| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Research Abstract |
H^+, K^+-ATPase is the proton pump responsible for gastric acid secretion. This pump consists of a catalytic α-subunit and a non-catalytic β-subunit. We studied the functional sites on the α- and β-subunits by using site-directed mutagenesis and chimera construction.
(1) We identified Glu-345 on the 4th transmembrane (M4) segment of the α-subunit as the K^+-recognition site which is involved in the K^+-dependent dephosphorylation step of H^+, K^+-ATPase.
(2) We found that the lysine/glycine cluster structure located on the N-terminus of the α-subunit is not directly involved in the function of pump activity as an ion filter.
(3) We found that the first extracellular loop (loop 1) of the α-subunit is not the binding site of proton pump inhibitor, SCH 28080. We newly prepared a series of chimeric pumps between H^+, K^+-ATPase and Na^+, K^+-ATPase α-subunits, and found that one of the chimeras was inhibited by both SCH 28080 and ouabain, indicating that the binding sites of these two inhibitors are separate.
(4) We constructed chimeric β-subunits between H^+, K^+-ATPase and Na^+, K^+-ATPase, and studied the compatibility of these β-subunits for the functional expression of proton pump. The whole cytoplasmic and transmembrane segments were interchangeable between H^+, K^+- and Na^+, K^+-ATPases. We newly identified the "^<76>QLK^<79>S" motif in the extracellular segment of H^+, K^+-ATPase β-subunit which is specific for the functional expression of H^+, K^+-ATPase.
(5) H^+, K^+-ATPase β-subunit contains seven suger chains. We progressively removed these sugar chains from the β-subunit, and studied the roles of sugar chains on the function of proton pump. We found that each sugar chain is not involved in the catalytic activity, α/β assembly and the cell surface delivery of the α- and β-subunits. We also found that the cell surface delivery mechanism is more dependent on the sugar chains than the expression of H^+, K^+-ATPase activity and α/β assembly.
|
