Determinants of triad junction reformation: Identification and isolation of an endogenous promotor for junction reformation in sketal muscle

Summary

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: (1) ammonium sulfate fractionation, (2) adsorption chromatography, and (3) molecular sieve chromatography. SDS-PAGE showed that the protein was purified to homogeneity and had a subunitM _r of 34,000 daltons. This protein has the following characteristics: (1) it exists in 0.1m KCl as a polymeric substance with an estimatedM _r =123,000 on molecular sieve chromatography and aM _r =155,000 on sedimentation equilibrium; (2) it promotes the formation of triadic vesicles from isolated organelles in a low ionic strength medium; (3) 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; (4) it appears to be identical to an extrinsic constituent of terminal cisternae, which has been described as a protein ofM _r =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.