TIME-RESOLVED FLUORESCENCE OF ERYTHROCYTE PLASMA-MEMBRANE CA2+-ATPASE IN DIFFERENT FUNCTIONAL-STATES

被引:2
|
作者
COELHOSAMPAIO, T [1 ]
FERREIRA, ST [1 ]
机构
[1] UNIV FED RIO DE JANEIRO,INST CIENCIAS BIOMED,DEPT BIOQUIM,BR-21944 RIO DE JANEIRO,BRAZIL
关键词
ERYTHROCYTE PLASMA MEMBRANE; CA2+-ATPASE; CONFORMATIONAL STATES; CALMODULIN; FLUORESCENCE LIFETIMES;
D O I
10.1016/0301-4622(92)80016-X
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The fluorescence decay of the plasma membrane calmodulin-activated Ca2+-ATPase from the erythrocyte was measured for the first time. The availability of a novel procedure for on-line blank subtraction in frequency-domain lifetime data acquisition (G.G. Reinhart, B. Feddersen, D. Jameson and E. Gratton, Biophys. J. 57 (1990) 189a) permitted the elimination of background interference from detergent-solubilized purified plasma membrane ATPase samples. The fluorescence decay of the erythrocyte Ca2+-ATPase was measured in the absence of Ca2+, or in the presence of Ca2+ or Ca2+ plus calmodulin. In the three different experimental conditions the fluorescence decay was very heterogeneous and could be best described by Lorentzian distributions of lifetime values. In the absence of Ca2+ the decay was described by a broad lifetime distribution centered at 4.4 ns with a width of 3.2 ns, indicating heterogeneity of tryptophan microenvironments in the ATPase. Calcium ion binding promoted an 11% increase in the center and a 27% decrease in the width of the distribution. By contrast, addition of calmodulin in the presence of Ca2+ caused a 15% decrease in the center of the distribution, revealing structural difference between calmodulin-activated and Ca2+-activated states of the ATPase. These results indicate the usefulness of on-line blank subtraction in frequency-domain lifetime measurements to investigate conformational changes in detergent-solubilized membrane protein samples.
引用
收藏
页码:243 / 248
页数:6
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