Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin

Abstract

Proteolysis by plasmin inactivates bovine ADP-ribosyltransferase; therefore, enzymatic activity depends exclusively on the intact enzyme molecule. The transferase was hydrolyzed by plasmin to four major polypeptides, which were characterized by affinity chromatography and N-terminal sequencing. Based on the cDNA sequence for human ADP-ribosyltransferase enzyme [Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M., & Sugimura, T. (1987) Biochem. Biophys. Res. Commun. 148, 617-622], a polypeptide map of the bovine enzyme was constructed by superposing the experimentally determined N-terminal sequences of the isolated polypeptides on the human sequence deduced from its cDNA. Two polypeptides, the N-terminal peptide (Mr 29,000) and the polypeptide adjacent to it (Mr 36,000), exhibited binding affinities toward DNA, whereas the C-terminal peptide (Mr 56,000), which accounts for the rest of the transferase protein, bound to the benzamide-Sepharose affinity matrix, indicating that it contains the NAD+-binding site. The fourth polypeptide (Mr 42,000) represents the C-terminal end of the larger C-terminal fragment (Mr 56,000) and was formed by a single enzymatic cut by plasmin of the polypeptide of Mr 56,000. The polypeptide of Mr 42,000 still retained the NAD+-binding site. The plasmin-catalyzed cleavage of the polypeptide of Mr 56,000-42,000 was greatly accelerated by the specific ligand NAD+. Out of a total of 96 amino acid residues sequenced here, there were only 6 conservative replacements between human and bovine ADP-ribosyltransferase.

A Third Zinc-binding Domain of Human Poly(ADP-ribose) Polymerase-1 Coordinates DNA-dependent Enzyme Activation

“…For example, a 36-kDa fragment of PARP-1 (residues 233-525) resulting from plasmin digestion interacts with DNA (43,44); however, unlike the N-terminal zinc fingers, this DNA binding activity is not structure-specific (e.g. strand breaks or hairpins) and requires long segments of DNA (ϳ222 base pairs).…”

Poly(ADP-ribose)polymerase: a novel finger protein

“…Examination of the recently published human poly(ADP-ribose)polymerase sequence deduced from the cloned cDNA (10, I 1) reveals two repeated putative zinc finger motifs in the N-terminal part of the DNA binding domain at position 2-97 and 106-207. The remarkable degree of conservation of the primary structure observed when one compare the partial sequences known for the calf thymus enzyme (22,26) to the full length human enzyme makes it likely that two zinc finger motifs exist in the N-terminal 29 Kd fragment of the bovine poly(ADP-4696 Nucleic Acids Research ribose)polymerase also. This in turn would correlate perfectly with the presence of the two zinc ions we have detected by EDXRF, and which would be implicated in the proper folding of this polypeptide for DNA binding.…”

Apparent Role of Adenosine Diphosphoribosyl Transferase in the Development of Mytilus edulis and the Inhibition of Differentiation by Ligands of the Enzyme Protein

“…separation of mono-ADP-ribosylated ADPRT of Mytilus is shown when 100 n M NAD was the substrate concentration (5,17,18) and the activation of mono-ADP-ribosylation by octamer C is illustrated in Figure 2, Lane 2. In the presence of 200 pMNAD and octamer C, the enzyme was poly(ADP-ribosylated) and its migration retarded due to oligomeric (ADPR)n, as apparent from Figure 2, Lane 3.…”

“…trophoretic enzyme assays were the same as described earlier (17,18). Coenzymic DNA (2-to 4-kb doublestranded DNA) was the byproduct of enzyme isolation (1 7) and was further purified by phenol extraction.…”