Arata Ichiyama

Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence

The complete amino acid sequence of the porcine cardiac muscarinic acetylcholine receptor has been deduced by cloning and sequencing the cDNA. The tissue location of the RNA hybridizing with the cDNA suggests that this muscarinic receptor species represents the M2 subtype.

Studies on the biosynthesis of nicotinamide-adenine dinucleotide in the brain☆

1. 1. The biosynthesis of NAD in the brain was investigated in vivo by following the metabolism of radioactive nicotinate, nicotinamide and their derivatives after injection into mice and rats. Although pyridine derivatives injected intraperioneally were incorporated efficiently into NAD in the liver and kidney, they could not readily penetrate into the brain, presumably due to the blood-brain barrier. 2. 2. Nicotinate injected into the cisternal cavity of rats was rapidly and efficiently converted to NAD in the brain by way of nicotinate ribonucleotide and deamido-NAD. 3. 3. Nicotinate ribonucleotide and deamido-NAD, administered in this way, were not directly utilized as precursors of NAD but were degraded to nicotinate ribonucleoside. The latter compound was phosphorylated again to the ribonucleotide and was converted to NAD. 4. 4. Nicotinate ribonucleoside was readily utilized as a precursor of NAD without prior degradation to nicotinate. 5. 5. Radioactive nicotinamide was also incorporated into NAD in the brain. This incorporation seems to be due to an exchange reaction with the nicotinamide moiety of NAD and does not represent de novo synthesis of NAD.

A New 30-kDa Ubiquitin-related SUMO-1 Hydrolase from Bovine Brain

SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates,125I-SUMO-1-αNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-35S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity of SUMO-1 hydrolase was almost completely inhibited byN-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1-precursor.

Flux of the l-Serine Metabolism in Rabbit, Human, and Dog Livers SUBSTANTIAL CONTRIBUTIONS OF BOTH MITOCHONDRIAL AND PEROXISOMAL SERINE:PYRUVATE/ALANINE:GLYOXYLATE AMINOTRANSFERASE

l-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mm l-serine and 0.25 mmpyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment withl-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis froml-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.

Control of Oxalate Formation from L-Hydroxyproline in Liver Mitochondria

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) is largely located in mitochondria in carnivores, whereas it is entirely found within peroxisomes in herbivores and humans. In rat liver, SPT/AGT is found in both of these organelles, and only the mitochondrial enzyme is markedly induced by glucagon. Although SPT/AGT is a bifunctional enzyme involved in the metabolism of both L-serine and glyoxylate, its contribution to L-serine metabolism is independent of mitochondrial or peroxisomal localization (Xue HH et al., J Biol Chem 274: 16028-16033, 1999). Therefore, the species-specific and food habit-dependent organelle distribution might be required for proper metabolism of glyoxylate at the subcellular site of its formation. Glyoxylate formation from glycolate and that from L-hydroxyproline have been shown to occur in peroxisomes and mitochondria, respectively. The present study found that urinary excretion of oxalate was markedly increased when a large dose of L-hydroxyproline or glycolate was administered to rats. Oxalate formation from L-hydroxyproline but not that from glycolate was significantly reduced when mitochondrial SPT/AGT had been induced by glucagon. The hydroxyproline content of collagen is 10 to 13%, and collagen accounts for about 30% of total animal protein; therefore, these results suggest that an important role of mitochondrial SPT/AGT in carnivores is to convert L-hydroxyproline-derived glyoxylate into glycine in situ, preventing undesirable overflow into the production of oxalate.

Flux of the l-Serine Metabolism in Rat Liver THE PREDOMINANT CONTRIBUTION OF SERINE DEHYDRATASE

l-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by l-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mm l-serine and 0.25 mm pyruvate) was about 1 10 of that through SDH. Flux through GCS accounted for only several percent of the amount ofl-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from l-serine were evaluatedin vivo based on the principle that 3H at the 3 position of l-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10–20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of l-serine in rat liver.

Phosphorylation by Protein Kinase C of the Muscarinic Acetylcholine Receptor

Abstract: 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.

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.

Agonist and Antagonist Binding of Muscarinic Acetylcholine Receptors Purified from Porcine Brain: Interconversion of High- and Low-Affinity Sites by Sulfhydryl Reagents

Abstract: 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. In particular, molecules carrying (an) intact, probably intramolecular, disulfide bond(s) show high affinity for agonists, whereas those carrying the reduced state of the relevant thiol group(s) show low affinity for agonists.

In vitro synthesis of a putative precursor of serine: pyruvate aminotransferase of rat liver mitochondria.

Abstract Serine:pyruvate aminotransferase [EC 2.6.1.51] of rat liver, an enzyme induced by glucagon in mitochondria, was synthesized in cell-free protein synthesizing systems derived from nuclease-treated rabbit reticulocyte lysate and wheat germ extract as a putative precursor which was approximately 2,000 daltons larger than the subunit of mature enzyme. The hepatic level of translatable messenger RNA coding for the putative precursor was approximately 40 times higher in rats received a glucagon administration 3.5 h before sacrifice than in control animals.