ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca(2+)-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin.
Sarcoplasmic reticulum Ca(2+)-ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca(2+). Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P(i) and phosphorylated state analogues produced by the binding of F(-) or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg(505)). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues.
Danko
S
S
Department of Biochemistry, Asahikawa Medical College, Midorigaokahigashi, Asahikawa, Japan.
Daiho
T
T
Yamasaki
K
K
Kamidochi
M
M
Suzuki
H
H
Toyoshima
C
C
D000244:Adenosine Diphosphate / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D010750:Phosphoproteins; D014638:Vanadates; D000244:Adenosine Diphosphate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D019286:Endopeptidase K; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005459:Fluorides; D002118:Calcium