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)

We have shown that nuclei isolated by two methods contain grossly different amounts of cyclic AMP-dependent histone kinase activity. Repeated washing of the isolated nuclei with a low ionic strength buffer removed the majority of the cyclic AMP-dependent histone kinase and cyclic AMP binding activity. Nuclear cyclic AMP-dependent histone kinase activity accounted for only 0.42% of the total cytoplasmic enzyme activity. Similarly, the lactate dehydrogenase activity associated with liver nuclei represented only 0.07% of the total cytoplasmic activity. The isolated liver nuclei contained only 0.27% of the total homogenate glutamate dehydrogenase activity and 1.7%of the total homogenate glucose-6-phosphatase activity. The cyclic AMP-dependent histone kinase behaves as a cytoplasmic rather than a nuclear enzyme. We have also shown that using crude extracts, one can achieve separation of the two nuclear casein kinases, NI and NII, on sucrose density gradients in the presence of 0.5M NaCl. Nuclear casein kinases NI and NII had sedimentation coefficients of 3.0 and 593 S, respectively, in the presence of 0.5 M NaCl. Under conditions of low ionic strength, all of the casein kinase activity in the crude nuclear extract sedimented as one peak with a seminentation coefficient of 7.3 S. The aggregation-disaggregation which occurred in the crude extract was reversible and was mainly due to the aggregative and disaggregative properties of casein kinase NII. The two nuclear casein kinases have different affinities for chromatin. When nuclei were disrupted in a hypotonic solution and extracted with a buffercontaining 0.14 M NaCl, casein kinase NII could be completely extracted from the viscous nuclear material. Although a significant amount of casein kinase NI was extracted by the buffer containing 0.14 M NaCl, re-extraction of the nuclear material with a buffer containing 0.5 M NaCl yielded substantial amounts of casein kinase NI, and a final extraction with a buffer containing 1.0 M NaCl yielded measurable amounts of casein kinase NI. No casein kinase NII activity could be detected in the 0.5 M and 1.0M NaCl extracts.
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PMID:Rat liver nuclerar protein kinases. 16 84

Results of this study demonstrate that vasopressin activates protein kinase in intact renal medullary cells as detected by measurement of the (-cyclic AMP/+cyclic AMP) protein kinase activity ratios in freshly prepared tissue extracts (40,000 X g supernates) from bovine renal medullary slices. The activation of protein kinase was specific for vasopressin since parathyroid hormone, histamine, angiotensin II, or the inactive analog of vasopressin did not activate protein kinase. There was a direct correlation between the extent of protein kinase activation and the elevation in tissue levels of cyclic AMP elicited by increasing doses of vasopressin or with an increase in incubation time. The elevation of tissue cyclic AMP level and maximum activation of protein kinase reached maximum level at a vasopressin concentration of about 2 X 10(-9) M. Incubation of slices with vasopressin caused a dose-dependent decrease in the cyclic AMP-dependent protein kinase activity in the 40,000 X g supernate of homogenate from the renal medullary slices. This effect of vasopressin was specific for protein kinase since activity of lactate dehydrogenase or a specific [3H]colchicine-binding activity was not affected, and the decrease in the protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase activity extracted from 40,000 X g pellets of homogenate prepared from slices exposed to vasopressin. Results thus provide evidence that cyclic AMP-mediated protein kinase activation in the intact cells is an integral part of cellular response of the mammalian renal medulla to vasopressin.
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PMID:Regulation of protein kinase by vasopressin in renal medulla in situ. 18 20

The involvement of calcium, ATP, and cyclic AMP-dependent protein kinase activity in the release of amylase from rat parotid glands was examined. Pretreatment of the glandular tissue in 11.25 mM Ca2+ medium potentiated the secretory responses to: dibutyryl cyclic AMP, elevation of the extracellular K+ concentration, reduction of the H+ concentration, La3+, and caffeine. Uncoupling of oxidative phosphorylation blocked release induced by dibutyryl cyclic AMP, K+, and reduction of H+, but had no effect on La3+, caffeine or tolbutamide-stimulated release. Inhibition of cyclic AMP-dependent protein kinase activity blocked only dibutyryl cyclic AMP-induced release and did not inhibit the responses to K+, reduction of H+ or caffeine. The loss of lactate dehydrogenase was used to access the integrity of the tissue during amylase release. No significant increase in the release of lactate dehydrogenase was observed during the secretory responses to: dibutyryl cyclic AMP, La3+, caffeine, or tolbutamide. Triton X-100 and ethanol increased the efflux of both amylase and lactate dehydrogenase. The differential involvement of Ca2+, ATP, and cyclic AMP-dependent protein kinase activity in amylase release induced by the various secretagogues suggests that three types of reactions are involved in the release of amylase.
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PMID:Amylase release from rat parotid glands. I. General characteristics. 22 Oct 43

The hypothesis was tested that it is possible to influence cellular responses of intact cells using synthetic peptide substrates, pseudosubstrates, and inhibitors of protein kinases. Using cytotoxic T-cells (CTL), we demonstrate here that some basic amino acid-containing synthetic peptide substrates of protein kinases [e.g., of cGMP-dependent protein kinase (peptide PKG-S), synthetic peptide inhibitor of cGMP-dependent protein kinase (peptide PKG-I), and peptide corresponding to the tyrosine phosphorylation site in pp60src (peptide RR-src)] were strongly inhibitory in T-cell receptor (TCR) and T-cell growth factor, interleukin 2 (IL-2)-triggered proliferation of CTL. These peptides also inhibited other cellular responses of CTL. Peptides which contain basic amino acids, but do not have substrate specificity determinants for protein kinase, were not inhibitory. The inhibition with peptides is not due to their toxicity, since no cell death was observed by the trypan blue exclusion test and by lactate dehydrogenase release. Use of the granule exocytosis assay provided opportunities to clarify the mechanism of the peptide action. Tested peptides inhibited not only cell-surface ligand-induced CTL activation, but also affected cell-surface receptor-independent CTL activation (granule exocytosis and gamma-interferon secretion) induced by the synergistic action of the protein kinase C activator (PMA) and ionophore A23187. It was found that minor changes in amino acid composition or amino acid position in the synthetic peptides dramatically change their ability to affect lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of the effector functions of cytolytic T-lymphocytes with synthetic peptide inhibitors of protein kinases. 161 67

We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B-50 (GAP-43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration-dependent manner by elevating the free Ca2+ concentration from 10(-8) to 10(-5) M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2(+)-induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2(+)-induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2(+)- and phorbol ester-induced release were not additive at higher phorbol ester concentrations (greater than 10(-7) M). We compared the sensitivities of Ca2(+)- and phorbol ester-induced release of noradrenaline to the protein kinase inhibitors H-7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B-50. Ca2(+)-induced release was inhibited by B-50 antibodies and polymyxin B, but not by H-7; phorbol ester-induced release was inhibited by polymyxin B and by H-7, but only marginally by antibodies to B-50.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Noradrenaline release from streptolysin O-permeated rat cortical synaptosomes: effects of calcium, phorbol esters, protein kinase inhibitors, and antibodies to the neuron-specific protein kinase C substrate B-50 (GAP-43). 182 43

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, we developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, "semipermeabilization," increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with gamma-[32P]ATP resulted in incorporation of 32Pi into two major protein bands [band "A" of apparent molecular mass (Mr) approximately equal to 66 kDa, and band "B" of Mr approximately equal to 45 kDa] detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in 32Pi incorporation into multiple protein bands. Incubation of MCT with 1 microM AVP resulted in increased 32Pi radioactivity in band A and decreased 32Pi radioactivity in band B. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.
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PMID:In situ phosphorylation of proteins in MCTs microdissected from rat kidney: effect of AVP. 283 21

Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.
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PMID:Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements. 302 81

A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.
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PMID:Protein phosphorylation in permeabilized pancreatic islet cells. 389 30

It is known that the ratio of isoenzyme 1 to total lactate dehydrogenase (LD, EC 1.1.1.27) in serum is increased in all patients with acute myocardial infarction within 24 h of the infarct. We now show that the LD-1/LD-2 ratio for serum more promptly indicates acute myocardial infarction, being for most patients equivalent to measurement of creatine kinase (EC 2.7.3.2) isoenzyme 2 (CK-2, CK-MB) in serum. Of 128 patients with a confirmed diagnosis of myocardial infarction, 66 had normal values for all "cardiac" enzymes at the time of admission, but greater than 75% of them showed a parallel increase in values for CK-2 and the LD-1/LD-2 ratio. Of the 26 patients who had one or more abnormal values for cardiac enzymes on admission, 95% showed a parallel increase in CK-2 and the LD-1/LD-2 ratio, the median time for the beginning of these changes being 9 h from the onset of chest pain. The remaining 36 patients were excluded from the study because CK-2 decreased after admission or because the time of onset of chest pain was uncertain.
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PMID:Changes in the ratio of lactate dehydrogenase isoenzymes 1 and 2 during the first day after acute myocardial infarction. 404 27

Enolase, lactate dehydrogenase, and phosphoglycerate mutase have previously been found to contain phosphotyrosine in fibroblasts transformed by Rous sarcoma virus, which encodes a tyrosine-specific protein kinase. However, these phosphorylations are not stoichiometric, and their significance for any aspect of the transformed phenotype is unknown. We show here that enolase and lactate dehydrogenase are each phosphorylated chiefly at a single tyrosine in Rous sarcoma virus-transformed cells. The purified enzymes can also be phosphorylated at the same tyrosine in vitro when incubated with an immunoprecipitated retroviral transforming protein having associated tyrosine protein kinase activity. The phosphorylated tyrosine in lactate dehydrogenase is amino acid 238. The phosphorylated tyrosine in enolase lies in a sequence homologous to that surrounding histidine 43 in yeast enolase. Although the phosphorylated sequence in lactate dehydrogenase shows some homology to those sequences surrounding phosphotyrosines found in retroviral transforming proteins, the phosphorylated sequence in enolase is quite different.
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PMID:Phosphorylation sites in enolase and lactate dehydrogenase utilized by tyrosine protein kinases in vivo and in vitro. 633 85


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