Changes in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ ATPase enzyme in the presence of terbium: a time-resolved x-ray diffraction study.

The design of the time-resolved x-ray diffraction experiments reported in this and an accompanying paper was based on direct measurements of enzyme phosphorylation using [gamma-32P]ATP that were employed to determine the extent to which the lanthanides La3+ and Tb3+ activate phosphorylation of the Ca2+ATPase and their effect on the kinetics of phosphoenzyme formation and decay. We found that, under the conditions of our experiments, the two lanthanides are capable of activating phosphorylation of the ATPase, resulting in substantial levels of phosphoenzyme formation and they slow the formation and dramatically extend the lifetime of the phosphorylated enzyme conformation, as compared with calcium activation. The results from the time-resolved, nonresonance x-ray diffraction work reported in this paper are consistent with the enzyme phosphorylation experiments; they indicate that the changes in the profile structure of the SR membrane induced by terbium-activated phosphorylation of the ATPase enzyme are persistent over the much longer lifetime of the phosphorylated enzyme and are qualitatively similar to the changes induced by calcium-activated phosphorylation, but smaller in magnitude. These results made possible the time-resolved, resonance x-ray diffraction studies reported in an accompanying paper utilizing the resonance x-ray scattering from terbium, replacing calcium, to determine not only the location of high-affinity metal-binding sites in the SR membrane profile, but also the redistribution of metal density among those sites upon phosphorylation of the Ca2+ATPase protein, as facilitated by the greatly extended lifetime of the phosphoenzyme.     

Moderate resolution profile structure of the sarcoplasmic reticulum membrane under low temperature conditions for the transient trapping of E1 approximately P.

The calcium uptake reaction kinetics of isolated sarcoplasmic reticulum (SR) vesicles have previously been shown to be at least biphasic over a range of temperatures (26 to 10 degrees C) with a fast phase identified with the formation of E1 approximately P and calcium occlusion and a slow phase with Ca2+ translocation across the membrane and turnover of the Ca2+ ATPase ensemble. At "low" temperatures, namely 0 degrees C or lower, E1 approximately P formation is slowed and E1 approximately P is transiently trapped for at least several seconds, as indicated by the absence of the slow phase for 6 s or more. We now report that a reversible, temperature-induced structural transition occurs at about 2-3 degrees C for the isolated SR membrane. We have investigated the nature of this structural transition utilizing meridional and equatorial x-ray diffraction studies of the oriented SR membrane multilayers in the range of temperatures between 7.5 and -2 degrees C. The phase meridional (lamellar) diffraction has provided the profile structure for the SR membrane at the highest vs. lowest temperature at the same moderate resolution of 16-17 A while the equatorial diffraction has provided information on the average lipid chain packing in the SR membrane plane in the two cases. To identify the contribution of each membrane component in producing the differences between the profile structures at 7.5 and -2 degrees C, step-function models have been fitted to the moderate resolution electron density profiles. Lipid lateral phase separation may be responsible for inducing the structural change in the Ca2+ ATPase, thereby resulting in the slowing of E1 approximately P formation and the transient trapping of E1 approximately P at the "lower" temperatures.     

Changes in the relative occupancy of metal-binding sites in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ATPase enzyme in the presence of terbium: a time-resolved, resonance x-ray diffraction study.

Time-resolved, terbium resonance x-ray diffraction experiments have provided the locations of three different high-affinity metal-binding/transport sites on the Ca2+ATPase enzyme in the profile structure of the sarcoplasmic reticulum (SR) membrane. By considering these results in conjunction with the known, moderate-resolution profile structure of the SR membrane (derived from nonresonance x-ray and neutron diffraction studies), it was determined that the three metal-binding sites are located at the "headpiece/stalk" junction in the Ca2+ATPase profile structure, in the "transbilayer" portion of the enzyme profile near the center of the membrane phospholipid bilayer, and at the intravesicular surface of the membrane profile. All three metal-binding sites so identified are simultaneously occupied in the unphosphorylated enzyme conformation. Phosphorylation of the ATPase causes a redistribution of metal density among the sites, resulting in a net movement of metal density toward the intravesicular side of the membrane, i.e., in the direction of calcium active transport. We propose that this redistribution of metal density is caused by changes in the relative binding affinities of the three sites, mediated by local structural changes at the sites resulting from the large-scale (i.e., long-range) changes in the profile structure of the Ca2+ATPase induced by phosphorylation, as reported in an accompanying paper. The implications of these results for the mechanism of calcium active transport by the SR Ca2+ATPase are discussed briefly.     

Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction.

Resonance x-ray diffraction measurements on the lamellar diffraction from oriented multilayers of isolated sarcoplasmic reticulum (SR) membranes containing a small concentration of lanthanide (III) ions (lanthanide/protein molar ratio approximately 4) have allowed us to calculate both the electron density profile of the SR membrane and the separate electron density profile of the resonant lanthanide atoms bound to the membrane to a relatively low spatial resolution of approximately 40 A. Analysis of the membrane electron density profile and modeling of the separate low resolution lanthanide atom profile, using step-function electron density models based on the assumption that metal binding sites in the membrane profile are discrete and localized, resulted in the identification of a minimum of three such binding sites in the membrane profile. Two of these sites are low-affinity, low-occupancy sites identified with the two phospholipid polar headgroup regions of the lipid bilayer within the membrane profile. Up to 20% of the total lanthanide (III) ions bind to these low-affinity sites. The third site has relatively high affinity for lanthanide ion binding; its Ka is roughly an order of magnitude larger than that for the lower affinity polar headgroup sites. Approximately 80% of the total lanthanide ions present in the sample are bound to this high-affinity site, which is located in the "stalk" portion of the "headpiece" within the profile structure of the Ca+2 ATPase protein, approximately 12 A outside of the phospholipid polar headgroups on the extravesicular side of the membrane profile. Based on the nature of our results and on previous reports in the literature concerning the ability of lanthanide (III) ions to function as Ca+2 analogues for the Ca+2 ATPase we suggest that we have located a high-affinity metal binding site in the membrane profile which is involved in the active transport of Ca+2 ions across the SR membrane by the Ca+2 ATPase.     

Time-resolved x-ray diffraction studies of the sarcoplasmic reticulum membrane during active transport.

X-ray and neutron diffraction studies of oriented multilayers of a highly purified fraction of isolated sarcoplasmic reticulum (SR) have previously provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10-A resolution. These studies used biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via phospholipid transfer proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have detected significant changes in the membrane profile structure associated with phosphorylation of the Ca2+-ATPase within a single turnover of the Ca2+-transport cycle. These studies used the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100-500-ms data collection times, and double-beam spectrophotometry to monitor the Ca2+-transport process directly in the oriented SR membrane multilayer.     

Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction.

A number of studies have indicated that Ca(2+)-ATPase, the integral membrane protein of the sarcoplasmic reticulum (SR) membrane, undergoes some structural change upon Ca2+ binding to its high affinity binding sites (i.e., upon conversion of the E1 to the CaxE1 form of the enzyme). We have used x-ray diffraction to study the changes in the electron density profile of the SR membrane upon high-affinity Ca2+ binding to the enzyme in the absence of enzyme phosphorylation. The photolabile Ca2+ chelator DM-nitrophen was used to rapidly release Ca2+ into the extravesicular spaces throughout an oriented SR membrane multilayer and thereby synchronously in the vicinity of the high affinity binding sites of each enzyme molecule in the multilayer. A critical control was developed to exclude possible artifacts arising from heating and non-Ca2+ photolysis products in the membrane multilayer specimens upon photolysis of the DM-nitrophen. Upon photolysis, changes in the membrane electron density profile arising from high-affinity Ca2+ binding to the enzyme are found to be localized to three different regions within the profile. These changes can be attributed to the added electron density of the Ca2+ bound at three discrete sites centered at 5, approximately 30, and approximately 67 A in the membrane profile, but they also require decreased electron density within the cylindrically averaged profile structure of the Ca(2+)-ATPase immediately adjacent (< 15 A) to these sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that lipid lateral phase separation induces functionally significant structural changes in the Ca+2ATPase of the sarcoplasmic reticulum.

We have studied lipid lateral phase separation (LPS) in the intact sarcoplasmic reticulum (SR) membrane and in bilayers of isolated SR membrane lipids as a function of temperature, [Mg+2], and degree of hydration. Lipid LPS was observed in both the intact membrane and in the bilayers of isolated SR lipids, and the LPS behavior of both systems was found to be qualitatively similar. Namely, lipid LPS occurs only at relatively low temperature and water content, independently of the [Mg+2], and the upper characteristic temperature (th) for lipid LPS for both the membrane and bilayers of its isolated lipids coincide to within a few degrees. However, at similar temperatures, isolated lipids show more LPS than the lipids in the intact membrane. Lipid LPS in the intact membrane and in bilayers of the isolated lipids is fully reversible, and more extensive for samples partially dehydrated at temperatures below th. Our previous x-ray diffraction studies established the existence of a temperature-induced transition in the profile structure of the sarcoplasmic reticulum Ca+2ATPase which occurs at a temperature corresponding to the [Mg+2]-dependent upper characteristic temperature for lipid LPS in the SR membrane. Furthermore, the functionality of the ATPase, and in particular the lifetime of the first phosphorylated enzyme conformation (E1 approximately P) in the Ca+2 transport cycle, were also found to be linked to the occurrence of this structural transition. The hysterisis observed in lipid LPS behavior as a function of temperature and water content provides a possible explanation for the more efficient transient trapping of the enzyme in the E1 approximately P conformation observed in SR membranes partially dehydrated at temperatures below th. The observation that LPS behavior for the intact SR membrane and bilayers of isolated SR lipids (no protein present) are qualitatively similar strongly suggests that the LPS behavior of the SR membrane lipids is responsible for the observed structural change in the Ca+2ATPase and the resulting significant increase in E1 approximately P lifetime for temperatures below th.     

Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane.

Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.     

Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes

Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of calcium, temperature and phospholamban phosphorylation on the dynamics of the calcium-stimulated ATPase of canine cardiac sarcoplasmic reticulum

Highly purified sarcoplasmic reticulum (SR) has been prepared from dog hearts and has been incubated with the triplet probe erythrosinyl isothiocyanate to specifically label the Ca2+-stimulated ATPase (Ca2+-ATPase) of the SR. The rotational mobility of the Ca2+-ATPase has been studied in this erythrosin-labelled SR using time-resolved phosphorescence polarization. Qualitatively, the mobility of the cardiac Ca2+-ATPase resembles that of skeletal muscle SR Ca2+-ATPase. Addition of Ca2+ to SR affects the mobility of the Ca2+-ATPase in a way consistent with a segment of the ATPase altering its orientation relative to the plane of the membrane. Phosphorylation of phospholamban in cardiac SR by the purified catalytic subunit of cAMP-dependent protein kinase, which is known to increase the activity of the Ca2+-ATPase by deinhibition, also alters measured anisotropy. The changes observed are not compatible with dissociation of the Ca2+-ATPase from phospholamban after the latter is phosphorylated. The data are more consistent with phospholamban associating with the Ca2+-ATPase following phosphorylation, or more complex models in which only the hydrophilic domain of phospholamban binds with and dissociates from the Ca2+-ATPase.     

Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin.

The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric titration of the membrane bound enzyme with TG. When Ca2+ binding to the enzyme was measured in the absence of ATP, or when one cycle of Ca(2+)-dependent enzyme phosphorylation by ATP was measured under conditions preventing turnover, protection against TG by Ca2+ was observed. The protection by Ca2+ disappeared if the phosphoenzyme was allowed to undergo turnover, indicating that a state reactive to TG is produced during enzyme turnover, whereby a dead end complex with TG is formed. Enzyme phosphorylation with Pi, ATP synthesis, and Ca2+ efflux by the ATPase in its reverse cycling were also inhibited by TG. However, under selected conditions (millimolar Ca2+ in the lumen of the vesicles, and 20% dimethyl sulfoxide in the medium) TG permitted very low rates of enzyme phosphorylation with Pi and ATP synthesis in the presence of ADP. It is concluded that the mechanism of ATPase inhibition by TG involves mutual exclusion of TG and high affinity binding of external Ca2+, as well as strong (but not total) inhibition of other partial reactions of the ATPase cycle. TG reacts selectively with the state acquired by the ATPase in the absence of Ca2+. This state is obtained either by enzyme exposure to EGTA, or by utilization of ATP and consequent displacement of bound Ca2+ during catalytic turnover.     

Two functional states of sarcoplasmic reticulum ATPase.

The "total" ATPase activity of rabbit sarcoplasmic reticulum (SR) vesicles includes a Ca2+-independent component ("basic") and Ca2+-dependent component ("extra"). Only the "extra" ATPase is coupled to Ca2+ transport. These activities can be measured under conditions in which the observed rates approximate maximal velocities. The "basic" ATPase is predominant in one of the various SR fractions obtained by prolonged density-gradient centrifugation of SR preparations already purified by repeated differential centrifugations and extractions at high ionic strength. This fraction (low dnesity, high cholesterol) has a protein composition nearly identical with that of other SR fractions in which the "extra" ATPase is predominant. In these other fractions the ratio of "extra" to "basic" ATPase activities is temperature dependent, being approximately 9.0 at 40 degrees C and 0.5 at 4 degrees C. In all the fractions and at all temperatures studied, similar steady-state levels of phosphorylated SR protein are obtained in the presence of ATP and Ca2+. Furthermore, in all cases the "basic" (Ca2+-independent) ATPase acquires total Ca2+ dependence upon addition of the nonionic detergent Triton X-100. This detergent also transforms the complex substrate dependence of the SRATPase into a simple dependence, displaying a single value for the apparent Km. The experimental findings indicate that the ATPase of rabbit SR exists in two distinct functional states (E1 and E2), only one of which (E2) is coupled to Ca2+ transport. The E1 in equilibrium E2 equilibrium is temperature-dependent and entropy-driven, indicative of its relation to the physical state of the ATPase protein in its membrane environment. Thenonlinearity of Arrhenius plots of Ca2+-dependent ("extra") ATPase activity and Ca2+ transport is explained in terms of simultaneous contribtuions from both the free energy of activation of enzyme catalysis and the free energy of conversion of E1 to E2. Thermal equilibrium between the two functional states is drastically altered by factors which affect membrane structure and local viscosity.     

A direct analysis of lamellar x-ray diffraction from hydrated oriented multilayers of fully functional sarcoplasmic reticulum.

The profile structure of functional sarcoplasmic reticulum (SR) membranes was investigated by X-ray diffraction methods to a resolution of 10 A. The lamellar diffraction data from hydrated oriented multilayers of SR vesicles showed monotonically increasing widths for higher order lamellar reflections, indicative of simple lattice disorder within the multilayer. A generalized Patterson function analysis, previously developed for treating lamellar diffraction from lattice-disordered multilayers, was used to identify the autocorrelation function of the unit cell electron density profile. Subsequent deconvolution of this autocorrelation function provided the most probable unit cell electron density profile of the SR vesicle membrane pair. The resulting single membrane profile possesses marked asymmetry, suggesting that a major portion of the Ca++ -ATPase resides on the exterior of the vesicle. The electron density profile also suggests that the Ca++-dependent ATPase penetrates into the lipid hydrocarbon core of the SR membrane. Under conditions suitable for X-ray analysis, SR vesicles prepared as partially dehydrated oriented multilayers are shown to conserve most of their ATP-induced Ca++ uptake functionality, as monitored spectrophotometrically with the Ca++ indicator arsenazo III. This has been verified both in resuspensions of SR after centrifugation and slow partial dehydration, and directly in SR multilayers in a partially dehydrated state (20-30 percent water). Therefore, the profile structure of the SR membrane that we have determined may closely resemble that found in vivo.     

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.     

Developmental changes in cardiac sarcoplasmic reticulum in sheep

Physiologic studies suggest that the myocardium from fetal and newborn sheep functions at a higher contractile state with decreased contractile reserve when compared to the myocardium of adult sheep. To investigate the role of Ca2+ transport by the sarcoplasmic reticulum (SR) in this phenomenon, we studied functional properties and protein composition of cardiac SR vesicles isolated from fetal and maternal sheep. Active accumulation of Ca2+ and the density of the Ca2+ pump protein were decreased 60% (p less than 0.01) in fetal SR vesicles; however Ca2+-dependent ATPase activity was decreased only 30% (p less than 0.01). This decreased difference in Ca2+-dependent ATPase activities was accounted for by the higher turnover number measured for the Ca2+ pump of fetal SR vesicles (1.6-fold increased, p less than 0.01). Ryanodine, an alkaloid which blocks Ca2+ efflux from cardiac SR vesicles, stimulated Ca2+ uptake more effectively in fetal SR vesicles, suggesting that these vesicles had a higher passive Ca2+ permeability during conditions of active Ca2+ transport. Protein compositional studies showed that the content of phospholamban was decreased in fetal SR vesicles and was correlated with the decrease in the density of Ca2+ pumps. In contrast, the content of calsequestrin and the density of [3H]nitrendipine-binding sites were increased approximately 2-fold in fetal SR vesicles. These functional and compositional differences between SR vesicles isolated from fetal and maternal sheep may indicate that there is relatively more junctional SR in fetal hearts. Since the SR regulates muscle contraction by modulating intracellular Ca2+ concentration, it is possible that developmental alterations in cardiac SR may contribute to the decreased myocardial contractile reserve noted in fetal sheep.     

Incorporation of synthetic phospholipids into the membranes of the sarcoplasmic reticulum from the rabbit muscle

The hydrophobic spin label used in ESR showed that the iminoxyl radical rotation in the native membrane of sarcoplasmatic reticulum (SR) occurred much faster than in the membranes, modified by a synthetic lipid. Such effect was observed throughout the whole temperature range (7-40 degrees). Experimental technique for the modification of the SR membrane and the lipid by ultrasonic treatment has been developed. Synthetic lipids without ultrasonic treatment did not inhibit the activity of Ca2+-ATPase. The change in both the enzyme activity and its ability to transport the Ca2+ ions through the membrane vesicules was observed after the phospholipids incorporation into the SR membrane. The investigation of the temperature dependence (in Arrhenius coordinates) of native and modified by lecithin Ca2+-ATPase after ultrasonic treatment and also of a "pure enzyme" showed the presence of two sharp breaks at 20 degrees and 40-42 degrees. It was shown tha the break of an Arrhenius anamorphosis was caused by a lipid environment of ATPase, "melting" of a phospholipid bilayer. The break at 20-22 degrees was observed in all cases and even after the incorporation of all the lipids into the SR membrane. This phenomenon can be explained by the distortion of the protein-lipid interaction, affecting the conformation mobility of protein and the geometry of its catalytically active center.     

Mechanism of the stimulation of calcium ion dependent adenosine triphosphatase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP) dependent protein kinase on a 22 000-dalton protein. Phosphorylation enhances the initial rate of Ca2+ uptake and Ca2+-ATPase activity. To determine the molecular mechanism by which phosphorylation regulates the calcium pump in SR, we examined the effect of cAMP-dependent protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Cardiac sarcoplasmic reticulum was preincubated with cAMP and cAMP-dependent protein kinse in the presence (phosphorylated SR) and absence (control) of adenosine 5'-triphosphate (ATP). Control and phosphorylated SR were subsequently assayed for formation (4-200 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase in media containing 100 microM [ATP] and various free [Ca2+]. cAMP-dependent phosphorylation of SR resulted in pronounced stimulation of initial rates and levels of E approximately P formed at low free [Ca2+] (less than or equal to 7 microM), but the effect was less at high free Ca2+ (greater than or equal to 10 microM). This stimulation was associated with a decrease in the dissociation constant for Ca2+ binding and a possible increase in Ca2+ sites. The observed rate constant for E approximately P formation of calcium-preincubated SR was not significantly altered by phosphorylation. Phosphorylation also increased the initial rate of E approximately P decomposition. These findings indicate that phosphorylation of cardiac SR by cAMP-dependent protein kinase regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the calcium pump observed at steady state.     

Coordination of cardiac sarcoplasmic reticulum and myofibrillar function by protein phosphorylation

Adrenergic stimulation alters functional dynamics of the heart by mechanisms most likely involving cyclic AMP (cAMP)-dependent protein phosphorylation. In vitro studies indicate that the myofibrils and sarcoplasmic reticulum (SR) may act as effectors of the adrenergic stimulation. cAMP-dependent phosphorylation of troponin I (TnI), one of the regulatory proteins of cardiac myofibrils, results in a decreased steady-state affinity of troponin C (TnC) for calcium, an increase in the off-rate for Ca2+ exchange with TnC, and a rightward shift of the relation between free Ca2+ and myofibrillar force or ATPase. Phosphorylation of phospholamban, a regulatory protein of cardiac SR, results in an increased velocity of Ca2+ transport by SR vesicles, an increased affinity of the transport protein for Ca2+, and an increased turnover of elementary steps of the ATPase reaction. These in vitro findings support the hypothesis that the inotropic response of the heart to catecholamine stimulation involves phosphorylation of TnI and phospholamban. Our in vivo studies with perfused rabbit hearts show that during the peak of the inotropic response to isoproterenol there is a simultaneous phosphorylation of TnI and an 11,000-dalton protein in the SR, most likely the monomeric form of phospholamban.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

Ca2+ regulation of 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate formation and hydrolysis on sarcoplasmic-reticular Ca2+-transport ATPase. A new principle of phospholipid turnover regulation.

Lipid phosphorylation was shown to occur on the isolated sarcoplasmic-reticulum (SR) Ca2+-transport ATPase. More than 95% of the radioactivity incorporated on incubation of the SR ATPase with [gamma-32P]ATPMg can be extracted with acidic organic solvents and was identified as 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate (PtdIns4P) [Varsányi, Toelle, Heilmeyer, Dawson & Irvine (1983) EMBO J. 2, 1543-1548]. This lipid phosphorylation is only observed at nanomolar concentrations of free Ca2+; in the presence of micromolar free Ca2+ PtdIns4P disintegrates rapidly. Also, upon blockade of the kinase reaction PtdIns4P decomposes, indicating a PtdIns/PtdIns4P turnover. The PtdIns4P concentration is dependent on the free Ca2+ concentration, being half-maximal at 35 nM-Ca2+. PtdIns4P hydrolysis is catalysed by a PtdIns4P phosphomonoesterase; accordingly no diacylglycerol is formed, which would be a product of a phosphodiesteratic cleavage. Fluoride inhibits this phosphomonoesterase. Ca2+ does not influence directly either the PtdIns kinase or the PtdIns4P phosphomonoesterase. PtdIns4P forms a tight complex with the transport ATPase, from which it can be removed only by chromatography on heparin-agarose in the presence of Triton X-100. It is concluded that Ca2+ regulates the PtdIns/PtdIns4P turnover by availability of substrate, depending on the Ca2+-transport-ATPase conformation, which traps or exposes the respective lipid head groups.