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M Ui

Publications and source records attributed to M Ui.

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

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.

ADP Ribose Transferases↗
PubMed1991

The repression of the reverse-oriented transcription from the adenovirus terminus by NFI in competition with TFIID.

Nuclear factor I (NF) represses the transcription which is promoted by the cloned adenovirus (Ad) type 5 DNA replication origin and is reverse-oriented with respect to the direction of the replication. The mechanism of this repression by NFI was investigated. In the cell-free transcription system, the repression was observed only when NFI was present during the formation of the transcription initiation complex. From the results of DNase I protection experiments, it was indicated that NFI bound to its binding site in the Ad replication origin prevents TFIID from proper binding to the adjacent AT-rich region and consequently represses the transcription.

Adenoviridae↗
PubMed1991

Interaction of guanine-nucleotide-binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL-60 cells.

Human leukemic HL-60 cells were differentiated into neutrophil-like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM,N-formylmethionine) and guanine-nucleotide-binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet-activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high-affinity binding to vesicle preparations from the Me2SO- and Bt2cAMP-induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP-sensitive G proteins in each of the two vesicle types. However, the high-affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP-induced cells than in those from Me2SO-induced cells, although the amount of IAP-substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi-2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi-2 from rat brain was reconstituted into the two IAP-treated vesicles, high-affinity fMLF binding was restored in a similar manner in both. IAP-substrate G proteins partially purified from the two differentiated HL-60 cells were also effective in restoring high-affinity fMLF binding to the IAP-treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G-protein-rich fraction from Bt2cAMP-induced cells than with that from Me2SO-induced cells, with each of the two IAP-treated vesicle types. These results suggest that the different high-affinity binding of fMLF observed in the two differentiated HL-60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi-2.

Bucladesine↗
PubMed1991

Human ubiquitin-activating enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome.

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 degrees C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 degrees C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 degrees C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 degrees C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.

Animals↗
PubMed1991

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

Adrenergic Agonists↗
PubMed1991

Direct interactions of mastoparan and compound 48/80 with GTP-binding proteins.

The effects of mastoparan and compound 48/80 on the activities of alpha beta gamma-trimeric GTP-binding proteins (G proteins) were studied with purified Go and Gi-1 which had been reconstituted into phospholipid vesicles. Pertussis toxin-catalyzed ADP-ribosylation of Go or Gi-1 was inhibited by mastoparan or compound 48/80, suggesting that the G proteins were dissociated into their constituent alpha- and beta gamma-subunits in the presence of these compounds. The steady-state rate of GTP hydrolysis catalyzed by Go or Gi-1 was stimulated by the two compounds. Both the stimulations were due to increases in the rate of the GDP-GTP exchange reaction occurring on the G proteins. However, the modes stimulation of the GTPase activity depended on the type of G protein used, and the stimulations caused by the two compounds were differently affected by pertussis toxin-catalyzed ADP-ribosylation of G proteins. Moreover, the mastoparan-induced stimulation of the GTPase activity was partially inhibited by compound 48/80. Thus, the two histamine secretagogues mastoparan and compound 48/80 appear to activate G proteins differently, though they interact with the signal-transducing proteins, at least partly, at a common binding site.

Adenosine Diphosphate Ribose↗
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↗
PubMed1991

Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication.

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.

Animals↗
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

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein.

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.

Animals↗
PubMed1990

Purification and characterization of mouse DNA polymerase alpha devoid of primase activity.

A simple method was developed for the isolation of primase-free DNA polymerase-alpha from the DNA polymerase-alpha-primase complex of mouse FM3A cells. The polymerase was separated from primase subunits by chromatography on a single-stranded DNA-cellulose column in the presence of 50% etylene glycol. The primase-free DNA polymerase-alpha contained two polypeptides with molecular masses of 180,000 and 68,000. Analysis of the DNA products with poly(dA)-oligo(dT)10 as template-primer revealed that both primase-free DNA polymerase-alpha and the DNA polymerase-alpha-primase complex predominantly synthesized short DNA with less than 30 nucleotides, but that the DNA polymerase-alpha-primase complex also synthesized some longer DNA with more than 300-400 nucleotides.

Animals↗
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

Peptide mapping of the four subunits of the mouse DNA polymerase alpha-primase complex.

We report a simple, two-step method (phosphocellulose and immunoaffinity column chromatographies) for purification of the mouse DNA polymerase alpha-primase complex. The advantages of this method over other procedures are its simplicity and rapidity, with little loss by proteolysis. Sedimentation analysis in a glycerol density gradient of the immunoaffinity-purified fraction revealed that four polypeptides with molecular weights of 180,000, 68,000, 54,000 and 46,000 in the enzyme fraction form a physical complex. Peptide mapping by reversed phase-high performance liquid chromatography demonstrated unequivocally that these four polypeptides constituting the complex are different entities.

Animals↗
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↗