scholarly journals ADP-ribosylation of a 21-24 kDa eukaryotic protein(s) by C3, a novel botulinum ADP-ribosyltransferase, is regulated by guanine nucleotide

1987 ◽  
Vol 247 (2) ◽  
pp. 363-368 ◽  
Author(s):  
K Aktories ◽  
J Frevert
Keyword(s):  
Protein S ◽  
Inhibitory Effect ◽  
The Novel ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Adp Ribosylation ◽  
Type C ◽  
Guanine Nucleotide Binding ◽  
Adp Ribosyltransferase ◽  
C2 Toxin

Besides botulinum C2 toxin, Clostridium botulinum type C produces another ADP-ribosyltransferase, which we termed ‘C3’. ADP-ribosyltransferase C3 has a molecular mass of 25 kDa and modifies 21-24 kDa protein(s) in platelet and brain membranes. C3 was about 1000 times more potent than botulinum C1 toxin in ADP-ribosylation of membrane proteins. C3-catalysed ADP-ribosylation of the 21-24 kDa protein(s) was decreased by stable guanosine triphosphates, with the potency order GTP[S] much greater than p[NH]ppG greater than p[CH2]ppG. GTP[S] inhibited the ADP-ribosylation caused by C3 by maximally 70-80%, with half-maximal and maximal effects occurring at 0.3 and 10 microM-GTP[S] respectively. The concomitant addition of GTP decreased the inhibitory effect of GTP[S]. GTP[S]-induced inhibition of ADP-ribosylation was resistant to washing of pretreated platelet membranes. The data suggest that the novel botulinum ADP-ribosyltransferase C3 modifies eukaryotic 21-24 kDa guanine nucleotide-binding protein(s).

Hepatocyte beta-adrenergic responsiveness and guanine nucleotide-binding regulatory proteins

1989 ◽  
Vol 256 (2) ◽  
pp. C384-C389 ◽  
Author(s):  
J. A. Garcia-Sainz ◽  
M. E. Huerta-Bahena ◽  
C. C. Malbon
Keyword(s):  
Binding Protein ◽  
Nucleotide Binding ◽  
Regulatory Proteins ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Beta Adrenergic ◽  
Adp Ribosylation ◽  
Liver Membranes ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Protein

Hepatocytes isolated from hypothyroid, adrenalectomized, or partially hepatectomized rats display an enhanced beta-adrenergic responsiveness as compared with cells from control animals. The enhanced beta-adrenergic responsiveness is evidenced by both increased ureagenesis and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to isoproterenol. The role of stimulatory guanine nucleotide-binding protein (Gs) and inhibitory guanine nucleotide-binding protein (Gi) in the enhanced responsiveness was studied. It was observed, contrary to what would have been anticipated, that the level of Gs [as reflected by cholera toxin-catalyzed ADP ribosylation, 5'-guanosine gamma-thiotriphosphate (GTP gamma S)-stimulated adenylate cyclase activity, and a functional reconstitution assay] was decreased in liver membranes from adrenalectomized and partially hepatectomized rats as compared with the controls. Furthermore, the level of Gi was increased in these conditions as reflected by pertussis toxin-catalyzed ADP ribosylation. The data suggest that changes in beta-adrenergic receptor levels rather than the levels of guanine nucleotide-binding (G) regulatory proteins predominate in regulation of hepatic beta-adrenergic responses by hypothyroidism, adrenalectomy, or partial hepatectomy.

scholarly journals Effects of Site-Directed Mutagenesis ofEscherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

1999 ◽  
Vol 67 (1) ◽  
pp. 259-265 ◽  
Author(s):  
Linda A. Stevens ◽  
Joel Moss ◽  
Martha Vaughan ◽  
Mariagrazia Pizza ◽  
Rino Rappuoli
Keyword(s):  
Cholera Toxin ◽  
Nucleotide Binding ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Guanine Nucleotide ◽  
Adp Ribosylation ◽  
Heat Labile ◽  
Guanine Nucleotide Binding ◽  
Catalyzed Reactions ◽  
Adp Ribosyltransferase

ABSTRACT Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.

scholarly journals Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes

1987 ◽  
Vol 248 (3) ◽  
pp. 749-754 ◽  
Author(s):  
P A Watkins ◽  
Y Kanaho ◽  
J Moss
Keyword(s):  
Nucleotide Binding ◽  
Rod Outer Segments ◽  
Guanine Nucleotide ◽  
Gtpase Activity ◽  
Adp Ribosylation ◽  
Outer Segments ◽  
Retinal Rod ◽  
Guanine Nucleotide Binding ◽  
Adp Ribosyltransferase ◽  
Retinal Rod Outer Segments

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical ‘helper subunits’ T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.

Sign in / Sign up

Export Citation Format

Share Document