Affinity labeling of the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by adenosine triphosphopyridoxal: identification of the reactive lysyl residue.
Adenosine triphosphopyridoxal (AP3PL) was used as an affinity label directed toward the ATP binding site of the Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum (SR). The reagent inhibited the ATPase activity competitively with ATP, Ki = 20 microM. Incubation of SR membranes with 100 microM AP3PL followed by treatment with NaBH4 resulted in 90% inactivation of the E-P forming activity as well as of the Ca2+-transporting activity. Adenosine di- and tetraphosphopyridoxals had similar but less pronounced effects on the Ca2+-transport system. AP3PL was bound to ATPase in a one-to-one stoichiometry in parallel with the loss of the enzymatic activities. ATP and ADP prevented the binding of AP3PL and thereby protected the enzyme from inactivation. The SR membranes were labeled with [3H]AP3PL and then digested with thermolysin in order to identify the attachment site of the affinity label. A 3H-labeled peptide (Val-Glu-Pro-Ser-His-Lys* 684-Ser-Lys) was purified to homogeneity by Sephadex LH-20 chromatography and C18-reversed phase HPLC (Lys* denotes the binding site of [3H]AP3PL). These results indicate that the SR-ATPase peptide is folded in such a manner that Lys684 and Asp351, the phosphorylation site, are located very close to each other, since the distance between the 4-formyl group reacting with Lys684 and the gamma-phosphoryl group of the ATP moiety of AP3PL is rather small.
Yamamoto
H
H
Department of Pure and Applied Science, College of Arts and Sciences, University of Tokyo.
Tagaya
M
M
Fukui
T
T
Kawakita
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006358:Hot Temperature; D008239:Lysine / Q000032:analysis; D010455:Peptides / Q000032:analysis; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000345:Affinity Labels; D010455:Peptides; C049517:adenosine triphosphopyridoxal; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008239:Lysine