Effects of ions on the intrinsic activities of c‐H‐ras protein p21

Abstract

The influence of the ionic environment on the intrinsic GTPase activity and the guanine-nucleotide interaction of Ha-rus protein p21 were studied in various experimental conditions and compared with the behaviour of elongation factor (EF) Tu. To this purpose, nucleotide-free p21 was prepared, which is much more stable than by any other reported method. Specific differences between p21 and EFTu were found in the action of divalent anions which strongly enhance the dissociation rate of p21 . GDP without affecting that of EF-Tu. Unlike EF-Tu, the GTPase activity of p21 is only slightly dependent on the presence and concentration of monovalent cations. The concentrations of Mgz + influencing the dissociation rate of the p21 . GDP complex are much higher than for the intrinsic GTPase activity, an effect also observed for EF-Tu. These results point to two distinct roles of Mg2+ : as a conformational regulator of the interaction with the substrate and as a key element for the hydrolysis of GTP. The GTPase activity of p21 is not affected by changes in pH over the range 6-9.2, different from that of EF-Tu. However, stabilization by kirromycin confers a pH independence to the GTPase of EF-Tu in the pH range 6.5 -10, suggesting that the bell-shaped behaviour of this activity in the absence of the antibiotic is due to denaturation. This implies similar properties in the catalytic mechanism of these two guanine-nucleotide-binding proteins.The Ha-rus protein p21 is one of the best studied guaninenucleotide-binding proteins, which are conserved switches adapted to the control of a large variety of fundamental functions in the cell. (Barbacid, 1987;Bourne et al., 1990). As for all the GTPases, GTP and GDP respectively induce the active and inactive conformation of protein H a m s p21 regulating an as-yet-unknown pathway controlling cell growth. Consequently, the GTPase and GDP/GTP exchange activity of Ras proteins are the key reactions which determine the functional state of the pathway and are modulated by a GTPaseactivating protein and a guanine-nucleotide-releasing factor (Hall, 1990). The prerequisite to understand the common mechanisms and the specific diversities of these proteins is the precise characterization of their intrinsic activities. In spite of the abundant literature dealing with the properties of p21, there are still several aspects which remain to be examined in more detail and compared with other members of this class of proteins under well-defined experimental conditions. In this work, we have carried out an analysis of the ionic effects on the intrinsic activities of p21, taking as a reference the elongation factor (EF) Tu, not only because of our long-time experience with this multifunctional protein, but because EFTu and p21 are among the most studied guanine-nucleotidebinding proteins and the only two with a known tertiary Correspondence to A. Parmeggiani,

Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

“…In fact, early biochemical data suggested a moderate pH dependence of the intrinsic GTPase activity of EF-Tu (43). For quantitative analysis, we measured the intrinsic GTPase activity within the pH range of 6-8.5 (Fig.…”

Properties of the Catalytic Domain of Sdc25p, a Yeast GDP/GTP Exchange Factor of Ras Proteins

“…Monovalent and divalent salts are known to influence specifically the Ras p2l/nucleotide interaction (Mistou et al, 1992a). Increasing concentrations of NH,Cl, KC1 or (NH,),SO, up to 100 mM inhibited the activity of Sdc25p-C (Fig.…”

“…MgC1, is required for stabilizing the interaction between Ras and guanine nucleotides (Pai et al, 1990;Mistou et al, 1992a;John et al, 1993).…”

“…The Ras proteins used were pure truncated (29 kDa) wild-type Ras2p (CrCchet et al, 1990b), [R80D, N81DIRas2p (a gift from Dr 0. Fasano, University of Palermo), [S24N]Ras2p, and intact human c-H-ras p21 (Mistou et al, 1992a). For the construction of [S24N]Ras2p, the EcoRl-Pstlfragment of pR2S (Fasano et al, 1988) was subcloned into pEMBL9' (Dente et al, 1983).…”

The β-Subunit of the Signal Recognition Particle Receptor Is a Novel GTP-binding Protein without Intrinsic GTPase Activity

“…Therefore we presume that this population consists of SR␤ that has become structurally unstable in the absence of bound GTP, and the resulting loss of conformation prevented the uptake of exogenous GTP. A structurally unstable empty state is a common feature of Ras-type GTPases (31,33,34). Addition of 10 M GTP to the buffers used during purification did not decrease the percentage of empty SR␤ (data not shown), suggesting that this population does not arise from dissociation of nucleotide during purification.…”