Three-dimensional (type I) microcrystals of detergent-solubilized membrane-bound gastric (H+, K+)-ATPase enzyme from hog and rabbit stomachs.

Multilamellar 3-dimensional (Type I) microcrystals of detergent-solubilized crude microsomes or purified protein preparations of membrane-bound gastric (H+, K+)-ATPase from rabbit or hog stomachs develop in media consisting of 0.1 M KCl, 20 mM imidazole, 5 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml DTBpC and 20-40% glycerol, using nonionic detergent of C12E8 or BRIJ 36 for solubilization. Crystals developed in a pH-range of 6.0-7.25, during 3-10 days of incubation, at 2 degrees C. For C12E8, the most effective detergent:protein ratio for crystallization varied between (1.8-2.0):1 for the microsomes and between (0.25-0.75):1 for the purified preparations. The results of biochemical and structural analysis of the (H+, K+)-ATPase crystals showed close resemblance to those of the sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle and plasmamembrane (Na+, K+)-ATPase from kidney (J. Biol. Chem., 269, 10107-111, 1994). Based on these identities and the high (62%) overall sequential homology to the (Na+, K+)-ATPase, we conclude that the new crystals of the (H+, K+)-ATPase could also contain only the alpha-chain of the alpha beta heterodimers found in the native membrane. High-resolution electron microscopy of frozen-hydrated crystalline (H+, K+)-ATPase samples are in progress to give unit cell dimensions and molecular packing of the new crystals.