PURIFICATION, AMINO-TERMINAL SEQUENCE AND FUNCTIONAL-PROPERTIES OF A 64 KDA CYTOSOLIC PROTEIN FROM HEART-MUSCLE CAPABLE OF MODULATING CALCIUM-TRANSPORT ACROSS THE SARCOPLASMIC-RETICULUM IN-VITRO

In previous studies we have described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by the sarcoplasmic reticulum (SR); further this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al., Biochim. Biophys. Acta. 735: 53-66, 1983; Can. J. Physiol. Pharmacol. 67: 999-1006, 1989). Here we report the complete purification of the antagonist protein (AP) and characterization of its functional properties. AP was purified to homogeneity from rabbit heart cytosol using two procedures, one utilizing sequential DE52-cellulose and hydroxylapatite chromatography, and the other utilizing anion exchange chromatography on Mono Q(TM) HR 5/5 column in a Pharmacia FPLC system. The purified AP has an apparent molecular weight of 64 kDa; it is made up of about 43% hydrophobic and 57% hydrophilic residues with the following amino-terminal sequence: E-A-H-K-S-E-I-A-H-R-F-N-D-V-G-E-E-H-F-I-G-L-V-L-I-T-F-S-Q-Y-L-Q-K-X-P-Y-E-E-H-A. This partial amino acid sequence data indicate strong sequence homology to serum albumin (sequence homology: 85% to rat serum albumin and 74% to sheep and bovine serum albumin). The purified AP caused concentration-dependent-blockade of the inhibition of Ca2+ uptake by SR observed in the presence of the cytosolic Ca2+ uptake inhibitor protein. This antagonist action of AP was markedly potentiated by calmodulin. AP did not influence the Ca2+ uptake activity of SR measured in the absence of the inhibitor protein and calmodulin. These observations suggest a likely physiological role for AP in the regulation of Ca2+ cycling by SR through a calmodulin-dependent mechanism.