ATP binding properties of the nucleotide-binding folds of SUR1.
Pancreatic beta cell ATP-sensitive potassium (K(ATP)) channels regulate glucose-induced insulin secretion. The activity of the K(ATP) channel, composed of SUR1 and Kir6.2 subunits, is regulated by intracellular ATP and ADP, but the molecular mechanism is not clear. To distinguish the ATP binding properties of the two nucleotide-binding folds (NBFs) of SUR1, we prepared antibodies against NBF1 and NBF2, and the tryptic fragment of SUR1 was immunoprecipitated after photoaffinity labeling with 8-azido-[(32)P]ATP. The 35-kDa fragment was strongly labeled with 5 microM 8-azido-[(32)P]ATP even in the absence of Mg(2+) and was immunoprecipitated with the antibody against NBF1. The 65-kDa fragment labeled with 100 microM 8-azido-[alpha-(32)P]ATP in the presence of Mg(2+) was immunoprecipitated with anti-NBF2 and anti-C terminus antibodies. These results indicate that NBF1 of SUR1 binds 8-azido-ATP strongly in a magnesium-independent manner and that NBF2 binds 8-azido-ATP weakly in a magnesium-dependent manner. Furthermore, the 65-kDa tryptic fragment was not photoaffinity-labeled with 8-azido-[gamma-(32)P]ATP at 37 degrees C, whereas the 35-kDa tryptic fragment was, suggesting that NBF2 of SUR1 may have ATPase activity and that NBF1 has none or little.
Matsuo
M
M
Laboratory of Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan.
Kioka
N
N
Amachi
T
T
Ueda
K
K
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D008970:Molecular Weight; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D014357:Trypsin / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D015221:Potassium Channels; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin