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)

A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.
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PMID:A microtiter-based assay for the detection of protein tyrosine kinase activity. 170 96

An inhibitor of protein kinases, staurosporine (ssp), was found to affect the endocytic pathway of asialoglycoproteins subsequent to endocytosis in monolayer cultures of rat hepatocytes. The effect of 5 or 10 microM staurosporine on the internalization of a synthetic ligand (galBSA-HRP: bovine serum albumin exposing galactose, horseradish peroxidase conjugates) prebound to the cell surface was minimal. The presence of 5, 7, or 10 microM ssp during a 1-h chase period resulted in the ligand remaining in a low density (1.04-1.05 g/ml), nonlysosomal subcellular fraction in a Percoll gradient. The ligand, arrested by 7 microM ssp, was further processed to the lysosome during subsequent incubation in the absence of ssp. Cells maintained the ability to internalize ligand at 37 degrees C for 1 h in the presence of these concentrations of ssp. During a 1-h continuous uptake of 0-50 micrograms/ml nonlabeled ligand, the presence of 7 microM ssp did not cause any decrease in the amount of asialoglycoprotein receptor at the cell surface, which indicates receptor recycling occurred normally. These results suggest a possible involvement of protein kinase(s), which can be inhibited by ssp, in the delivery of endocytosed ligand to the lysosome, but not in ligand endocytosis and receptor recycling.
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PMID:An inhibitory effect of a protein kinase inhibitor, staurosporine, on delivery of endocytosed asialoglycoprotein to lysosome in monolayer culture of rat hepatocytes. 195 54

We reported previously that, following phosphorylation by cyclic AMP-dependent protein kinase, tyrosine hydroxylase in rat corpus striatal extracts is inactivated in a time-dependent and apparently irreversible fashion. Removal of low molecular weight substances from these extracts by gel filtration attenuates this inactivation. We tried to determine the identity of endogenous metabolites that promote inactivation of tyrosine hydroxylase under our experimental conditions. In the present study, we report that the reducing co-substrate tetrahydrobiopterin and its analogues promoted this irreversible inactivation. The concentration that produced a 50% loss of activity (at 20 min) of the phosphorylated enzyme was 0.7 microM and that for the unphosphorylated enzyme was 420 microM. Using enzyme purified from a rat pheochromocytoma, we found that tyrosine, alpha-methyl-p-tyrosine, and a 3-iodotyrosine protected the phosphorylated enzyme against the inactivation produced by tetrahydrobiopterin. Catecholamines (dopamine, norepinephrine, epinephrine, and some of their analogues) also nullified inactivation. In contrast, the product of the reaction, dihydroxyphenylalanine, failed to attenuate the inactivation process. We performed several studies to ascertain the mechanism of inhibition by tetrahydrobiopterin. We considered the possibility that it formed reactive free radicals that produced inhibition. Free radical scavengers, however, failed to block the inhibition produced by tetrahydrobiopterin. Superoxide dismutase, catalase, and peroxidase also failed to protect tyrosine hydroxylase against inactivation. Moreover, when the experiments were performed under anaerobic conditions, the inactivation process was unaffected. These results suggest that reactive oxygenated species were not required for inactivation by tetrahydrobiopterin.
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PMID:Inactivation of tyrosine hydroxylase by pterin substrates following phosphorylation by cyclic AMP-dependent protein kinase. 197 41

Monoclonal antibody YC10 showed specificity for the phosphorylated form of human, bovine and porcine glial fibrillary acidic proteins (GFAPs) and negligible reactivity towards the dephosphorylated form of the GFAPs. Analysis of species specificity and of the epitope, determined using synthetic phosphopeptides, indicated that this antibody recognized the local phosphorylation-site sequence Thr-phosphoSer-Ala-Ala-Arg-Arg (residues 7-12 of GFAP). Making use of this antibody we developed a non-radioactive method to measure protein kinase activities. After incubation of a protein kinase with non-radioactive ATP in ninety-six wells coated with the synthetic peptide Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser-Cys (residues 3-13 of GFAP), the phosphorylated product was detected by using this mouse antibody and peroxidase-labeled goat anti-mouse IgG. This method proved to be equally as sensitive as the radioactive method for the measurement of protein kinase activities and was less affected by concentrations of ATP present in the reaction mixture.
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PMID:A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities. 202 46

We have demonstrated that anterior pituitary corticotropes can be identified cytochemically by their capacity to bind potent biotinylated analogs of CRH. In addition, 50-80% of corticotropes bind biotinylated arginine vasopressin (AVP). The percentage of CRH-bound cells is rapidly reduced after 1-h exposure to glucocorticoids. However, the rapid effects of glucocorticoids on AVP binding by corticotropes have not been tested. The first aim of this study was to examine the binding capacity of small and large corticotropes enriched to 90% by counterflow centrifugation. Biotinylated analogs of CRH or AVP were detected cytochemically on the cells by avidin-biotin-peroxidase complex protocols. At least 80% of the cells bound CRH after 1 day of culture. More large corticotropes bound AVP (93%) than small corticotropes (80%). AVP pretreatment of large corticotropes stimulated an increase in CRH-bound cells to over 90%, but it had no effect on CRH binding by small corticotropes. Corticosterone pretreatment (100 nM) for 10 min caused a 50% reduction in the percentage of cells that bound CRH and in the levels of ACTH released in response to biotinylated CRH. After 30 and 60 min of pretreatment, the percentages of CRH-bound cells were reduced by 75%, and ACTH levels remained low. No reduction in percentages of AVP-bound cells was evident at any time point after corticosterone pretreatment. These studies stimulated further tests based on previous reports that showed that AVP or its activated second messengers enhanced CRH binding. We reasoned that this potentiation might promote a recovery in CRH binding to corticosterone-inhibited cells. However, 1-h stimulation by AVP or activation of calcium channels (by Bay K 8644) or protein kinase-C by 12-O-tetradecanoyl-phorbol-13-acetate did not restore CRH binding. AVP evoked a partial recovery in ACTH release. Furthermore, Bay K and 12-O-tetradecanoyl-phorbol-13-acetate pretreatment effectively blocked the fast feedback effects of corticosterone on CRH-mediated ACTH release. Thus, these studies demonstrate that glucocorticoids rapidly inhibit CRH-receptor binding in a domain that is not affected by AVP potentiation of ACTH release. Perhaps they immobilize transport processes needed to bring unoccupied CRH receptors to the surface for binding and cytochemical detection.
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PMID:Rapid corticosterone inhibition of corticotropin-releasing hormone binding and adrenocorticotropin release by enriched populations of corticotropes: counteractions by arginine vasopressin and its second messengers. 215 74

Tryptophan hydroxylase is activated in a crude extract by addition of ATP and Mg2+. This activation is reversible and requires in addition both Ca2+ and calmodulin. Thus, phosphorylation by an endogenous calmodulin-dependent protein kinase has long been suspected. Now that we have prepared a specific polyclonal antibody to rat brain tryptophan hydroxylase, we have been able to prove that this hypothesis is correct. After incubation of purified tryptophan hydroxylase with Ca2+/calmodulin-dependent protein kinase together with [gamma-32P]ATP, Mg2+, Ca2+, and calmodulin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting of the enzymes onto nitrocellulose sheets, we could label the band of tryptophan hydroxylase by the antiserum and the peroxidase technique and show by autoradiography that 32P was incorporated into this band. By measuring the radioactivity, we calculated that about 1 mol of phosphate was incorporated per 8 mol of subunits of the enzyme (2 mol of native enzyme). Because the concentration of ATP which we employed (50 microM) gives about half-maximal activation in crude extract compared to saturating ATP conditions (about 1 mM), this result indicates that the incorporation of at least 1 mol of phosphate/mol of tetramer of native tryptophan hydroxylase is required for maximal activation.
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PMID:Formal demonstration of the phosphorylation of rat brain tryptophan hydroxylase by Ca2+/calmodulin-dependent protein kinase. 254 52

Isolated yeast nuclei were able to incorporate 3H-labeled pJDB219 DNA in vitro in the presence of ATP and Mg2+. The number of plasmid molecules incorporated into each nucleus was calculated to be 60 under the conditions we used. Enzyme-histochemical staining of the incorporated biotinylated pJDB219 with streptavidin-biotinylated-peroxidase complex indicated a uniform distribution of the incorporated plasmids within each nucleus. After intranuclear incorporation, substrate pJDB219 DNAs (open and closed circular forms) were changed to the linear form and were weakly digested over the longer incubation period (over 60 min). Facile release of the once-incorporated plasmid DNA was never observable; discharge of the incorporated [3H]pJDB219 during a 60-min incubation was less than 5%. The addition of adenylyl-imidodiphosphate, N,N'-dicyclohexylcarbodiimide (DCCD), or quercetin inhibited in vitro DNA uptake reaction. DCCD and quercetin inhibited the nuclear ATPase and apparent protein kinase, respectively; hence, the involvement of these enzymes in the nuclear DNA transport system was suggested.
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PMID:Characterization of a DNA uptake reaction through the nuclear membrane of isolated yeast nuclei. 282 13

This is the first report to show that pancreatic islet cells generate H2O2 and this H2O2 generation is regulated synergistically by cytoplasmic free calcium ([Ca2+]i) and protein kinase-C. Effects of calcium ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate (TPA), a tumor promoter, on H2O2 generation were studied in whole pancreatic islets obtained from male Wistar rats. We employed A23187 to elevate cytoplasmic free calcium, and TPA to activate protein kinase-C and monitored continuously their effects on H2O2 generation, measured using homovanillic acid and horseradish peroxidase. A23187 stimulates H2O2 generation. TPA, which activates protein kinase-C, augments this A23187-stimulated H2O2 generation. H2O2 generation is stimulated by an increase in [Ca2+]i and regulated synergistically by [Ca2+]i and protein kinase-C.
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PMID:Hydrogen peroxide generation in whole rat pancreatic islets; synergistic regulation by cytoplasmic free calcium and protein kinase-C. 313 92

Purified bovine brain calmodulin was biotinylated with biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidin-alkaline phosphatase procedures. Purified calcineurin was subjected to one- and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulin-binding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulin-binding proteins.
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PMID:A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin. 386 79

The authors investigated the immunohistochemical localization of S-100 protein in normal human brain and glioblastoma tissues by the peroxidase anti-peroxidase method of Sternberger. In normal human brain the positive immunoperoxidase reaction for S-100 protein was observed in astrocytes, oligodendrocytes, ependymal cells, Bergmann's glial cells and epithelial cells of choroid plexus. No positive staining was revealed in any cortical neurons. Immunoelectron-microscopically, the electron dense positive reaction for S-100 protein was seen throughout the cytoplasm, nucleoplasm and cell processes of astrocyte as well as oligodendrocyte. The positive reaction for S-100 protein was demonstrated occasionally in association with cytoplasmic membrane or the membrane constituting cell organelles. We suspect that this observation indicates the existence of membrane-bound form of S-100 protein. In glioblastoma cells, the positive reaction for S-100 protein was relatively weak in intensity as compared with astrocytes, and the degree of positive staining varied from cell to cell. Subcellular localization of S-100 protein in glioblastoma seemed to be essentially similar to that of normal astrocyte. There are some recent reports concerning immunohistochemical localization of alpha and beta subunits of S-100 protein. As compared with these reports, the present immunohistochemical results indicate that the rabbit anti-S-100 antibody embloyed in the present study is mainly against beta subunit of S-100 protein. Although there have been many reports concerning immunohistochemical localization of S-100 protein, the biological role of S-100 protein is still speculative. Some hypotheses are advocated in connection with the possible biological role of S-100 protein. For example, the modulation of synaptic transmission by S-100 protein, the participation of S-100 protein in hormonal secretion and in transport of cations through lipid membrane, the activation of protein kinase and the promotion of disassembly of microtubules by S-100 protein are postulated. It is hard to assume the biological role of S-100 protein based on the immunohistochemical results alone. The present study clearly indicates that S-100 protein exists widely in the cytoplasm, nucleoplasm, cytoplasmic membrane, outer membranes of cell organelles and cell processes of glial cells as well as glioblastoma cells. From these results we assume that S-100 protein plays an important role of intracellular transport of cations as one of the calcium binding proteins.
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PMID:[Immunohistochemical study of S-100 protein in the normal human brain and glioblastoma]. 391 99


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