Infrared spectroscopic signals arising from ligand binding and conformational changes in the catalytic cycle of sarcoplasmic reticulum calcium ATPase.
Fourier transform infrared spectroscopy was used to investigate ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum during the catalytic cycle. The ATPase reaction was started in the infrared sample by release of ATP from the inactive, photolabile ATP derivative P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP). Absorption spectroscopy in the visible spectral region using the Ca2(+)-sensitive dye Antipyrylazo III ensured that the infrared samples were able to transport Ca2+ in spite of their low water content, which is required for mid-infrared measurements (1800-950 cm-1). Small, but characteristic and highly reproducible infrared absorbance changes were observed upon ATP release. These infrared absorbance changes exhibit different kinetic properties. Comparison with model compound infrared spectra indicates that they are related to photolysis of caged ATP, hydrolysis of ATP in consequence of ATPase activity and to molecular changes in the active ATPase. The absorbance changes due to alterations in the ATPase were observed mainly in the region of Amide I and Amide II protein absorbance and presumably reflect the molecular processes upon phosphoenzyme formation. Since the absorbance changes were small compared to the overall ATPase absorbance, no major rearrangement of ATPase conformation as the result of catalysis could be detected.
Barth
A
A
Institut für Biophysik und Strahlenbiologie, Universität Freiburg, F.R.G.
Mäntele
W
W
Kreutz
W
W
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D006868:Hydrolysis; D008024:Ligands; D009282:Naphthalenesulfonates; D010782:Photolysis; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013055:Spectrophotometry, Infrared / Q000379:methods
D008024:Ligands; D009282:Naphthalenesulfonates; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C016238:antipyrylazo III; D002118:Calcium