Synthesis of a radioactive azido derivative of thapsigargin and photolabeling of the sarcoplasmic reticulum ATPase.
A thapsigargin C8-derivative (ZTG) was synthesized by acylating debutanoylthapsigargin with 4-azido[carboxyl-14C]benzoic acid. ZTG retains the inhibitory activity of thapsigargin (TG) with respect to the Ca2+ ATPase of sarcoplasmic reticulum (SR). Covalent ATPase labeling was obtained by photoactivation of the ZTG azido moiety under conditions optimized to reduce nonspecific association of ZTG with SR vesicles and to approximate a matching ZTG:ATPase stoichiometry. Specific photolabeling of the Ca2+ ATPase with ZTG was obtained with 30% efficiency and was competitively inhibited by TG. Analysis of the labeled protein and its proteolytic fragments demonstrates that the ZTG label is associated covalently with the membrane-bound portion of tryptic subfragment A1, which spans the sequence between Leu253 and Arg324 and includes segments of S3 and S4 in the stalk, the M3 and M4 transmembrane helices, and the intervening lumenal loop. This finding is in agreement with previous spectroscopic observations and mutational analysis.
Hua
S
S
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Inesi
G
G
D000215:Acylation; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D006868:Hydrolysis; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019284:Thapsigargin / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D019284:Thapsigargin; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases