Partial purification and characterization of the components of the neutral ribonucleage II – inhibitor system of normal and dystrophic mouse skeletal muscle

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Abstract
The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.1.27.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in purification behavior, apparent MW of .apprx. 50,000, thermal denaturation (50% loss of activity in 5 min at 73.5.degree. C), isoelectric points (pH 4.8) and procedures for reversible resolution into free inhibitor and free RNase II. The free RNase II species are similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent MW of 11,500 compared with 13,700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95.degree. C. The active RNases II prepared directly from muscle, by resolution of inhibitor complexes or by organic mercurial treatment of the inhibitor complexes, all have identical pH-activity profiles in 200 mM KCl with an optimum near pH 7.0. In comparison RNase A has an optimum pH near 7.5 and its activity decreases more rapidly as KCl concentration is increased above 50 mM KCl. RNase II inhibitor obtained by resolution of the purified complexes or by direct isolation in the free form from normal muscle extracts has an apparent MW of 42,000 and is very sensitive to heat; it loses all activity at 40.degree. C in 5 min. These studies: provide methods for obtaining useful amounts of the components of the neutral RNase II-inhibitor system for muscle; provide the 1st method reported for the reversible resolution of RNase II-inhibitor complexes; fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice; establish interesting differences between the apparently homologous enzymes; murine muscle neutral RNase II and bovine pancreatic RNase A; and provide a substantially lower MW estimate for RNase II inhibitor from muscle than was reported for the inhibitor from liver, kidney and placenta.