Does fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole incorporated into sarcoplasmic reticulum ATPase monitor putative E1-E2 conformational transition?
When a purified preparation of sarcoplasmic reticulum Ca2(+)-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 1 mM AMPPNP and 1 mM CaCl2 at 25 degrees C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD-inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C12E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2+ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2(+)-ATPase reaction.
Wakabayashi
S
S
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.
Imagawa
T
T
Shigekawa
M
M
D009327:4-Chloro-7-nitrobenzofurazan; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D010069:Oxadiazoles; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D010069:Oxadiazoles; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D002118:Calcium