title: Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation.
abstract: Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically 'frozen' during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca2+ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromatography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18--20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3--0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 microM epinephrine.
mesh_terms: D000818:Animals; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D009210:Myofibrils / Q000032:analysis* / Q000201:enzymology; D009218:Myosins / Q000302:isolation & purification*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis; D011817:Rabbits; D013552:Swine
chemical_list: D046911:Macromolecular Substances; D010770:Phosphotransferases; D010744:Phosphoric Monoester Hydrolases; D009218:Myosins