Isolation And Identification Of An Endogenous Promotor For The Reformation Of The Triad Junction In Skeletal Muscle
Date of Award
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.
Corbett, Adrian Marie, "Isolation And Identification Of An Endogenous Promotor For The Reformation Of The Triad Junction In Skeletal Muscle" (1984). Dissertations from ProQuest. 1444.