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Role of regucalcin as an activator of sarcoplasmic reticulum Ca 2+ ‐ATPase activity in rat heart muscle
Author(s) -
Yamaguchi Masayoshi,
Nakajima Rie
Publication year - 2002
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.10209
Subject(s) - thapsigargin , endoplasmic reticulum , vanadate , western blot , microsome , dithiothreitol , atpase , digitonin , enzyme assay , chemistry , enzyme , activator (genetics) , egta , microbiology and biotechnology , biology , calcium , biochemistry , medicine , endocrinology , organic chemistry , gene
The expression of regucalcin, a regulatory protein of Ca 2+ signaling, and its effect on Ca 2+ pump activity in the microsomes (sarcoplasmic reticulum) of rat heart muscle was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in heart muscle using rat regucalcin‐specific primers. Results with Western blot analysis showed that regucalcin protein was present in the cytoplasm, although it was not detected in the microsomes. Microsomal Ca 2+ ‐ATPase activity was significantly increased in the presence of regucalcin (10 −10 –10 −8 M) in the enzyme reaction mixture. This increase was not seen in the presence of thapsigargin (TP) (10 −5 M), a specific inhibitor of the microsomal Ca 2+ pump enzyme. Regucalcin (10 −10 –10 −8 M) significantly stimulated ATP‐dependent 45 Ca 2+ uptake by the microsomes. The effect of regucalcin (10 −8 M) in increasing microsomal Ca 2+ ‐ATPase activity was completely prevented in the presence of digitonin (10 −3 or 10 −2 %), which has a solubilizing effect on membranous lipid, or N ‐ethylmaleimide (NEM), a modifying reagent of sulfhydryl (SH) groups. Dithiothreitol (DTT; 5 mM), a protecting reagent of SH groups, increased markedly Ca 2+ ‐ATPase activity. In the presence of DTT (5 mM), regucalcin could not significantly enhance the enzyme activity. Also, the effect of regucalcin in increasing Ca 2+ ‐ATPase activity was completely inhibited by the addition of vanadate (1 mM), an inhibitor of phosphorylation of enzyme. In addition, the effect of regucalcin on Ca 2+ ‐ATPase activity was not significantly modulated in the presence of dibutyryl cyclic AMP (10 −4 M), inositol 1,4,5‐trisphosphate (10 −3 M), or calmodulin (5 μg/ml) which is an intracellular signaling factor. The present study demonstrates that regucalcin can activate Ca 2+ pump activity in rat heart microsomes, and that the protein may act the SH groups of Ca 2+ ‐ATPase by binding to microsomal membranes. J. Cell. Biochem. 86: 184–193, 2002. © 2002 Wiley‐Liss, Inc.