Effects of ionic strength on endogenous nuclease activity in chelated and nonchelated chromatin.
Calf thymus chromatin, isolated using a standard (low ionic strength, but nonchelating) isolation protocol, dialyzed against either Tris-PMSF or Tris-EDTA, was reconstituted in a high salt compacting buffer (COM) or a low salt dispersing buffer (DIS) prior to digestion with endogenous nucleases. A greater level of enzyme activity occurred when chromatin was in a condensed state (COM buffer) and not chelated prior to digestion. In contrast, chromatin chelated by dialysis against Tris-EDTA prior to digestion showed higher levels of enzyme activity in the dispersed state (DIS buffer). Nonchelated undigested chromatin contained 0.280 +/- 0.16 ug copper/mg DNA and and 0.305 +/+- 0.09 ug zinc/mg DNA. Chelation removed about 78% of copper per mg DNA and approximately 65% of zinc per mg DNA. In COM buffer after a 20 min digestion, the solubilized fraction was enriched in copper showing about 20 X more metal per mg DNA than nonchelated chromatin. Approximately the same amount of zinc was found in both chelated and nonchelated chromatin while there was less zinc in chelated chromatin solubilized in DIS buffer. Thus, chelation has important effects on the digestibility of chromatin and on the type of ionic environment that provides the most favorable conditions for endogenous nuclease activity.
Saucier
M A
MA
Department of Biological Sciences, University of New Orleans, Louisiana 70148.
Wang
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X
Re
R N
RN
Brown
J
J
Bryan
S E
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D000818:Animals; D002021:Buffers; D002417:Cattle; D002614:Chelating Agents; D002843:Chromatin / Q000201:enzymology*; D003300:Copper / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D004492:Edetic Acid; D009994:Osmolar Concentration; D010664:Phenylmethylsulfonyl Fluoride; D012995:Solubility; D013950:Thymus Gland / Q000648:ultrastructure; D014325:Tromethamine; D015032:Zinc / Q000378:metabolism
D002021:Buffers; D002614:Chelating Agents; D002843:Chromatin; D014325:Tromethamine; D010664:Phenylmethylsulfonyl Fluoride; D003300:Copper; D004247:DNA; D004492:Edetic Acid; D003851:Deoxyribonucleases; D015032:Zinc