Pressure effects on sarcoplasmic reticulum

The irreversible effects of pressure (1-2000 atm) upon the enzymatic activity and structure of the Ca2+-ATPase of sarcoplasmic reticulum were investigated. Sarcoplasmic reticulum vesicles suspended in a medium of 0.1 M KCl, 10 mM imidazole, pH 7.0, 5 mM MgCl2, and 0.5 mM EGTA irreversibly lose their Ca2+ transport and Ca2+-stimulated ATPase activities on exposure to pressures of 800-2000 atmospheres. The pressure-induced inactivation of Ca2+-ATPase is accompanied by inhibition of the formation of phosphorylated enzyme intermediate, an increase in the passive Ca2+ permeability of the membrane, and structural changes in the Ca2+-ATPase as shown by disruption of Ca2+-ATPase membrane crystals, increased susceptibility to tryptic digestion, unmasking of SH groups, and loss of the conformational responses to Ca2+ and vanadate. The sensitivity to pressure is influenced by enzyme conformation. Ca2+ or vanadate + EGTA protect the Ca2+-ATPase against pressure-induced inactivation, implying a greater stability of the enzyme in the E1 and E2 states than in the conformational equilibrium that prevails at low [Ca2+] in the absence of vanadate. Protection against pressure inactivation was also observed in the presence of sucrose, glycerol, ethylene glycol and 1 M KCl, suggesting that water density modifying groups significantly affect the stability of Ca2+-ATPase under pressure.     

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.     

Inactivation of detergent-solubilized sarcoplasmic reticulum ATPase

Inactivation of sarcoplasmic ATPase in the solubilized state was studied in the absence and presence of Ca2+, Mg2+ and glycerol. The effects of the detergents octa(ethyleneglycol) mono-n-dodecyl ether (C12E8), 1-O-tetradecylpropanediol-(1,3)-3-phosphorylcholine and myristoylglycerophosphocholine were compared. All three detergents caused a rapid decline of the dinitrophenyl phosphatase activity of the unprotected enzyme. The stabilizing effect of Ca2+ ions was kinetically analysed. It was found that the stability of the solubilized enzyme depends on the Ca2+ concentration in a manner which is best explained by assuming rapid inactivation of Ca2+-free enzyme accompanied by slow inactivation of a calcium-enzyme complex (E1Ca). The apparent affinity constants obtained are in the order of 10(6)M-1, suggesting that high-affinity Ca2+ binding must be involved. No indications of a contribution were found, either of low-affinity Ca2+-binding sites of the conformational state E2 or of the high-affinity calcium complex E1Ca2. If Ca2+ was replaced by Mg2+, which exerts a weaker protection, the apparent affinity constants for Mg2+ are in the range of 1 mM-1. The stoichiometry of the effect of Mg2+ depends on the detergent.     

Calcium efflux from sarcoplasmic reticulum vesicles

Ca²⁺ efflux was studied in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In experimental conditions in which the Ca²⁺ pump is reversed, the rate of Ca^2? efflux varies with the ADP, orthophosphate and Mg²⁺ concentrations of the assay medium and is inhibited by Na⁺.     

Spin-labelling studies of fragmented sarcoplasmic reticulum

Vesicular fragments of sarcoplasmic reticulum (SR) were spin labelled with 2,2,6,6-tetramethyl, 4-isothiocyanate piperidine-1-oxyl (probe A) and 2,2,6,6-tetramethyl, 4-amino (N-iodoacetamide) piperidine-1-oxyl (probe B). Two to five moles of probe A or B were covalently bound to 10⁶g of membrane protein, with minimal loss of activity (ATPase, Ca²⁺, uptake). The EPR spectra of labelled SR were then studied in various experimental conditions.Strongly acid or alkaline pH, protein denaturation with ura, and membrane solubilization with deoxycholate produced marked alterations of the EPR spectra of spin-labelled SR, indicating changes in the local environment surrounding the probes, and the occurrence of conformational changes.A reversible modification of the EPR spectra of probe A and an accelerated efflux of accumulated Ca²⁺ were produced by increasing the temperature of SR suspensions from 30° to 40° C. Such a parallel behavior indicates that reversible structural transitions may control membrane permeability and Ca²⁺ efflux.ATP modifies the EPR spectra of probe B, suggesting that ATP binding to the membrane induces a structural change involving the local environment of certain sulfhydryl groups. The ATP concentration required for this effect is comparable to that requied for activation of ATPase. ADP and ITP are also effective, while pyrophosphate, AMP, and cyclic AMP are not. The effect of ATP is reversible.In other experiments, 2,2,6,6-tetramethylpiperidine-1-oxyl (probeC) was equilibrated with concentrated suspensions of SR. The EPR spectra obtained thereafter indicate that probe C binds to the membrane fragments, still maintaining a high degree of motional freedom. These spectra were markedly changed by deoxycholate solubilization of the membrane fragments, while they were little affected by protein denaturation with guanidine. These results confirm the hypothesis that the region of distribution of probe C into SR, is prevalently constituted by low-viscosity lipids.Supported by research grants from USPHS (HE 09878), the American Heart Association (66742), and the Muscular Distrophy Association of America.     

Ultrastructure of dystrophic mouse sarcoplasmic reticulum

A freeze-fracture study of normal mouse muscle revealed the following ultrastructural features of the sarcoplasmic reticulum (SR) and the T-tubule system: The SR cytoplasmic leaflets possess 8- to 9-nm particles at a density of 3000/μm². The SR luminal leaflets possess few particles. The T tubules possess 11- to 13-nm particles, primarily on the luminal leaflet, at a density of 1800/μm².Thin sections of dystrophic mouse muscle showed areas of SR dilatation and fragmentation. The T tubules were uninvolved until late in the disease process. Freeze fractures showed these dilated areas to be relatively particle-free. Areas of SR free of swelling or fragmentation did not differ from normal in freeze-fracture ultrastructure. Dystrophic mouse muscle T tubules displayed no abnormalities in freeze-fracture replicas.Enriched preparations of fragmented SR vesicles were isolated. On a sucrose density gradient, light SR from dystrophic mice was shown to be of purity equal to the normal preparation by ultrastructural (thin section, freeze fracture, and negative stain) criteria. Differences in yield of the heavier fraction were shown to be explainable by postulating a subpopulation of vesicles with increased buoyancy. The purest fraction appeared to consist of sarcoplasmic reticulum vesicles (about two-thirds of the material) and vesicles from another source, possibly T tubules.     

Investigation of sarcoplasmic reticulum SH-groups]

The amount and the reaction capacity of the thiol groups in the sarcoplasmic reticulum containing up to 86% of Ca-ATPase were determined using 7-chloro-4-nitrobenzo-2-hydroxo-1,3-diazole (NBD-chloride). The total amount of SH-groups interacting with NBD-chloride is about 9 moles/10(5) g of protein as determined in the excess of NBD-chloride (750 micrometers). With respect to their sensitivity to NBD-chloride the SH-groups may be divided into two classes: slow and fast ones (5,3 and 3,5 moles/10(5) g of protein, respectively). The modification constants for the fast and slow SH-groups are 0,16 and 0,015min-1. ATP (30 micrometers) decreases the number of fast groups by 1 mole/10(5) g of protein. At higher concentrations of ATP (1--3 mM) the amount of fast SH-groups is decreased by 3 moles/10(5) g of protein, their modification rate constant being decreased 2-fold. ATP at concentration of 1 mM, decreases the rate constant for the Ca-ATPase inactivation by NBD-chloride from 0.68 down to 0,073 min-1, which coincides with the modification rate constant for fast SH-groups (0,071 min-1) under the same conditions. Ca2+ at concentration of 10(-4) M increases the amount of fast thiol groups by 1 mole/10(5) g of protein, the rate constant of their modification by NBD-chloride being increased 2-fold. A half-maximal effect was observed in the presence of 5.10(-7) M Ca2+ . Mg2+ did not affect the total amount of fast thiol groups; however, it decreased their modification rate constant.