Radiation inactivation analysis of oligomeric structure of the H,K-ATPase.
The oligomeric size of the H,K-ATPase was determined in frozen gastric microsomal vesicles irradiated with high energy electrons. Target sizes of various catalytic activities associated with H,K-ATPase function fell into two distinct groups. The lower group of target sizes described the radiation-induced loss of steady-state phosphoenzyme and structural monomer: the MgATP-dependent formation of a beta-aspartyl phosphate exhibited a size range of 133-147 kDa; the size range for the structural measurement (i.e. loss of H,K-ATPase monomer on sodium dodecyl sulfate-polyacrylamide gels) was 92-143 kDa. In contrast, a larger group of target sizes described the loss of full cycle catalytic activities (i.e. K+-dependent stimulation of p-nitrophenyl phosphate and ATP hydrolysis). The K+-phosphatase and K+-stimulated ATPase exhibited target sizes fo 200 +/- 13 and 232 +/- 23 kDa, respectively. The lower target size group represents the first evidence that a monomer of the catalytic subunit maintains partial enzyme function. The larger group of target sizes describing K+-phosphatase and ATPase activities suggest that subunit interactions contribute to full cycle catalytic activity. Subunit interactions appear to be involved in all ion transport activities. Passive Rb+ exchange and active H+ transport in reconstituted proteoliposomes exhibited target sizes of 233n = 2 and 388 +/- 48 kDa, respectively. H+ transport appears to require a subunit arrangement more complex than that associated with catalytic activity or passive ion transport.
Rabon
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EC
Center for Ulcer Research and Education, Veterans Administration Center, Wadsworth Division, Los Angeles, California 90073.
Gunther
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RD
Bassilian
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Kempner
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ES
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000528:radiation effects*; D000818:Animals; D004307:Dose-Response Relationship, Radiation; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D013270:Stomach / Q000201:enzymology; D013552:Swine
D046911:Macromolecular Substances; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase