Stabilization and crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum.

S Pikula,N Mullner,L Dux,A Martonosi

doi:10.1016/s0021-9258(18)60712-4

Abstract

Conditions were developed for the long-term stabilization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum, purified Ca2+-ATPase, and purified-delipidated Ca2+-ATPase preparations. The standard storage medium contains 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Preparations stored under these conditions at 2 degrees C in a nitrogen atmosphere retain significant Ca2+-stimulated ATPase activity for periods of 5-6 months or longer when assayed in the presence of asolectin. The same conditions are also conducive for the formation of three-dimensional microcrystals of Ca2+-ATPase. Of the 49 detergents tested for solubilization, optimal crystallization of Ca2+-ATPase was obtained in sarcoplasmic reticulum solubilized with octaethylene glycol dodecyl ether at a detergent/protein weight ratio of 2, and with Brij 36T, Brij 56, and Brij 96 at a detergent/protein ratio of 4. Similar Ca2+-induced crystals of Ca2+-ATPase were obtained with purified or purified delipidated ATPase preparations at lower detergent/protein ratios. The stabilization of the ATPase activity in the presence of detergents is the combined effect of high Ca2+ (20 mM) and a relatively high glycerol concentration (20%). Ethylene glycol, glucose, sucrose, or myoinositol can substitute for glycerol with preservation of ATPase activity for several weeks in the presence of 20 mM Ca2+.Ca2+-induced association between ATPase molecules may be an essential requirement for preservation of enzymatic activity, both in intact sarcoplasmic reticulum and in solubilized preparations.

Highlights

Exposure of sarcoplasmic reticulum vesicles to 0.1 mM Ca2+ or t o stoichiometric amountsof lanthanides at pH8.0 induces theformation of PI typecrystals(Dux et al, 1985) that contain CaZ+-ATPase monomeirns the El conformation
Sarcoplasmic reticulum was solubilized with ClzEs at a detergent/protein weight ratio of 4 in the standard storage medium containing 20% glycerol and 20 mMCaC1, at pH 6.0 (Fig. 1).The Ca2+-modulatedATPase activity decreased only slightly during storage at 2 "C under nitrogen atmosphere for at least 86 days when assayed in the presence of a large excess of asolectin
Omission of glycerol from the standardstorage medium causes irreversible loss of ATPase activity at a rate that is only slightly slower than that observed with 0.2 mM Ca2+,either solubilized preparations were incubated at 2 "C under nitrogen for the times indicated on the abscissa

Summary

Introduction

Exposure of sarcoplasmic reticulum vesicles to 0.1 mM Ca2+ or t o stoichiometric amountsof lanthanides at pH8.0 induces theformation of PI typecrystals(Dux et al, 1985) that contain CaZ+-ATPase monomeirns the El conformation. Sarcoplasmic reticulum was solubilized with ClzEs at a detergent/protein weight ratio of 4 in the standard storage medium containing 20% glycerol and 20 mMCaC1, at pH 6.0 (Fig. 1).The Ca2+-modulatedATPase activity decreased only slightly during storage at 2 "C under nitrogen atmosphere for at least 86 days when assayed in the presence of a large excess of asolectin.

Results

Conclusion