A plasma membrane-enriched preparation from giant barnacle muscle fibers.
We describe a procedure for obtaining a highly enriched plasma membrane (sarcolemmal) preparation from muscle fibers of the giant barnacle (Balanus nubilus). The sarcolemmal-enriched portion migrated as a light fraction (F1) at the 10-24% sucrose interface. This fraction displayed saturable ouabain binding (Kd = 0.119 microM) that was enriched 10 times compared to that in the original homogenate. F1 was also prepared using muscle fibers previously labeled with 1,2-ditritio-1,2(2,2'-disulfo-4,4'-diisothiocyano)diphenylet hane, disodium salt [( 3H]-H2DIDS). F1 was enriched 25-fold in [3H]H2DIDS binding sites with respect to the homogenate. Ca2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca2+ uptake. Compared to membranes of sarcoplasmic reticulum origin, F1 was enriched in sarcolemma membranes by about 45-fold while it was enriched approximately 30-fold over mitochondrial membranes. Thus, F1 provides an extremely pure source of external muscle membranes.
Altamirano
A A
AA
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
Hamilton
S L
SL
Russell
J M
JM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D008854:Microscopy, Electron; D008931:Mitochondria, Muscle / Q000201:enzymology; D010042:Ouabain / Q000378:metabolism; D012508:Sarcolemma / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013347:Subcellular Fractions / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism; D001468:Thoracica
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D010042:Ouabain; C018564:dihydro-DIDS; D013385:Succinate Dehydrogenase; D000252:Calcium-Transporting ATPases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium