ACTIVATION AND ENZYME CHARACTERISTICS OF A DNA-RESTRAINED PHOSPHATASEIN CHROMATIN-ASSOCIATED COMPLEXES

DNA-bound polypeptide complexes composed of several non-histone polype ptides that resisted harsh DNA deproteinization procedures were charac terized. The three major polypeptides of these complexes have molecula r masses of 62, 52, and 40 kDa. They constitute supramolecular structu res that reside on isolated DNA in dense clusters. The supramolecular complexes were released from DNA as globular 12.8 +/- 0.8-nm particles : these particles were gradually disassembled to form smaller supramol ecular structures. The DNA-bound complexes comprise of an encrypted ad enosinetriphosphatase/phosphatase activity, which is a minor but intri nsic component of the complexes. The enzyme remained inactive as long as the complexes were bound to DNA. However, the enzyme was activated concomitantly with the progression of DNA digestion, which indicated t hat DNA was involved in the downregulation of the enzyme. The inactive DNA-restrained complex could not be restored in vitro, which indicate d its non-trivial nature. Once released from DNA, the enzyme was inact ivated over a period of several hours. However, in the DNA-associated complexes its potential to become activated during DNA digestion was c onserved for several months. In the activated state, the enzyme showed an optimum activity at pH 9.5, was stimulated by Mg2+, inhibited by v anadate and EDTA but was not significantly inhibited by okadaic acid. The active enzyme, which consists of two subunits of 56 kDa and 59 kDa , can be released from the supramolecular structures by agarose gel el ectrophoresis. A regulatory mechanism therefore exists for the downreg ulation of this phosphatase by DNA.