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A Ichiyama

Publications and source records attributed to A Ichiyama.

At least 55 records · Page 3Linked to original sources
PubMed1990

Interaction of deglycosylated muscarinic receptors with ligands and G proteins.

Endoglycosidase F was used to investigate the role of the carbohydrate moiety of muscarinic acetylcholine receptors in antagonist and agonist binding, and the interaction with G proteins. The receptors were purified from porcine cerebrum, treated with endoglycosidase F and then covalently labeled with [3H]propylbenzilylcholine mustard [( 3H]PrBCM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]PrBCM-labeled receptors showed that the endoglycosidase F treatment caused a decrease in apparent Mr from 70 to 51 kDa, the Mr predicted for the peptide portions of M1, M2 and M4 subtypes. Endoglycosidase F-treated receptors had essentially the same affinities for both agonists and antagonists as those of control receptors. In addition, treated receptors that had been reconstituted in lipid vesicles with G proteins showed guanine nucleotide-sensitive high affinity for agonists. These results suggest that the carbohydrate moiety of muscarinic acetylcholine receptors is not involved in their interaction with muscarinic ligands and G proteins.

Animals↗
PubMed1990

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase.

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.

Amino Acid Sequence↗
PubMed1990

The formation of oxalate from glycolate in rat and human liver.

In this study, we attempted to elucidate the metabolic pathway and enzymes actually involved in oxalate formation from glycolate in rat and human liver. In rat liver, the formation of oxalate from glycolate appeared to take place predominantly via glyoxylate. The oxalate formation from glycolate observed with crude enzyme preparations was almost entirely accounted for by the sequential actions of glycolate oxidase and xanthine oxidase (XOD) or lactate dehydrogenase (LDH). Under the conditions used, no significant activity was attributable to glycolate dehydrogenase, an enzyme reported to catalyze the direct oxidation of glycolate to oxalate. Among the three enzymes known to catalyze the oxidation of glyoxylate to oxalate, glycolate oxidase and XOD showed much lower activities (a higher Km and lower Vmax) toward glyoxylate than those with the respective primary substrates. As to LDH, none of the LDH subunit-deficient patients examined showed profoundly lowered urinary oxalate excretion. Based on the results obtained, the presumed efficacies in vivo of individual enzymes, as catalysts of glyoxylate oxidation, and the in vivo conditions assumed to allow their catalysis of oxalate production are discussed.

Alcohol Oxidoreductases↗
PubMed1990

Generation from a single gene of two mRNAs that encode the mitochondrial and peroxisomal serine:pyruvate aminotransferase of rat liver.

In rat liver there are two types of serine:pyruvate aminotransferase (SPT) whose natures are indistinguishable but whose subcellular localization are different. One is a mitochondrial enzyme (SPTm) and the other a peroxisomal enzyme (SPTp). We compared, in this study, the structure of mRNAs encoding SPTm and SPTp by comparison of the sizes after removal of poly(A) tail by ribonuclease H and by means of RNA blot analysis and S1 nuclease protection assay. No differences were detected between these two mRNAs other than that about 100 nucleotides of the 5'-terminal sequence of SPTm mRNA are lacking in SPTp mRNA, and the length of the poly(A) tail is different. Southern blot analysis of rat genomic DNA showed that the SPT gene is single. Primer extension and S1 nuclease mapping analyses, using a DNA fragment of a genomic clone, revealed that the SPTm and SPTp mRNAs are transcribed from different initiation sites, about 70 nucleotides apart, in the same exon, exon 1. Ribonuclease protection assay performed with RNA hybridization probe corresponding to 5'-terminal portion of SPTm mRNA also showed that the 5'-terminal sequence of SPTp mRNA is about 70 nucleotides shorter than that of hormone-responsive SPTm mRNA. These results indicate that the different organelle distribution of SPTm and SPTp, the products of the same SPT gene, arises from transcription from different initiation sites, conferring N-terminal extension peptide, the mitochondrial targeting signal, only on the translation product of SPTm mRNA.

Amino Acid Sequence↗
PubMed1990

Carbachol-induced potentiation and inhibition of acid secretion by guinea pig gastric gland.

The effects of muscarinic ligands on acid secretion were examined by estimating the accumulation of [14C]aminopyrine in gastric glands isolated from guinea pigs. The accumulation of [14C]aminopyrine in the presence of 0.1 mM histamine was potentiated by 1 microM carbachol but suppressed by 1 mM. These two effects of carbachol were abolished by atropine, pirenzepine and AF-DX 116. Assuming that the binding of carbachol to one site (Site 1) increases [14C]aminopyrine accumulation but its binding to the other site (Site 2) reduces [14C]aminopyrine accumulation, we analysed the dose-response curves for the carbachol effects in the absence and presence of different concentrations of atropine, pirenzepine and AF-DX 116. The dissociation constants determined for these ligands at Sites 1 and 2 were as follows: carbachol, 0.28 and 7.1 microM; atropine, 0.28 and 0.54 nM; pirenzepine, 45 and 560 nM; and AF-DX 116, 380 and 4400 nM, respectively. The binding of [3H]N-methylscopolamine to the gastric glands indicated the presence of two populations of binding sites with different affinities for the above ligands, other than atropine. The apparent dissociation constants, which were estimated by analysing the displacement curves for [3H]N-methylscopolamine binding, were as follows: carbachol, 0.18 microM (10%) and 31 microM (90%); atropine, 1.24 nM; pirenzepine, 15 nM (16%) and 220 nM (84%); and AF-DX 116, 370 nM (10%) and 2970 nM (90%). These results suggest that there are two kinds of muscarinic acetylcholine receptors in the guinea pig gastric gland, one potentiating and the other inhibiting the acid secretion induced by histamine.

Aminopyrine↗
PubMed1990

Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins.

Purified porcine atrial muscarinic acetylcholine receptors were reconstituted into lipid vesicles with three different G proteins (Gi, Go and Gn)1 purified from porcine cerebrum. All the G proteins interacted with the receptor as evidenced by GTP-sensitive high affinity binding with acetylcholine, and stimulation by acetylcholine of GTP gamma S binding and GTPase activities. The curves of displacement by acetylcholine of [3H]QNB binding were explained by assuming two sites with the same affinity for [3H]QNB but different affinities for acetylcholine. The proportion of the high affinity site increased from 3 to 7% up to 82 to 83% of total binding sites with increasing G protein concentration, and essentially the same results were obtained with the three G proteins. The GTPase activities of Gi, Go and Gn in the reconstituted vesicles were 2.7-, 1.7- and 1.6-times higher, respectively, in the presence of 1 mM acetylcholine than those in the presence of 10 microM atropine. An obvious enhancement by acetylcholine of the GTP gamma S binding was observed in the presence of 10 to 100 microM GDP, while the enhancement was minimal, if at all, in the absence of GDP. When the molar ratios of reconstituted Gi, Go and Gn to muscarinic receptors were 54, 84 and 107, respectively, the acetylcholine-induced increase in the [35S]GTP gamma S binding was as much as 12, 35 and 27 mol with Gi, Go and Gn, respectively, per mole of the receptor molecule, indicating that the muscarinic receptors interact with G proteins catalytically.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1990

Phosphorylation by protein kinase C of the muscarinic acetylcholine receptor.

Muscarinic acetylcholine receptors purified from porcine cerebrum were phosphorylated by protein kinase C purified from the same tissue. More than 1 mol of phosphate was incorporated per mole of receptor, with both serine and threonine residues being phosphorylated. Neither the degree nor the rate of the phosphorylation was affected by the presence or absence of acetylcholine. GTP-sensitive high-affinity binding with acetylcholine was observed for muscarinic receptors reconstituted with GTP-binding proteins (Gi or Go), irrespective of whether muscarinic receptors or the GTP-binding proteins had been phosphorylated by protein kinase C or not. This indicates that the interaction between purified muscarinic receptors and purified GTP-binding proteins in vitro is not affected by their phosphorylation.

Animals↗
PubMed1990

Location in muscarinic acetylcholine receptors of sites for [3H]propylbenzilylcholine mustard binding and for phosphorylation with protein kinase C.

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.

Animals↗
PubMed1989

Induction of mitochondrial serine:pyruvate aminotransferase of rat liver by glucagon and insulin through different mechanisms.

Studies were performed in the rat liver to examine whether or not insulin as well as glucagon causes the induction of mitochondrial serine:pyruvate aminotransferase (SPTm) [EC 2.6.1.51] and if so, whether the mechanisms of induction are similar or different for the two hormones. Not only glucagon but also insulin induced SPTm. Cell-free translation assaying and RNA blot analysis showed that both hormones cause an increase in the hepatic level of mRNA for the precursor of SPTm. Their effects were virtually additive, and the time course of the increase in the mRNA level differed between the hormones. The maximal increase induced by glucagon was observed 3.5 h after the hormone injection while that by insulin was found after 6 h. The increase in the mRNA due to insulin was completely inhibited by the co-administration of cycloheximide, while that due to glucagon was not. The finding suggests that a newly synthesized, insulin-dependent protein(s) is involved in the regulation of the mRNA level by insulin. On the other hand, hydrocortisone treatment selectively suppressed the increase in the mRNA due to glucagon. These data indicate that the synthesis of the mRNA for SPTm is regulated by glucagon and insulin through different mechanisms. The size of the hormone-induced mRNA for SPTm gradually decreased with time, but the cell-free translation products did not exhibit size alteration. RNase H digestion to remove the poly(A) tail of the mRNA indicated that shortening of the poly(A) sequence might be responsible for the time-dependent size alteration of the mRNA.

Animals↗
PubMed1989

Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli.

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.

Amino Acid Sequence↗
PubMed1989

Intracellular metabolism of biogenic amines in paraneurons.

Serotonin is one of the representative biogenic amines produced from tryptophan in the gastrointestinal tract as well as in the brain. The pineal gland also synthesizes serotonin, but as an intermediate in the biosynthesis of melatonin. The first enzymic step in the biosynthesis of serotonin is the hydroxylation of L-tryptophan catalyzed by tryptophan 5-monooxygenase. The 5-hydroxy-L-tryptophan (L-5HTP) formed is then decarboxylated to serotonin. Since the second step is catalyzed by non-specific aromatic L-amino acid decarboxylase, whether or not a given cell has the capacity to synthesize serotonin depends on the existence of tryptophan monooxygenase, and regulation of the serotonin biosynthesis is achieved mainly by modulating the activity of the first step enzyme. The activity of tryptophan monooxygenase was detected in extracts of the mouse stomach and intestines, although this activity was as low as approximately 1/20 of that in similar extract of the mouse brainstem. The upper small intestine and colon had a higher level of activity than other parts, and in the upper small intestine the enzyme was found to reside primarily in enterochromaffin cells of the mucosa. The intestinal tryptophan monooxygenase shared a common antigenic character with the enzyme from murine mastocytoma and was immunologically different from the brain enzyme. The enzymic properties were also similar to those of mastocytoma tryptophan monooxygenase which requires loosely-bound functional iron (Fe2+) for the activity. It seemed likely that the activity of tryptophan monooxygenase in paraneurons such as mastocytoma and enterochromaffin cells depends on available ferrous iron, and is therefore regulated, at least in part, by the cytosolic level of chelatable iron.

Animals↗
PubMed1989

Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components.

A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.

Animals↗
PubMed1989

Effect of the lipid environment on the differential affinity of purified cerebral and atrial muscarinic acetylcholine receptors for pirenzepine.

Muscarinic acetylcholine receptors (mAChRs) of porcine cerebral membrane (predominantly M1 subtype) and porcine atrial membrane (M2 subtype) showed the same affinity for the muscarinic antagonist [3H]quinuclidinylbenzylate [( 3H]QNB). In contrast, the affinity for pirenzepine (another muscarinic antagonist) of 86% of binding sites in the cerebral membrane (H sites) was 34-fold higher than that in the atrial membrane. After purification of mAChRs by affinity chromatography, this difference was less than 3-fold. This phenomenon was fully reversed by insertion of purified mAChRs into either cerebral or atrial membranes whose native muscarinic binding sites had been alkylated with propylbenzilycholine mustard, indicating that the purified receptors recovered their original affinities for pirenzepine upon interaction with membrane components. To examine the effect of the interaction between receptors and lipid components on the affinities for [3H]QNB and pirenzepine, binding experiments were carried out with mAChRs inserted into various lipid preparations. When purified cerebral and atrial mAChRs were inserted into cholesteryl hemisuccinate, their affinities for [3H]QNB and pirenzepine became close to the membrane values and were 7- and 50- to 60-fold higher than those of receptors inserted into phosphatidylcholine, respectively. When insertion was carried out into either cholesteryl hemisuccinate, phosphatidylcholine, or cholesteryl hemisuccinate/phosphatidylcholine mixtures, (80:20 and 50:50, w/w), the affinity of cerebral H sites for pirenzepine was only 3- to 5-fold higher than that of atrial receptors, but it became 20- and 60-fold higher when the receptors were inserted in a cholesteryl hemisuccinate/phosphatidylcholine mixture (20:80, w/w) and in a cholesteryl hemisuccinate/phosphatidylcholine/phosphatidylinositol mixture (4:48:48, w/w), respectively. These results suggest that the affinities of mAChRs for antagonists, in particular the differential affinities of cerebral and atrial mAChRs for pirenzepine, are modulated by the lipid environment.

Alkylation↗
PubMed1988

Cervical dislocation of mice induces rapid accumulation of platelet serotonin in the lung.

As much as 3-8 micrograms/g wet weight of serotonin (5-hydroxytryptamine, 5HT) has been detected in mouse lung 10 sec after the animal was sacrificed by cervical dislocation. Perfusion of the lung was carried out to determine where the majority of 5HT was located. Of the total 5HT, 84.3% was measured in the perfusate in which 90-98% was recovered in the platelet-rich fraction. When lungs were dissected from mice without cervical dislocation, either after instantaneous killing by irradiation with high power microwaves or under anesthesia, 5HT in the lung was 0.40-1.60 microgram/g tissue. These results show that cervical dislocation induces the entrapment of platelets in the pulmonary capillaries within 10 sec. The pulmonary accumulation of 5HT induced by cervical dislocation was approximately 50% inhibited by both prazosin and yohimbine, suggesting that the alpha-adrenergic receptor plays some part in the process. Norepinephrine but not isoproterenol augmented by 180% the response of pulmonary 5HT to cervical dislocation.

Animals↗
PubMed1988

Agonist and antagonist binding of muscarinic acetylcholine receptors purified from porcine brain: interconversion of high- and low-affinity sites by sulfhydryl reagents.

The affinity for muscarinic ligands of a preparation of muscarinic acetylcholine receptors purified from porcine brain was examined by means of competitive binding of [3H]quinuclidinylbenzylate and unlabeled ligands, followed by computer-assisted nonlinear regression analysis. The displacements by antagonists fitted a single-site model. In contrast, the displacements by agonists did not fit the single-site model and could be explained by assuming two populations of binding sites. The proportion of the sites with high affinity for muscarinic agonists (H-sites) ranged from 25 to 35% of the total number of sites. GTP had no effect on the displacements by agonists, a finding indicating that H-sites did not result from interaction between receptors and GTP-binding proteins. In the presence of dithiothreitol, the affinity for muscarinic ligands decreased. The largest effects were observed on the affinity for pirenzepine and that of H-sites for carbachol. Preincubation of the preparation with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in an increase in the proportion of H-sites to 75% of the total number of binding sites. The results of sucrose density gradient centrifugation of the preparation indicated apparent heterogeneity as to molecular size of the receptors, but this heterogeneity did not correlate with that of the affinity for agonists. In addition, the receptors were detected as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation, regardless of the presence or absence of disulfide-reducing reagents. These results suggest that the redox state of thiol groups in the receptor molecules is relevant to their affinities for ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The interaction between D-2 dopamine receptors and GTP-binding proteins.

D-2 dopamine receptors were solubilized from porcine striatal membranes with 0.3% sodium cholate/1 M NaCl and separated from the bulk of the guanine nucleotide-binding regulatory proteins (G-proteins) by Ultrogel AcA34 gel filtration chromatography. The partially purified D-2 receptors were reconstituted in phospholipid vesicles with Gi or Go purified from porcine brain. The dissociation constant (Kd) of the D-2 receptors in the reconstituted vesicles for [3H]spiperone binding was 82-89 pm, which was not affected by the presence or absence of G-proteins. The displacement curve for [3H]spiperone/dopamine was analyzed, assuming that there are two populations of binding sites. The Kd values for the binding sites with high affinity for agonists (HAS) and that for the binding sites with low affinity for agonists (LAS) were approximately 1 microM and 100 microM, respectively. The proportion of HAS was 8% when the receptor preparation was reconstituted into phospholipid vesicles without G-proteins, but it increased to 58-64% with increasing G-protein concentrations. The potency of Go was a little higher than that of Gi. The proportion of HAS in the presence of G-proteins decreased to about 11% on addition of GTP. When G-proteins were treated with islet-activating protein, GTP-sensitive HAS were not observed. These results indicate that at least 50% of the partially purified D-2 receptors interact with both Gi and Go.

Animals↗