Identification of a new subpopulation of triad junctions isolated from skeletal muscle; morphological correlations with intact muscle.
It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca2(+)-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, "weak," and breakage resistant, "strong," triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.
Kim
K C
KC
Department of Pharmacology, University of Miami, School of Medicine, Florida 33101.
Caswell
A H
AH
Brunschwig
J P
JP
Brandt
N R
NR
D000818:Animals; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels; D002458:Cell Fractionation / Q000379:methods; D066298:In Vitro Techniques; D017275:Isradipine; D007700:Kinetics; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure; D009132:Muscles / Q000378:metabolism / Q000648:ultrastructure*; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010069:Oxadiazoles / Q000378:metabolism; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D014357:Trypsin
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010069:Oxadiazoles; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D019837:Ryanodine Receptor Calcium Release Channel; D014357:Trypsin; D017275:Isradipine