Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody.

Abstract

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 x 10-8 M for GTP and 1.0 x 10-8 M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

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

“…As shown in Table 1, our purified p21 shows a very high affinity for GDP and GTP (K',, = lOpM), which is more than three orders of magnitude stronger than reported for p21 purified using denaturing agents (Hattori et al, 1985;Sigal et al, 1986;Clanton et al, 1986;Feig and Cooper, 1988) and in good agreement with results reported by other authors using methods avoiding denaturing agents Neal et al, 1988). (NH,),SO, strongly increases the dissociation rate of p21 (Table2), as already observed by Satoh et al (1988).…”

Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.

“…One report (23) claimed that Y13-259 did not interfere with either the GTP-binding or the GTPase activity of v-Ha-ras protein produced in E. coli. On the other hand, it has been claimed that the same antibody blocked [3H]GTP binding and autophosphorylation of v-Ha-ras p21 (12,18) severely hampered the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides, and decreased the intrinsic GTPase of c-Ha-ras (17,20). These discrepancies probably come from the fact that in one case the protein was not completely renatured (23).…”

Inhibition of activated Ras-induced neuronal differentiation of PC12 cells by the LIM domain of LIM-kinase 1

“…The bgalactosidase cDNA was excised out from pSV-b-galactosidase (Promega) by HindIII ± ApaI digestion, and subcloned into the pRc-CMV vector (pRc-CMV-b-Gal). The c-Ha-Ras V12 cDNA was cut out from pMT-cHr-2 (provided by Dr S Hattori) (Hattori et al, 1985) by BglII digestion, and subcloned into pRc-CMV with a NotI linker (pRc-CMV-Ras V12 ). SRa456 -XenopusD(32 ± 51) -S218E/S222E-MAPKK (DNSESE, provided by Drs Y Gotoh and E Nishida) (Gotoh et al, 1995) was digested with BglII and the BglII sites in both ends were substituted by NotI sites.…”