Determinants of triad junction reformation: identification and isolation of an endogenous promotor for junction reformation in skeletal muscle.
The junction of isolated triads can be mechanically broken by passage through a French press and subsequently reformed by incubation of the isolated organelles with certain salts of weak acids (e.g., K cacodylate, K propionate, and K butyrate). In contrast, other salts (e.g., KCl, K phosphate, and K benzoate) are ineffective in promoting triad formation. An endogenous factor obtained from a muscle homogenate acts in the same manner as these artificial compounds. When rabbit skeletal muscle is homogenized in a KCl solution and centrifuged to remove large cellular components and membrane fractions, an endogenous factor is extracted into the high speed supernatant which promotes the reformation of mechanically broken triads. A three-stage purification of this factor has been achieved using: ammonium sulfate fractionation, adsorption chromatography, and molecular sieve chromatography. SDS-PAGE showed that the protein was purified to homogeneity and had a subunit Mr of 34,000 daltons. This protein has the following characteristics: it exists in 0.1 M KCl as a polymeric substance with an estimated Mr = 123,000 on molecular sieve chromatography and a Mr = 155,000 on sedimentation equilibrium; it promotes the formation of triadic vesicles from isolated organelles in a low ionic strength medium; Both this protein and cacodylate share the property of specifically catalyzing the association and aggregation of junctional proteins which had previously been dissolved by neutral detergent and salt; it appears to be identical to an extrinsic constituent of terminal cisternae, which has been described as a protein of Mr = 34K. It is not clear, however, whether this protein is a necessary and integral component of the junctional feet or whether it exerts predominantly a catalytic role in the formation of the triad junction.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D008854:Microscopy, Electron; D008861:Microsomes / Q000648:ultrastructure; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification* / Q000502:physiology; D009132:Muscles / Q000502:physiology / Q000648:ultrastructure*; D009940:Organoids / Q000648:ultrastructure*; D011817:Rabbits; D012492:Salts
D009124:Muscle Proteins; D012492:Salts