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M Tagaya

Publications and source records attributed to M Tagaya.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of Lys277 at the active site of Escherichia coli glycogen synthase. Application of affinity labeling combined with site-directed mutagenesis.

Lys15 in Escherichia coli glycogen synthase, which is specifically labeled by adenosine diphosphopyridoxal, is mainly involved in binding of the substrate ADP-glucose (Furukawa, K., Tagaya, M., Tanizawa, K., and Fukui, T. (1993) J. Biol. Chem. 268, 23837-23842). We have found that the mutant glycogen synthase in which Lys15 is replaced by Gln via site-directed mutagenesis is inactivated by adenosine diphosphopyridoxal at concentrations higher than those required for the inactivation of the wild-type enzyme. ADP and ADP-glucose offered protective effects on inactivation, suggesting that the label binds to the ADP-glucose-binding site in the mutant enzyme. Sequence analysis of the labeled peptide revealed that the labeled residue is Lys277. This lysyl residue is conserved in maize starch synthase, which shows about 30% amino acid identity to E. coli glycogen synthase. Substitution of Gln for Lys277 by site-directed mutagenesis resulted in a 140-fold decrease in the kcat value with little changes in the Km values for ADP-glucose and glycogen. These results suggest that Lys277 at the active site participates in the catalytic reaction rather than binding of substrate. The present study shows the usefulness of the combined application of affinity labeling and site-directed mutagenesis.

Affinity Labels↗
PubMed1994

A new gerbil model of hindbrain ischemia by extracranial occlusion of the bilateral vertebral arteries.

A new gerbil model of hindbrain ischemia was induced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Carbon black studies, performed at 5 min after occlusion, revealed that the pons-medulla oblongata, and the cerebellum were quite ischemic in all animals. Cardiovascular changes in mean arterial blood pressure (MABP) and heart rate were recorded until 30 min after occlusion, and revealed that the typical cerebral ischemic response (i.e., abrupt increase in MABP, bradycardia, and apnea) was elicited in all animals (n = 10). Thirty minutes after occlusion, animals (n = 4) were decapitated and immersion-fixed. Brain sections were stained with hematoxylin-eosin (HE) and also immunostained for microtubule-associated protein 2 in order to evaluate ischemic neuronal damage from 30 min of ischemia. By HE staining, ischemic lesions were detected bilaterally in the oculomotor, the trigeminal motor, the lateral vestibular, and the cerebellar interpositus nucleus. In addition, immunostaining revealed ischemic lesions in several other hindbrain areas. In conclusion, we could successfully establish a new gerbil model of hindbrain ischemia. Carbon black perfusion and hemodynamic studies revealed that severe and reproducible hindbrain ischemia was produced. By histopathological examination, we could also clearly demonstrate symmetrical ischemic lesions in several hindbrain areas.

Animals↗
PubMed1994

Effects of recombinant superoxide dismutase on manganese superoxide dismutase gene expression in gerbil hippocampus after ischemia.

BACKGROUND AND PURPOSE: We reported that recombinant human superoxide dismutase ameliorates delayed neuronal death in the postischemic gerbil hippocampus. Since postischemic induction of copper-zinc superoxide dismutase messenger RNA was abolished by this treatment, oxygen radicals generated on reperfusion may induce the expression of this gene. In the present study we examined whether oxygen radicals also induce the expression of manganese superoxide dismutase messenger RNA in the postischemic brain. METHODS: We induced transient cerebral ischemia by occluding the bilateral common carotid arteries of gerbils. Recombinant human superoxide dismutase (8 x 10(5) U/kg) or apo-superoxide dismutase was administered intravenously 1 minute before a 5-minute occlusion of the carotid arteries. We analyzed both copper-zinc and manganese superoxide dismutase RNA by in situ hybridization histochemistry and by Northern and dot blot analyses using radioisotope-labeled oligonucleotide probes. RESULTS: Hybridization with the manganese superoxide dismutase messenger RNA occurred at the limit of detection in normal CA1 neurons. We observed striking increases in the labeling of CA1 up to 24 hours after 5 minutes of ischemia. The hybridization occurred anew in glial cells of the CA1 layer during 3 to 7 days. Pretreatment with recombinant human superoxide dismutase had no effect on the postischemic induction of manganese superoxide dismutase messenger RNA, whereas the same treatment significantly attenuated (P < .01) the increase in copper-zinc superoxide dismutase messenger RNA. CONCLUSIONS: Our results demonstrated temporal postischemic induction of manganese superoxide dismutase messenger RNA. The inducer may not be superoxide radicals but may be other chemical mediators such as cytokines.

Amino Acid Sequence↗
PubMed1993

Role of the conserved Lys-X-Gly-Gly sequence at the ADP-glucose-binding site in Escherichia coli glycogen synthase.

Although bacterial and mammalian glycogen synthases differ in the primary structure and specificity for glucosyl donor, lysyl residues identified at their substrate-binding sites by affinity labeling are present in a conserved tetrapeptide sequence, Lys-X-Gly-Gly, where X is a residue not conserved (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260, 6670-6676; Furukawa, K., Tagaya, M., Inouye, M., Preiss, J., and Fukui, T. (1990) J. Biol. Chem. 265, 2086-2090). To elucidate the functional role of this conserved sequence, Lys-15, Gly-17, and Gly-18 in Escherichia coli glycogen synthase have been replaced by other amino acid residues via site-directed mutagenesis. Kinetic analyses of the Lys-15 mutant enzymes showed that the epsilon-amino group of Lys-15 is mainly involved in binding of the phosphate moiety adjacent to the glycosidic linkage in the substrate ADP-glucose, presumably through an ionic interaction. The mutant enzyme in which Ala was substituted for Gly-17 had a catalytic rate constant 3 orders of magnitude smaller than that of the wild-type enzyme with a slightly increased Michaelis constant for ADP-glucose, whereas the Gly-18-->Ala mutant showed a rate constant only 3.2-fold smaller. In addition, mutations of Gly-17 and Gly-18 resulted in marked changes in the reactivity of Lys-15 with affinity labeling reagents. These results suggest that the 2 glycyl residues in the conserved Lys-X-Gly-Gly sequence, in particular the one closer to the ADP-glucose-binding lysyl residue, participate in catalysis by assisting conformational change(s) of the active site or stabilizing the transition state.

Adenosine Diphosphate Glucose↗
PubMed1993

Correlation between phospholipase A2 activity and intra-Golgi protein transport reconstituted in a cell-free system.

A wide variety of phospholipase A2 inhibitors blocks intra-Golgi protein transport reconstituted in a cell-free system. Phospholipase A2 activity detectable under the protein transport assay conditions is actually inhibited by the inhibitors. There is a good correlation between the inhibition of protein transport and that of phospholipase A2 activity. Prolactin secretion from GH3 cells is also blocked by a membrane-permeable phospholipase A2 inhibitor, suggesting the physiological relevance to inhibition of protein transport in vitro by phospholipase A2 inhibitors.

Acetylglucosamine↗
PubMed1993

Domain structure of an N-ethylmaleimide-sensitive fusion protein involved in vesicular transport.

N-Ethylmaleimide-sensitive fusion protein (NSF) is an essential component for protein transport between Golgi cisternae. Sequence analysis and proteolytic dissection reveal that NSF contains two tandem "ATP domains," each containing the consensus sequence for the binding of nucleotide. When Escherichia coli-produced Chinese hamster ovary NSF is purified, it exhibits a low, but significant, ATPase activity. The ATPase activity of NSF is sensitive to N-ethylmaleimide and influenced by monoclonal antibodies against recombinant NSF.

Adenosine Triphosphatases↗
PubMed1993

In vitro fusion of rabbit liver Golgi membranes with liposomes.

Fusion of Golgi membranes isolated from rabbit liver with liposomes was studied by lipid mixing of fluorescent lipid analogues and internal content mixing and by electron microscopic observation of transfer of horseradish peroxidase from liposomes into Golgi membranes. A monoclonal antibody was used to confirm fusion of Golgi membranes but not other contaminating vesicles. Fusion was rapid and efficient, reaching about 20% of the maximum after a 5-min incubation using small or large unilamellar dioleoylphosphatidylcholine vesicles. The fusion was dependent on temperature, decreasing at lower temperatures, and becoming nearly zero below 10 degrees C. The addition of ATP, GTP, cytosolic factors, or N-ethylmaleimide did not affect fusion. Treatments of Golgi membranes with 0.1 M Na2CO3 or 1 M KCl did not cause any changes in fusion. However, treatment with proteases inhibited fusion. These results suggest that Golgi integral membrane protein(s) are involved in fusion. Changing the medium to an isoosmotic substance, sucrose, in place of KCl or NaCl inhibited fusion. The binding assay of fluorescent liposomes to Golgi membranes showed that lowering the temperature or replacing salts with sucrose did not affect binding. However, treatment of Golgi membranes with proteases inhibited binding. Addition of phosphatidylserine or phosphatidylethanolamine to dioleoylphosphatidylcholine liposomes caused a 2-fold increase in binding and fusion. Fusion between Golgi membranes by themselves did not occur. These results provide some information on the mechanism of intracellular vesicular transport.

Animals↗
PubMed1993

Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.

Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.

Affinity Labels↗
PubMed1993

Exploring the active site in UDP-glucose pyrophosphorylase by affinity labelling and site-directed mutagenesis.

UDP-glucose pyrophosphorylase catalyses the reversible uridylyl transfer from UDP-glucose to MgPPi, forming glucose 1-phosphate and MgUTP. We have identified, by using uridine di- and tri-phosphopyridoxals, five lysyl residues located at or near the UDP-glucose-binding site. Another reactive analogue, pyridoxal diphosphoglucose, also modified the same set of lysyl residues. Based on the amounts of labels incorporated into each lysyl residue, we have provided a hypothetical model for possible locations of the five lysyl residues around the substrates bound to the enzyme. This model was consistent with the results of the mutagenesis studies. Lys367 is essential for the catalysis, whereas Lys263 may participate in the binding of PPi and/or glucose 1-phosphate in the binary complex. On the other hand, the results of affinity labelling with pyridoxal 5'-diphosphate suggest the role of Lys329 in the direct interaction with PPi in the enzyme-UDP-glucose complex.

Affinity Labels↗
PubMed1992

The characteristics of blood-brain barrier in three different conditions--infarction, selective neuronal death and selective loss of presynaptic terminals--following cerebral ischemia.

We investigated the extravasation of serum albumin using immunohistochemistry in three different conditions, i.e., infarction, selective neuronal death and selective loss of presynaptic terminals following cerebral ischemia in gerbils. In selective neuronal death, which is typically found in the CA1 neurons of the hippocampus after 5-min bilateral cerebral ischemia, selective damage of postsynaptic components with intact presynaptic sites was demonstrated by immunohistochemical examination for microtubule-associated protein 2 and synapsin I, and albumin extravasation did not become apparent before postsynaptic structures were destroyed. In cerebral infarction, which was consistently observed in the thalamus after 15-min forebrain ischemia, massive albumin extravasation was visible early after ischemia due probably to the ischemic endothelial necrosis. In selective loss of presynaptic terminals, which was detected at the molecular layer of the dentate gyrus in the contralateral, nonischemic hippocampus after unilateral cerebral ischemia, immunoreaction for albumin was not visualized. Since endothelium and glial cells were intact in morphological aspects in selective damage of both pre- and postsynaptic sites, it was thought that extravasation was facilitated by the stimulation of endothelial cells and glial cells with unknown factors that were induced by the destruction of post- but not presynaptic elements.

Albumins↗
PubMed1992

Recombinant human superoxide dismutase can attenuate ischemic neuronal damage in gerbils.

The effects of recombinant human superoxide dismutase (r-hSOD) on ischemic neuronal injury were examined. Cerebral ischemia was produced in Mongolian gerbils by occluding bilateral common carotid arteries for 5 min. Preischemic treatment with r-hSOD clearly reduced hippocampal neuronal damages while postischemic treatment did not. This result suggests that oxygen free radicals play an important role in selective vulnerability to ischemia and r-hSOD has a potential clinical usefulness against cerebral ischemia.

Analysis of Variance↗
PubMed1992

The synapsin I brain distribution in ischemia.

We examined the distribution of synapsin I in the gerbil brain and investigated ischemic damage of presynaptic terminals immunohistochemically by using this protein as a marker protein of synaptic vesicles. The reaction for synapsin I in normal gerbil brain is exclusively localized in the neuropil, and other brain structures such as neuronal soma, dendrites, axon bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, ischemic loss of synapsin I immunoreactivity in the affected hemisphere was confined to the area exhibiting overt infarction, where the breakdown of this protein was also confirmed by the immunoblot analysis, and noted much later than that of microtubule-associated protein 2 immunoreactivity, which was demonstrated in neuronal soma and dendrites. In the non-affected hemisphere, selective damage of presynaptic terminals due to Wallerian degeneration and subsequently occurring resynaptogenesis at the molecular layer of the dentate gyrus were clearly demonstrated as a loss and recovery of immunoreaction for synapsin I, respectively. In a gerbil model of bilateral cerebral ischemia, immunoreaction for synapsin I was persistently preserved after seven days to two months recirculation following a brief period of global forebrain ischemia in the CA1 region of the hippocampus, where delayed neuronal death was consistently observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

'Ischemic tolerance' phenomenon detected in various brain regions.

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the 'ischemic tolerance' phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of 'ischemic tolerance'.

Animals↗
PubMed1991

Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals.

Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.

Affinity Labels↗
PubMed1991

Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae.

We report that brefeldin A prevents the assembly of non-clathrin-coated vesicles from Golgi cisternae in a cell-free system. This finding provides a simple molecular explanation for the primary effect of this remarkable compound in blocking constitutive secretion. We further report that when coated vesicle assembly is blocked, extensive tubule networks form that connect previously separate cisternae and stacks into a single topological unit, allowing the intermixing of contents of Golgi cisternae, presumably by lateral diffusion. Formation of the tubule networks requires ATP, cytosol, and the general fusion protein NSF. Tubule networks may be related to the membrane tubules mediating retrograde transport in vivo.

Animals↗