Nuclear DNA ligase and its action on chromatin DNA in neuronal, glial, and liver nuclei isolated from adult guinea pigs.

Abstract

Abstract: DNA ligase activities were measured in neuron‐rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (Km= 46–48 μM) and bivalent cation (Mg2+ or Mn2+) were required for the maximal activities in cerebral and liver nuclei. β‐Mercaptoethanol did not affect the activities, but N‐ethylmaleimide and p‐chloromercuribenzoate completely inhibited the activities. Deoxyadenosine‐5′‐triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na+ and Mg2+ on the enzyme activities was observed. A high concentration (200 mM) of Na+ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM‐NaCl. Apparent Km for the substrate [32P]phosphoryl DNA was determined according to a modification of the Michaelis‐Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar Km values (about 20 μg of [32P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher Km value (54 μg of [32P]phosphoryl DNA/ml). The modified Michaelis‐Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′‐phosphomonoester converted into a phosphatase‐resistant form per min at 37°C) per μg of nuclear DNA] was 11‐fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].