High Affinity Calcium Binding Research Articles

Calcium binding to a human factor IXa derivative lacking γ-carboxyglutamic acid: Evidence for two high-affinity sites that do not involve β-hydroxyaspartic acid

A derivative of human blood clotting factor IXaβ lacking γ-carboxyglutamic acid (Gla) residues was prepared by limited proteolysis with chymotrypsin, and subsequently examined for its ability to bind calcium ions. By amino acid analysis, Gla-domainless human factor IXaβ contained 0.3–0.4 moles of β-hydroxyaspartic acid per mole of protein. Equilibrium dialysis experiments demonstrated that Gla-domainless human factor IXaβ retained two high-affinity calcium binding sites (K d=52 μM), a finding essentially identical to that observed for Gla-domainless bovine factor IX that contains 0.8–0.9 moles of β-hydroxyaspartic acid per mole of protein. These data strongly suggest that the β-hydroxyaspartic acid residue in these proteins does not participate in their high affinity calcium sites.

External calcium inhibits the efflux of calcium from isolated retinal rod outer segment disks.

Increasing the concentration of calcium in the external buffer flowing past isolated, intact bovine retinal rod outer segment disks immobilized in a flow system reduced the rate of radioactive calcium efflux from within the disks in the dark. We interpret these results as extradiskal calcium acting at an inhibitory binding site to block the calcium efflux. A Scatchard analysis of the external calcium dependence of the efflux yields an apparent dissociation constant of 50 microM, which further suggests that the inhibition is mediated by a specific membrane binding site. The observed inhibition of calcium efflux may represent a functional role for the high-affinity calcium binding site which has been identified by others in previous physical studies of the disk membrane. This external calcium inhibited permeability may explain some of the discrepancies in the reported calcium transport properties of disks. Variations in the external calcium concentration may alter the calcium content of isolated disks, thereby indirectly affecting other transport functions including the measured light-induced release of calcium. No evidence was found for either Na/Ca or Ca/Ca exchange processes across the disk membrane. Lanthanum was even more effective than calcium in inhibiting calcium efflux in the dark. Neither lanthanum nor calcium inhibited the light-induced efflux of calcium from disks, which implies either that light and extradiskal calcium regulate separate permeability processes in the disk membrane or that light greatly reduces the affinity of the inhibitory site for calcium and lanthanum.

Dependence on membrane lipids of the effect of vanadate on calcium and ATP binding to sarcoplasmic reticulum ATPase.

The affinity of the sarcoplasmic reticulum transport ATPase for calcium and ATP is not affected by lipid deprivation while vanadate binding is completely abolished. Lipid substitution restores vanadate binding as well as the vanadate induced disappearance of the enzyme's high affinity calcium and nucleotide binding sites. Nucleotide binding is simultaneously restored with the displacement of vanadate from the enzyme following the occupation of its low affinity calcium binding sites.

Nd3+ and Co2+ binding to sarcoplasmic reticulum CaATPase. An estimation of the distance from the ATP binding site to the high-affinity calcium binding sites.

Nd3+ binding to sarcoplasmic reticulum (SR) was detected by inhibition of ATPase activity and directly by a fluorimetric assay. Both methods indicated that Nd3+ inhibited the ATPase activity by binding in the high-affinity Ca2+ binding sites. The stoichiometry of binding was about 11 nmol of Nd3+ bound per mg of SR proteins at pNd = 6.5. At higher [Nd3+], substantial nonspecific binding occurred. The association constant for Nd3+ binding to the high-affinity Ca2+ binding sites was estimated to be near 2 X 10(9) M-1. When the CaATPase was inactivated with fluorescein isothiocyanate (FITC), 5.3 nmol were bound per mg of SR protein. This fluorescent probe is known to bind in the ATP binding site. The stoichiometry of Nd3+ binding to FITC-labeled CaATPase was the same, within experimental error, as to the unlabeled CaATPase. Fluorescence energy transfer between FITC in the ATP site and Nd3+ in the Ca2+ sites was found to be very small. This donor-acceptor pair has a critical distance of 0.93 nm and the distance between the ATP site and the closest Ca2+ was estimated to be greater than 2.1 nm. Parallel measurements with FITC-labeled SR and Co2+, an acceptor with a critical distance 1.2 nm, suggested the ATP and Ca2+ binding sites are greater than 2.6 nm apart.

Identification of a novel hepatic calcium binding protein

Calcium binding activity in the 100,000 X g supernatant of bovine liver has been isolated by a procedure involving DEAE cellulose and Sephadex G-100 chromatography. In addition to calmodulin, two new high affinity calcium binding proteins have been identified. On gel filtration chromatography these proteins migrate with apparent molecular weights of 83,700 and 51,400; whereas by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the two proteins migrate identically with Mr 63,000. In the presence of millimolar Mg2+, both proteins bind up to one mol Ca2+/mol protein. Half-maximal binding occurs at approximately 0.1 microM Ca2+. Amino acid compositional analysis reveals that both proteins are acidic, and contain about 40% glx and asx. Peptide mapping procedures suggest that these proteins may be highly homologous or multiple forms of a single protein. The results show the existence of calcium binding protein(s) other than calmodulin in hepatic cytosol.

Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis.

A new simple method of detecting calcium binding proteins in a protein mixture is described. A sample which might include calcium binding proteins was subjected to SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred to a nitrocellulose membrane. The membrane was then incubated with 45Ca to detect calcium binding proteins as radioactive bands by autoradiography. Purified troponin-C, calmodulin, myosin DTNB light chain, and parvalbumin were clearly identified by this method. In the whole homogenate of chicken skeletal muscle, myosin DTNB light chain, troponin-C, and 55K calcium binding protein were found to be radioactive. In the frog skeletal muscle, small molecular weight proteins of approximately 13-15K and 70K protein appeared to be the calcium binding proteins. In the case of the carp skeletal muscle, small molecular weight proteins including parvalbumin and two proteins of about 80K seemed to bind calcium ion. Two high molecular weight calcium binding proteins were present in the scallop striated muscle. The procedure described can be completed within 24 h and can detect as little as 2 micrograms of calcium binding protein in the starting sample. Under appropriate conditions it was possible to detect only high affinity calcium binding proteins.

Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase.

The phosphorylation of sarcoplasmic reticulum ATPase with Pi in the absence of Ca2+ was studied by equilibrium and kinetic experimentation. The combination of these measurements was then subjected to analysis without assumptions on the stoichiometry of the reactive sites. The analysis indicates that the species undergoing covalent interaction is the tertiary complex E X Pi X Mg formed by independent interaction of the two ligands with the enzyme. The binding constant of Pi or Mg2+ to either free or partially associated enzyme is approximately equal to 10(2) M-1, and no significant synergistic effect is produced by one ligand on the binding of the other; the equilibrium constant (Keq) for the covalent reaction E X Pi X Mg E-P X Mg is approximately equal to 16, with kphosph = 53 s-1, and khyd = 3-4 s-1 (25 degrees C, pH 6.0, no K+). The phosphorylation reaction of sarcoplasmic reticulum ATPase with Pi is highly H+ dependent. Such a pH dependence involves the affinity of enzyme for different ionization states of Pi, as well as protonation of two protein residues per enzyme unit in order to obtain optimal phosphorylation. The experimental data can then be fitted satisfactorily assuming pK values of 5.7 and 8.5 for the two residues in the nonphosphorylated enzyme (changing to 7.7 for one of the two residues, following phosphorylation) and values of 50.0 and 0.58 for the equilibrium constants of the H2(E X HPO4) in equilibrium with H(E-PO3) + H2O and H(E X HPO4) in equilibrium with E-PO3 + H2O reactions, respectively. In addition to the interdependence of H+ and phosphorylation sites, an interdependence of Ca2+ and phosphorylation sites is revealed by total inhibition of the Pi reaction when two high affinity calcium sites per enzyme unit are occupied by calcium. Conversely, occupancy of the phosphate site by vanadate (a stable transition state analogue of phosphate) inhibits high affinity calcium binding. The known binding competition between the two cations and their opposite effects on the phosphorylation reaction suggest that interdependence of phosphorylation site, H+ sites, and Ca2+ sites is a basic mechanistic feature of enzyme catalysis and cation transport.

Initial rate of calcium uptake by fragmented sarcoplasmic reticulum(FSR) from guinea pig heart and competition between Ca 2+, H + and Mg 2+ for high-affinity calcium binding sites

Initial rate of calcium uptake by fragmented sarcoplasmic reticulum(FSR) from guinea pig heart and competition between Ca 2+, H + and Mg 2+ for high-affinity calcium binding sites

Effect of divalent cation bound to the ATPase of sarcoplasmic reticulum. Activation of phosphoenzyme hydrolysis by Mg2+.

In order to study the mechanism for activation of ATP hydrolysis by Mg2+, the stoichiometry of the high affinity calcium-binding sites with respect to each form of reaction intermediate of sarcoplasmic reticulum ATPase was determined at 0 degrees C and pH 7.0 in the presence and absence of added Mg2+ using the purified ATPase preparation. High affinity calcium binding to the enzyme-ATP complex and to ADP-sensitive (E1P) and ADP-insensitive (E2P) phosphoenzymes occurred with stoichiometric ratios of 2, 2, and 0, and 3, 3, and 1 in the presence and absence of added Mg2+, respectively. The results were interpreted to indicate that in addition to 2 mol of calcium bound to the transport sites of the ATPase, 1 mol of divalent cation, which is derived from the metal component of the substrate, the metal-ATP complex, remains bound to each mole of the enzyme at least until E2P is hydrolyzed. As activation of phosphoenzyme hydrolysis by Mg2+ was blocked by the low concentrations of Ca2+ used in the calcium binding experiments, it was concluded that it is the magnesium derived from MgATP that is responsible for rapid hydrolysis of the phosphoenzyme intermediate.

A conformational transition of the sarcoplasmic reticulum calcium transport ATPase induced by vanadate.

Vanadate binding to sarcoplasmic reticulum vesicles results in the loss of the externally located high affinity calcium binding sites of the calcium transport ATPase. Conversely the occupation by calcium of the internally located low affinity sites in the vanadate enzyme complex leads to the release of vanadate. Since the total number of calcium binding sites is not diminished by vanadate binding but slightly increases we conclude that vanadate binding induces a transition of the enzymes external high to internal low affinity calcium binding sites. The transposition of external to internal calcium binding sites is accompanied by a definite change in the structure of the sarcoplasmic reticulum membranes. On vanadate binding the asymmetrically arranged electron dense protein particles become symmetrically distributed.

Diffuse structural alterations in cell membranes of spontaneously hypertensive rats.

Plasma membranes from heart, nerve endings, and liver were compared in 3-week-old male spontaneously hypertensive rats from the Okamoto substrain (SHR) and normotensive Wistar/Kyoto control rats (WKY) [systolic blood pressure 105 +/- 4 and 95 +/- 4 mm Hg, respectively (1 mm Hg = 133 Pa)] according to two criteria: calcium binding at physiological intracellular concentrations and polarization of an embedded fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Whatever the tissue of origin, the density of high-affinity calcium binding sites was lower in SHR than in WKY plasma membranes, and the polarization of diphenylhexatriene fluorescence was constantly higher in SHR than in WKY membranes. These membrane abnormalities are similar to those previously described in the erythrocyte membrane from SHR. The presence of diffuse structural alterations in cellular membrane from young spontaneously hypertensive rats when blood pressure is still in the normotensive range suggests a genetic origin. Such inherited abnormalities may by themselves participate in the rise in blood pressure.

Hydrogen-1 nuclear magnetic resonance investigation on bovine cardiac troponin C. Comparison of tyrosyl assignments and calcium-induced structural changes to those of two homologous proteins, rabbit skeletal troponin C and bovine brain calmodulin.

The effect of Ca2+ binding on the 270-MHz proton nuclear magnetic resonance spectrum of bovine cardiac troponin C (cTnC) has been examined. Assignment of resonances in the aromatic spectral region to tyrosine residues 10, 111, and 150 has been made for apo-cTnC and calcium-bound cTnC on the basis of decoupling experiments, pH titrations, temperature-induced changes, and gadolinium broadening experiments. The sequence homology which these tyrosine residues display with residues in two previously studied proteins, rabbit skeletal troponin C (sTnC) [Seamon, K. B., Hartshorne, D. J., & Bothner-By, A. A. (1977) Biochemistry 16, 4039] and bovine brain calmodulin [Seamon, K. B. (1980) Biochemistry 19, 207], was also used in assignments. High-affinity calcium binding (up to 2 mol/cTnC) causes large alterations in the environments of tyrosines-10 and -150, indicating that the N terminus is probably buried in the protein interior. The evidence suggests that the environment of tyrosine-150 in calcium-saturated cTnC must closely resemble that of tyrosine-138 in calmodulin in that it experiences the hydrophobic core of the protein. However, there is no similarity between these environments in the apoproteins. Dramatic alterations in phenylalanine resonances are seen during the binding of the third mole of calcium, corresponding to filling the sole low affinity site. Comparison of the spectral calmodulin reveals many structural similarities which stem from their high degree of primary sequence homology.

Pressure-induced inactivation of sarcoplasmic reticulum adenosine triphosphatase during high-speed centrifugation.

Sarcoplasmic reticulum vesicles were found to be highly sensitive to high-speed centrifugation in metal-deprived mediums at low temperature (4 degrees C). The irreversible modifications induced were easily detected from observation of the environment-sensitive spectrum of an iodoacetamide spin-label bound to the ATPase. Centrifugation also resulted in vesicle aggregation and inhibition of calcium transport, ATPase activity, and phosphoenzyme formation. These denaturation-like phenomena were prevented in the presence of sucrose, or by nucleotide binding, or, again, by cation binding to the ATPase high-affinity calcium binding sites and were only present when centrifugation was performed at low temperature. The crucial parameter during this process was found to be the hydrostatic pressure which developed in the centrifuge tube. SR vesicles exposed to 800 bars in a pressure bomb displayed the same features. It is suggested that irreversible denaturation takes place after one or both of the two following well-documented effects of pressure: a rise in the lipid order/disorder transition temperature or dissociation of the oligomeric structure of the calcium pump.

Calcium binding alteration in plasma membrane from various tissues of spontaneously hypertensive rat.

Calcium binding properties of plasma membrane enriched fractions from various tissues were studied in young spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). In all tissues tested (heart, liver, nerve endings, erythrocytes), high affinity calcium binding sites were characterized. Their KD values were in the range of the cytosolic free calcium concentration : (10(-8)-10(-7)). Whatever the tissue, plasma membrane enriched fraction from SHR exhibited a reduced calcium binding capacity but no significant change in affinity. This decrease which averaged 15-25% was also observed in the presence of the calcium ionophore A23187 and thus does not result from a modified accessibility of calcium to its binding sites. It is suggested that this abnormality may be of genetic origin and is possibly implicated in the pathogenesis of hypertension.

Hybrid formation between scallop myofibrils and foreign regulatory light-chains

Scallop myofibrils ( Placopecten magellanicus) from which regulatory light-chains had been completely removed by EDTA treatment at 30 °C were hybridized with regulatory light-chains of different myosins. Pure hybrids, containing only foreign regulatory light-chains with a stoichiometry of two moles per myosin, were readily formed with all the light-chains tested. Some of the regulatory light-chains restored regulatory functions to desensitized myofibrils by selectively inhibiting the actin activated Mg-ATPase in the absence of calcium. Light-chains from Mercenaria, Spisula Loligo and Urechis behaved as scallop regulatory light-chains, were inhibitory in the absence of calcium, and restored high-affinity calcium binding sites. Regulatory light-chains of Limulus, cricket, chicken gizzard and platelet were also inhibitory; however, calcium binding was restored with a lowered affinity and the hybrids required higher calcium concentrations for ATPase activation. Hybrids formed with the regulatory light-chains of vertebrate striated (rabbit, chicken, skate), bovine cardiac and lobster tail and claw muscles remained insensitive to calcium, their ATPase activity was not selectively depressed in the absence of calcium and specific high-affinity calcium binding sites were not restored. Phosphorylation of the light-chains (rabbit, cardiac and gizzard) has no effect on ATPase activity. The behaviour of the hybrids supports the interpretation that in vertebrate striated muscles myosin does not function as a regulatory switch. Foreign regulatory light-chains ( Spisula, Loligo, Mercenaria, rabbit) bind to desensitized myofibrils with a similar or slightly higher affinity as scallop regulatory light-chains. The two light-chain binding sites of myosin are equivalent and differences in affinity appear to be the result of an interaction between the two halves of the myosin molecules.

Sickle red cell calcium metabolism: studies on Ca2+-Mg2+ATPase and Ca-binding properties of sickle red cell membranes.

Sickle (Hb SS) red cells, preloaded with 45Ca by reversal of hemolysis, exhibit an incomplete 45Ca extrusion, retaining approximately four times more 45Ca than normal cells. Studies indicated that neither the reduction in Hb SS cell Ca2+-Mg2+ ATPase activity (84% of normal) nor the activation of Ca2+-Mg2+ ATPase by calmodulin was sufficiently different from normal cells to attribute a major role to the calcium pump in 45Ca retention. These results suggested that 45Ca retention may reflect an alteration in the calcium-binding properties of Hb SS cell membranes. Low-affinity calcium-binding (freely dissociable) was similar in normal and Hb SS cell membranes. However, the total calcium bound with high-affinity (tightly bound) was four-to-five times greater in Hb SS cell membranes than in normal membranes. These results are compatible with the hypothesis that Hb SS cell 45Ca retention reflects an exchange of a fraction of the total 45Ca with a tightly bound calcium pool, larger in Hb SS cell membranes than in normal membranes. A comparable degree of red cell 45Ca retention, which did not correlate with the reticulocyte population, was observed in other chronic anemic states. These findings suggest that the increased high-affinity calcium binding by the membrane may be a consequence of cellular changes induced by the anemic condition.

Localization of the high affinity calcium binding protein and an intrinsic glycoprotein in sarcoplasmic reticulum membranes.

Several proteins in sarcoplasmic reticulum preparations move in a band with a mobility, in sodium dodecyl sulfate-polyacrylamide gels (0.1 M phosphate buffer, pH 7.0), corresponding to a molecular mass of about 55,000 daltons. Only one of these proteins is the high affinity calcium binding protein. An intrinsic glycoprotein is also present in this band, and it is this glycoprotein which is found in vesicles reconstituted after dissolution of sarcoplasmic reticulum in deoxycholate. Both of these proteins are found in rather constant ratios with the ATPase in light, intermediate, and heavy sarcoplasmic reticulum vesicles. Transverse tubular vesicles can be isolated from the heavy sarcoplasmic reticulum vesicles after disruption of the membrane in a French pressure cell (Lau, Y.H., Caswell, A.H., and Brunschwig, J.P. (1977) J. Biol. Chem. 252, 5565-5574). These vesicles are enriched in their content of the high affinity calcium binding and depleted of the intrinsic glycoprotein. Cycloheptaamylose . fluorescamine complex (CFC) labels the intrinsic glycoprotein heavily indicating that it is at least partially exposed on the cytoplasmic surface of sarcoplasmic reticulum membranes. Since the carbohydrate component of the protein must lie in luminal spaces, it is inferred that the intrinsic glycoprotein is a transmembrane protein. The high affinity calcium binding protein is not labeled by CFC indicating that it is not exposed on the cytoplasmic surface of sarcotubular vesicles. The protein is also not affected by proteolytic digestion of sarcoplasmic reticulum vesicles and can be isolated intact from trypsin-digested vesicles. It is not removed from sarcoplasmic-reticulum vesicles by washing with buffers containing Chelex 100 or ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). These data show that the high affinity calcium binding protein is localized in the interior of the sarcotubular system and suggest that it might be common to both sarcoplasmic reticulum and transverse tubular membranes.

Assembly of the sarcoplasmic reticulum. Biosynthesis of the high affinity calcium binding protein in rat skeletal muscle cell cultures.

Temporal patterns of biosynthesis of the high affinity calcium binding protein from the sarcoplasmic reticulum were determined and compared with rates of ATPase ane cells. Cells at various stages of differentiation were incubated for 2 h with [35S]methionine. Specific proteins were isolated from detergent extracts of cells by incubation with antibodies specific against the various proteins and immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Radioactivity incorporated into specific bands was analyzed by counting gel slices and incorporation data were used to obtain relative rates of individual protein sys found to be indistinguishable from that of calsequestrin when cells were grown in standard medium, in medium containing 60 microM Ca2+ which prevented fusion of cells, or in enriched medium which delayed cell fusion. The high affinity calcium binding protein had a relatively high turnover rate with a half-life of about 10 h. These studies suggest that synthesis of calsequestrin and the high affinity calcium binding protein are coordinated even though calsequestrin is a glycoprotein, whereas the high affinity calcium binding protein is not glycosylated.

Ionized and Bound Calcium Inside Isolated Sarcoplasmic Reticulum of Skeletal Muscle and Its Significance in Phosphorylation of Adenosine Triphosphatase by Orthophosphate

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5--15 mM) on ice gives calcium loads of 50--60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid], but almost completely released by ionophore X-537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non-saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half-saturation constant of 5,8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 microliter/mg protein, respectively. Calcium-dependent phosphorylation of sarcoplasmic reticulum calcium-transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium-dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively. It is further suggested that the saturation of the low-affinity calcium-binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium-dependent phosphorylation than in calcium-independent phosphorylation from orthophosphate. Maximum calcium-dependent phosphoprotein formation at 20 degrees C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.

The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum

In the absence of cyclic nucleotides heart microsomes have two classes of calcium binding sites with binding constants of 0.69 and 0.071 micron-1 and capacities of 2.2 and 9.7 nmol/mg protein, respectively. Neither cyclic AMP nor monobutyryl cyclic AMP affect binding but cyclic GMP and monobutyryl cyclic GMP cause the complete loss of the high affinity calcium binding sites, Cyclic GMP (but not monobutyryl cyclic GMP) also causes a decrease in the binding constant of the low affinity binding sites. AMP, GMP and Tris-butyrate do not affect calcium binding. The effects of the cyclic nucleotides are direct and are not mediated by protein phosphorylation. Phosphorylation of microsomal proteins increases the binding constant but not the capacity of the high affinity calcium binding sites. The capacity and also, perhaps, binding constant of the low affinity sites is also increased by phosphorylation. In additon to their effects on calcium binding the cyclic nucleotides also affect the movements of calcium into and out of the microsomes. The effects are again direct and not mediated by protein phosphorylation. Cyclic GMP decreases the rate of Ca2+ efflux from preloaded cardiac microsomes and also appears to decrease the rate of uptake of Ca2+ by cardiac microsomes though this effect is less clear cut than the action on efflux. The cyclic nucleotide has a half maximal effect at a concentration of 100 microns. By contrast cyclic AMP increases the rate of influx of Ca2+ into heart microsomes and the rate of efflux of Ca2+ from preloaded preparations. The effect is, however, rather slight. It is suggested that the most obvious interpretation of these results is that cyclic GMP decreases the Ca2+ permeability of the cardiac microsomal membrane while cyclic AMP increases the permeability. In contrast to the results found with membrane preparations from certain other tissues phosphorylation of cardiac microsomal proteins does not appear to alter Ca2+ efflux or influx out of, or into, cardiac microsomal preparations. It is thus concluded that phosphorylation of cardiac microsomal proteins does not affect the Ca2+ permeability of the microsomal membrane.