Stoichiometric photolabeling of two distinct low and high affinity nucleotide sites in sarcoplasmic reticulum ATPase.
Highly purified 3'-arylazido-ATP (aATP) was obtained by high performance liquid chromatography. In the dark, this photoactivatable ATP analog was a competitive inhibitor of ATP hydrolysis catalyzed by purified sarcoplasmic reticulum (SR) ATPase with a Ki of 10 microM. The analog itself was hydrolyzed by the enzyme in the dark. A biphasic curve of velocity of hydrolysis of the analog versus aATP concentration was obtained, indicating the presence of high and low affinity sites with K0.5 of approximately 10 microM and 300 microM, respectively. Upon irradiation with visible light, a biphasic curve was obtained for the level of covalent photolabeling of the enzyme versus [beta-32P]aATP concentrations. Levels of 6.5-9 nmol of analog/mg of protein and 20-22 nmol of analog/mg of protein were obtained when labeling with 20-30 or with 400 microM aATP, respectively, showing the existence of 1 mol of high affinity sites/mol of ATPase and 1-1.5 mol of low affinity sites/mol of enzyme. The rate of light-dependent incorporation of [beta-32P]aATP was decreased by the presence of ATP, Pi, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene-ATP, or Ca2+ in the illumination media. Photolabeling of the high affinity sites had little effect on the velocity of ATP hydrolysis but significantly inhibited the splitting of additional aATP added in the dark. Photolabeling the low affinity sites caused irreversible inhibition of the ATPase activity. The inhibition was prevented by having ATP in the illumination medium, which protected it from labeling. Gel filtration chromatography in the presence of detergent showed that radioactive photolabel was incorporated in the SR ATPase protein. The results indicate that aATP is a useful tool for stoichiometrically labeling and probing the nucleotide binding domains of the SR ATPase.
Carvalho-Alves
P C
PC
Oliveira
C R
CR
Verjovski-Almeida
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D006868:Hydrolysis; D007700:Kinetics; D010777:Photochemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
C012761:3'-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)adenosine-5'-triphosphate; D000345:Affinity Labels; D001386:Azides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases