Regulation of the Rate and Extent of Phospholipase C 2 Effector Activation by the 
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- 作者：Loren W. Runnels and Suzanne F. Scarlata
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：November 3, 1998
- 年：1998
- 卷：37
- 期：44
- 页码：15563 - 15574
- 全文大小：278K
- 年卷期：v.37,no.44(November 3, 1998)
- ISSN：1520-4995
文摘
The activity of mammalian phosphoinositide-specific phospholipase C 
2 (PLC-2) is regulatedby the 
q family of G proteins and by 
subunits. We measured the affinity between the laterallyassociating PLC-2 and G on membrane surfaces by fluorescence resonance energy transfer. Using asimple model, we translated this apparent affinity to a bulk or three-dimensional equilibrium constant(Kd) and obtained a value of 3.2 
M. We confirmed this Kd by separately measuring the on and off (kfand kr) rate constants. The kf was slower than a diffusion-limited value, suggesting that conformationalchanges occur when the two proteins interact. The off rate shows that the PLC-2·G complexes arelong-lived (~123 s) and that activation of PLC-2 by G would be sustained without a deactivatingfactor. The addition of i1(GDP) subunits failed to physically dissociate the complex as determined byfluorescence. However, enzyme activity studies performed under similar conditions show that the additionof Gi1(GDP) results in reversal of PLC-2 activation by G during the time of the assay (30 s). Fromthese results, we propose that G(GDP) subunits can bind to the PLC-2·G complex to allow for rapiddeactivation without complex dissociation. In support of this model, we show by fluorescence thatGi1(GDP)·G·PLC-2 can form.
2 (PLC-2) is regulatedby the q family of G proteins and by subunits. We measured the affinity between the laterallyassociating PLC-2 and G on membrane surfaces by fluorescence resonance energy transfer. Using asimple model, we translated this apparent affinity to a bulk or three-dimensional equilibrium constant(Kd) and obtained a value of 3.2 M. We confirmed this Kd by separately measuring the on and off (kfand kr) rate constants. The kf was slower than a diffusion-limited value, suggesting that conformationalchanges occur when the two proteins interact. The off rate shows that the PLC-2·G complexes arelong-lived (~123 s) and that activation of PLC-2 by G would be sustained without a deactivatingfactor. The addition of i1(GDP) subunits failed to physically dissociate the complex as determined byfluorescence. However, enzyme activity studies performed under similar conditions show that the additionof Gi1(GDP) results in reversal of PLC-2 activation by G during the time of the assay (30 s). Fromthese results, we propose that G(GDP) subunits can bind to the PLC-2·G complex to allow for rapiddeactivation without complex dissociation. In support of this model, we show by fluorescence thatGi1(GDP)·G·PLC-2 can form.