Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin

• Published in: Biochemistry (Easton) Vol. 27; no. 16; pp. 5990 - 5995
• Main Authors: Buki, Kalman G, Kun, Ernest
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.08.1988
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Cattle; Enzymes and enzyme inhibitors; Fibrinolysin; Fundamental and applied biological sciences. Psychology; Humans; Molecular Sequence Data; Molecular Weight; Peptide Fragments - isolation & purification; plasmin; Poly(ADP-ribose) Polymerases - isolation & purification; Species Specificity; Transferases; Proteins; Vertebrata; Mammalia; Affinity chromatography; Gel electrophoresis; Enzyme; N terminal-Sequence; ADP-ribosyltransferase; Enzymatic digestion; Plasmin; Aminoacid sequence
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00416a024

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.