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T Katada

Publications and source records attributed to T Katada.

At least 145 records · Page 8Linked to original sources
PubMed1991

Purification and characterization of a new GTP-binding protein of Mr 24,000 in bovine brain membranes.

A GTP-binding protein with an Mr of 24,000 was purified from a cholate extract of bovine brain membranes in addition to the previously reported alpha beta gamma-trimeric GTP-binding proteins (G proteins). Partial amino acid sequence analysis of the purified 24-kDa protein revealed that it was not identical to any of the low Mr GTP-binding proteins already reported, but similar to the rac-gene products serving as the substrate of an ADP-ribosyltransferase (C3) purified from the culture medium of Clostridium botulinum type C. However, the 24-kDa protein was not ADP-ribosylated by the botulinum C3 enzyme. The 24-kDa protein was purified as a nucleotide-free form and characterized by the following unique properties distinct from those of alpha beta gamma-trimeric G proteins. (1) Mg2+ was essentially required for nucleotide binding to the 24-kDa protein; there was a progressive increase in its binding affinity for nucleotides as the concentration of the divalent cation was increased. (2) Nucleotides previously bound to the 24-kDa protein were rapidly dissociated from the protein in Mg(2+)-free medium, in accord with the fact that the protein was indeed purified as a nucleotide-free form with Mg(2+)-free solutions. (3) The 24-kDa protein apparently exhibited much lower GTPase activity than do alpha beta gamma-trimeric G proteins because the product GDP was released from the 24-kDa protein in exchange for the substrate GTP only at a very low rate. Based on these findings, a possible role of the 24-kDa protein in cellular signalling is discussed in comparison with well characterized alpha beta gamma-trimeric G proteins.

ADP Ribose Transferases↗
PubMed1990

Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins.

A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.

Adenosine Triphosphate↗
PubMed1990

Identification of sites for alkylation by N-ethylmaleimide and pertussis toxin-catalyzed ADP-ribosylation on GTP-binding proteins.

An alpha beta gamma-trimeric GTP-binding protein (Go) serving as the substrate of pertussis toxin-(IAP) catalyzed ADP-ribosylation was purified from rat brain membranes. The constituent alpha-subunit (alpha o) was alkylated with N-ethylmaleimide (NEM), and the functionally important sulfhydryl groups were investigated. There were at least two cysteine residues highly reactive to NEM on the GDP-bound form of alpha o. These alkylations resulted in loss of its ability to be ADP-ribosylated by IAP and to associate with beta gamma, but leaving the GTP-binding site of alpha o intact. The reacted cysteine residues were identified by the sequencing of tryptic fragments of alpha o. One of the alkylation sites was Cys-351, which was four amino acid residues away from the carboxyl-terminus of the molecule. The Cys-351 was proven to be also a site for IAP-catalyzed ADP-ribosylation. Possible roles of cysteine residues on the alpha-subunit of Go are discussed in the functions of the signal transducing protein.

Adenosine Diphosphate Ribose↗
PubMed1990

Possible interaction of alpha 1-adrenergic receptor with pertussis-toxin-sensitive guanine-nucleotide-binding regulatory proteins (G proteins) responsible for phospholipase C activation in rat liver plasma membranes.

Islet-activating protein (IAP; pertussis toxin) was employed to test the hypothesis that IAP-sensitive GTP-binding regulatory proteins (G proteins) are coupled with alpha 1-adrenergic receptor in rat liver plasma membranes. The high-affinity state of the binding of alpha 2-adrenergic agonist, which is known to be coupled with IAP-sensitive G protein, was abolished in IAP-treated plasma membranes. IAP treatment of plasma membranes could also diminish the high-affinity state of the alpha 1-adrenergic receptor for the agonist. Restoration of the high-affinity state of the alpha 1-adrenergic receptor for the agonist occurred on reconstitution of the bovine brain IAP-sensitive G proteins. The alpha 1-adrenergic receptor agonist stimulated inositol triphosphate (InsP3) production from [3H]inositol-labeled liver plasma membranes in a concentration-dependent manner. IAP treatment also decreased alpha 1-adrenergic-agonist-induced InsP3 production but not completely. From these results, we concluded that there is a possibility that both IAP-sensitive and IAP-insensitive G proteins were involved in alpha 1-adrenergic-receptor-stimulated phospholipase C activation in rat liver plasma membranes.

Adenosine Diphosphate Ribose↗
PubMed1990

Distinctive regulation of the functional linkage between the human cation-independent mannose 6-phosphate receptor and GTP-binding proteins by insulin-like growth factor II and mannose 6-phosphate.

The rat insulin-like growth factor II (IGF-II) receptor develops transmembrane signaling functions by directly coupling to a guanine nucleotide-binding protein (G protein) having a 40-kDa alpha subunit, Gi-2, whereas recent studies have indicated that the IGF-II receptor is a molecule identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), a receptor implicated in lysosomal enzyme sorting. In this study, by using vesicles reconstituted with the clonal human CI-MPR and G proteins, we indicated that the CI-MPR could stimulate guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding and GTPase activities of Gi proteins in response to IGF-II. The stimulatory effect of IGF-II on Gi-2 depended on the reconstituted amount of the CI-MPR; it could not be found in vesicles reconstituted with Gi-2 alone; and it was also observed on Gi-1 reconstituted with the CI-MPR in phospholipid vesicles. Of interest, such stimulatory effect was not reproduced by Man-6-P in CI-MPR vesicles reconstituted with either G protein. Furthermore, the affinity for Man-6-P-mediated beta-glucuronidase binding to several kinds of native cell membranes was not reduced by 100 microM GTP gamma S. Instead, however, Man-6-P dose-dependently inhibited IGF-II-induced Gi-2 activation with an IC50 of 6 microM in vesicles reconstituted with the CI-MPR and Gi-2. The action of 100 nM IGF-II was completely abolished by 1 mM Man-6-P. Such an inhibitory effect of Man-6-P was reproduced by 4000 times lower concentrations of beta-glucuronidase or similar concentrations of fructose 1-phosphate, but not by mannose or glucose 6-phosphate. These results indicate that the human CI-MPR has two distinct signaling functions that positively or negatively regulate the activity of Gi-2 in response to the binding of IGF-II or Man-6-P.

Animals↗
PubMed1990

A simple structure encodes G protein-activating function of the IGF-II/mannose 6-phosphate receptor.

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/man6PR) can directly interact with and activate Gi-2, a GTP binding protein (G protein). We found that the segment of residues 2410-2423 in the human IGF-II/man6PR activates Gi-2 in a manner similar to G-coupled receptors. We observed a hierarchy of the segment action when tested on various G proteins, with an order of Gi-2 greater than Gi-1 approximately Gi-3 greater than Go. The segment had no effect on Gs or low molecular weight G proteins. The segment action depended on its primary structure and was potentiated when the segment was connected with a part of the receptor transmembrane region. Finally, the Gi-2-activating function of the human IGF-II/man6PR could be blocked by an antibody against the segment, indicating a critical role for this small region of the receptor.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of five different alpha subunits of guanine-nucleotide-binding proteins in bovine brain membranes. Their physiological properties concerning the activities of adenylate cyclase and atrial muscarinic K+ channels.

We have purified five different alpha subunits of guanine-nucleotide-binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). All the purified alpha subunits were interacted with beta gamma subunits and served as a substrate for pertussin-catalyzed ADP-ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various alpha subunits of G proteins, three of them were identified as alpha i-1, alpha i-2 and alpha i-3, and the other two were classified into alpha o type. One of the alpha o-type proteins was the most abundant in the brain membranes (termed alpha o), and the other (alpha o2) appeared to differ from alpha o in its proteolytic digestion data. The physiological properties of these purified GTP[gamma S]-bound alpha subunits towards adenylate cyclase and atrial muscarinic K+ channels were studied. The nucleotide-bound forms of alpha i-1, alpha i-2, alpha i-3 and alpha o2 inhibited the adenylate cyclase activity of S49 cyc- membranes which had been reconstituted with GTP[gamma S]-treated Gs; this inhibition appeared to be mainly competitive with the activated Gs, alpha i-1 having the most potent inhibitory activity among them. GTP[gamma S]-bound alpha o, however, could not inhibit the Gs-stimulated activity at all. On the other hand, all the GTP[gamma S]-bound alpha subunits activated atrial muscarinic K+ channels, accompanied by a lag time, at picomolar concentrations. The beta gamma subunits resolved from G proteins also activated the K+ channels without a lag time at nanomolar concentration. The maximum activation by the beta gamma subunits appeared to be more potent than that by any of the alpha subunits. These results suggest that alpha and beta gamma subunits might activate the K+ channels by mechanisms different from each other.

Adenosine Diphosphate Ribose↗
PubMed1990

Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, Gi1 and Gi2, but not G0 in reconstitution experiments.

The receptor-mediated inhibition of intrinsic activities of GTP-binding proteins (G-proteins) was studied. Pertussis toxin (IAP)-substrate G-protein, Gi1, Gi2 or G0, was prelabeled with [alpha-32P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G-proteins were evaluated by the release of [alpha-32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U-50,488H (1 nM-10 microM), a specific kappa-subtype of opioid receptor agonist, inhibited the [alpha-32P]GDP release in exchange for added 1 microM GppNHp in Gi1-reconstituted preparations in a concentration-dependent manner. On the other hand, the kappa-opioid agonist at 10 microM increases the Km values of GppNHp, but not GDP in exchange for [alpha-32P]GDP release in preparations reconstituted with Gi1 or Gi2, but not with G0. These findings indicate that kappa-opioid receptor is coupled to inhibition of intrinsic activities of Gi1 and Gi2, but not G0, in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G proteins, but not by a change in affinity of GDP.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1990

Phosphorylated mu-opioid receptor purified from rat brains lacks functional coupling with Gi1, a GTP-binding protein in reconstituted lipid vesicles.

The effects of phosphorylation of a mu-opioid receptor on signal transduction to G-protein were studied. The mu-opioid receptor purified from rat whole brains was reconstituted with purified Gi1 in phosphatidylcholine vesicles. DAGO, a mu-opioid agonist at 1 microM-1 mM increased GTPase activity by 10-110% of control, in a concentration-dependent manner. When the mu-opioid receptor was phosphorylated by cyclic AMP-dependent protein kinase prior to reconstitution with Gi1, the DAGO-stimulation was markedly reduced (20% increase at 1 mM DAGO).

Animals↗
PubMed1990

Characterization of four G0-type proteins purified from bovine brain membranes.

Recently we reported there were at least four types of G0 or G0-like proteins in bovine brain membranes based on their elution profiles from Mono Q columns and their immunological reactivities; one of the proteins was purified as an alpha-monomeric form, and the others as alpha beta gamma-trimers. The four proteins, of which alpha-subunits were confirmed to be a family of G0-type by an immunoblot analysis, were thus referred to as alpha (0)1, G(0)2, G(0)3 and G(0)4, respectively, in order of their elutions from the column. Immunostained peptide mappings arising from proteolytic digestions of the four alpha-subunits, together with their fragmentation patterns containing radiolabeled ADP-ribose that had been incorporated by pertussis toxin-catalyzed ADP-ribosylation, suggested that the four G0-alpha were classified into either of two groups such as alpha (0)1 and G(0)2-alpha, or G(0)3-alpha and G(0)4-alpha. The kinetic parameters of their GTPase activities, however, revealed that there were different properties between alpha (0)1 and G(0)2-alpha or G(0)3-alpha and G(0)4-alpha. Thus, the four G0-type proteins appeared to be different entities from one another.

Amino Acid Sequence↗
PubMed1990

Botulinum ADP-ribosyltransferase activity as affected by detergents and phospholipids.

GTP-binding proteins with Mr values of 22,000 and 25,000 in bovine brain cytosol were ADP-ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3-catalyzed ADP-ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3-catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP-ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADP-ribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other.

ADP Ribose Transferases↗
PubMed1990

Molecular heterogeneity of the subclasses of islet-activating protein (pertussis toxin)-sensitive GTP-binding proteins in porcine thyroid tissue.

From porcine thyroid cell membranes, we purified five GTP-binding proteins (G-proteins); Nos. 1 to 3 have 41-kDa alpha-subunits, and Nos. 4 and 5 have 40-kDa alpha-subunits. They were chromatographically (Mono Q) separable and served as specific substrates for islet-activating protein (pertussis toxin). G-proteins 1 and 2 were indistinguishable from porcine brain Gi1 with respect to three criteria, i.e., mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI of the ADP-ribosylated alpha-subunit, and immunoreactivity. G-protein 3 was identified as Gi3 by immunoreactivity. The SDS-PAGE and isoelectric focusing (IEF) analyses identified G-proteins 4 and 5 as being chromatographically heterogeneous subtypes of Gi2 in comparison with a pure porcine brain preparation. The IEF analysis also disclosed that each of the Gi1, Gi2, and Gi3 subspecies isolated in the present study has a minor component characterized by a slightly lower pI of its alpha-subunit. We conclude that porcine thyroid tissue contains at least Gi1, Gi2, and Gi3, and that each is made up of heterogeneous populations.

Animals↗
PubMed1990

Selective coupling of purified alpha-subunits of pertussis toxin-substrate GTP-binding proteins to endogenous receptors in rat brain membranes treated with N-ethylmaleimide.

The membrane fraction prepared from rat brain was incubated with 0.5 mM N-ethylmaleimide (NEM) for 10 min. 3H-labelled agonist binding to muscarinic, A1-adenosine, opiate and alpha 2-adrenergic receptors was markedly inhibited by this NEM treatment of membranes, which interfered with the subsequent ADP-ribosylation of endogenous G-proteins by pertussis toxin. This indicated that the toxin target cysteine residues of the G-protein were modified by NEM. The NEM-induced inhibition of agonist bindings was mostly reversed by reconstitution of the alpha-subunits of purified Gi or Go into the membranes. The NEM-induced inhibition, together with the reversal by the G alpha reconstitution, was due to changes in the relative number of high- to low-affinity receptors solely without change in the total (high- plus low-affinity) receptor number. Thus, in NEM-treated membranes endogenous G-proteins become uncoupled from receptors, which were coupled to either Gi alpha or Go alpha. Reconstitution of NEM pre-treated membranes showed that Go acted in preference to Gi in interaction with muscarinic receptors and vice versa in interaction with three other types of receptor. The possible involvement of Go in mediating phospholipase C activation and Gi in mediating adenylate cyclase inhibition is discussed.

Adenosine Diphosphate Ribose↗
PubMed1990

Functional reconstruction of purified Gi and Go with mu-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide.

Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-opioid agonist [( D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO)] caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.

Adenylate Cyclase Toxin↗
PubMed1990

[Experimental studies on the influences of physical properties of foods on the appearance of silent period in discharge of masticatory muscles].

UNLABELLED: It is considered that the appearance of Silent Period (SP) during food crushing may relieve tissue injury caused by sudden change of maxillo-mandibular impactive force. In connection with this phenomenon, the relation between changes in vertical velocity of mandibular movement and the appearance of SP during food crushing was investigated. In addition, efforts were made to clarify the influences of lack of periodontal pressoreceptive information on the appearance of SP. Subjects were ten males (aged 24 to 31) with natural dentition and no masticatory dysfunction. Electromyograms were recorded on 4 lead locations: the central parts of each masseter and the anterior bundles of each temporal muscle. A Mandibular Kinesiograph and electromyograph were used to record simultaneously mandibular movement and myoelectric discharge during food crushing for the sake of comparing changes in mandibular position, vertical velocity of mandibular movement and muscular activity. Eight kinds of foods of various textures were selected for use in the examinations: dry macaroni, rice cracker, raw carrot, pickled thistle root, pickled radish, raisin, synthetic rubber block and softened chewing gum. The food was positioned on the occlusal table of the lower first molar on the habitual masticatory side of each subject. Then the subject was required to close his mouth rapidly to crush the food at a single masticatory stroke. Two experimental conditions were established in order to clarify the significance of periodontal pressoreceptive information on the appearance of SP: (1) normalcy and (2) infiltration anesthesia of the periodontal tissues of the upper and lower first-molar region on the habitual masticatory side. Results 1. SP appeared during a single masticatory stroke in response to sudden decreases or increases of vertical velocity of mandibular movement. 2. The frequency of the appearance of SP depends on the crushing characteristics of the food which is tested. For softened chewing gum and the synthetic rubber block, only one SP occurred; two SPs occurred in the cases of pickled radish and raisin; three SPs in the cases of raw carrot and pickled thistle root; and more than four SPs in the cases of dry macaroni and rice cracker. 3. Lack of periodontal pressoreceptive information resulting from anesthesia had no effect on the appearance of SP. CONCLUSIONS: 1. Sudden changes in vertical velocity of mandibular movement may depend on the crushing characteristics of the food which is crushed.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗