Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(ADP-ribose) synthetase has been purified 2,000-fold to apparent homogeneity from human placenta. The purification procedure involves affinity chromatography with 3-aminobenzamide as the ligand. The purified enzyme absolutely requires DNA for the catalytic activity and catalyzes poly(ADP-ribosyl)ation of the synthetase itself (automodification) and histone H1. Mg2+ enhances both the automodification and poly(ADP-ribosyl)ation of histone H1. The enzyme is a monomeric protein with a pI of 10.0 and an apparent molecular weight of 116,000. The sedimentation coefficient and Strokes radius are 4.6 S and 5.9 nm, respectively. The frictional ratio is 1.82. Amino acid analysis and limited proteolysis with papain and alpha-chymotrypsin indicate that the human placental enzyme is very similar to the enzyme from calf thymus, although some differences are noted. Mouse antibody raised against the placental enzyme completely inhibits the activity of enzymes from human placenta and HeLa cells and cross-reacts with the enzymes from calf thymus and mouse testis. Immunoperoxidase staining with this antibody demonstrates the intranuclear localization of the enzyme in human leukemia cells. All these results indicate that molecular properties as well as antigenic determinants of poly(ADP-ribose) synthetase are highly conserved in various animal cells.
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PMID:Purification and characterization of poly (ADP-ribose) synthetase from human placenta. 243 82

Poly(ADP-ribose) synthetase of Mr = 120,000 is cleaved by limited proteolysis with alpha-chymotrypsin into two fragments of Mr = 54,000 (54K) and Mr = 66,000 (66K). When the native enzyme is modified with 3-(bromoacetyl)pyridine, both portions of the enzyme are alkylated; however, alkylation of the 54K portions of the enzyme is protected by the addition of the substrate, NAD, or its analog, nicotinamide, suggesting that the substrate-binding site is localized in the 54K fragment. When the enzyme previously automodified with a low concentration of [adenine-U-14C] NAD is digested with alpha-chymotrypsin, the radioactivity is detected exclusively in the 66K fragment. The 66K fragment thus labeled is further cleaved with papain into two fragments of Mr = 46,000 and Mr = 22,000. With these two fragments, the label is detected only in the 22K fragment, but not in the 46K fragment. The 46K fragment binds to a DNA-cellulose column with the same affinity as that of the native enzyme, while the 22K fragment and the 54K fragment have little affinity for the DNA ligand. These results indicate that poly (ADP-ribose) synthetase contains three separable domains, the first possessing the site for binding of the substrate, NAD, the second containing the site for binding of DNA, and the third acting as the site(s) for accepting poly(ADP-ribose).
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PMID:Poly (ADP-Ribose) synthetase. Separation and identification of three proteolytic fragments as the substrate-binding domain, the DNA-binding domain, and the automodification domain. 632 8