Isolation of three types of Gi from bovine spleen.
We have previously reported the purification of two alpha subunits of G proteins, Gi2 and Gi3, from bovine spleen. However, it recently became clear that the preparation of Gi3 alpha contained a significant amount of Gi1 alpha by the immunoblot analysis using specific antibodies. In this study, we purified these G proteins as a trimer form from bovine spleen, and obtained following results. (1) Gi3 was separated from Gi1 using Mono Q column chromatography. Isoelectric focusing was employed to distinguish Gi3 from Gi1 in the column eluates. (2) Purified Gi2 and Gi3 retained much higher activities to bind GTP gamma S or to be ADP-ribosylated by pertussis toxin than the alpha subunits purified previously. (3) Using these spleen Gi2 and Gi3 and bovine brain Gi1, the parameter of GTP gamma S binding to the three types of Gi was compared. Three Gis showed different rates of GTP gamma S binding but showed the similar Kd values.
Morishita
R
R
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
Kobayashi
T
T
Kato
K
K
Asano
T
T
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D037342:Pertussis Toxin; D011485:Protein Binding; D013154:Spleen / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins