Investigation of Chromatin Bound Enzymes

Citation

Chong, Ming Ta (1974) Investigation of Chromatin Bound Enzymes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/18PT-FD16. https://resolver.caltech.edu/CaltechETD:etd-11152005-155825

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Part I. Rat liver chromatin contains a neutral protease with a marked preference for chromosomal proteins as substrates. The enzyme has been purified 705-fold from chromatin by salt extraction, chromatography on Bio-Rex 70, Sepharose 6B, calcium phosphate gel and QAE Sephadex. The enzyme has a molecular weight of 200,000 with two identical subunits of molecular weight 100,000. It attacks rat liver histones, NHC proteins and L-poly-lysine preferentially, is essentially inactive with rat liver cytosol proteins, and slowly degrades caseine, L-poly-arginine and protamines. The [...] for histones is 0.5 mg/ml; for NHC proteins [...] is 1 mg/ml. The enzyme is quite stable when stored at -20[degrees] for 4 months. Activity is diminished to 50% by heating to 62[degrees] for 15 min and totally destroyed at 70[degrees]. The enzyme has an optimal pH at 7.0 and a half maximal activity at pH 6.0, suggesting that a histidine residue is involved in catalysis. It is inhibited by DFP and PMSF, suggesting that a serine residue is involved. Hg++ inhibits enzyme activity, suggesting sulfhydryl group is important for enzyme activity. High salt (above 1M NaCl) inhibits enzyme activity completely but reversibly. The enzyme needs divalent ions as activators; especially potent is Mn++ (6-8 mM) which stimulates activity about 2 fold. The isolated enzyme appears to be similar to that responsible for the endogeneous degradation of histones in chromatin. The susceptibility of the five histone fractions to proteolysis is critically dependent upon whether or not the histones are complexed with DNA. In the intact nucleohistone four major histones are rather resistant to proteolytic attack, while histone I is rapidly attacked. If histones are freed from DNA all the histone molecules are attacked at about the same rate except histone I, which is relatively resistant. Part II. Rat liver chromatin also contains a nonspecific esterase which cleaves the artificial substrate--[...]-tosyl arginine methyl ester (TAME). The enzyme has been purified to 510 fold from chromatin by salt extraction, chromatography on Bio-Rex 70, Sephadex G-200, calcium phosphate gel and SE Sephadex. The molecular weight of the purified enzyme is estimated to be about 15,000. The enzyme has an optimal pH at 8.2 and half maximal activities at 6.9 and 10.5, suggesting that histidine and lysine residue might be involved in catalysis. It is inhibited by DFP and PMSF, suggesting that a serine residue is involved. At high substrate concentrations inhibition is noted. The [...] for TAME is 0.16 mM. Part III. Ferritin tagged c-RNA molecules were hybridized to rat ascites nuclear DNA in an effort to map the arrangement of these sequences in the rat genome by electron microscopy. The ferritin acts as an electron dense marker. The interferritin distance is also the inter RNA distance and c-RNA hybridizes specifically to middle repetitive DNA sequences. Thus measuring the distances between ferritin molecules attached to c-RNA reveals that middle repetitive DNA sequences (about 300 nucleotides in length) were arranged either singly or in tandem and immediately followed by a unique sequence about 500-1500 nucleotides in length.

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