Affinity labeling of GTP‐binding proteins in cellular extracts

Abstract

GTP-binding pro&s in cellular extracts from &schrrickio co/i, Thrrmtrs rhennophl/us, yeast. wheat germ or calf thymus were identified using in situ pcriodate-oxidized [@P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiffs base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamidc gel clectrophoresis and visualized by autoradiography. In addition cross-linking of [t@P]GTP with G'TP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-rus ~21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. Awr~ophihts and initiation factor 2 (lF2) from T. rhernwphi/us. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initibcion factors, and heterolrimeric signal transducing G-proteins.

G proteins as drug targets

“…When bound to the h-subunit, oGTP is hydrolyzed to oGDP; hence, oGTP can only support one round of G protein activation and subsequently traps the h-subunits in the inactive conformation [70,71]. Because the guanine nucleotide binding pocket is highly conserved among various classes of GTP-binding proteins, these also bind oGTP and related periodateoxidized guanine nucleotides [72][73][74]; hence, even if membrane permeable analogues of oGTP can be generated, they will presumably also block a host of other GTP-dependent processes such as ras-dependent cell growth, cytoskeletal dynamics, protein synthesis and translocation through the endoplasmic reticulum and the nuclear membrane as well as vesicle transport. Thus, although oxidized guanine nucleotides are useful experimental tools to dissect reaction pathways [75], the low selectivity that is inherent in their mechanism of action casts doubt on their usefulness in the search for therapeutically relevant G-protein antagonists.…”

Detection and characterization of GTP‐binding proteins in the chloroplast envelope of spinach (Spinacia oleracea)

“…The second method involved crosslinking of GTP after periodate/borohydride treatment, prior to separation by SDS‐PAGE. This method is based on the formation of a Schiff base on the ribose unit of GTP that is cross‐linked to a lysine in the conserved GTP‐binding domain, NKXD, mainly found in GTPases belonging to the classes of ras ‐proteins, elongation and initiation factors and heterotrimeric G‐proteins (Löw et al. 1992).…”

“…GTP-affinity labelling was modified from a previous protocol (Löw et al 1992). Chloroplast subfractions (15 mg protein) were incubated at 20aeC in 120 ml buffer (25 m HEPES/KOH pH 7.0, 1.0 mM Mg-acetate, 0.10% (w/v) Triton X-100, 0.5 mM (DTT) containing either 185 kBq [a-32 P]GTP (110 TBq mmol ª1 ), 185 kBq [g-32 P]GTP (157 TBq mmol ª1 ) or 18.5 kBq [ 35 S]GTPgS (43 TBq mmol ª1 ).…”