Expression and assembly of Torpedo californica (Na,K)ATPase alpha-subunit truncated at N-terminal end in Xenopus oocytes.
cDNAs for mutant alpha-subunits of Torpedo californica (Na,K)ATPase variously truncated at the N-terminal end were constructed and transcribed in vitro. Each of the mRNAs thus synthesized was co-injected into Xenopus oocytes together with mRNA for wild-type beta-subunit. Truncation of the alpha-subunit at trypsin accessible site T2(removal of the N-terminal 36 residues, alpha delta K37) led to a decrease in ouabain-sensitive ATPase activity and ouabain-binding capacity, leaving the amount of immunoprecipitable alpha-subunit unchanged. The Km values for Na+ and K+ of alpha delta K37 were about 10mM and 2mM, respectively, and fall in the same range for the wild-type ATPase. Truncation of the alpha-subunit leaving lysine-54(alpha delta K54) or alanine-79(alpha delta A79) resulted in the loss of the ATPase activity as well as a substantial decrease in the amount of immunoprecipitable alpha-subunit. Since the beta-subunit assembles with and thereby stabilizes the alpha-subunit, which is otherwise degraded rapidly, these results suggest that the segment of the alpha-subunit between lysine-37 and lysine-54 is involved in the assembly with the beta-subunit leading to the formation of the stable and active alpha beta complex.
Mutoh
Y
Y
Department of Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
Noguchi
S
S
Ohta
T
T
Higashi
K
K
Takasugi
M
M
Kuroiwa
A
A
Kawamura
M
M
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005260:Female; D015870:Gene Expression; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000201:enzymology; D010042:Ouabain / Q000378:metabolism; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014101:Torpedo / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014981:Xenopus
D012333:RNA, Messenger; D010042:Ouabain; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase