Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 2 cysteine residues present in the A subunit of pertussis toxin form a disulfide bond in the conformation of the toxin secreted from the bacteria. Previous studies have shown that reduction of this bond is necessary for activation of the enzyme. We have found that reduction of this bond also alters the conformation of the A subunit such that it no longer readily associates with the B oligomer of the toxin, a finding which may have implications concerning the form of the toxin found within the eukaryotic cell. In addition, we have demonstrated that reduction of the disulfide bond of the purified A subunit followed by treatment with sulfhydryl-modifying reagents such as N-ethylmaleimide or 5,5'-dithiobis-(2-nitrobenzoic acid) results in inhibition of the NAD glycohydrolase activity of the protein. When a tryptic fragment of the A subunit which contains only 1 of the cysteine residues (Cys-41) of the native protein was reacted with N-ethylmaleimide, the NAD glycohydrolase activity of this fragment was substantially reduced. These data indicate that Cys-41 may be in a region of the molecule which is critical for the enzymatic activity of the toxin.
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PMID:Role of cysteine 41 of the A subunit of pertussis toxin. 253 46

Sulfhydryl-alkylating reagents are known to inactivate the NAD glycohydrolase and ADP-ribosyltransferase activities of the S1 subunit of pertussis toxin, a protein which contains two cysteines at positions 41 and 200. It has been proposed that NAD can retard alkylation of one of the two cysteines of this protein (Kaslow, H.R., and Lesikar, D.D. (1987) Biochemistry 26, 4397-4402). We now report that NAD retards the ability of these alkylating reagents to inactivate the S1 subunit. In order to determine which cysteine is protected by NAD, we used site-directed mutagenesis to construct analogs of the toxin with serines at positions 41 and/or 200. Sulfhydryl-alkylating reagents reduced the ADP-ribosyltransferase activity of the analog with a single cysteine at position 41; NAD retarded this inactivation. In contrast, sulfhydryl-alkylating reagents did not inactivate analogs with serine at position 41. An analog with alanine at position 41 possessed substantial ADP-ribosyltransferase activity. We conclude that alkylation of cysteine 41, and not cysteine 200, inactivates the S1 subunit of pertussis toxin, but that the sulfhydryl group of cysteine 41 is not essential for the ADP-ribosyltransferase activity of the toxin. These results suggest that the region near cysteine 41 contributes to features of the S1 subunit important for ADP-ribosyltransferase activity. Using site-directed mutagenesis, we found that changing aspartate 34 to asparagine, arginine 39 to lysine, and glutamine 42 to glutamate had little effect on ADP-ribosyltransferase activity. However, substituting an asparagine for the histidine at position 35 markedly decreased, but did not eliminate, ADP-ribosyltransferase activity. Chou-Fasman analysis predicted no significant modifications in secondary structure of the S1 peptide with the change of histidine 35 to asparagine. Thus, histidine 35 may interact with a substrate of the S1 subunit without being essential for catalysis.
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PMID:Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin. 270 95

The combination of ATP, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), and DTT (dithiothreitol) is known to promote the expression of the NAD glycohydrolase activity of pertussis toxin, which resides in the toxin's S1 subunit. By monitoring changes in electrophoretic mobility, we have found that ATP and CHAPS act by promoting the reduction of the disulfide bond of the S1 subunit. In addition, ATP, CHAPS, and DTT allowed sulfhydryl-alkylating reagents to inactivate the NAD glycohydrolase activity. In the presence of iodo[14C]acetate, the combination of ATP, CHAPS, and DTT increased 14C incorporation into only the S1 subunit of the toxin, indicating that alkylation of this subunit was responsible for the loss of activity. If iodoacetate is used as the alkylating reagent, alkylation can be monitored by an acidic shift in the isoelectric point of the S1 peptide. Including NAD in alkylation reactions promoted the accumulation of a form of the S1 peptide with an isoelectric point intermediate between that of native S1 and that of S1 alkylated in the absence of NAD. This result suggests that NAD interacts with one of the two cysteines of the S1 subunit. In addition, we found the pH optimum for the NAD glycohydrolase activity of pertussis toxin is 8, which may reflect the participation of a cysteine in the catalytic mechanism of the toxin.
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PMID:Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin. 282 91

This paper investigates protein mono(ADP-ribosylation) in rat liver mitochondria. In isolated inner mitochondrial membranes, in the presence of both ADP-ribose and NAD+, a protein is mono-(ADP-ribosylated) with high specificity. The reaction apparently consists of enzymatic NAD+ glycohydrolysis and subsequent binding of free ADP-ribose to the acceptor protein. In terms of chemical stability, the resulting bond is unique among the ADP-ribose linkages thus far characterized. Formation of a Schiff base adduct between free ADP-ribose and the acceptor protein is excluded. In intact mitochondria at least three classes of proteins are ADP-ribosylated in vivo. One ADP-ribose-protein linkage is of the carboxylate ester type as indicated by its lability in neutral buffer. Another class of ADP-ribosylated proteins requires hydroxylamine for release of ADP-ribose. The third class is stable in hydroxylamine but labile to alkali, similar to the ADP-ribose-cysteine linkage in transducin formed by pertussis toxin.
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PMID:Mono(ADP-ribosylation) in rat liver mitochondria. 283 67

The observation that virtually all of the somatostatin-like immunoreactivity in the stomach consists of somatostatin-14 (S14), to the exclusion of somatostatin-28 (S28), suggests a unique pattern of prosomatostatin posttranslational processing. In order to examine the mechanisms by which S14 is produced from its precursor in the stomach, we investigated the biosynthesis of somatostatin in isolated canine fundic D cells. D cells pulse-labeled with [35S]cysteine revealed a cycloheximide inhibitable time-dependent incorporation of radioactivity into S14. A small fraction of radioactivity was incorporated into S28 but not into larger precursors. However, when the cells were incubated with monensin (1 microM), incorporation of radioactivity into a presumed somatostatin precursor was noted. Upon transfer of [35S]cysteine prelabeled cells to radioactivity-free medium, no conversion of S28 to S14 could be detected and the decrease of labeled S14 in cells correlated with a complimentary increase in the culture medium. Exogenous somatostatin inhibited somatostatin biosynthesis in a fashion that could be blocked by pertussis toxin pretreatment. Stimulation of prelabeled D cells with tetradecanoyl phorbol 13-acetate (10(-7) M) or forskolin (10(-4) M) for 2 h resulted in release of 41 and 33% of the newly synthesized radioactive S14, respectively, while only 9 and 6% of the total cell content of radioimmunoassayable somatostatin was secreted. These data suggest that: (a) somatostatin is synthesized in fundic D cells primarily as S14, (b) S14 is produced by rapid processing of a larger precursor but there is little, if any, conversion of S28 to S14, (c) somatostatin biosynthesis is autoregulated, and (d) newly synthesized S14 is preferentially released from D cells in response to stimulation.
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PMID:Biosynthesis of somatostatin in canine fundic D cells. 289 59

The guanine nucleotide-binding proteins which mediate hormonal inhibition of adenylate cyclase as well as hormonal regulation of other membrane functions are alpha, beta, and gamma heterotrimers which are structurally homologous to each other. In brain, the predominant guanine nucleotide-binding component is a 39-kDa protein whose physiological role is as yet unknown. We have used N-ethylmaleimide to define functionally important sulfhydryl groups on alpha 39. Three cysteine residues in the molecule are reactive in unliganded alpha 39. Alkylation of two of these is reduced when guanosine 5'-(3'-O-thio)triphosphate (GTP gamma S) is bound. We have isolated and sequenced tryptic peptides containing the three reactive cysteines. The octapeptide containing the GTP gamma S-insensitive cysteine is at a position equivalent to amino acids 106-113 of the transducin alpha subunit (Lochrie, M. A., Hurley, J. B., and Simon, M. I. (1985) Science 228, 96-99). However, the equivalent peptide in transducin does not contain a cysteine residue. Alkylation of this cysteine blocks ADP-ribosylation of cysteine 351 by pertussis toxin. However, alkylation does not prevent association of alpha with the beta X gamma subunits nor does it inhibit GTPase activity. The two GTP gamma S-sensitive cysteines are at positions equivalent to cysteines 139 and 286 of the transducin alpha subunit. Alkylation of these residues inhibits GTPase activity. Neither of these GTP gamma S-sensitive cysteines are in those regions of alpha 39 which are highly homologous to the GTP-binding site of elongation factor Tu (Jurnak, F. (1985) Science 230, 32-36). However, both are present in the brain 41-kDa guanine nucleotide-binding protein and in the two transducins. The conservation of these cysteine residues suggests that they are important for the function of the subunits.
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PMID:Reactive sulfhydryl groups of alpha 39, a guanine nucleotide-binding protein from brain. Location and function. 310 18

An NAD:cysteine ADP-ribosyltransferase designated ADP-ribosyltransferase C was purified approximately 35,000-fold from human erythrocytes with an 11% yield. The purified ADP-ribosyltransferase C exhibited one predominant protein band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight (Mr) of 28,500. The Km values for NAD and cysteine methyl ester were determined to be 65 and 4,400 microM, respectively. By using human erythrocyte inside-out membrane vesicles, the transferase C was found to ADP-ribosylate the alpha subunit (Mr = 41,000) of Gi, which is a substrate for pertussis toxin. The ADP-ribosylation of Gi alpha catalyzed by ADP-ribosyltransferase C was inhibited by pre-ADP-ribosylation with pertussis toxin. The linkage of ADP-ribose-Gi alpha in the membranes formed by ADP-ribosyltransferase C was as stable to hydroxylamine as that formed by pertussis toxin. These data represent the first demonstration that eukaryotic cells contain an ADP-ribosyltransferase which can catalyze the ADP-ribosylation of a cysteine residue in Gi alpha.
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PMID:Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein. 312 40

Thiols such as cysteine and dithiothreitol are substrates for the ADP-ribosyltransferase activity of pertussis toxin. When cysteine was incubated with NAD+ and toxin at pH 7.5, a product containing ADP-ribose and cysteine (presumably ADP-ribosylcysteine) was isolated by high-performance liquid chromatography, and characterized by its composition and release of AMP with phosphodiesterase. Cysteine has a Km of 105 mM at saturating NAD+ concentration. The ability of thiols to act as a substrate is one explanation for the very high concentrations (250 mM or greater) that have been observed to enhance the apparent NAD glycohydrolase activity of the toxin.
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PMID:Thiol reagents are substrates for the ADP-ribosyltransferase activity of pertussis toxin. 313 46

A G protein alpha subunit gene has been isolated from a Drosophila melanogaster genomic library using a combination of bovine rod and cone transducin alpha subunit cDNAs as a probe under reduced stringency conditions. The gene, DG alpha 1, encodes a protein with an amino acid sequence 78% identical to bovine Gi alpha 1. However, unlike all reported Gi alpha subunit the DG alpha 1-encoded protein is not expected to be a pertussis toxin substrate, because it lacks a cysteine at the appropriate site. The protein coding region of the gene is split by four introns. The sequence of a head tissue cDNA clone, as well as amino acid similarities to mammalian G proteins, confirms this exon/intron structure. Northern blots of total cellular RNA reveal a major 2.3-kilobase transcript and a less abundant 1.7-kilobase transcript. These transcripts are most abundant in RNA from embryos and pupae. The DG alpha 1 gene is located on band 65C on the left arm of the third chromosome, on the basis of in situ hybridizations to Drosophila salivary gland polytene chromosomes.
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PMID:A Drosophila melanogaster G protein alpha subunit gene is expressed primarily in embryos and pupae. 313 72

We attempted to characterize ADP-ribose-amino acid bonds formed by various bacterial toxins. The ADP-ribose-arginine bond formed by botulinum C2 toxin in actin was cleaved with a half-life of about 2 h by treatment with hydroxylamine (0.5 M). In contrast, the ADP-ribose-cysteine bond formed by pertussis toxin in transducin and the ADP-ribose-amino acid linkage formed by botulinum ADP-ribosyltransferase C3 in platelet cytosolic proteins were not affected by hydroxylamine. HgCl2 cleaved the ADP-ribose-amino acid bond formed by pertussis toxin in transducin but not those formed by botulinum C2 toxin or botulinum ADP-ribosyltransferase C3 in actin and platelet cytosolic proteins, respectively. NaOH (0.5 M) cleaved the ADP-ribose-amino acid bonds formed by botulinum C2 toxin and pertussis toxin but not the one formed by botulinum ADP-ribosyltransferase C3. The data indicate that the ADP-ribose bond formed by botulinum ADP-ribosyltransferase C3 differs from those formed by the known bacterial ADP-ribosylating toxins.
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PMID:Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin. 314 Aug 13


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