocardium. Thus, in membrane preparations from patients with terminal heart failure due to idiopathic dilated cardiomyopathy (NYHA IV. heart transplants) and control hearts (NF), SERCA 2a activity was measured with an NADH coupled assay with as well as without stimulation with protein kinase A (PKA). The protein expression of SERCA 2a, PLB and calsequestrin as well as the phosphorylation status of PLB (Back-phosphorylation technique: Serine-16-PLB specific antibody) were analysed using Western blotting technique and specific antibodies. In NF, the maximal activity (Vmax) and the Ca(2+)-sensitivity of SERCA 2a activity were significantly higher compared to NYHA IV. Protein expression of SERCA 2a, PLB and calsequestrin were unchanged, whereas both, the phosphorylation status of PLB as well as serine-16-PLB-phosphorylation, were significantly reduced in NYHA IV. After stimulation with PKA only the Ca(2+)-sensitivity, but not Vmax increased concentration-dependently. Therefore, in human myocardium, the Ca(2+)-sensitivity but not the Vmax of SERCA 2a is regulated by cAMP-dependent phosphorylation of phospholamban at position serine-16. Threonine-17-PLB-phosphorylation or direct phosphorylation of SERCA 2a may be candidates for regulation of maximal SERCA 2a activity in human myocardium.
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PMID:Reduced Ca(2+)-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation. 1019 80

It is unclear whether decreased protein expression of SERCA2 (SR-Ca(2+)-ATPase) and phospholamban (PLB), or alterations in the phosphorylation state of PLB leading to increased inhibition of SERCA2 are responsible for the reduced SERCA2 function in failing human myocardium. In crude membrane preparations from patients with terminal heart failure due to idiopathic dilated cardiomyopathy (DCM) and control hearts (NF), SERCA2 activity was measured with a NADH coupled assay. Protein expression of SERCA2 and PLB and the phosphorylation state at the two phosphorylation sites, serine-16-PLB and threonine-17-PLB, were investigated with specific (phosphorylation) antibodies and Western blot technique. In NF, the Vmax and the Ca2+ sensitivity of SERCA2 activity were significantly higher compared to DCM. Protein expression of SERCA2 and PLB were unchanged, whereas the phosphorylation status at both serine-16-PLB and threonine-17-PLB were significantly reduced in DCM. The native phosphorylation status of PLB measured by the back-phosphorylation technique was reduced in DCM as well. After stimulation with protein kinase A only the Ca2+ sensitivity, but not Vmax, increased. The reduced phosphorylation state of PLB may lead to decreased Ca2+ sensitivity of SERCA2 in failing human myocardium. The altered regulation of the SR-CA(2+)-ATPase in human heart failure may offer an opportunity for an improvement in the therapy of heart failure.
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PMID:cAMP-dependent protein kinase A-stimulated sarcoplasmic reticulum function in heart failure. 1060 52

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a Km of 2 mM for 5'-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the Km of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD+, whereas, the Km was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in Vmax with no change in Km. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.
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PMID:Isolation and regulation of piglet cardiac AMP deaminase. 1063 Jun 34


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