Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues.
Hydrolysis of fluorescent GTP analogues BODIPY FL guanosine 5 '-O-(thiotriphosphate) (BGTPgammaS) and BODIPY FL GTP (BGTP) by Galpha(i1) and Galpha was characterized using on-line capillary electrophoresis (o) laser-induced fluorescence assays in order that changes in sub-strate, substrate-enzyme complex, and product could be monitored separately. Apparent k values (V /[E]) (max cat) steady-state and K(m) values were determined from assays for each substrate-protein pair. When BGTP was the substrate, maximum turnover numbers for Galpha and Galpha(i1) were 8.3 +/- 1 x 10(-3) and 3.0 +/- 0.2 x 10(-2) s(-1), respectively, and K(m) values were 120 +/- 60 and 940 +/- 160 nm. Assays with BGTPgammaS yielded maximum turnover numbers of 1.6 +/- 0.1 x 10(-4) and 5.5 +/- 0.3 x 10(-4) s(-1) for Galpha and Galpha(i1); K(m) values were 14 (o)(+/-)8 and 87 +/- 22 nm. Acceleration of Galpha GTPase activity by regulators of G protein signaling (RGS) was demonstrated in both steady-state and pseudo-single-turnover assay formats with BGTP. Nanomolar RGS increased the rate of enzyme product formation (BODIPY(R) FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199 -778% in pseudo-single-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38-81% by 40 mum YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that Galpha subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTPgammaS hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe.
Jameson
Emily E
EE
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Roof
Rebecca A
RA
Whorton
Matthew R
MR
Mosberg
Henry I
HI
Sunahara
Roger K
RK
Neubig
Richard R
RR
Kennedy
Robert T
RT
D001671:Biochemistry / Q000379:methods; D001896:Boron Compounds / Q000494:pharmacology; D002021:Buffers; D002138:Calibration; D019075:Electrophoresis, Capillary; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D013050:Spectrometry, Fluorescence; D013997:Time Factors
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002021:Buffers; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go