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T Kuno

Publications and source records attributed to T Kuno.

At least 127 records · Page 7Linked to original sources
PubMed1989

The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A.

A complementary DNA (cDNA) clone encoding the catalytic subunit of calcineurin (calcineurin A) has been isolated from a rat brain cDNA library. The primary structure of the cDNA consists of 2,337 nucleotides including the entire coding region for 521 amino acids, and the calculated molecular mass is 58,643 Da. The calcineurin A is strikingly homologous to protein phosphatases 1 and 2A, approximately 50% of the amino acids over an internal 250-residue region between residues 78 and 329 being identical. Twenty four amino acid-residue region between residues 391 and 414 shows the consensus structural features for a calmodulin-binding domain. These data suggest that the allosteric character of this chimeric enzyme is generated by gene fusion of two separate protein families.

Amino Acid Sequence↗
PubMed1989

Activation of type I cyclic AMP-dependent protein kinases is impaired by a point mutation in cyclic AMP binding sites.

The type I regulatory (R-I) subunit of cyclic AMP-dependent protein kinase (A-kinase) was expressed in E. coli, and a single amino acid substitution in cyclic AMP binding sites A or B was introduced by site-directed mutagenesis. The cyclic AMP binding activity and cyclic AMP-stimulated phosphotransferase activity of the holoenzymes formed by wild-type or mutant R-Is and the purified bovine catalytic subunit of A-kinase were then examined. The wild-type holoenzyme was activated by low concentrations of cyclic AMP, a finding in accord with its high-affinity binding to cyclic AMP. In contrast, although the two mutant holoenzymes showed high-affinity cyclic AMP binding at their non-mutated sites, both holoenzymes were resistant to activation by cyclic AMP. Thus, binding of cyclic AMP to the non-mutated cyclic AMP binding site is not sufficient to dissociate the catalytic subunit from the mutant R-Is upon cyclic AMP binding. These results suggest that both A and B cyclic AMP binding sites are required for efficient coupling between cyclic AMP binding and activation of the enzyme.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence.

Protein kinase C normally has a tandem repeat of a characteristic cysteine-rich sequence in C1, the conserved region of the regulatory domain. These sequences resemble the DNA-binding zinc finger domain. For the gamma subspecies of rat brain protein kinase C, various deletion and point mutants in this domain were constructed, and the mutated proteins were expressed in Escherichia coli by using the T7 expression system. Radioactive phorbol 12,13-dibutyrate binding analysis indicated that a cysteine-rich zinc-finger-like sequence was essential for protein kinase C to bind phorbol ester and that one of two sequences was sufficient for the phorbol ester binding. Conserved region C2, another region in the regulatory domain, was apparently needed for the enzyme to require Ca2+ for phorbol ester binding activity.

Amino Acid Sequence↗
PubMed1989

A rapid filtration assay for cAMP.

The receptor-binding assay for cAMP was improved by using polyethylenimine-treated glass filters. A polyethylenimine-treated glass filter has high protein binding capacity. This high capacity allows an increase in the amount of protein per assay tube and the use of a crude preparation, such as a beef heart extract, as specific binding protein instead of a purified protein, which has been used in the classical filtration assays involving cellulose ester filters. Since the time required for the separation of the protein-cAMP complex and the free nucleotide can be shortened by the use of polyethylenimine-treated filters, the dissociation of the bound ligand during the separation procedure, which is a serious problem with other modified assay methods involving charcoal adsorption, is minimized. Filtration through polyethylenimine-treated glass filters also gives low blanks and prevents the loss of protein or ligand due to breakage of the filters, which is often observed with fragile cellulose ester membranes. In consequence, this simple and rapid filtration assay allows more accurate and reproducible determinations.

Animals↗
PubMed1989

Molecular cloning of cDNA for rat brain metallothionein-II and regulation of its gene expression.

A rat brain metallothionein-II (MT-II) complementary DNA (cDNA) clone was isolated from a cDNA plasmid library, which was prepared from non-treated rat brain mRNA, by a colony screening procedure using 32P-labeled synthetic oligonucleotide probes. It is deduced that the clone encodes for a protein of 61 amino acids comprising 20 cysteines, which is highly homologous to MT-IIs in other species. Northern blot analysis demonstrated major mRNA species in the brain, liver and kidneys (approximately 350 b in size), which is induced in response to dexamethasone, zinc, cadmium and mercury but not to methyl mercury. These findings confirm that MT-II genes are expressed and regulated both by steroid and heavy metals in the brain as well as in peripheral organs.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung.

A complementary DNA clone corresponding to the 70 kDa subunit of soluble guanylate cyclase (EC 4.6.1.2) of rat lung has been isolated. The primary structure of the cDNA consisted of 3063 nucleotides including a 1857-nucleotide coding region for 619 amino acids, and the calculated molecular weight was 70476. Blot hybridization of total poly(A)+RNAs from rat tissues detected a mRNA of about 3.4 kilobases. The amount of mRNA was abundant in lung, cerebrum and cerebellum, moderate in heart and kidney, and low in liver and muscle. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated the presence of one gene in the rat haploid genome. The amino acid sequence of the 70 kDa subunit has partial homology with particulate guanylate cyclase from sea-urchin sperm, and protein phosphatase inhibitor I.

Amino Acid Sequence↗
PubMed1988

Characterization of cyclic GMP-binding sites of cyclic GMP-dependent protein kinase by rapid filtration assay.

The kinetics of cyclic [3H]GMP binding to the purified cyclic GMP-dependent protein kinase (cG kinase) were studied by using the rapid filtration assay method with polyethyleneimine-treated glass filters (method A), and the data were compared with those of the (NH4)2SO4 precipitation procedure (method B), which has been used for many previous studies on cyclic GMP binding to cG kinase. Each method gave a similar stoichiometry of approx. 2 mol of cyclic GMP/mol of cG kinase subunit; however, other binding kinetics obtained with these two methods were different. The dissociation of bound cyclic [3H]GMP from the kinase showed a single slow component when method A was used, whereas rapid and slow dissociation components were observed with method B. The Scatchard plot of cyclic [3H]GMP binding with method A was linear with a Kd value of 11 +/- 2 nM, suggesting that the two intrachain binding sites have similar high affinity for cyclic GMP. Results obtained on cyclic nucleotide analogue specificity of the two intrachain cyclic GMP-binding sites were also different between these two methods. These findings suggest that cG kinase has two high-affinity cyclic GMP-binding sites per subunit in the native state, and that when (NH4)2SO4 is added, ostensibly to stop the binding reaction, one low-affinity site is created from one high-affinity site.

Animals↗
PubMed1988

Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Immunohistochemical localization of gamma-aminobutyric acid- and aspartate-containing neurons in the rat deep cerebellar nuclei.

The immunohistochemical localization of gamma-aminobutyric acid (GABA)- and aspartate-containing neurons was evidenced in the rat deep cerebellar nuclei, using purified antisera to GABA and aspartate, respectively. Most GABA-containing neurons had small cell bodies and were scattered unevenly throughout the deep cerebellar nuclei. The medial cerebellar nucleus had a few GABA-containing cell bodies and interpositus and lateral nuclei contained many GABA-containing cell bodies. GABA-containing terminals were distributed throughout the nuclei. A large number of various-sized aspartate-containing cells were present in the deep cerebellar nuclei and most of these cells were large or medium-size. Aspartate-containing cells in the medial, interpositus and lateral nucleus were surrounded by GABA-like immunoreactive terminals, thereby suggesting the modulation of aspartate-containing neurons by GABA-ergic fibers from Purkinje cells.

Animals↗
PubMed1988

Novel type of monoclonal antibodies against cyclic GMP and application to immunocytochemistry of the rat brain.

A novel type of monoclonal antibodies against cyclic GMP were produced to study the immunocytochemical distribution of cyclic GMP in the rat brain. Cyclic GMP conjugated to bovine serum albumin with glutaraldehyde was used as an immunogen, and monoclonal antibodies were produced. The one monoclonal antibody which did not crossreact against other nucleotides was applied to the immunocytochemistry of the rat brain. Cyclic GMP immunoreactivities were distributed unevenly in the rat brain. The cerebellar cortex, hippocampus and cerebral cortex contained a high degree of cyclic GMP immunoreactivity, while most of the white matter was not stained. In the cerebellar cortex, stellate cells and Golgi cells showed intense immunoreactivities, but granule cells showed weak immunoreactivities. Approximately 60-80% of the Purkinje cells showed intense immunoreactivities, while the remaining ones showed only weak staining. The pyramidal cells in the cerebral cortex and hippocampus also showed intense immunostaining. Some glial cells adjacent to the Purkinje cells also stained. The nuclei of cyclic GMP-immunoreactive cells were not stained. These immunocytochemical distributions of cyclic GMP are in fairly good agreement with reported the biochemical data and the immunocytochemical distribution of guanylate cyclase. These monoclonal antibodies should be helpful for elucidating the physiological role of cyclic GMP in the brain.

Animals↗
PubMed1987

Molecular cloning and cDNA structure of the regulatory subunit of type I cAMP-dependent protein kinase from rat brain.

Complementary DNA (cDNA) clones encoding the regulatory subunit of the type I cAMP-dependent protein kinase (R-I) were isolated by screening of rat brain cDNA libraries. A 1.5-kilobase (kb) cDNA insert containing the entire coding region was sequenced and full amino acid sequence has been deduced from the nucleotide sequence. The clone encodes for a protein of 380 amino acids that shows 97% homology to the bovine R-I subunit. Northern blot analysis demonstrated two major mRNA species (2.8 and 4.4 kb in size) in rat brain and liver.

Amino Acid Sequence↗
PubMed1987

Distribution of beta-adrenoceptors associated with cAMP-generating system in cat colon.

The localization of beta-adrenoceptor associated with the adenosine 3',5'-cyclic monophosphate (cAMP)-generating system was determined by an assessment of the smooth muscle cell contractility and by assay of cAMP in individual layers of the cat colon. Isoproterenol inhibited the acetylcholine (ACh)-induced contractions of isolated longitudinal and circular muscle cells in a dose-dependent manner. The response of the longitudinal muscle cells to isoproterenol was double that seen in the circular muscle cells, and this inhibition was antagonized by sotalol. The basal content of cAMP was 3.42 pmol/mg protein in longitudinal muscle layers, 3.09 pmol/mg protein in circular muscle layers, and 2.75 pmol/mg protein in Auerbach's plexus. Isoproterenol produced a dose-dependent elevation of cAMP levels in longitudinal and circular muscle layers, which were antagonized by sotalol and potentiated by theophylline. By contrast, isoproterenol had no effect on cAMP in Auerbach's plexus. Phenylephrine did not alter the cAMP levels in any layer. Therefore, beta-adrenoceptors associated with the cAMP-generating system probably exist in the longitudinal and circular muscles of the cat colon.

Acetylcholine↗
PubMed1987

Antimuscarinic effects of antihistamines: quantitative evaluation by receptor-binding assay.

Quantitative evaluation of antimuscarinic effects of antihistamines (H1- and H2-receptor antagonists) was carried out using a receptor-binding assay. The -inhibition constants (Ki values) of twenty seven H1-receptor antagonists, one related antidepressant and three H2-receptor antagonists at H1-receptors and muscarinic receptors in the bovine cerebral cortex were determined. All the H2-receptor antagonists examined showed very low affinity for the muscarinic receptors. On the other hand, some H1-receptor antagonists (mequitazine, cyproheptazine, clemastine, diphenylpyraline, promethazine, homochlorcyclizine and alimemazine) had high affinity for the muscarinic receptors (Ki = 5.0-38 nM). Another group of H1-receptor antagonists (mepyramine, terfenadine, metapyrilen, azelastine, hydroxyzine and meclizine) had low affinity for the muscarinic receptors (Ki = 3,600-30,000 nM). Thus, a broad range of antimuscarinic potencies among the antihistamines was demonstrated. These results should provide helpful information with regard to the clinical and experimental use of antihistamines.

Animals↗
PubMed1986

Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells.

In a previous study, we found that atriopeptin I was much weaker (EC50 greater than 500 nM) than atrial natriuretic factor (ANF-(8-33)) (EC50 = 0.3 nM) at increasing cyclic GMP in cultured endothelial cells. In this study, we used the cross-linking reagent disuccinimidyl suberate to investigate whether the differences in activity were due to the presence of multiple ANF receptors. When 98% of the ANF-binding sites on endothelial cells were occupied by tyrosine-atriopeptin I after cross-linking, there was no difference in the concentration-response curve to ANF-(8-33) with regard to cyclic GMP accumulation. In contrast, when 96% of the binding sites were occupied by cross-linked ANF-(8-33), a 60% decrease in the maximal cyclic GMP response was observed after the readdition of ANF-(8-33). These results suggest that ANF-(8-33) is binding to an additional site that atriopeptin I does not effectively bind. Affinity cross-linking of 125I-ANF to intact endothelial cells resulted in the labeling of two sites of Mr approximately 66,000 and approximately 130,000. Approximately 94% of the 125I-ANF binding sites had an Mr approximately 66,000. Labeling of this site was inhibited by both tyrosine-atriopeptin I (KI = 0.9 nM) and ANF-(8-33) (KI = 0.09 nM). Although 0.1 microM tyrosine-atriopeptin (AP I) inhibited labeling of the 66,000-dalton site to nearly the same degree as ANF-(8-33), it produced only a 4-fold increase in cyclic GMP compared to a 400-fold increase with ANF-(8-33). These results suggest that the 66,000-dalton site is not coupled to guanylate cyclase and cyclic GMP formation. Tyrosine-AP I (KI greater than 10 nM) was much weaker at competing for the 130,000-dalton site than ANF-(8-33) (KI = 0.075 nM). Because the EC50 for cyclic GMP stimulation for tyrosine-AP I (greater than 100 nM) and ANF-(8-33) (0.4 nM) is closer to the KI values for the 130,000-dalton protein, this site probably mediates the marked stimulation of cyclic GMP. Our results demonstrate that endothelial cells contain two binding sites for ANF-(8-33) and suggest that only the less abundant site (Mr approximately 130,000) is the receptor coupled to the activation of guanylate cyclase.

Animals↗
PubMed1986

Soluble guanylate cyclase from rat lung exists as a heterodimer.

The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.

Animals↗