G-PROTEIN BETA-GAMMA-SUBUNIT - PHYSICAL AND CHEMICAL CHARACTERIZATION

The betagamma subunits of heterotrimeric G proteins play a central rol e in regulating the function of the G protein alpha subunits and in mo dulating the activity of several enzymes and ion channels. We have use d the signature tryptic cleavage pattern of native betagamma from bovi ne brain as a starting point for our analysis of its physical and chem ical properties. Digestion of bovine brain betagamma with trypsin yiel ds only 2 beta-derived fragments, with relative mobilities on SDS-PACE of 14 kDa (amino terminal) and 27 kDa (carboxyl terminal), despite th e presence of .32 potential tryptic cleavage sites in the beta1 subuni t. Trypsin-cleaved betagamma remains in a complex that has the same ap parent sedimentation coefficient as intact betagamma, and retains its ability to associate functionally with the alpha(o) subunit. Compariso n of the incorporation of [C-14] iodoacetamide into reduced denatured beta and unreduced denatured beta showed that there are no disulfide b onds 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 be tagamma shows that gamma is cross-linked to the 14-kDa amino-terminal fragment of the beta subunit. On the basis of its primary sequence, th e beta subunit is predicted to form a repetitive structure encompassin g the 27-kDa fragment and part of the 14-kDa fragment. Analysis of the thermal denaturation of trypsin-cleaved betagamma supports this predi ction and confirms that both fragments retain stable tertiary structur es following tryptic cleavage. The stability of the betagamma subunit may reflect strong noncovalent interactions among repetitive structura l units of beta.