Identification of hydrophobic regions of the calcium-transport ATPase from sarcoplasmic reticulum after photochemical labeling with adamantane diazirine.
The Ca-ATPase from skeletal muscle sarcoplasmic reticulum was labeled with [3H]adamantane diazirine. Adamantane diazirine is a hydrophobic photoactivated probe that partitions into the cell membrane and can be used to identify regions of proteins that are embedded within the membrane. Digestion of the labeled protein with trypsin and separation of the labeled tryptic fragments by SDS-polyacrylamide-gel electrophoresis indicated that all of the major tryptic fragments were labeled by the probe. The presence of glutathione in the sample buffer during photolysis did not alter the pattern of labeling, indicating that adamantane diazirine labeled the Ca-ATPase from within the lipid bilayer. These results indicate that the Ca-ATPase polypeptide must cross the membrane at least 3 times.
D000218:Adamantane / Q000031:analogs & derivatives*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D010777:Photochemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin
D003840:Deoxycholic Acid; C024080:adamantane diazirine; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D005978:Glutathione; D000218:Adamantane