Homogeneity of myosin subfragments by equilibrium centrifugation.
A number of enzymes are currently in use for obtaining proteolytic subfragments of rabbit skeletal muscle myosin. Subfragment-1 can be obtained by papain digestion of polymeric myosin in the presence (Mg-S1) or absence (EDTA-S1) of divalent cations [Margossian, S.S., Lowey, S., & Barshop, B. (1975) Nature (London) 258, 163-166]. Subfragment-1 prepared by chymotrypsin is readily fractionated according to its alkali light-chain content into S1(A1) and S1(A2) [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. Digestion of soluble myosin by trypsin or chymotrypsin leads to heavy meromyosin (HMM) and light meromyosin (LMM). Many of these subfragments show extensive cleavages in the heavy- and/or light-chain region by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In view of the widespread use of proteolytic subfragments in kinetics and structural studies, it was of interest to establish the extent of heterogeneity of these preparations under nondenaturing conditions by equilibrium centrifugation. Analysis of the fringe displacements by the computer programs of Roark & Yphantis [Roark, D.E., & Yphantis, D.A. (1969) Ann. N.Y. Acad. Sci. 164, 245-278] showed that for three initial loading concentrations, the molecular weight averages Mn, Mw, M2, were superimposable across the entire solution column for all S1 and HMM species. The same applied for the initial molecular weight averages of LMM and rod, except that with these highly asymmetric molecules, a small drop in molecular weight was observed toward the cell bottom as would be expected from excluded volume effects. We conclude that the subfragments of myosin are remarkably homogeneous in benign solvents, despite the existence of some cleavages in their primary structure.
D000818:Animals; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis; D015879:Myosin Subfragments / Q000032:analysis; D009218:Myosins; D010446:Peptide Fragments / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D011817:Rabbits; D014461:Ultracentrifugation / Q000379:methods
D015879:Myosin Subfragments; D010446:Peptide Fragments; D010455:Peptides; D002918:Chymotrypsin; D009218:Myosins