Assembly of ATPase protein in sarcoplasmic reticulum membranes.

Three specimen preparation techniques for electron microscopy were used to investigate the incorporation of the ATPase polypeptide chains in the membranes of fragmented sarcoplasmic reticulum (SR) obtained from rabbit skeletal muscle. Observations were made of both normal vesicles and vesicles exposed to trypsin, which is known to cleave the ATPase protein and to alter the ultrastructure of the vesicles in predictable ways. Freeze-fracture replicas reveal the typical 90-A particles on the concave (PF) faces with a density of 5,730 +/- 520/mum2. On the other hand both negatively stained and deeply etched preparations display outer projections, which are absent on trypsin-incubated vesicles. The etched specimens afford for the first time top views of the vesicles in the absence of any stain. These views reveal outer projections on the PS surface with a density of 21,000 +/- 3,900/mum2, a value nearly approximating the density of the ATPase polypeptide chains (106,000 mol wt) calculated on the basis of protein and membrane area determinations. On the other hand, this value is three to four times higher than that found for the density of the 90-A particles on the concave fracture faces. Since both outer projections and 90-A particles are identified with the ATPase protein, it is suggested that the ATPase polypeptide chains are amphiphilic molecules, with polar ends protruding individually as outer projections on the surface of the vesicles, and hydrophobic ends appearing as 90-A particles on the concave fracture faces. The discrepancy between the densities of the outer projections and the 90-A particles may be attributed either to variable penetration of the polypeptide chains into the membrane bilayer, or to formation of oligomers containing three or four hydrophobic ends and appearing as single 90-A particles. Each ATPase chain forms a complex with 20-30 phospholipid molecules. The remaining phospholipids (approximately 70% of the total SR phospholipids) account for less than half the membrane volume. It is proposed that the outer leaflet of the SR membrane is prevalently composed of the ATPase lipoprotein complex, and the inner leaflet is mostly a phospholipid monolayer.     

Effect of tetraphenylboron on the calcium-dependent ATPase activity of sarcoplasmic reticulum

The apparent effect of tetraphenylboron on the Ca2+-dependent ATPase activity is rather complex being dependent of the physical state of the membrane. In leaky vesicles, tetraphenylboron decreases the affinity of the enzyme for Ca2+, and high concentrations (100 microM) inhibit the enzyme. These effects are mediated by the membrane structure. In native vesicles, tetraphenylboron induces an apparent stimulation of the maximum hydrolytic activity and a decrease of the Ca2+ gradient formed due to specific Ca2+ releasing activity of the mentioned drug.     

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.     

Effect of temperature on Ca-ATPase from sarcoplasmic reticulum membranes: ESR studies

Using spin-labeled fatty acid derivatives and maleimide, the effect of temperature on the structural state of various parts of the lipid bilayer of sarcoplasmic reticulum (SR) membranes and the segmental motion of the Ca-ATPase molecule were investigated. The mobility of the spin probes localized in the hydrophobic zone and the outer part of the SR membrane was shown to increase with a rise in temperature from 4 to 44 degrees C, the temperature of 20 degrees C being critical for these changes. In the presence of ATP, critical changes in the spin probe mobility occur at lower temperatures, while in the presence of ATP and Ca2+ they are observed at 20 degrees C for a spin probe localized in the outer part of the SR membrane. The mobility of a spin probe localized in the hydrophobic part of the membrane increases linearly with a rise in temperature. In the absence of ligands, the segmental motion of Ca-ATPase changes linearly within a temperature range of 10-30 degrees C. However, when ATP alone or ATP and Ca2+ are simultaneously added to the incubation mixture, the protein mobility undergoes critical changes at 20 degrees C. The Arrhenius plots for ATPase activity and Ca2+ uptake rate in SR membrane preparations also have a break at 20 degrees C. It is assumed that changes in the structural state of membrane lipids produce conformational changes in the Ca-ATPase molecule; the enzyme seems to be unsensitive to the structural state of the membrane lipid matrix in the absence of the ligands.     

Substrate regulation of the sarcoplasmic reticulum ATPase. Transient kinetic studies.

The rate of phosphorylation of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles by ITP and ATP was studied using a millisecond mixing and quenching device. The rate of phosphorylation was slower when the vesicles were preincubated in a Ca2+-free medium than when preincubated with Ca2+, regardless of the substrate used and of the pH of the medium. When the vesicles were preincubated with Ca2+ at pH 7.4 an overshoot of phosphorylation was observed in the presence of ITP. The overshoot was abolished when the pH of the medium was decreased to 6.0 or when the vesicles were preincubated in a Ca2+-free medium. Using vesicles preincubated with Ca2+ the apparent Km for ITP found was 2.5 mM at pH 6.0 and 1.0 mM at pH 7.4. The Vmax observed (77 mumol g-1 s-1) did not change with the pH of the medium. Both at pH 6.0 and 7.4 the apparent Km for ATP was 3 microM when preincubated in a Ca2+-free medium. At pH 6.0 the Vmax for ATP varied from 96 to 33 mumol g-1 s-1 depending on whether the vesicles were preincubated in the presence or absence of Ca2+. At pH 7.4 the Vmax for ATP was 90 mumol g-1 s-1 in both conditions. The rate of phosphorylation of the vesicles was dependent on the relative Ca2+ and Mg2+ concentrations of the reaction medium regardless of the substrate used.     

Role of sarcoplasmic reticulum phospholipids in calcium ion binding activity

The relationship between sarcoplasmic reticulum phospholipid and Ca(2+) binding by sarcoplasmic reticulum membranes was explored. Ca(2+) bound in the absence of ATP was defined as "ATP-independent Ca(2+) binding," and the additional amount of Ca(2+) bound in the presence of ATP was defined as "ATP-dependent Ca(2+) binding." The latter was found to be very sensitive to the loss of sarcoplasmic reticulum phospholipid; the amount of Ca(2+) bound was reduced when as little as 3% of the phospholipid was destroyed by phospholipase C. Further destruction of membrane phospholipid up to a 40% loss caused little or no further reduction of this Ca(2+) binding. However, when the destruction of phospholipid exceeded 40%, further loss of this Ca(2+) binding occurred, and there was an almost complete loss of this function when more than 60% of the sarcoplasmic reticulum phospholipid was destroyed.     

Cross-linking of the sarcoplasmic reticulum ATPase protein.

Treatment of rabbit sarcoplasmic reticulum vesicles with the cross-linking agent, cupric phenanthroline, causes production of high-molecular weight bands on SDS-gel electrophoresis. A plot of log mol wt $\text{vs}$ mobility indicates that the main band produced from the ATPase (mol wt = 10⁵) has a mol wt of 4 × 10⁵ and thus suggests formation of a tetramer. Notably, bands corresponding to dimers, trimers, pentamers, etc., are absent. The bands attributable to calsequestrin and calcium binding protein are unchanged by cupric phenanthroline. With extended treatment, the tetramer itself is polymerized (mol wt>10⁶). Partial disruption of the membranes with deoxycholate or Triton X-100 before cross-linking favors tetramer formation; the presence of sodium dodecyl sulfate, on the other hand, prevents intermolecular cross-linking. Our results suggest that the ATPase is at least partially associated within the membrane as a tetramer.     

Localization of the amino phospholipids in sarcoplasmic reticulum membranes revealed by trinitrobenzene-sulfonate and fluorodinitrobenzene

The chemical probes for amino compounds 2,4,6-trinitrobenzenesulfonate (TNBS) and 1-fluoro-2,4-dinitrobenzene (FDNB) were utilized to determine the localization of the amino phospholipids in the sarcoplasmic reticulum membranes. At low concentrations (<1 mM), TNBS does not penetrate the sarcoplasmic reticulum membrane, while FDNB readily penetrates it. The results show that about 70% of the total phosphatidylethanolamine is located on the external surface of the membrane, about 20% is on the internal surface and 10% is probably strongly interacting with the proteins since it is not accessible to the probes. In contrast, most of the phosphatidylserine is located on the inner surface of the membrane. This molecular distribution of the amino phospholipids supports a structural assymmetry of the sarcoplasmic reticulum membrane.     

Kinetics and regulation of sarcoplasmic reticulum ATPase.

The measurement of ATP binding to the sarcoplasmic reticulum membrane reveals that the calcium pump possesses one high affinity (Kd = 2--3 muM) site. Competition with substrate analogs show the high specifity of that site. At high ATP concentration another class of site can be detected with a much higher dissociation constant (Kd approximately 500 muM). This class of sites is of low specificity and ATP is easily displaced by other polyphosphates. The steady state rate of ATP cleavage is measured in the presence of ATP analogs. It is shown that the catalysis is due to the high affinity site. The activation of the hydrolysis rate at high substrate concentration may be related to the effect of binding of ATP to the weak sites. The effect of ATP analogs for various ATP concentration supports this hypothesis.     

Effects of phospholipase A2 and albumin on the calcium-dependent ATPase and the lipid composition of sarcoplasmic membranes.

1. The calcium-dependent ATPase activity of phospholipase-A2-digested sarcoplasmic vesicles decreases concomitantly with the contents of residual lysophospholipids and fatty acids when increasing albumin concentrations are applied. 2. Delipidated albumin preferentially removes unsaturated fatty acids and lysophosphatidylcholine. A complete removal of the phospholipids by albumin does not occur. 3. The membrane-bound lysophospholipids were analysed with respect to type of phospholipid, plasmalogen content and fatty acid chains by means of thin-layer chromatography and gas chromatography. 4. While the fatty acid composition of the lysophospholipids is independent of the degree of delipidation, the composition of the residual free fatty acids is found to change with the albumin concentration. 5. Reactivation of the Ca2+-ATPase by oleate leads to reasonable activities at room temperature as long as a minimum of about 30 lysophospholipid molec-les per ATPase is left. The course of the residual Ca2+-ATPase activity with the degree of delipidation is related to the presence of unsaturated fatty acids. 6. No specific role of either sphingomyelin or the plasmalogens has been found.     

The effect of phospholipids on the thermostability of the ATPase from locust and crayfish sarcoplasmic reticulum

The temperature dependence of delipidated and phospholipid-replaced ATPase from crayfish and locust sarcoplasmic reticulum (SR) was studied. Removal of approx. 85% of the phospholipids present in locust SR considerably decreased the temperature optimum of the ATPase, indicating that the thermostability of the ATPase is dependent on the phospholipids surrounding the enzyme. Addition of distearoylphosphatidylcholine to the delipidated ATPase from locust and crayfish SR increased the thermostability, whereas a decrease could be observed upon addition of dilinoleoyl- and dimyristoylphosphatidylcholine. These results suggest that the thermostability of the ATPase might be affected by the conformation of the fatty acyl chains in the microenvironment of the enzyme.     

Two populations of phospholipids exist in sarcoplasmic reticulum and in recombined membranes containing Ca-ATPase

Phosphorus nuclear magnetic resonance spectra of sarcoplasmic reticulum membranes from rabbit muscle and of recombined membranes containing the calcium-dependent adenosinetriphosphatase (Ca-ATPase) of sarcoplasmic reticulum reveal two distinguishable, overlapping resonances. One resonance resembles a normal phospholipid bilayer resonance, and the other is much broader. The broader component is not seen in protein-free phospholipid vesicles. In recombined membranes of the Ca-ATPase, the intensity found in the broad component was proportional to the concentration of protein in the vesicles. The two-component spectra are interpreted to arise from at least two different domains of phospholipids, one of which is motionally restricted by the Ca-ATPase. Phospholipids exchange between these two domains at a rate less than 10(3) s-1. A model for protein-lipid interactions in membranes containing the Ca-ATPase is proposed in which some of the phospholipid head groups of the membrane interact directly with the protein.     

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.     

Localization of ATPase protein in sarcoplasmic reticulum membrane.

The substrate specificity of an extensively purified flavanone synthase from light-induced cell suspension cultures of Petroselinum hortense was investigated. p-Coumaroyl-CoA was found to be the only efficient substrate for flavanone synthesis, producing naringenin (5,7,4′-trihydroxyflavanone). Besides 4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975) Arch. Biochem. Biophys.169, 84–90) two further release products of the synthase reaction in vitro were identified as 4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p-hydroxybenzalacetone. The apparent K_m values for malonyl-CoA and p-coumaroyl-CoA in the reaction leading to naringenin, and for p-coumaroyl-CoA in the reaction leading to the styrylpyrone derivative were 35, 1.6, and 2.6 μm, respectively. With caffeoyl-CoA as substrate only a very small amount of eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) was formed besides relatively large amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone derivatives. No flavanone formation was observed with feruloyl-CoA as substrate, but again appreciable amounts of the three types of short-chain release products were formed. No reaction at all took place with cinnamoyl-CoA, p-methoxycinnamoyl-CoA, isoferuloyl-CoA, or p-hydroxybenzoyl-CoA.None of the styrylpyrone, dihydropyrone, and benzalacetone derivatives has been detected in the cell cultures in vivo. The present results suggest that naringenin is the only natural product of the synthase reaction and that further substitution in the B-ring of the flavonoids occurs in parsley at or after the flavanone stage. The nature of the smaller release products is consistent with the assumption of a stepwise addition of acetate units from malonyl-CoA to the acyl moiety of the starter molecule, p-coumaroyl-CoA.     

Role of cholesterol in the Ca uptake and ATPase activity of fragmented sarcoplasmic reticulum

With the use of continuous sucrose density gradient a highly purified preparation of vesicles of fragmented sarcoplasmic reticulum was obtained and its lipid content was determined.     

Multilayer planar membranes of sarcoplasmic reticulum.

Multilayer planar membranes were constructed between a pair of cellulose sheets from fragmented sarcoplasmic reticulum (FSR) as well as a mixture of egg yolk lecithin and the Ca2+-ATPase purified from FSR. Since sodium deoxycholate was used instead of organic solvents in order to dissolve phospholipids in the process of the membrane preparation, the total activity of the Ca2+-ATPase was still preserved in the planar membrane of FSR. It was also indicated using a spin label technique that the orientation of phospholipids in the planar membrane of FSR was considerably disturbed by the presence of proteins such as the Ca2+-ATPase included in FSR.