ABSTRACT
The role of poly ADP-ribosylation in DNA excision repair was studied in experimental models of various complexities. In intact cells in vivo, the unfolding of chromatin during DNA excision repair apparently requires the presence of a functional poly-ADP-ribosylation system. In vitro studies involving a reconstituted poly-ADP-ribosylation system show that the enzyme poly(ADP-ribose)polymerase has the capacity to shuttle core histones on a core DNA fragment of 146 bp. Under these conditions, the polymerase operates in a strictly processive mode. Furthermore, the polymerase adapts to different shuttling targets by producing very distinct polymer patterns. We conclude that the eukaryotic poly-ADP-ribosylation system has the capacity to regulate DNA-protein interactions and this may be an essential part of the unfolding mechanism of chromatin during excision repair in vivo.
Subject(s)
Chromatin/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Animals , DNA Repair/physiology , Models, BiologicalABSTRACT
Efflux of various amino acids from rat brain slices was determined under resting or depolarizing conditions. Slices of neocortex, hippocampus, striatum, cerebellum, mesodiencephalon, pons-medulla, and spinal cord were depolarized by K+ (50 mM) or veratrine (33 micrograms/ml). The 4-N,N-dimethylamino-azobenzene-4'-isothiocyanate (DABITC) derivatization method of Chang [Biochem. J. 199, 537-545 (1981)] for HPLC was adapted for analysis of amino acids and peptides in superfusion solutions. It allowed the separation and simultaneous detection of the sulfur-containing amino acids cysteine sulfinic acid (CSA), cysteic acid (CA), homocysteine sulfinic acid (HCSA), and homocysteic acid (HCA) at the picomole level. All four were shown to be released on depolarization in a Ca2+-dependent manner from brain slices. CSA and HCSA were released from cortex, hippocampus, mesodiencephalon, and, for HCSA only, striatum. HCA release, observed in all regions, was most prominent in cortex and hippocampus. CA was slightly increased by depolarization in hippocampus and mesodiencephalon. These sulfur-containing amino acids have been shown to exert an excitatory action on CNS neurons. The fact that these sulfur-containing amino acids are released as endogenous substances from nervous tissue supports the hypothesis that they play a role in CNS neurotransmission.
Subject(s)
Amino Acids, Sulfur/metabolism , Brain/metabolism , Animals , Brain/drug effects , Cysteic Acid/metabolism , Cysteine/analogs & derivatives , Cysteine/metabolism , Homocysteine/analogs & derivatives , Homocysteine/metabolism , In Vitro Techniques , Male , Neurotransmitter Agents , Potassium/pharmacology , Rats , Tissue Distribution , Veratrine/pharmacologyABSTRACT
Inner membrane vesicles have been prepared by cholate treatment of rat liver mitoplasts. The vesicles can actively accumulate Ca2+ in the absence or presence of inorganic phosphate. The uptake is inhibited by ruthenium red and uncouplers of oxidative phosphorylation. Like in intact mitochondria the driving force for the uptake reaction seems to be the negative inside membrane potential generated during the oxidation of substrates. The level of antimycin-A-sensitive reduction of ferricyanide by succinate indicates that the cholate inner membrane vesicles are about 70% right side out. Using cytochrome-c-extracted inner membrane vesicles it can be shown that only those which have the same right-side-out polarity as intact mitochondria can actively accumulate Ca2+.
