Role of regucalcin as an activator of sarcoplasmic reticulum Ca2+-ATPase activity in rat heart muscle.
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 microg/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.
Yamaguchi
Masayoshi
M
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. yamaguch@u-shizuoka-ken.ac.jp
Nakajima
Rie
R
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D015238:Sulfotransferases; D019284:Thapsigargin / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D020536:Enzyme Activators; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D003994:Bucladesine; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D005033:Ethylmaleimide; D002118:Calcium; D004229:Dithiothreitol