Isolation And Identification Of An Endogenous Promotor For The Reformation Of The Triad Junction In Skeletal Muscle

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Doctor of Philosophy (Ph.D.)




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 proprionate, and K butyrate. 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 subunit molecular weight of 34,000 daltons. This protein has the following characteristics: (1) it exists in 0.1 M KCl as a polymeric substance with an estimated M(,r) = 123,000 on molecular sieve chromatography and a M(,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; (5) it appears to be identical to an extrinsic constituent of terminal cisternae which has been described as a protein of M(,r) = 34,000, (6) amino acid analysis indicated that the protein was glyceraldehyde 3-phosphate dehydrogenase, and (7) it appears to associate with a lumenal protein of terminal cisternae, calsequestrin. It is not clear, however, whether this protein is a necessary and integral component of the junctional feet or whether it exerts predominately a catalytic role in the formation for triad junction.


Biophysics, General

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