Chimeric Domain Analysis of the Compatibility between H+,K+-ATPase and Na+,K+-ATPase β-Subunits for the Functional Expression of Gastric H+,K+-ATPase

Mutational Study on the Roles of Disulfide Bonds in the β-Subunit of Gastric H+,K+-ATPase

“…In the cells co-expressing the wild-type ␣-and ␤-subunits, two ␤-subunit bands reacting with antibody 2B6 were observed, one with a molecular mass of 60 -70 kDa (␤ m ) representing the ␤-subunit modified with complex-type carbohydrate chains and the other with a molecular mass of 48 kDa (␤ c ) representing the ␤-subunit modified with high mannose-type carbohydrate chains as shown in the previous study (32) (Fig. 5B, lane 1).…”

“…2A, lanes 1 and 5). This was due to the stabilization of the ␣-subunit in the membrane (9,32). ␤-Subunit mutants C10S, C21S, and C58S also significantly increased the expression of the ␣-subunit compared with that in the absence of the ␤-subunit ( Fig.…”

“…Immunoprecipitation-Membrane fractions of HEK cells stably expressing the ␣␤ complex were incubated in 1 ml of lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, at 4°C for 30 min as described previously (32). The solubilized fraction was incubated with anti ␣-subunit antibody Ab1024 and ImmunoPure immobilized protein A at 4°C for 12 h. After centrifugation, the pellet was washed 4 times with the lysis buffer followed by 2 washes in 0.1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM TrisHCl, pH 7.4.…”

Structural Analysis of Late Intermediate Complex Formed between Plasmid ColIb-P9 Inc RNA and Its Target RNA

“…G-327 and G-328 at the top of the loop were strongly cleaved by RNase T1. These two bases are most critical for both the pseudoknot formation (13,14) and Inc RNA binding (19).…”

“…Specifically, they block the translation of a rep leader peptide (RLP) coupled to the translation of rep (5)(6)(7)(8)(9), by binding to the vicinity of the ribosome-binding site (RBS) for the RLP (10 -12). In the case of IncI␣ ColIb-P9 and IncB pMU720 plasmids, the translation of RLP (repY and repB) induces formation of a pseudoknot between the target stem-loop and a sequence preceding the rep (repZ and repA, respectively) RBS, which is required for rep translation (13)(14)(15). The antisense RNA additionally inhibits the pseudoknot formation by binding to the loop of the target stem-loop structure (16).…”