Configuration of unsheared nucleohistone. Effects of ionic strength and of histone F1 removal.

Nucleohistone solubilized from rabbit thymus nuclei by an endogenous nuclease has in 0.15 M salt an exceptionally low intrinsic viscosity and very high sedimentation velocity. A fully reversible expansion of configuration occurs on lowering ionic strength. When [eta] is plotted against I-1/2 and extrapolated to high I, [eta] = 0 is reached at I = 0.4-1 M and [eta] at I = infinity is negative, contrary to the behavior of DNA and of the great majority of polyelectrolytes, which extrapolate to a positive [eta] at I = infinity. This behavior demands that the configuration of nucleohistone depends not only on electrostatic expansive forces but also on contracting forces which are not electrostatic and do not go to zero in any accessible configuration. Intramolecular hydrophobic bonds might provide such contracting forces. Increasing I above 0.15 M leads to precipitation near 0.3 M and redissolution with dissociation of F1 and expansion in 0.6 M. The expansion is largely but not completely reversed on return to 0.15 M. Much further expansion occurs in I = 1.2 M. Nucleohistone exposed to 1.2 M could not be redissolved in the original medium. Nucleohistone depleted of F1 exhibits a similar expansion as ionic strength is reduced, at higher viscosities throughout. On extrapolation to I = infinity both positive and negative viscosities were observed, on different lots, perhaps reflecting variable extraction of other histones. Circular dichroism spectra are very little affected by ionic strength (0.6 M and lower) or F1 removal, despite tenfold changes in viscosity.

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.