Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. (25/44)

Pseudomonas aeruginosa exoenzyme S is an adenosine diphosphate ribosyltransferase distinct from Pseudomonas toxin A. Exoenzyme S catalyzes the transfer of radioactivity from all portions of radiolabeled NAD+ except nicotinamide. Digestion of the radiolabeled product(s) formed in the presence of [adenine-14C]NAD+ and exoenzyme S with snake venom phosphodiesterase yields only AMP, suggesting that ADP-ribose is present as monomers and not as poly(ADP-ribose). Exoenzyme S does not catalyze the transfer of ADP-ribose from NAD+ to elongation factor 2, as do toxin A and diphtheria toxin, but to one or more other proteins present in crude extracts of wheat germ or rabbit reticulocytes and in partially purified preparations of elongation factor I. The ADP-ribosyltransferase activity of exoenzyme S is distinct from toxin A by several tests: it is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. These latter observations and the substrate specificity suggest that exoenzyme S is different from any previously described prokaryotic ADP-ribosyltransferase.  (+info)

Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase. (26/44)

An ADP-ribosyltransferase was purified approximately 500-fold from the supernatant fraction of turkey erythrocytes. The enzyme hydrolyzed [carbonyl-(14)C]NAD to ADP-ribose and [carbonyl-(14)C]nicotinamide at a low rate. Nicotinamide formation from NAD was enhanced by arginine methyl ester > D-arginine approximately L-arginine > guanidine; lysine, histidine, and citrulline were ineffective. Incubation of [adenine-U-(14)C]NAD and arginine methyl ester or arginine with the purified enzyme resulted in the formation of new compounds that contained (14)C, reacted with ninhydrin, and quenched background fluorescence of thin-layer plates viewed in ultraviolet light. Their mobilities on thin-layer chromatograms were indistinguishable from those of ADP-ribosylarginine methyl ester and ADP-ribosylarginine formed during incubation of choleragen with NAD and arginine methyl ester or arginine, respectively [Moss, J. & Vaughan, M. (1977) J. Biol. Chem. 252, 2455-2457]. The purified transferase also catalyzed the incorporation of label from [adenine-(14)C]-NAD into lysozyme, histones and polyarginine. When the (14)C-labeled lysozyme was incubated with snake venom phosphodiesterase, the radioactivity was released and, on thin-layer chromatograms, exhibited a mobility indistinguishable from that of 5'-AMP, as would be expected of an ADP-ribosylated protein, but not of a poly(ADP-ribosylated) product. The purified transferase activated rat brain adenylate cyclase and, as is the case with choleragen, activation was absolutely dependent on NAD. The presence in the avian erythrocyte of a protein that, like choleragen and Escherichia coli heat-labile enterotoxin, apparently activates adenylate cyclase and possesses ADP-ribosyl transferase activity is consistent with the view that the mechanisms through which the bacterial toxins produce pathology are not entirely foreign to vertebrate cells, at least some of which may possess and employ an analogous mechanism for activation of adenylate cyclase.  (+info)

ADP ribosylation of rat liver nucleosomal core histones. (27/44)

When nucleosomal core histones were isolated from rat liver nuclei incubated with [14C]NAD+ and fractionated into the individual components (H2A, H2B, H3, and H4), [14C]adenosine diphosphate ribose (ADP-Rib) was found to be associated with all of them. However, while about 15% of the H2B molecules were modified, less than 2% of the other fractions contained radioactive ADP-Rib. The nucleotide attached to H2B was identified as a single monomer of ADP-Rib. On subjectint H2B to electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid, one single band of H2B with 5% less mobility than the unomdified control was obtained. The linkage between H2B and ADP-Rib was rapidly hydrolyzed with 0.1 N NaOH or with 1 M neutral hydroxylamine. Hydrolysis of ADP-ribosylated H2B with trypsin generated a single peptide linked to ADP-Rib, which corresponded to the sequence Pro-Glu-Pro-Ala-Lys. We were able to dansylate the NH2-terminal proline, which proved that the imino group of this amino acid was not substituted. These findings, together with the chemical properties of the linkage, which were typical of those of an ester-like bond, strongly suggest that the ADP-Rib residue was linked to the gamma-COOH group of the glutamic acid in position 2 of H2B.  (+info)

ADP ribosylation of rat liver lysine-rich histone in vitro. (28/44)

Purified rat liver nuclei were incubated with [14C]-NAD+ and the various nuclear protein fractions were separated. Forty per cent of the total radioactivity incorporated was associated with the histone fraction. Of this, about 50% was extracted with H1, in 0.5 N perchloric acid. When crude H1 was purified and fractionated into five different subfractions by chromatography on Bio-Rex 70, it was found that all the H1 subfractions contained radioactivity. This radioactive material was identified as oligomers of adenosine diphosphate ribose (ADP-Rib) with an average chain length which corresponded to trimers. The extent of the modification was dependent on the concentration of NAD+. About 60% of the H1 molecules were modified with a concentration of 1 mM NAD+. The presence of these oligomers of ADP-Rib introduced a large degree of microheterogeneity to H1 as detected by electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid. Bands of H1 with 10 to 20% less mobility than the unmodified H1 were present. Also, as a consequence of large content of ADP-Rib, the absorption maximum shifted from 275 to 259 nm. The half-life of the bond between the oligomers of ADP-Rib and H1 was about 3 min at 37 degrees C in the presence of 0.1 N NaOH, and 10 m1 were modified. The site of ADP ribosylation in the NH2-terminal half was localized in the tryptic peptide extending from the NH2-terminal end to lysine 15. The site of modification of the COOH-erminal half was localized in the tryptic peptide which contained the only glutamic acid residue in this fragment of H1...  (+info)

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. I. The tryptic peptides from the maleylated protein. (29/44)

Six tryptic peptides ranging in size from 3 to 126 residues were isolated from maleylated Fragment A of diphtheria toxin after tryptic hydrolysis. These peptides accounted for all 193 residues found by amino acid analysis. After demaleylation, the six peptides were purified by chromatography on Sephadex G-50, coupled with paper chromatography and electrophoresis, and were analyzed by various methods. The compositions and properties of the peptides are reported. Almost 70% of the residues were positioned within these peptides.  (+info)

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides. (30/44)

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).  (+info)

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. III. The chymotryptic peptides, the peptides derived by cleavage at tryptophan residues, and the complete sequence of the protein. (31/44)

Fragment A (21,145 daltons in its longest known form) may be derived from diphtheria toxin (60,000 daltons) by mild tryptic digestion and reduction. Purified Fragment A consists of a mixture of 3 molecules of 190, 192, and 193 residues; the first 190 residues are in common and correspond to the NH2-terminal region the toxin. All three species of Fragment A are active in catalyzing ADP ribosylation of elongation factor 2, an essential component of protein synthesis. This reaction inactivates the factor and is responsible for the toxin's action in inhibiting protein synthesis in animal cells. It is believed that Fragment A or similar enzymically active fragments released into the cytosol of toxin-treated cells mediate this inhibition. The complete amino acid sequence of Fragment A has been determined from 32 chymotryptic peptides, three peptides derived by chemical cleavage of Fragment A at its 2 tryptophan residues, five cyanogen bromide peptides, and six tryptic peptides from the maleylated protein.  (+info)

Enzymic activity of cholera toxin. II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition. (32/44)

Cholera toxin containing intact A chain (Mr = 29,000) was isolated, and its enzymic properties were characterized. The "unnicked" form of the toxin, produced by a protease-deficient, hypertoxinogenic mutant of Vibrio cholerae 569B, had greatly reduced activity in catalyzing the NAD+-glycohydrolase and ADP-ribosyltransferase reactions as compared to the naturally nicked form commonly isolated. In the latter, the intact A chain has been cleaved by bacterial proteases to yield disulfide-linked A1 and A2 chains (Mr = 23,000 and 6,000, respectively). Digestion of unnicked toxin with trypsin or elastase yielded a nicked form similar to or identical with the naturally nicked toxin, but chymotryptic digestion did not. Disulfide bond reduction was necessary for expression of enzymic activity by naturally nicked or trypsin-nicked toxin, or the A1A2 protomer. Fractionation of thiol-treated, nicked cholera toxin by ion exchange, molecular exclusion, or affinity chromatography gave results suggesting that the reduced toxin displays enzymic activity while remaining structurally intact.  (+info)