Surface charge and calcium binding in sarcoplasmic reticulum membranes

Vesicles of fragmented sarcoplasmic reticulum membranes have been adsorbed on to 2.68 latex spheres. Observation of these vesicle containing spheres in the presence of an electric field allows a calculation of the electrophoretic mobility of the vesicles Following this determination, the net membrane surface charge has been estimated. The mobility of sarcoplasmic reticulum membranes exhibited a dependency on pH. At an ionic strength of 0.10 a mobility (pH=7.0) of –0.67±0.10/sec/volt/cm was observed. At pH=7.0 and /2=0.150 the net excess negative charge density was 2.0×10^–2 coul/m². This is equivalent to one charge per 10³ A² (assuming a uniform charge distribution). With an average vesicle volume of 2.8×10⁸ A³ and a surface area of 2×10⁶ A² the surface of one vesicle would contain a net of approximately 2×10³ negative charges. While the mobility did not change during uptake of calcium by the vesicles, both glutaraldehyde fixation and lecithin extraction by phospholipase C greatly altered the mobility of the vesicle membrane. Calcium binding and uptake both exhibited a dependence on pH.     

Chemical modification of sarcoplasmic reticulum membranes

The usefulness of chemical cross-linking and ¹²⁵I-labeling techniques in the analysis of protein-protein interactions and membrane polarity was evaluated on sarcoplasmic reticulum membranes. Treatment of fragmented sarcoplasmic reticulum vesicles with glutaraldehyde, dimethylsuberimidate, or copper-phenanthroline leads to the formation of high molecular weight aggregates of the Ca²⁺ transport ATPase; intermediate polymers of functionally and structurally interesting sizes accumulated only occasionally and in amounts of questionable significance. Coupling of membrane proteins with tolylene 2,4-diisocyanate-albumin inhibited tht ATPase activity and caused the appearance of high molecular weight aggregates and a band of about 160 000 dalton which corresponds to the ATPase-albumin complex.Even after the 100 000 dalton band of the Ca²⁺-transport ATPase was severely diminished by cross-linking with copper-phenanthroline or toluene diisocyanate-albumin, the Ca²⁺ binding proteins of sarcoplasmic reticulum remained unreacted. A consistent finding was the presence of dimers of the Ca²⁺ transport ATPase in aged preparations of sarcoplasmic reticulum which were converted upon reduction with β-mercaptoethanol into 100 000 dalton units.Microsomes were labeled with ¹²⁵I in the presence of lactoperoxidase, glucose oxidase, and glucose and the radioactivity oft he various protein components was measured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of calsequestrin was many times greater than that of the Ca⁺ transport ATPase suggesting that it is exposed on the outside surface may be sterically hindered from access by bulky reagents (tolylene diisocyanate-albumin, ferritin-labeled anti-calsequestrin antibodies, proteolytic enzymes, etc.), as calsequestin becomes highly reactive with these agents only after its release from the membrane.     

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.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000–1130 cm^?1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the same technique. The fluidity of the membrane also manifests itself in the amide I portion of the membrane spectrum with a strong 1658 cm^?1 band characteristic of CC stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H₂O and in ²H₂O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm^?1 attributable to membrane-associated carotenoids.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes.

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000-1130 cm-1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the I portion of the membrane spectrum with a strong 1658 cm-1 band characteristic of C=C stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm-1 attributable to membrane-associated carotenoids.     

Sulfhydryl group modification of sarcoplasmic reticulum membranes.

Modification of calcium-translocating sarcoplasmic reticulum membranes (SR) with 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) reveals four classes (kinetic sets) of sulfhydryl groups. Of the 25 mol/1.5 X 10(5) G OF SR protein (i.e., containing 1 mol of ATPase protein) estimated in the presence of sodium dodecyl sulfate, 8 mol are unreactive, while 7, 8, and 2 mol display pseudo-first-order rate constants (k1) of 0.16, 0.68, and 8.3 min(-1), respectively (25 decrees C, pH 7.8, 4 MM Nbs2). Under these conditions, the Ca-ATPase activity is lost with k1 = 0.73 min(-1), whereas the Ca-independent ATPase activity is essentially unchanged. These results are little changed by the presence of Mg2+ or Ba2+ in the modification mixture, while Ca2+ or Sr2+ causes all 16-17 reactable sulfhydryls to be modified with k1 = 0.50 and 0.53 min(-1), respectively. The corresponding values for the loss of Ca-ATPase activity are 0.53 and 0.67 min(-1); this suggests that blocking of only one of the 16-17 SH groups inactivates the enzyme, i.e., that there is a single "essential" SH group. The midpoint of the transition between the Ca2+-free and Ca2+-modification patterns occurs at a free Ca2+ concentration of about 0.9 muM, implying that it is Ca2+ binding at the active sites (KD = 0.1 muM), rather than at the low-affinity nonspecific sites, that effects a conformation change in the ATPase protein (which contains greater than 90% of the cysteines). A calcium-induced conformation change is also suggested by increased ultraviolet absorbance spectrum of the purified ATPase protein upon calcium binding. If protein-lipid interaction is disrupted with deoxycholate or Triton X-100 (which does not destroy the Ca-ATPase activity and hence presumably leaves the tertiary structure of the ATPase protein largely intact), 95% of the sulfhydryls react with Nbs2 considerably faster; thus, at 2 mg/ml o- deoxycholate, 14 groups react with k1 greater than 20, 5 with k1 = 2.3, and 5 with k1 = 0.4 min(-1). These results suggest that the inaccessibility of SH groups in the absence of detergents is due to extensive interaction of the bilayer phospholipids with the ATPase protein.     

Relationship among the surface potential, Donnan potential and charge density of ion-penetrable membranes

Calculation of the potential distribution across a uniformly charged ion-penetrable membrane that we developed previously is extended to derive a relationship among the surface potential, Donnan potential and charge density of an ion-penetrable membrane in a mixed solution of 2-1 and 1-1 electrolytes. We also present an approximate method for calculating the surface potential/Donnan potential/charge density relationship for membranes with an arbitrary distribution of membrane-fixed charges.     

Membrane potential and surface charge densities as possible generalized regulators of membrane protein activities

On the basis of experimental evidence accumulated in studies of coupling and other membranes a theoretical discussion of a “breathing” membrane structure dependent upon a changing transmembrane electrical (electrochemical) gradient is presented. In the case of certain membranes difference of electrical potentials across the membrane (Δψ) is considered as one of important driving forces for some membrane protein folding and/or the arrangement of subunits within some oligomeric proteins. Δψ changes may induce synchronous reversible energetically favourable transitions in the three-dimensional architecture of several spatially separated proteins of the biomembrane. A polyfunctional regulation of levels of activities of spatially separated membrane proteins may be achieved; in some cases alterations of Δψ and surface charge densities may fulfil function of a link in the chain of events triggering membrane-bound activities in response to external stimuli.     

A model for cation content of plants based on surface potentials and surface charge densities of plant membranes

The model presented takes into account the interaction between the negatively charged membranes and macromolecules and the cations. A central postulate is that a constant average surface charge density (σ) as well as a constant average surface potential (Ψ) is conserved under different ionic conditions. The model makes it possible to predict the size of σ and Ψ from measurements of Na, K, Mg and Ca content in plant tissues of the same age but grown under two different ionic conditions (e.g. high and low K⁺). Assumptions were made about the relative amounts of free and bound Ca²⁺ and σ and Ψ were calculated from values in the literature. In all cases σ (and Ψ) are predicted to be higher for shoot (−29 to −96 mC m⁻²) than for root membranes (−14 to −27 mC m⁻²). In most cases the predicted σ falls within the range determined experimentally for biological membranes.     

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes

The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca²⁺ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography.     

Molecular arrangement of phosphatidylcholine and sphingomyelin in sarcoplasmic reticulum membranes

The distribution of phosphatidylcholine and of sphingomyelin in sarcoplasmic reticulum membranes was studied by using phospholipases. Treatment of intact membranes with phospholipase A from Vipera russeli, at 35 °C, causes breakdown of about 50–55% of the total phosphatidylcholine present in the sarcoplasmic reticulum, whereas about 90–95% degradation is obtained under the same conditions in membranes disrupted by sodium deoxycholate. On the other hand, in intact membranes, sphingomyelinase hydrolyzes only 20% of the sphingomyelin, which is largely hydrolyzed by the enzyme after disrupting the membranes with deoxycholate. The results suggest that phosphatidylcholine is similarly distributed on both layers of the membrane (~50% on each side), whereas most of the sphingomyelin (~80%) is internally localized and, therefore, asymmetrically distributed in the sarcoplasmic reticulum membranes.     

Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes

A transition in the temperature dependences of Ca²⁺ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca²⁺ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition.Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca²⁺ accumulation and ATPase activity are uncoupled, and Ca²⁺ accumulation is inhibited, while ATPase activity and passive Ca²⁺ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.     

Ionic permeability of sarcoplasmic reticulum membranes damaged by x-rays

As early as 1 and 24 h following single local X-irradiation (0.21 C/kg) of rabbit hindlimbs an increase was noted in the permeability of skeletal muscle sarcoplasmic reticulum membranes for Ca2+, K+ and Na+. The effect was maximum 1 h after irradiation and more pronounced for K+ and Na+ than Ca2+.