GDP binding

There are, however, two possible complications the existence of multiple affinity sites (or unspecific sites), and the question of unmasking. 

A very-high-affinity site 0.1 jaM) has been claimed to exist by Rothwell 

Unmasking [52] is the term used for an apparent increase in GDP-binding without a parallel increase in amount of thermogenin. It is still not certain whether this 

Further, manipulations of the mitochondria, such as osmotic swelling or shrinkage (Fig. 10.11), or a pH cycle [53], may change the number of GDP binding sites found. This indicates that if non-GDP-binding thermogenin may be found, it is not necessarily a chemically modified form of thermogenin, but rather it may be physically inaccessible to the assay. 

As the degree-of-activation of mitochondria is said to influence their physical appearance, it may be suggested that some of the observed unmasking phenomena may be due to the actual state of the brown fat mitochondria at the time of isolation, and/or to the incubation procedure, rather than reflecting true changes in the in situ qualities of thermogenin itself. This, however, remains to be investigated. 

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Gq-GDP binds to the Gpy dimer through its GTPase domain in a region of the p propeller opposite to where G is bound (Figure 13.15). There are, therefore, no contacts between Gq and Gy in the heterotrimeric G py complex. The... 

Rab is a family of small G proteins involved in membrane vesicle trafficking. Mammalian tissues contain around 30 forms of Rab, which specifically associate with the various types of membrane vesicles and organelles that exist in cells [30,31]. Rab proteins, named originally as ras-related proteins in brain, are isoprenylated and associate with membranes, as do isoprenylated Ras and G protein y subunits. However, unlike these other G proteins, the GTP and GDP binding to Rab appears to regulate its association with membrane compartments. 

It is worthwhile to note that abrogation of interaction as detected in biological assays, often reported for mutants or other modifications, does not really mean complete loss of binding. Ras GDP binds Raf-RBD 1000 times less strong-... 

The sequences of events that occur during activation of adenylate cyclase after receptor occupancy are shown in Figure 6.3. This scheme thus shows activation of a Gofc-type protein (i.e. a process that leads to the activation of adenylate cyclase), whereas similar processes will occur with a Ga protein, except that the interaction with adenylate cyclase will result in its inactivation. In the same way, activation of phospholipases by mobile Ga-type subunits will occur via similar mechanisms. In the unstimulated state, Gas (or Gcq) is bound to GDP. Binding of the receptor with its agonist induces a conformational change in the receptor that activates its G-protein. This stim-... 

The G-proteins are a family of guanine nucleotide (GTP or GDP) binding proteins that are a component of several hundred hormone-effector systems or hgand-binding... 

Fig. 1.56. Control of eIF-2 by phosphorylation. Phosphorylated eIF-2 GDP binds strongly to eIF-2B without nucleotide exchange occurring. Initiation of protein biosynthesis is not possible in this case.In reticulocytes, eIF-2 is subject to phosphorylation by the heme-regulated eIF-2-kinase (HRI). The activity of the dimeric HRI is regulated via the heme concentration. Another protein kinase that can phosphorylate and regulate eIF-2 is the RNA-dependent eIF2a-kinase (PKR). The latter is induced by interferons and activated by double stranded RNA.

Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998). A novel mutation in the switch 3 region of Gsa in a patient with albright hereditary osteodystrophy impairs GDP binding and receptor activation. J. Biol. Chem. 273,... 

Malan, A., and E. Mioskowski (1988). pH-tempera-ture interactions on protein function and hibernation GDP binding to brown adipose tissue mitochondria. J. Comp. Physiol. B. 158 487-493. Martin, D.D., R.A. Ciulla, and M.F. Roberts (1999). Osmoadaptation in Archaea. Appl. Environ. Microbiol. 65 1815-1825. 

This reversible activation/deactivation process can be summarized as follows H + PM R— H-R— H-R-Ga-GDP-G(3-Gy interaction— H-R + Ga-GTP + G 3 Gy complex— active Ga-GTP activates effector proteins —> downstream effects deactivation occurs via the GTPase activity of Ga so that Ga-GTP — Ga-GDP + P — Ga GDP binds G(3-Gy — the inactive GDP-Ga-Gfi Gy complex is re-formed. 

With our present understanding, the thermogenic qualities of brown adipose tissue mitochondria are a consequence of the existence in the mitochondrial inner membrane of a polypeptide, thermogenin, uniquely [13-15] found in brown adipose tissue. (For technical and historical reasons, thermogenin is also known under several other names, such as the GDP-binding protein, the 32000 protein, the purine-nucleotide-binding protein (NbP), the uncoupling protein (UCP), the proton conductance pathway, etc.)... 

Since it was shown by Cannon et al. [23] that ADP acted from the outside of the mitochondria to induce respiratory control, a specific site of interaction could be envisaged. Such a site was characterized by Nicholls [33] by binding of [ H]GDP. Further, by labelling brown fat mitochondria with [ P]azido-ATP, Heaton et al. [34] demonstrated that — besides the ATP/ADP-translocase at 30 kDa — a specific band with a molecular weight of 32000 was labelled, and this was identical with the GDP-binding site. This protein (i.e., thermogenin) had already been observed by Ricquier and Kader as the only protein the concentration of which was markedly altered in brown fat mitochondria isolated from cold-acclimated animals [35] (Fig. 10.8). 

In conclusion it would seem that GDP-binding is an adequate way of determining thermogenin concentration in brown fat mitochondria. It should however be added that for physiological studies, this is not the only relevant parameter. As brown adipose tissue hypertrophies when stimulated, increases in thermogenin content per animal are often markedly greater than increases in thermogenin concentration in... 

Fig. 10.11. The effect of osmolarity on the apparent number of GDP binding sites in brown adipose tissue mitochondria. The number of GDP-binding sites was measured in mitochondria from control and cold-exposed (24 h at 4°C) rats as earlier described (Sundin and Cannon [37]), but in media with the indicated concentrations of sucrose. Note that, if iso-osmotic sucrose is used (250 mM), a low GDP binding can be observed, especially in control rats. This may be related to the condensation phenomenon discussed in section 2.4. However, if 100 mM sucrose is used, the mitochondria swell, and the full number of binding sites is determined. (Our unpublished observations.)...

When changes in thermogenin concentration due to cold acclimation of hamsters were measured by GDP-binding, ELISA, GDP-sensitive swelling, and GDP-sensitive respiration, it was found that these parameters increased in parallel, indicating that all thermogenin was functionally active [38,57]. 

It has been suggested that free fatty acids in some way stimulate thermogenin by acting on a (putative) site on thermogenin other than the GDP-binding site. The argument for this has until now been rather indirect and simply related to the fact that brown fat mitochondria are more sensitive to free fatty acids as uncouplers than... 

The role of Arf in the in vivo activation of PLD remains unclear for Arfs 1-5, but there is more evidence for Arf6. Whereas Arfs 1-3 are associated with the Golgi, the subcellular distribution of Arfs 4 and 5 is unclear. In contrast, Arf6 is predominantly associated with the plasma membrane where it cycles between this membrane and the cytosol and a vesicular compartment as a function of its GTP/ GDP binding [105-109]. Stimulation of chromaffin cells induces translocation of Arf6 to the plasma membrane and an associated increase in PLD activity [109]. In addition, treatment of the cells with a myristoylated peptide corresponding to the N-terminal sequence of Arf6 inhibits PLD activation [109]. Similar results have been obtained wifh this peptide in myometrium [110]. 

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