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.
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F. Verzar International Laboratory for Experimental Gerontology, University Medical School, Debrecen, Hungary.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003460:Crystallization; D003902:Detergents / Q000494:pharmacology; D005753:Gastric Mucosa / Q000201:enzymology; D005990:Glycerol / Q000737:chemistry; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D011092:Polyethylene Glycols / Q000737:chemistry; D011817:Rabbits; D012995:Solubility; D013270:Stomach / Q000201:enzymology*; D013552:Swine
D003902:Detergents; D011092:Polyethylene Glycols; D017506:H(+)-K(+)-Exchanging ATPase; D005990:Glycerol