Extensive Purification of Nuclear Poly(ADP-ribose) Glycohydrolase

Abstract

Catabolism of poly(ADP-tibose) attached to specific chromosomal proteins has been shown to occur during distinct nuclear processes such as DNA replication, repair and transcription (1). Thus, de-poly(ADP-ribosyl)ation is an important response of nuclei that reflect various cellular signals. Two different types of enzymes have been thought to be involved in de-poly(ADP-ribosyl)ation of chromosomal proteins. One enzyme, poly(ADP-ribose) glycohydrolase catalyzes hydrolysis of glycosidic (1“-2’) linkages of poly(ADP-ribose) to give mono(ADP-ribosyl)-protein and free ADP-ribose (2-9). A second type is ADPribosyl-protein lyase, which is capable of splitting mono(ADP-ribose)-protein linkages (10). The glycohydrolase has been purified from nuclei (4) and post-nuclear fractions (cytoplasm) (6-9) of several tissues and cultured cells. To distinguish the nuclear glycohydrolase from the cytoplasmic glycohydrolase, the nuclear enzyme is designated as poly(ADP-ribose) glycohydrolase I, and the cytoplasmic enzyme, poly(ADP-ribose) glycohydrolase II (5, 7, 9). The biological relationship between the two forms of glycohydrolase remains to be determined. As yet no procedure that makes available nuclear poly(ADP-ribose) glycohydrolase with sufficient purity and quantity to determine amino acid composition or sequence has been reported. Here, we report a reproducible and efficient method for extensive purification of the major poly(ADP-ribose) glycohydrolase present in mammalian cell nuclei and characterization of its properties.