Rabbit cardiac myosin. II. Proteolytic fragmentation with insolubilized papain.
The substructure of the cardiac myosin molecule was examined by the limited proteolytic digestion of the parent molecule with (dialdehyde starch)-methylenedianiline-mercuripapain, S-MDA-mercuripapain, at low temperatures and neutral pH, using moderate enzyme to myosin rations. Pertinent properties of the insoluble enzyme complex were also examined. Kinetic, ultracentrifugal, and chromatographic observations of the fragmentation process revealed that a single type of lytic reaction occurs during the early stages, predominately releasing heavy meromyosin subfragment 1 (HMM-S1) and myosin rods. With further time digestion, the rods are additionally cleaved yielding light meromyosin and HMM-S2, and HMM-S1 is found to be partially degraded. The major proteolytic subfragments were isolated, purified, and characterized with respect to their enzymatic, optical, amino acid, and physicochemical properties. Only HMM-S1 exhibited Ca-2+-activated ATPase activity, and at a level three- to fourfold higher than that of native myosin. Moreover, its hydrohynamic properties suggest that it is globular in structure. On the other hand, light meromyosin-A (LMM-A) (which consists mainly of rods), and HMM-S2 appear to be highly asymmetric, rigid, alpha-helical molecules devoid of the amino acid proline. Strong similarities were evident in all aspects upon comparison of these results with documented information concerning the skeletal system. On the basis of the physical and chemical properties of the proteolytic subfragments relative to that of native myosin, it was further concluded that the cardiac myosin molecule is a double-stranded, alpha-helical rod ending in tow subfragment 1 globules, of which only one may be enzymatically active at a time.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008628:Mercury; D008667:Metalloproteins / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013696:Temperature; D013997:Time Factors; D014461:Ultracentrifugation
D000596:Amino Acids; D008667:Metalloproteins; D010446:Peptide Fragments; D010206:Papain; D000251:Adenosine Triphosphatases; D009218:Myosins; D008628:Mercury; D002118:Calcium