Further characterization of the 3-dimensional crystals of detergent-solubilized (Na+,K+)-ATPase from pig kidney.
Multilamellar 3-dimensional (Type I) crystals of detergent-solubilized, purified (Na+, K+)-ATPase enzyme of pig kidney grow in media consisting of 0.1 M KCl, 0.1 M NaCl, 20 mM imidazole pH: 7.5 (20 degrees C), 5 mM MgCl2, 5 mM DTT, 3 mM ZNaN3, 0.025 TIU/ml Aprotinin, 2 micrograms/ml BHT and 20-40% glycerol, using nonionic detergents of C12E8 or BRIJ 36 for solubilization. The refined crystallization protocol: the use of media containing 20% glycerol, the low detergent: protein ratio and preincubation at subzero temperature at the initial phase of crystallization resulted in a remarkable increase of the yield and overall dimension of the crystals (up to 3-4 microns), while the stacking of the crystalline sheets was dramatically reduced. Biochemical and structural analysis of these crystals revealed further similarities between the 3-d crystals of the (Na+, K+)-ATPase and the Ca2+ ATPase of skeletal muscle-SR (Taylor and Varga, J. Biol. Chem., 269, 10107-10111, 1994). Computer image processing of the electron micrographs of stacked crystalline sheets of (Na+,K+)-ATPase molecules gave unit cell dimensions: a = 166.2 +/- 3.8 A, b = 54.2 +/- 3.5 A, with an included angle of 90 degrees C. Based on the close identity of the filtered images in projection and of other data, we concluded that the 3-dimensional crystals of the (Na+, K+)-ATPase contain only the alpha catalytic subunits.
Varga
S
S
F. Verzár International Laboratory for Experimental Gerontology, Hungarian Section.
Szabolcs
M
M
D000818:Animals; D003460:Crystallization; D003902:Detergents / Q000494:pharmacology*; D005990:Glycerol / Q000737:chemistry; D007668:Kidney / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D012995:Solubility; D013552:Swine; D013696:Temperature
D003902:Detergents; D000254:Sodium-Potassium-Exchanging ATPase; D005990:Glycerol