Partial purification and characterization of the components of the neutral ribonuclease II-inhibitor system of normal and distrophic mouse skeletal muscle.
The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.127.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in (a) purification behavior, (b) apparent molecular weights of approximately 50 000, (c) thermal denaturation (50% loss of activity in 5 min at 73.5 degrees C), (d) isoelectric points (pH 4.8), and (e) procedures for reversible resolution into free inhibitor and free RNase II. The free RNase II species are also similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent molecular weights of 11 500 compared with 13 700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95 degrees C. The active RNase 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 KC1 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 KC1 concentration is increased above 50 mM KC1. 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 molecular weight of 42 000 and is very sensitive to heat; it loses all activity at 40 degrees C in 5 min. These studies (a) provide methods for obtaining useful amounts of the components of the neutral RNase II - inhibitor system from muscle, (b) provide the first method reported for the reversible resolution of RNase II - inhibitor complexes, (c) fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice, (d) establish interesting differences between the apparently homologous enzymes, murine muscle neutral RNase II, and bovine pancreatic RNase A, and (e) provide a substantially lower molecular weight estimate for RNase II inhibitor from muscle than has been reported for the inhibitor from liver, kidney, and placenta.
D000818:Animals; D004720:Endonucleases; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D005092:Exonucleases / Q000302:isolation & purification; D005095:Exoribonucleases; D005260:Female; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008297:Male; D051379:Mice; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D009137:Muscular Dystrophy, Animal / Q000378:metabolism*; D011189:Potassium Chloride; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011189:Potassium Chloride; D004720:Endonucleases; D005092:Exonucleases; D005095:Exoribonucleases; D012260:Ribonucleases; C022535:exoribonuclease II; D012259:Ribonuclease, Pancreatic