DNA ligases from rat liver. Purification and partial characterization of two molecular forms

• Published in: Biochemistry (Easton) Vol. 29; no. 25; pp. 6009 - 6017
• Main Authors: Elder, Rhoderick H, Rossignol, Jean Michel
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 26.06.1990
• Subjects: 550201 - Biochemistry- Tracer Techniques; Adenosine Monophosphate - metabolism; AMP; Analytical, structural and metabolic biochemistry; ANIMALS; ANTIBODIES; Antibodies - immunology; ATP; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; BODY; CHEMICAL COMPOSITION; DIGESTIVE SYSTEM; DNA; DNA Ligase ATP; DNA Ligases - immunology; DNA Ligases - isolation & purification; DNA Ligases - metabolism; ELECTROPHORESIS; ENZYME ACTIVITY; ENZYMES; Enzymes and enzyme inhibitors; FRACTIONATION; Fundamental and applied biological sciences. Psychology; GLANDS; ISOENZYMES; Isoenzymes - immunology; Isoenzymes - isolation & purification; Isoenzymes - metabolism; ISOTOPE APPLICATIONS; LIGASES; LIVER; Liver - analysis; Liver - enzymology; MAMMALS; Molecular Weight; NUCLEIC ACIDS; NUCLEOTIDES; ORGANIC COMPOUNDS; ORGANS; PEPTIDES; Phosphorus Radioisotopes; Polynucleotide Ligases - isolation & purification; POLYPEPTIDES; PROTEINS; Rabbits; RATS; RODENTS; SEPARATION PROCESSES; Sulfur Radioisotopes; TRACER TECHNIQUES; VERTEBRATES; Ligase; Purification; Radiolabelling; Rat; Gel electrophoresis; Liver; Rodentia; Chromatography; Characterization; Density gradient centrifugation; Vertebrata; Mammalia; DNA; Gel filtration; Immunoblotting assay
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00477a019

The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.