Rate Of ADP-ribosylation Research Articles

Time course of action of pertussis toxin to block the inhibition of stimulated insulin release by norepinephrine.

Male Sprague-Dawley rats were injected ip with 1 microgram pertussis toxin (PTX)/100 g BW. The rats were killed 24, 48, and 72 h after injection, and their pancreases were removed. At each time point, insulin secretion by isolated islets was measured under basal and glucose-stimulated conditions and in the absence and presence of norepinephrine. cAMP levels were measured under basal and forskolin-stimulated conditions in the absence and presence of norepinephrine. PTX-induced ADP ribosylation of Gi/Go proteins in vivo was monitored by ADP ribosylation in vitro using PTX and 32P-labeled NAD and also by Western blotting. At 24 h, 1 microM norepinephrine inhibited glucose-stimulated insulin secretion by 92% in the control islets, but by only 53% in the PTX-treated islets; at 48 h, norepinephrine still inhibited secretion (by 40%) in the PTX-treated islets; at 72 h, the inhibitory effect of norepinephrine was abolished. Therefore, contrary to recent suggestions, all of the effect of norepinephrine to inhibit insulin release is PTX sensitive. The effects of PTX on the ability of norepinephrine to lower cAMP levels were similar to those observed for the inhibition of insulin release. PTX partially blocked the effect of norepinephrine to lower cAMP levels at 24 and 48 h, and the block was complete after 72 h. The extent of the in vivo ADP ribosylation of the Gi/Go proteins, monitored at each time point by in vitro [32P]ADP-ribosylation and Western blotting, demonstrated a profound ADP ribosylation at 48 and 72 h. As detected by Western blotting, the rates of ADP ribosylation by PTX and the onset of decreased expression varied among the different G-proteins. G alpha o was virtually eliminated after only 24 h of PTX treatment. G alpha i2 was markedly affected by 48 h; G alpha i3 was little affected until 72 h.

Cysteine-specific ADP-ribosylation of actin.

Incubation of lysate from human polymorphonucleated neutrophils and human platelets with [32P]NAD resulted in the labeling of a 42-kDa protein. Phosphodiesterase (Crotalus durissus) released 5'-AMP from the radiolabeled protein. The 42-kDa protein was identified as actin by binding to DNAse-I, two-dimensional gel electrophoresis and partial proteolysis. The rate of ADP-ribosylation was greater with [32P]ADP-ribose than with [32P]NAD, indicating a non-enzymic modification. ADP-ribose also modified actin in the actin-DNAase-I complex, but denatured actin was not modified by ADP-ribose. Only cytoplasmic beta/gamma-actin isoforms were non-enzymically ADP-ribosylated but not muscle alpha-actin. The acceptor amino acid was identified as a cysteine residue whereas the bacterial ADP-ribosyltransferase C. perfringens iota toxin catalyzes incorporation of ADP-ribose to Arg177 of actin. Alkylation of cysteine residues of actin with N-ethylmaleimide prevented subsequent non-enzymic ADP-ribosylation but not the toxin catalyzed modification. Non-enzymically ADP-ribosylated actin was further modified by C. perfringens iota toxin. The F-actin stabilizing mycotoxin phalloidin blocked the non-enzymatic ADP-ribosylation and, conversely, ADP-ribosylation inhibited the phalloidin-induced polymerization of ADP-ribosylated actin. The data indicate that cytoplasmic actin is non-enzymically ADP-ribosylated by ADP-ribose at a cysteine residue to inhibit actin polymerization.

Modulation of endogenous ADP-ribosylation in rat brain

Endogenous ADP-ribosylation of proteins was measured in homogenates, membranes, and cytosol from rat brain regions. Several proteins were ADP-ribosylated in homogenates, especially a 49 kDa protein. Sodium nitroprusside, a source of nitric oxide, particularly enhanced the ADP-ribosylation of 47 kDa and 39 kDa proteins. Levels of basal and sodium nitroprusside-stimulated ADP-ribosylated proteins were similar, but not identical, in homogenates from the cerebral cortex, hippocampus, striatum, thalamus and cerebellum. In neonatal cerebral cortex, ADP-ribosylation of an additional 110 kDa protein was detected and this was also enhanced by sodium nitroprusside. ADP-ribosylation of the 110 kDa protein was evident one and two days after birth, but not at five days and later. Each protein demonstrated unique sensitivities to sodium nitroprusside and rates of ADP-ribosylation. Cyclic GMP did not mimic the effects of sodium nitroprusside. Mg 2+ inhibited ADP-ribosylation of the 49 kDa and 47 kDa proteins but had a smaller effect on the 39 kDa protein. ADP-ribosylation in the cytosol predominantly affected only a single protein of 39 kDa, and this was stimulated by sodium nitroprusside and by addition of cofactors necessary for activation of nitric oxide synthase. Several proteins in membranes were ADP-ribosylated and the 49 and 47 kDa proteins were released from the membranes coincidentally with ADP-ribosylation. The predominate substrates of endogenous ADP-ribosylation did not appear to be substrates for pertussis toxin-induced ADP-ribosylation. These and previously published results indicate that nitric oxide generated from sodium nitroprusside or endogenous sources may have modulatory effects through regulation of the endogenous ADP-ribosylation of proteins.

Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation

Reversible ADP-ribosylation of dinitrogenase reductase forms the basis of posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. This report describes the physiological effects of mutations in the genes encoding the enzymes that add and remove the ADP-ribosyl moiety. Mutants lacking a functional draT gene had no dinitrogenase reductase ADP-ribosyltransferase (DRAT, the draT gene product) activity in vitro and were incapable of modifying dinitrogenase reductase with ADP-ribose in vivo. Mutants lacking a functional draG gene had no dinitrogenase reductase-activating glycohydrolase (DRAG, the draG gene product) activity in vitro and were unable to remove ADP-ribose from the modified dinitrogenase reductase in vivo. Strains containing polar mutations in draT had no detectable DRAG activity in vitro, suggesting likely cotranscription of draT and draG. In strains containing draT and lacking a functional draG, dinitrogenase reductase accumulated in the active form under derepressing conditions but was rapidly ADP-ribosylated in response to conditions that cause inactivation. Detection of DRAT in these cells in vitro demonstrated that DRAT is itself subject to posttranslational regulation in vivo. Mutants affected in an open reading frame immediately downstream of draTG showed regulation of dinitrogenase reductase by ADP-ribosylation, although differences in the rates of ADP-ribosylation were apparent.

Comparative studies on structure and function of archaebacterial elongation factors indicate the phylogenetic diversity of the urkingdom

The sequences of ADP-ribosylated tryptic peptides from three archaebacterial elongation factors — aEF-2 from Methanobacterium thermoautotrophicum, from Sulfolobus acidocaldarius and from Desulfurococcus mucosus — were established. The rates of ADP-ribosylation by diphtheria toxin of these factors and of two others — aEF-2 from Methanococcus vannielii and from Thermoplasma acidophilum — were determined. Enzymatically active EF's and purified ribosomes from the two sulphur metabolizing strains were prepared. The phenylalanine polymerization activity of factors and ribosomes from these two strains and from T. acidophilum and Mc. vannielii in various combinations was analyzed. The results of these experiments allow, in connection with previously published findings (Klink et al., 1983; Gehrmann et al., 1985), the following statements and conclusions: Comparison of the ADP-ribosylatable domains of aEF-2 from six of seven archaebacterial orders and of the four known sequences of eEF-2 reveals greater differences within the archaebacterial sequences than within eukaryotic ones or between particular archaebacterial and eukaryotic sequences. Only the two sulphur metabolizing strains have identical peptides. The universal consensus sequence comprises only four of nine amino acids, although non-conservative exchanges occur only in two positions. No correlation has been found between sequence differences and ADP-ribosylation rates. Concerning the cooperation of both factor types - EF-1 and EF-2 - with heterologous ribosomes, restrictions are found to exist within the archaebacterial kingdom which have never been observed within the other kingdoms, with the exception of mitochondrial ribosomes. These restrictions confirm the deep hiatus between the sulphur metbolizing orders and the other archaebacteria, and also clearly separate the Sulfolobales from the Thermoproteales. Thermoplasma seems to be more closely related to the methanogenic/halophilic branch. In summary, the results reported here may indicate that the archaebacterial kingdom split up into several lines at a very early date, the two sulphur metabolizing orders being the most ancient branches.

Toxins which activate adenylate cyclase.

Cholera toxin and other heat-labile enterotoxins have the same subunit structure (A5B) and all catalyse the mono ADP-ribosylation of Ns, a regulator of adenylate cyclase, probably at an arginine residue. They also ADP-ribosylate a variety of other membrane and soluble proteins at much slower rates. The rates differ from protein to protein but it may be that every arginine residue in every protein is ADP-ribosylated at some slow rate. A guanine nucleotide triphosphate is required for the ADP-ribosylation of the major (Ns) and minor substrates alike. It used to be thought that all the substrates were GTP-binding proteins but this cannot be so. Rather, the GTP is required because it has to bind to some additional site on the membrane, termed 'S', in a cooperative event that involves a soluble protein called cytosolic factor (CF). If we expose erythrocyte membranes to CF and the GTP analogue Gpp(NH)p we can later extract in detergent a factor or complex that confers upon naive erythrocyte membranes the ability to be ADP-ribosylated. Pertussis toxin also has an A5B structure and acts on an intracellular substrate for ADP-ribosylation, namely the negative regulator of adenylate cyclase, called Ni. ADP-ribosylation prevents the reduction of cyclase activity by inhibitory hormones. The ADP-ribosylation of Ns or Ni does not affect the rate of ADP-ribosylation of the other protein.

Cholera toxin activation of adenylate cyclase. Roles of nucleoside triphosphates and a macromolecular factor in the ADP ribosylation of the GTP-dependent regulatory component.

Cholera toxin-catalyzed ADP ribosylation of the membrane-bound GTP-binding protein that regulates adenylate cyclase activity requires certain components of the cytosol. In broken cells it is prevented by depletion of endogenous nucleotides and is restored by the provision of GTP (1 to 3 PM half-maximum) or ITP (30 PM) although not by ATP, CTP, TTP, or UTP. An hydrolysis-resistant GTP analog, guanyl-5’-yl imidodiphosphate (Gpp(NH)p, 1 to 3 p ~ ) , also supports the toxin-catalyzed reaction, but adenyl-5’-yl imidodiphosphate is only weakly effective. The ability of nucleoside triphosphates to support toxin-catalyzed ADP ribosylation or the toxin-catalyzed activation of adenylate cyclase correlate well. Even with excess GTP or Gpp(NH)p, the rate of ADP ribosylation and adenylate cyclase activation depends upon the presence of a macromolecular component of the erythrocyte cytosol. If GTP is employed, ADP ribosylation requires the simultaneous presence of this nucleotide, cytosolic macromolecules, NAD’, toxin, and membranes. However, membranes may be predisposed to respond to the toxin by preincubating them with Gpp(NH)p and cytosolic macromolecules. Preincubated membranes, washed free of unbound nucleotide, can then be ADP ribosylated by incubation with cholera toxin and NAD+ without additional cofactors. Both Gpp(NH)p and the macromolecular fraction of the cytosol are required for such preactivation. It is complete in 15 min at 37°C and is about three times slower at 25°C. It requires magnesium, is accelerated by isoproterenol, and is inhibited by GTP or sodium fluoride. The conditions for preactivation resemble those for the specific binding of Gpp(NH)p to the regulatory component of adenylate cyclase and consequent quasi-permanent activation of the cyclase, except that direct activation of the cyclase does not depend upon a cytosolic macromolecule. We conclude that the substrate for cholera toxin is a complex of the GTP-binding protein with GTP, in some way modified by a macromolecule of the cytosol.

Effect of polyamines on ADP-ribosylation of nuclear proteins from rat liver

The effects of polyamines on the in vitro ADP-ribosylation of rat liver nuclei have been found to be dose dependent. These effects were detected in the absence of Mg 2+. Maximal increases occurred at 1 mM spermine, 5 mM spermidine, and 7.5 mM putrescine, respectively. The rate of ADP-ribosylation in the presence of 1 mM spermine was 2-fold higher than that in the presence of 10 mM Mg 2+, a concentration which maximally stimulates ADP-ribosylation. When the nuclei were incubated with NAD in the presence of spermine, ADP-ribosylation occurred predominantly in the non-histone protein fractions, but in the presence of Mg 2+, it occurred predominantly in the histone fractions. These results suggest that polyamines may have some regulatory effect on the ADP-ribosylation of non-histone chromatin proteins in rat liver.