Abstract
The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.
Highlights
From the ‡Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland Oregon 97201-3098 and the §Center for Ulcer Research, West Los Angeles Veterans Affairs Medical Center, Los Angeles, California 90073
Kϩ ions lead to the destabilization and selective release of the M5M6 hairpin from the membrane and (ii) if such release occurs, is the hairpin released to the cytoplasmic or extracellular compartment?
If release of M5M6 occurs to the extracellular space the vesicles will need to be disrupted in order for the M5M6 hairpin to be seen in the supernatant; if release is to the cytoplasmic space, disruption of the vesicles by detergent will not be required, and centrifugation alone will separate the released hairpin from the vesicles
Summary
Materials—TPCK-treated trypsin, NaCl, KCl, Na2ATP, bovine serum albumin, sucrose, ultra pure urea, Trizma (Tris) base, and Tricine were purchased from Sigma. -Mercaptoethanol, SDS, ammonium persulfate, and Coomassie Brilliant Blue R-250 were from Bio-Rad. 4-(2Aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) was from ICN. In experiments to determine the appropriate [SDS] required to permeabilize the vesicles, enzyme activity was measured at various ratios of SDS to protein (see Fig. 2) and compared with maximal activity with nigericin or NH4Cl. Trypsin Digestion—H,K-ATPase (1 mg/ml) was suspended in medium containing 1 mM EDTA, 200 mM KCl, 20 mM Tris/HCl (pH 6.8) and incubated at 4 °C for 16 –24 h. Pellets were resuspended (1 mg/ml) in the same buffer (including KCl and MgPi, respectively) with the addition of 0.05% SDS (w/w) and incubated (37 °C, 15 min) and centrifuged (436,000 ϫ g, 30 min, 4 °C). The pellet from the sample-containing KCl tube was resuspended in buffer (1 mg/ml), but MgPi was added instead of KCl. The suspension was incubated (37 °C, 15 min) and centrifuged (436,000 ϫ g, 30 min, 4 °C). Fluorescent CPM-labeled bands were cut from the PVDF and subjected to N-terminal amino acid sequencing
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