A time-resolved Fourier transformed infrared difference spectroscopy study of the sarcoplasmic reticulum Ca(2+)-ATPase: kinetics of the high-affinity calcium binding at low temperature.
We have used time-resolved Fourier transformed infrared difference spectroscopy to characterize the amplitude, frequency, and kinetics of the absorbance changes induced in the infrared (IR) spectrum of sarcoplasmic reticulum Ca(2+)-ATPase by calcium binding at the high-affinity transport sites. 1-(2-Nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis [(oxycarbonyl)methyl]-1,2-ethanediamine (DM-nitrophen) was used as a caged-calcium compound to trigger the release of calcium in the IR samples. Calcium binding to Ca(2+)-ATPase induces the appearance of spectral bands in difference spectra that are all absent in the presence of the inhibitor thapsigargin. Spectral bands above 1700 cm-1 indicate that glutamic and/or aspartic acid side chains are deprotonated upon calcium binding, whereas other bands may be induced by reactions of asparagine, glutamine, and tyrosine residues. Some of the bands appearing in the 1690-1610 cm-1 region arise from modifications of peptide backbone carbonyl groups. The band at 1653 cm-1 is a candidate for a change in an alpha-helix, whereas other bands could arise from modifications of random, turn, or beta-sheet structures or from main-chain carbonyl groups playing the role of calcium ligands. Only a few residues are involved in secondary structure changes. The kinetic evolution of these bands was recorded at low temperature (-9 degrees C). All bands exhibited a monophasic kinetics of rate constant 0.026 s-1, which is compatible with that measured in previous study at the same temperature in a suspension of sarcoplasmic reticulum vesicles by intrinsic fluorescence of Ca(2+)-ATPase.
Troullier
A
A
C.E.A., Laboratoire de Biophysique Moléculaire et Cellulaire, URA CNRS 520, Département de Biologie Moléculaire et Structurale, Grenoble, France.
Gerwert
K
K
Dupont
Y
Y
D000085:Acetates; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D002614:Chelating Agents; D005029:Ethylenediamines; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
D000085:Acetates; D002614:Chelating Agents; D005029:Ethylenediamines; C057014:DM-nitrophen; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium