phosphorus-radioisotopes and 1-6-bismaleimidohexane

The beta gamma subunits of heterotrimeric G proteins play a central role in regulating the function of the G protein alpha subunits and in modulating the activity of several enzymes and ion channels. We have used the signature tryptic cleavage pattern of native beta gamma from bovine brain as a starting point for our analysis of its physical and chemical properties. Digestion of bovine brain beta gamma with trypsin yields only 2 beta-derived fragments, with relative mobilities on SDS-PAGE of 14 kDa (amino terminal) and 27 kDa (carboxyl terminal), despite the presence of 32 potential tryptic cleavage sites in the beta 1 subunit. Trypsin-cleaved beta gamma remains in a complex that has the same apparent sedimentation coefficient as intact beta gamma, and retains its ability to associate functionally with the alpha o subunit. Comparison of the incorporation of [14C]iodoacetamide into reduced denatured beta and unreduced denatured beta showed that there are no disulfide bonds in the molecule to hold the complex together. The brain beta and gamma subunits can be cross-linked by 1,6-bis(maleimido)hexane to form a 46-kDa product on SDS-PAGE, and trypsin cleavage of cross-linked beta gamma shows that gamma is cross-linked to the 14-kDa amino-terminal fragment of the beta subunit. On the basis of its primary sequence, the beta subunit is predicted to form a repetitive structure encompassing the 27-kDa fragment and part of the 14-kDa fragment. Analysis of the thermal denaturation of trypsin-cleaved beta gamma supports this prediction and confirms that both fragments retain stable tertiary structures following tryptic cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carbon Radioisotopes; Cross-Linking Reagents; Cysteine; GTP-Binding Proteins; Hot Temperature; Iodoacetamide; Kinetics; Macromolecular Substances; Maleimides; Molecular Weight; NAD; Peptide Fragments; Phosphorus Radioisotopes; Protein Denaturation; Thermodynamics; Trypsin; Virulence Factors, Bordetella