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Studies on Conformational Transitions of Ca2+,Mg2+-Adenosine Triphosphatase of Sarcoplasmic Reticulum. I. Selective Labeling of Functionally Distinct Sulfhydryl Groups with Conformational Probes and Evidence for a Ca2+-Dependent Conformational Change

Studies on Conformational Transitions of Ca2+,Mg2+-Adenosine Triphosphatase of Sarcoplasmic... Abstract Several maleimide derivatives of potential usefulness as conformational probes were tested for reactivity toward SH groups of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum. These include three fluorescent labels, N-(1-anilinonaphthyl-4)male-imide (ANM), N-(p-(2-benzirnidazolyl)phenyl)maleimide (BIPM), and N-(7-dimethyl-amino-4-methyl-3-coumarinyl)maleimide (DACM), and a spin label, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (MSL). These reagents also exhibit a selective reactivity toward SH groups which is similar to that of N-ethylmaleimide, although these conformational probes were somewhat more reactive than N-ethylmaleimide. Based on the above finding, procedures were devised to specifically label either one of two reactive SH groups of the ATPase, namely one highly reactive but functionally nonessential (SHN) and the other, essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609–617], with any one of these conformational probes. Sarcoplasmic reticulum membranes labeled with ANM at either SHN or SHD showed a characteristic fluorescence whose intensity reversibly changed in response to the removal and readdition of Ca2+ ions in the range of 10−6 to 10−7 M. The change could be ascribed to a conformational change of the ATPase in response to dissociation and association of Ca2+ ions at the transport site. The Ca2+-dependent fluorescence change was quantitatively different, depending on whether the ATPase was labeled at SHN or SHD. Moreover, it was probe-specific in that BIPM and DACM fluorescence did not change in response to Ca2+. The possible significance of these observations is discussed. This content is only available as a PDF. Author notes 1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, Itoh Science Foundation, The Naito Foundation, and Yamada Science Foundation. 2 Present address: Institute of Medical Science, The University of Tokyo, Takanawa, Minato-ku, Tokyo 108. © 1983, by the Japanese Biochemical Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Studies on Conformational Transitions of Ca2+,Mg2+-Adenosine Triphosphatase of Sarcoplasmic Reticulum. I. Selective Labeling of Functionally Distinct Sulfhydryl Groups with Conformational Probes and Evidence for a Ca2+-Dependent Conformational Change

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Publisher
Oxford University Press
Copyright
© 1983, by the Japanese Biochemical Society
ISSN
0021-924X
eISSN
1756-2651
DOI
10.1093/oxfordjournals.jbchem.a134406
Publisher site
See Article on Publisher Site

Abstract

Abstract Several maleimide derivatives of potential usefulness as conformational probes were tested for reactivity toward SH groups of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum. These include three fluorescent labels, N-(1-anilinonaphthyl-4)male-imide (ANM), N-(p-(2-benzirnidazolyl)phenyl)maleimide (BIPM), and N-(7-dimethyl-amino-4-methyl-3-coumarinyl)maleimide (DACM), and a spin label, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (MSL). These reagents also exhibit a selective reactivity toward SH groups which is similar to that of N-ethylmaleimide, although these conformational probes were somewhat more reactive than N-ethylmaleimide. Based on the above finding, procedures were devised to specifically label either one of two reactive SH groups of the ATPase, namely one highly reactive but functionally nonessential (SHN) and the other, essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609–617], with any one of these conformational probes. Sarcoplasmic reticulum membranes labeled with ANM at either SHN or SHD showed a characteristic fluorescence whose intensity reversibly changed in response to the removal and readdition of Ca2+ ions in the range of 10−6 to 10−7 M. The change could be ascribed to a conformational change of the ATPase in response to dissociation and association of Ca2+ ions at the transport site. The Ca2+-dependent fluorescence change was quantitatively different, depending on whether the ATPase was labeled at SHN or SHD. Moreover, it was probe-specific in that BIPM and DACM fluorescence did not change in response to Ca2+. The possible significance of these observations is discussed. This content is only available as a PDF. Author notes 1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, Itoh Science Foundation, The Naito Foundation, and Yamada Science Foundation. 2 Present address: Institute of Medical Science, The University of Tokyo, Takanawa, Minato-ku, Tokyo 108. © 1983, by the Japanese Biochemical Society

Journal

The Journal of Biochemistry – Oxford University Press

Published: Jul 1, 1983

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