Abstract
G protein heterocomplex undergoes dissociation and association during its functional cycle. Quantitative measurements of alpha and betagamma subunit binding have been difficult due to a very high affinity. We used fluorescence flow cytometry to quantitate binding of fluorescein-labeled Gi1alpha (F-alpha) to picomolar concentrations of biotinylated G beta gamma. Association in Lubrol solution was rapid (kon = 0.7 x 10(6) M-1 s-1), and equilibrium binding revealed a Kd of 2.9 +/- 0.8 nM. The binding showed a complex dependence on magnesium concentration, but activation of F-alpha with either GDP/aluminum fluoride or guanosine 5'-O-(3-thiotriphosphate) completely prevented formation of the heterocomplex (Kd > 100 nM). The binding was also influenced by the detergent or lipid environment. Unlabeled betagamma reconstituted in biotinylated phospholipid vesicles (pure phosphatidylcholine or mixed brain lipids) bound F-alpha approximately 2-3-fold less tightly (Kd = 6-9 nM) than in Lubrol. In contrast, beta gamma in ionic detergents such as cholate and 3-[(cholamidopropyl)diethylammonio]-1-propanesulfonate exhibited substantially lower affinities for F-alpha. Dissociation of F-alpha from beta gamma reconstituted in lipid vesicles was observed upon addition of aluminum fluoride or excess unlabeled alpha subunit, indicating that myristoylated alpha subunit has only a weak interaction with lipids without the beta gamma subunit. The kinetics of aluminum fluoride-stimulated dissociation were slower than those of the alpha subunit conformational change detected by intrinsic fluorescence. These results quantitatively demonstrate G protein subunit dissociation upon activation and provide a simple but powerful new approach for studying high affinity protein/protein interactions in solution or in a lipid environment.
Highlights
Endoscopic diagnosis of a bleeding ileal carcinoid tumourW D W REES AND J BANCEWICZ From the Departments ofMedicine and Surgery, Hope Hospital, (University of Manchester School of Medicine), Salford
On entering the terminal ileum a 2 cm yellow polyp was observed 2 cm from the ileocaecal valve. The mucosa overlying this lesion was ulcerated (Figure) and a blood clot was visible within the ulcer
Massive gastrointestinal bleeding is a very rare complication of small bowel carcinoid tumour and we are currently aware of only 10 cases in th.e literature.[1,2,3]
Summary
W D W REES AND J BANCEWICZ From the Departments ofMedicine and Surgery, Hope Hospital, (University of Manchester School of Medicine), Salford. Local gastrointestinal complications occur infrequently and we report a case of repeated and massive gastrointestinal haemorrhage from a non-metabolically active carcinoid tumour of the distal ileum diagnosed by colonoscopy
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