Investigation of Myosin Heterogeneity Observed during Chromatography on Diethylaminoethyl Cellulose

Abstract

A procedure is described for obtaining a purified, stable, polyribonucleotide-free myosin fraction from skeletal muscle of chicken. The method involves extraction by pyrophosphate, differential ultracentrifugation of the extract, treatment with pancreatic ribonuclease, gel filtration of the treated extract, and chromatography on diethylaminoethyl cellulose. A myosin obtained by the pyrophosphate extraction and gel filtration appears to be homogeneous by electrophoresis or analytical ultracentrifugation. This material yields, on DEAE-cellulose chromatography, five fractions (I to V), eluting at ionic strengths corresponding to the following compositions of the elution buffers: I, 0.02 m potassium pyrophosphate; II, 0.125 m KCl-0.003 m potassium pyrophosphate; III, 0.18 m KCl-0.005 m potassium pyrophosphate; IV, 0.36 m KCl-0.01 m potassium pyrophosphate; and V, 0.90 m KCl-0.02 m potassium pyrophosphate. Fraction I consists mainly of myoglobin, Fractions II to IV show the characteristics of myosin, and Fraction V is a ribonucleoprotein. The two myosin fractions eluted at lower ionic strength contain polyribonucleotide and have a high Ca2+ adenosine triphosphatase activity, while Fraction IV, which is eluted at high ionic strength, is free of polyribonucleotide and has a low Ca2+ ATPase activity. The sedimentation values of the chromatographic myosin fractions are similar, but their electrophoretic behavior differs. All fractions give negative results for ATP sensitivity and Mg2+ ATPase activity. Results obtained following treatment with RNase or chelating agents of the extract or myosin eluted at lower ionic strength on DEAE-cellulose suggest that the chromatographic heterogeneity observed is due to the complex formation between polyribonucleotide and myosin and mediated by divalent ions. Pretreatment with these agents results in a single myosin fraction eluted from DEAE-cellulose at the same ionic strength as the myosin free from polyribonucleotide. Treatment of chromatographic myosin fractions containing polyribonucleotide by this same procedure gives the same polyribonucleotide-free fraction upon chromatography. Consideration of the results obtained by electrophoresis and ultracentrifugation of the Sephadex-excluded fractions in relation to the chromatographic behavior suggests that pyrophosphate extraction results in myosin which is in complex with polyribonucleotide by means of divalent ions. ATPase activity was found to be lowest in the myosin fraction which was free of polyribonucleotide when compared to the fractions which did contain polyribonucleotide. No explanation of this observation is given at the present time.