Enzymatic and nonenzymatic ADP-ribosylation of cysteine

Mono-ADP-ribosylation is a protein modification that occurs at a number of different amino acids, dictated by the specificity of the individual ADP-ribosyltransferases. A specific cysteine in several guanine nucleotide-binding regulatory proteins is ADP-ribosylated by the bacterial protein pertussis toxin. Recent purification of an ADP-ribosylcysteine hydrolase and NAD:cysteine ADP-ribosyltransferase, and detection of ADP-ribose-cysteine linkages in tissue samples has raised hope that an endogenous regulatory cysteine-specific ADP-ribosylation pathway exists. A current goal is the identification of such a pathway for ADP-ribosylation of cysteine within animal cells. Interpretation of the data in this field has been complicated by recent reports that revealed several unforeseen chemical reactions of NAD and its metabolites with free cysteine and cysteine in proteins. This mini-review covers the latest understanding of the ADP-ribosylation reactions associated with cysteine, and provides a set of criteria for future research to establish positively the existence of an endogenous cysteine-specific mono-ADP-ribosyltransferase.     

Mono(ADP-ribosylation) in rat liver mitochondria

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.     

Thiol reagents are substrates for the ADP-ribosyltransferase activity of pertussis toxin

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.     

Amino acid-specific ADP-ribosylation. Sensitivity to hydroxylamine of [cysteine(ADP-ribose)]protein and [arginine(ADP-ribose)]protein linkages

Hydroxylamine stability has been used to classify (ADP-ribose)protein bonds into sensitive and resistant linkages, with the former representing (ADP-ribose)glutamate, and the latter, (ADP-ribose)arginine. Recently, it was shown that cysteine also serves as an ADP-ribose acceptor. The hydroxylamine stability of [cysteine([32P]ADP-ribose)]protein and [arginine([32P] ADP-ribose)]protein bonds was compared. In transducin, pertussis toxin catalyzes the ADP-ribosylation of a cysteine residue, whereas choleragen (cholera toxin) modifies an arginine moiety. The (ADP-ribose)cysteine bond formed by pertussis toxin was more stable to hydroxylamine than was the (ADP-ribose)arginine bond formed by choleragen. The (ADP-ribose)cysteine bond apparently represents a third class of ADP-ribose bonds. Pertussis toxin ADP-ribosylates the inhibitory guanyl nucleotide-binding regulatory protein (Gi) of adenylate cyclase, whereas choleragen modifies the stimulatory guanyl nucleotide-binding regulatory protein (Gs). These (ADP-ribose)protein linkages are identical in stability to those formed in transducin by the two toxins, consistent with the probability that cysteine and arginine are modified in Gi and Gs, respectively. Bonds exhibiting differences in hydroxylamine-stability were found in membranes from various non-intoxicated mammalian cells following incubation with [32P]NAD, which may reflect the presence of endogenous NAD:protein-ADP-ribosyl-transferases.     

Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.     

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.     

Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin

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.     

Evidence of endogenous mono-ADP-ribosylation of cardiac proteins via anti-ADP-ribosylarginine immunoreactivity

Arginine-specific mono-ADP-ribosylation of proteins and arginine-specific mono-ADP-ribosyltransferase occur in heart. We developed a polyclonal antiserum, R-28, against ADP-ribosylpolyarginine that recognized mono-ADP-ribosylated proteins and identified the major mono-ADP-ribosylation products of quail heart. Treatment of Immobilon-bound ADP-ribosylated Gs protein with hydroxylamine under conditions that remove ADP-ribose from its arginines eliminated R-28 immunoreactivity to Gs. Also, R-28 immunoreactivity to quail heart proteins was removed by NaOH and phosphodiesterase I treatments. Similar treatment with mercuric chloride did not remove the immunoreactivity but did remove exogenously (via in vitro pertussis toxin treatment) added ADP-ribose from cysteine of cardiac Gi/Go proteins. The antiserum did not appear to react with ADP-ribosylasparagine of Rho (formed by C3 toxin), ADP-ribosyldiphthamide of elongation factor 2 (formed by diphtheria toxin) in quail heart preparations, or polyADP-ribosylated proteins of a neonate rat cardiac nuclear preparation. Thus, the R-28 antiserum appears to contain predominantly antibodies directed against ADP-ribosylarginine. To test the usefulness of R-28, immunoblotting of subcellular fractions of quail heart was performed. R-28 showed the greatest immunoreactivity in the sarcolemma with significant immunoreactivity in denser membrane fractions. The cytosol also contained an immunoreactive band distinct from those found in the membranes. Hydroxylamine treatment eliminated immunoreactivity in the sarcolemma and denser membrane fractions but not the cytosol, suggesting the membranous immunoreactive bands contain ADP-ribosylarginine. In conclusion, a polyclonal antiserum that recognizes ADP-ribosylarginine proteins has been raised. The usefulness of the antiserum is demonstrated by the characterization of endogenous arginine mono-ADP-ribosylation products in quail heart. The quail heart has several sarcolemmal and denser membrane fraction proteins that appear to be mono-ADP-ribosylated on arginines.     

Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein

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.     

Immunochemical detection of guanine nucleotide binding proteins mono-ADP-ribosylated by bacterial toxins

Rabbits immunized with ADP-ribose chemically conjugated to carrier proteins developed antibodies reactive against guanine nucleotide binding proteins (G proteins) that had been mono-ADP-ribosylated by bacterial toxins. Antibody reactivity on immunoblots was strictly dependent on incubation of substrate proteins with both toxin and NAD and was quantitatively related to the extent of ADP-ribosylation. Gi, Go, and transducin (ADP-ribosylated by pertussis toxin) and elongation factor II (EF-II) (ADP-ribosylated by pseudomonas exotoxin) all reacted with ADP-ribose antibodies. ADP-ribose antibodies detected the ADP-ribosylation of an approximately 40-kilodalton (kDa) membrane protein related to Gi in intact human neutrophils incubated with pertussis toxin and the ADP-ribosylation of an approximately 90-kDa cytosolic protein, presumably EF-II, in intact HUT-102 cells incubated with pseudomonas exotoxin. ADP-ribose antibodies represent a novel tool for the identification and study of G proteins and other substrates for bacterial toxin ADP-ribosylation.     

ADP-ribosyl proteins formed by pertussis toxin are specifically cleaved by mercury ions

Various types of ADP-ribosyl protein conjugates were synthesized and their chemical stability was compared with that of cysteine-linked ADP-ribosyl groups as formed by incubation of transducin or Gi/Go proteins with NAD and pertussis toxin. Treatment with 0.1 mM HgCl2 specifically cleaved the cysteine-linked conjugates. This may provide a tool for the quantitation of modified Gi/Go proteins as well as of other acceptors modified by ADP-ribose at cysteine residues in the presence of other ADP-ribosyl proteins.     

Common structure of the catalytic sites of mammalian and bacterial toxin ADP-ribosyltransferases

The amino acid sequences of several bacterial toxin ADP-ribosyltransferases, rabbit skeletal muscle transferases, and RT6.2, a rat T-cell NAD glycohydrolase, contain three separate regions of similarity, which can be aligned. Region I contains a critical histidine or arginine residue, region II, a group of closely spaced aromatic amino acids, and region III, an active-site glutamate which is at times seen as part of an acidic amino acid-rich sequence. In some of the bacterial ADP-ribosyltransferases, the nicotinamide moiety of NAD has been photo-crosslinked to this glutamate, consistent with its position in the active site. The similarities within these three regions, despite an absence of overall sequence similarity among the several transferases, are consistent with a common structure involved in NAD binding and ADP-ribose transfer.     

Characterization of NAD:arginine ADP-ribosyltransferases

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.     

An NAD:cysteine ADP-ribosyltransferase is present in human erythrocytes

A novel enzymatic activity, i.e., the catalysis of the formation of ADP-ribosylcysteine, was found in the cytosol of human erythrocytes. The NAD:cysteine ADP-ribosyltransferase was partially purified by sequential chromatographic steps on phenyl-Sepharose, phosphocellulose, and Sepharose CL-6B. The enzyme has an apparent molecular weight of 27,000 +/- 3,000, as determined by gel permeation. The formation of ADP-ribosylcysteine was associated with the stoichiometric release of nicotinamide from NAD. The enzyme was found to be highly specific toward cysteine and cysteine methyl ester as ADP-ribose acceptors. S-Benzoyl-L-cysteine, cystine, histidine, glutamic acid, arginine, arginine methyl ester, and agmatine were ineffective as acceptors for this enzyme.     

NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5'-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.     

A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.     

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.     

Regulation of acetyl-CoA carboxylase by ADP-ribosylation

Because of certain similarities between acetyl-CoA carboxylase (ACC) and tubulin, and the recent demonstration of the ADP-ribosylation of tubulin by cholera toxin, we have investigated a potential role for ADP-ribosylation in the regulation of ACC activity. Incubation of purified rat liver ACC with cholera toxin in the presence of millimolar concentrations of [adenylate-32P]NAD results in a time-dependent incorporation of ADP-ribose into ACC of greater than 2 mol/mol of enzyme subunit, accompanied by a marked inactivation of enzyme activity. This effect is not mimicked by pertussis toxin, ADP-ribose, or ribose 5-phosphate. Incubation of labeled ACC with snake venom phosphodiesterase and alkaline hydrolysis release 32P-products tentatively identified by high-performance liquid chromatography as 5'-[32P]AMP and [32P]ADP-ribose, respectively. These data are consistent with a mono-ADP-ribosylation of ACC catalyzed by cholera toxin. Phosphodiesterase treatment of inactivated ACC partially restores enzyme activity. The effects of ADP-ribosylation of ACC are expressed both as a decrease in the enzyme Vmax and as an increase in the apparent Ka for citrate. These results suggest that ACC might be a substrate for endogenous ADP-ribosyltransferases and that this covalent modification could be an important regulatory mechanism for the modulation of fatty acid synthesis in vivo.     

Adenosine diphosphate ribose transferase from baby-hamster kidney cells (BHK-21/C13). Characterization of the reaction and product.

Some properties of ADP-ribose transferase, and its reaction product, from BHK-21/C13 cells are described. Enzyme activity was found almost exclusively in nuclei (90%), with the remaining 10% located in the cytosolic fraction. The nuclear enzyme is chromatin-bound and requires bivalent cations, preferably Mg2+, a pH of 8.0 and a temperature of 25 degrees C for optimal activity. Chromatin preparations incorporated radioactivity from [14C]NAD+ into acid-insoluble material for about 60 min. Kinetics for substrate NAD+ utilization were not of Michaelis--Menten type; biphasic kinetics were shown from a double-reciprocal plot (1/reaction velocity against 1/[NAD+]) and from a 'Hofstee' plot (reaction velocity/[NAD+] against reaction velocity). The transferase is unstable in the absence of Mg2+ ions. It is inhibited by thymidine, nicotinamide and nicotinamide analogues, but not by ATP, which stimulates it at concentrations of 5 mM and above. The enzyme requires thiol groups for activity; it is readily inhibited by N-ethylmaleimide at 0.5 mM. The product of the reaction is stable under acid conditions at temperatures up to 25 degrees C, but it is hydrolysed by HClO4 at 70 degrees C. It is resistant to NaOH, but is cleaved from its attachment to protein with alkali into trichloroacetic acid-insoluble and -soluble components. On the basis of Cs2SO4- density-gradient analysis under denaturing conditions (gradients included urea and guanidinium hydrochloride), and analysis of the reaction product directly on hydroxyapatite, we conclude that most of the radioactive ADP-ribose residues are firmly bound to protein, presumably in covalent linkage. Hydroxyapatite-chromatographic analysis of ADP-ribose residues released from protein by alkaline digestion showed a spectrum of molecular sizes including mono-, oligo- and poly-(ADP-ribose), when chromatin was incubated initially with [14C]NAD+ for 10 min and then for a further 30 min after addition of excess non-radioactive NAD+, only about 10% of the radioactive mono-(ADP-ribose) could be 'chased' into longer-chain molecules. Hydroxyapatite analysis was also used to show that, whereas all ADP-ribose residues were released from protein with NaOH, only 50% of them were susceptible to hydroxylamine. These hydroxylamine-sensitive residues included all size classes, although mono-(ADP-ribose) predominated. Finally, there was an approximately equal distribution of ADP-ribose incorporated into HCl-soluble proteins (including the histones) and HCl-insoluble proteins (including the non-histone proteins) when chromatin was incubated with NAD+ up to 0.5 mM, but at higher NAD+ concentrations more ADP-ribose was incorporated into the HCl-soluble fraction (82% at 4.0 mM-NAD+).     

Polypeptide Hormones and Chromatin-Associated Proteins Act as Acceptors for Cholera Toxin-Catalyzed ADP-Ribosylation

Abstract: Cholera toxin catalyzed the ADP-ribosylation of the pituitary protein hormones thyrotropin (TSH), lutropin (LH), follitropin (FSH), human chorionic gonadotropin (hCG). and corticotropin (ACTH)_1–24, and ADP-ribosylation of the basic proteins histone subfraction H₁ and protamine. Casein and phosvitin, acidic nuclear proteins, did not act as acceptors for toxin-catalyzed ADP-ribosylation. The isolated TSH A and B subunits were tested for their ADP-ribose acceptor activity. The TSH A subunit showed fourfold greater ADP-ribose acceptor activity than the TSH B subunit. The ADP-ribose acceptor protein protamine was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis following incubation with cholera toxin under ADP-ribosylating conditions. [³H]ADP-ribose incorporated into protein from [³H]NAD migrated with the acceptor protein protamine. In the absence of added acceptor protein, the [³H]ADP-ribose incorporated into protein migrated with the A₁ fragment of cholera toxin. Cholera toxin A and B subunits were isolated and tested for their ability to catalyze the transfer of ADP-ribose to protamine. The cholera toxin A subunit showed 50-fold greater ADP-ribosyltransferase activity than the B subunit. Our data indicate that a variety of adenohypophyseal hormones and regulatory proteins act as acceptors for toxin-catalyzed ADP-ribosylation. These studies may help in understanding the role of endogenous ADP-ribosyltransferases and the physiological effects of this modification of protein.