Search PubMed⌕ Search

Biomedical subjects

A Ichiyama

Publications and source records attributed to A Ichiyama.

At least 91 records · Page 5Linked to original sources
PubMed1983

Muscarinic receptors in porcine caudate nucleus. I. Enhancement by nickel and other cations of [3H]cis-methyldioxolane binding to guanyl nucleotide-sensitive sites.

Muscarinic acetylcholine receptors in the synaptic membrane fraction of porcine caudate nucleus were characterized by using a radiolabeled agonist, [3H]cis-methyldioxolane [( 3H]CD) and an antagonist, [3H]quinuclidinyl benzilate [( 3H]QNB). Scatchard analysis of the specific binding of [3H]CD gave a single equilibrium dissociation constant of 8.1 nM when a concentration of less than 80 nM [3H]CD was used. The binding capacity was 390 fmoles/mg of protein and corresponded to about 10% of the binding sites of [3H]QNB. Agonist/[3H]CD competition binding experiments indicated that [3H]CD was selectively bound to the sites with a high affinity for agonists. [3H]CD binding was inhibited by Na+, K+, Mg2+, and Ca2+ with the half-maximal effect at 10-50 mM. Nickel ion showed biphasic effects on [3H]CD binding: a 2- to 3-fold enhancement of binding at 0.1-10 mM and inhibition above 10 mM. Other cations, including Co2+, Mn2+, and Zn2+, at 1 mM also increased [3H]CD binding by a factor of 1.5-1.8. Among 18 cations examined, only Cd2+, Hg2+, and Cu2+ caused significant inhibition of [3H]CD binding at 1 mM. [3H]CD binding was decreased to about 20% of the control value in the presence of guanylyl-5'-imidodiphosphate (GppNHp), GTP, and GDP with the half-maximal effect at 1.3, 32, and 45 microM, respectively. [3H]CD binding in the presence of Ni2+ was decreased by GppNHp to a level obtained in the presence of GppNHp alone. The increase caused by Ni2+ in [3H]CD binding was due to the increase in the maximal binding capacity (Bmax) without changes in the affinity for [3H]CD. We conclude that Ni2+ increases the proportion of a muscarinic receptor subclass (or state) that is sensitive to guanyl nucleotide.

Animals↗
PubMed1983

Muscarinic receptors in porcine caudate nucleus. II. Different effects of N-ethylmaleimide on [3H]cis-methyldioxolane binding to heat-labile (guanyl nucleotide-sensitive) sites and heat-stable (guanyl nucleotide-insensitive) sites.

Heat treatment of membranes from porcine caudate nucleus (50 degrees for 7 min) caused a marked decrease in [3H]cis-methyldioxolane [( 3H]CD) binding without affecting seriously the binding of [3H]3-quinuclidinyl benzilate [( 3H]QNB). Approximately 20% of the [3H]CD binding at 5 nM [3H]CD remained after the heat treatment. The remaining binding was not affected by 0.1 mM guanylyl-5'-imidodiphosphate (GppNHp) or by nickel or other cations at concentrations below 10 mM. Treatment of the membranes with trypsin (30 micrograms/mg of protein) at 20 degrees for 20 min also caused a marked decrease in [3H]CD binding without affecting seriously the binding of [3H]QNB. About 20% of the original [3H]CD binding remained in the presence of trypsin at a high concentration of protein (90 micrograms/mg). N-Ethylmaleimide (NEM) affected [3H]CD binding in two different ways: (a) preincubation of the membranes with NEM caused a marked reduction in heat- and GppNHp-sensitive [3H]CD binding, and (b) treatment with NEM caused an enhancement of heat-, GppNHp-, and trypsin-insensitive [3H]CD binding. Neither of the NEM effects required the coexistence of agonists. The concentration of NEM required for the first effect was 10 times lower than that for the second effect, indicating the existence of two NEM-binding sites with different affinities for NEM. The equilibrium dissociation constant (Kd) for [3H]CD after NEM treatment was 33 nM and was not affected by GppNHp, Ni2+, or heat treatment; the Kd was only 4 times higher than that (8 nM) without NEM treatment. These findings indicated the existence of two kinds of [3H]CD binding sites with high affinities for agonists: one is sensitive to guanyl nucleotide and is abolished by NEM and the other is induced by NEM and insensitive to guanyl nucleotide.

Animals↗
PubMed1982

Tryptophan hydroxylase from mouse mastocytoma P-815. Reversible activation by ethylenediaminetetraacetate.

Tryptophan hydroxylase from mouse mastocytoma P-815 is activated by ethylenediaminetetraacetate (EDTA). The activation proceeds in a pH-, temperature-, and time-dependent manner and leads to a 30-fold higher activity at maximum. The optimal EDTA concentration is 10 microns. The activation requires solely EDTA with desalted crude enzyme solution in the absence of any cellular metabolites. There are no indications that the activation is due to proteolysis, modification of protein-bound sulfhydryl groups or other covalent modifications such as phosphorylation and methylation. In the presence of appropriate stabilizing agents, the activated state is maintained after the removal of EDTA by gel-filtration. The activation is reversible under conditions in which bound metal(s) is dissociated from the complex with EDTA. These results imply that the role of EDTA is metal chelation. A model is proposed in which the enzyme has at least two interconvertible states, the activated state and ground state, corresponding to the free and metal-bound forms, respectively. The metal is probably derived from the cell. The assay method for tryptophan hydroxylase utilized a rapid and sensitive (5 pmol/injection) determination of 5-hydroxytryptophan by high performance liquid chromatography.

Animals↗
PubMed1981

The activity of rat pineal and brain tyrosine hydroxylase during the daily cycle of light and darkness as determined by the modified 14CO2 assay method.

A previous published assay method for tyrosine hydroxylase by the evolution of 14CO2 was modified to a two-step procedure to allow reliable measurement of large numbers of samples containing low tyrosine hydroxylase activity. The reliability of the method was examined in detail. Properties of rat brain and pineal tyrosine hydroxylase solubilized with 0.2% Triton X-100 were as follows. The apparent Km values of the brain enzyme for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-L-erythro-biopterin (BPH4) as cofactor and for BPH4 with 62 microM-L-tyrosine as substrate were approximately 25 microM and 85 microM, respectively. The Km's for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-6-methylpterin (6MPH4) as cofactor and for 6MPH4 with 210 microM-L-tyrosine as substrate were 68 microM and 270 microM, respectively. The marked substrate inhibition by high concentrations of L-tyrosine was observed only when BPH4 was used as cofactor. High concentrations of BPH4 inhibited the reaction slightly. The kinetic properties of tyrosine hydroxylase in the pineal extract were similar to those of the brain enzyme, except that a Lineweaver-Burk plot of reciprocal velocity versus the reciprocal concentration of BPH4 with 62 microM-L-tyrosine as substrate deviated downward at a BPH4 concentration of about 100 microM. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at two independent sites or with two forms (6L- and 6D-isomers) of the tetrahydrobiopterin cofactor, with apparent Km for BPH4 of 23 microM and 1025 microM, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. Based on the results obtained as reported above the standard assay conditions of tyrosine hydroxylase in tissue extracts were established. Using the assay method and conditions, the absence of the daily rhythmicity of tyrosine hydroxylase in rat pineal glands and three discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline turnover.

Animals↗
PubMed1980

Studies on biologically active pteridines. III. The absolute configuration at the C-6 chiral center of tetrahydrobiopterin cofactor and related compounds.

Tetrahydrobiopterin, the natural pteridine cofactor for aromatic amino acid hydroxylases, was produced stereopecifically with reference to the C-6 chiral center from 7,8-dihydrobiopterin by the action of dihydrofolate reductase. Similarly, 7,8-dihydro-6-methylpterin was reduced to tetrahydro-6-methylpterin having the same 6-configuration by the same enzyme. The absolute configuration of these tetrahydropterins at the C-6 chiral center was determined to be L, as in (2S)-1,2,3,4-tetrahydro-2-methylquinoxaline, which was servied from L-alanine, by comparison of the CD spectra.

Biopterins↗
PubMed1978

Immunochemical studies on induction of rat liver mitochondrial serine: pyruvate aminotransferase by glucagon.

The 7- to 10-fold increase in the rat liver serine:pyruvate aminotransferase activity after glucagon administration was shown to occur mainly in the mitochondrial matrix of parenchymal cells. The enzyme was purified from glucagon-treated rat liver mitochondria to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A specific rabbit antibody was prepared against the purified enzyme. Upon Ouchterlony double diffusion analysis, the mitochondrial extracts of glucagon-treated rat liver produced a single and fused precipitin line between the purified enzyme against the antibody. The supernatant fraction of glucagon-treated rat liver and the mitochondrial extracts of normal liver were also shown to make a single and fused precipitin line with the purified enzyme, when applied in large quantities. The quantitative immunotitration demonstrated that the glucagon-induced increase in the activity of liver serine:pyruvate aminotransferase were accompanied by the parallel increase in the amount of the enzyme antigen. Isotopic leucine incorporation studies showed that the relative rate of synthesis of the enzyme was increased approximately 10-fold by glucagon administration under the conditions employed. The rate of the degradation of the aminotransferase in the normal rat liver was a relatively slow process with a half-life of approximately 30 h. Thus the accumulation of serine:pyruvate aminotransferase in rat liver mitochondria by glucagon treatment can be ascribed mainly to the rise in the rate of enzyme synthesis.

Animals↗
PubMed1976

Some properties of bovine pineal tryptophan hydroxylase.

Bovine pineal tryptophan hydroxylase is a pterin-dependent aromatic amino acid monooxygenase with a broad substrate specificity and with low Km values for the amino acid substrates, L-tryptophan and L-phenylalanine. p-Chlorophenylalanine, an inhibitor of the tryptophan hydroxylation in the brain, also serves as a good substrate of the bovine pineal enzyme. The full activity of this enzyme is detected in vitro only after preincubation with dithiothreitol under reductive conditions. The enzyme is profoundly and more or less specifically inhibited by L-5HTP, the product of tryptophan hydroxylation, suggesting a possible regulatory role of this hydroxylated amino acid. The Km of the enzyme for tetrahydrobiopterin, a presumed natural cofactor, is significantly higher than the expected tissue concentration if the cofactor is assumed to be uniformly distributed in the tissue. These properties of bovine pineal tryptophan hydroxylase are distinguishable from those of the hydroxylase in the brain indicating that the hydroxylation of tryptophan in the brain leading to the synthesis of serotonin and the reaction in the pineal gland leading to the formation of melatonin are catalyzed by different tryptophan hydroxylases and are probably under separate regulatory mechanisms.

Animals↗
PubMed1975

Enzymic studies on adrenocortical deoxycorticosterone 11beta-hydroxylase system.

Radiometric methods for the assay of deoxycorticosterone 11beta-hydroxylase and for the determination of NADP on a microscale were developed. The determination of NADP was based on the quantitative conversion of 6-phospho[1-14C]gluconate to 14CO2 by the action of 6-phosphogluconate dehydrogenase. Using these methods NADPH oxidase activity of the adrenodoxin reductase-adrenodoxin system as well as kinetic properties of deoxycorticosterone 11beta-hydroxylase (cytochrome P-450) were investigated. The NADPH oxidase activity observed in the presence of adrenodoxin reductase, adrenodoxin, and O2, but in the absence of cytochrome P-450 and deoxycorticosterone, were functions of O2 and adrenodoxin concentrations and represented the autooxidation of reduced adrenodoxin which resulted in the production of H2O2. Due to the rapid autooxidizability of reduced adrenodoxin, only a small fraction of electrons conveyed from NADPH to adrenodoxin by way of adrenodoxin reductase was utilized for the deoxycorticosterone 11beta-hydroxylase reaction under the conditions employed.

Adrenal Cortex↗