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T M Hengeveld

Publications and source records attributed to T M Hengeveld.

2 recordsLinked to original sources
PubMed1993

Structural and functional studies on the G(o) protein.

Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitopes recognized by three monoclonal antibodies (MONO, 3C2, and 3E7) were mapped by expressing defined parts of the bovine G(o) alpha-cDNA in bacteria, followed by immunoblotting. All three antibodies recognize the recombinant bovine alpha o-protein, but at distinct sites. The epitopes of MONO and 3C2 were mapped between alpha o amino acids 80 and 145, and both antibodies recognize alpha o exclusively. Heterotrimeric G(o)-proteins as well as guanosine 5'-3-O-(thio)triphosphate-liganded free alpha o-subunits are readily immunoprecipitated by these monoclonal antibodies. Binding of MONO or 3C2 does not affect ADP-ribosylation of the alpha o-subunit by pertussis toxin. Apparently, the antibodies do not bind to or induce large conformational changes in regions of the alpha o-subunit that are involved in association with beta gamma-subunits or ADP-ribosylation. 3E7 behaves as an anti common alpha-subunit antibody when used in immunoblots. However, under nondenaturing conditions, 3E7 recognizes alpha o exclusively. After binding of 3E7, the pertussis toxin-dependent ADP-ribosylation of alpha o is effectively blocked, while the ADP-ribosylation of the various alpha i-subunits is not affected. The epitope of 3E7 was mapped between alpha o amino acids 13-88, a region which has been implicated in the interaction between alpha- and beta gamma-subunits. Possibly, the inhibitory effect of 3E7 on ADP-ribosylation of G(o) is secondary to the loss of beta gamma-subunits that is observed upon binding of 3E7.

Adenosine Diphosphate Ribose↗
PubMed1992

Subunit interactions of the Go protein.

The monoclonal antibody, MONO, recognizes an epitope on the G protein alpha o-subunit [van der Voorn et al., submitted] and readily immunoprecipitates heterotrimeric Go proteins from solubilized, crude bovine brain membranes, as well as from a purified bovine brain G protein preparation. Upon incubation of the immunoprecipitates with GTP gamma S, all beta gamma-subunits are released from the alpha o-subunit. Thus, binding of MONO to the Go protein does not appear to interfere with release of bound GDP, binding of GTP gamma S or GTP gamma S-induced subunit dissociation. However, we have been unable to induce a similar dissociation of Go using its physiological activator, GTP. Surprisingly, we did not observe any dissociation of Go (bound to MONO) upon dilution in a range from 500 to 5 nM. Since an apparent Kd of alpha o-GDP for binding beta gamma of 340-390 nM has been reported [(1989) J. Biol. Chem. 264, 20688-20696] our results would suggest that binding of MONO to the alpha o-subunit induces an increased affinity of alpha o-GDP for beta gamma. Alternatively, these results could be explained if, under the conditions used, the Kd of alpha o-GDP for beta gamma were at least two orders of magnitude lower than estimated previously.

Animals↗