Biomedical subjects

Y Ohoka

Publications and source records attributed to Y Ohoka.

23 records · Page 2Linked to original sources

PubMed1991

Functional modification by cholera-toxin-catalyzed ADP-ribosylation of a guanine-nucleotide-binding regulatory protein serving as the substrate of pertussis toxin.

The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21,394-21,400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.

Adenosine Diphosphate Ribose↗

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

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

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↗

PubMed1989

Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells.

A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.

ADP-Ribosylation Factors↗