Regulation of the ATP dependent calcium uptake activity of heart sarcolemmal vesicles by endogenous cytosolic proteins

We have studied the activities of Ca2(+)-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca2(+)-stimulated ATPase in sarcolemma exhibited two activation sites--one with low affinity (Km = 0.70 +/- 0.2 mM; Vmax = 10.0 +/- 2.2 mumol Pi/mg/h) and the other with high affinity (Km = 0.16 +/- 0.7 mM; Vmax = 4.6 +/- 0.8 mumol Pi/mg/h) for Mg2+ ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca2(+)-stimulated ATPase without affecting its sensitivity to Ca2+ or Mg2+ ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5-10 mM concentrations caused a 4-5 fold activation of Ca2(+)-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg2+ ATP. DTT increased the affinity of the enzyme to Mg2+ ATP at the high affinit site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca2(+)-stimulated ATPase to Ca2+. DTT protected the Ca2(+)-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca2(+)-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca2(+)-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca2(+)-stimulated ATPase activity in sarcolemmal preparations.

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

Seppet

Dhalla

1989

Ontogeny of cytosolic proteins capable of modulating sarcoplasmic reticulum calcium transport in heart muscle

Donat

Narayanan

1991

In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by 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). The present study investigated the ontogenetic expression of the activities of Ca2+ transport inhibitor and inhibitor antagonist in heart cytosol during fetal and postnatal development of the rat. The SR Ca2+ transport inhibitor activity was undetectable in the cytosol of fetal (15- or 20-days gestation) rat heart but was manifested in the cytosol as early as one day after birth and increased progressively thereafter to reach almost adult levels within the first two weeks of postnatal development. The activity of the SR Ca2+ transport inhibitor antagonist was barely detectable in the near-term (20 days gestation) fetus but increased substantially during early postnatal development, in parallel with the rise in activity of the inhibitor. The ontogenetic appearance and increase in the activities of the Ca2+ transport inhibitor and its antagonist correlated well with the concurrent appearance and increase in the amounts of two polypeptides of apparent molecular weights 43 kDa and 64 kDa, which we have tentatively identified as the inhibitor and inhibitor antagonist, respectively. The co-ordinated expression of both the inhibitor and inhibitor antagonist activities in the cytosol during the early postnatal period parallels the morphogenesis and functional maturation of SR in cardiac muscle suggesting likely involvement of these cytosolic proteins in the physiological regulation of SR function.

Purification, amino-terminal sequence and functional properties of a 64 kDa cytosolic protein from heart muscle capable of modulating calcium transport across the sarcoplasmic reticulumin vitro

Narayanan

1994

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 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.(ABSTRACT TRUNCATED AT 250 WORDS)