Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.2.30 (PARP)
 13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.
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PMID:Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase. 1019 6

Clostridium botulinum C2 enterotoxin consists of two unlinked proteins designated as C2II, which recognizes a cell-surface glycoprotein and translocates an ADP-ribosyltransferase, C2I, into the cytosol of a targeted cell. Fluorescence-activated cytometry was used to study the cellular interactions of Alexa488-labeled C2I (C2I-A488) and proteolytically activated C2II (C2IIa-A488). The binding of C2IIa-A488 (4 degrees C/10 min) to Chinese hamster ovary (CHO) and African green monkey kidney (Vero) cells yielded a signal/noise ratio of 7:1 and 4:1, respectively. C2I-A488 binding required C2IIa and resulted in a 4:1 (CHO) and 10:1 (Vero) signal/noise ratio that was readily competed by unlabeled C2I. Neither C2I nor C2IIa bound to a CHO line (RK14), lacking the receptor for C2IIa. C2I-A488 did not dock with the heterologous cell-binding component (iota b) of Clostridium perfringens iota toxin, a binary toxin closely related to C2. Pretreatment of wild-type CHO or Vero cells with pronase or papain (37 degrees C/30 min) prevented a cell-associated C2IIa-specific signal. However, CHO and Vero cells pretreated with papain at 25 degrees C had a 1.5- to 2.3-fold increase in C2IIa-specific fluorescence versus untreated cells incubated with C2IIa-A488. Overall, these studies further demonstrated the utility of fluorescence-activated cytometry for studying the binding characteristics of bacterial binary toxins like C2.
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PMID:Clostridium botulinum C2 toxin: binding studies with fluorescence-activated cytometry. 1216 16