Amino acid sequence determination of the novel forms of G_oα purified from bovine brain membranes

Abstract

Previously we have reported that there are at least four different forms of Goα in bovine brain membranes which can be distinguished by their elution profiles from Mono Q column and their immunological reactivities. The four α‐subunits are referred to as αo 1, αo 2, αo 3 and αo 4 in their elution orders from the column. Partial amino acid sequences of the purified αo 1 and αo 2 were determined and compared with the predicted sequences of two classes of Goα cDNAs, termed Goα‐1 and Goα‐2. There were at least two unique fragments corresponding with the predicted amino acid sequence of the Goα‐2 cDNA but different from that of the Goα‐1 cDNA upon tryptic digestion of αo 1‐ or αo 2‐subunit. The αo 3‐ and αo 4‐subunits, but not αo 1‐ and αo 2‐subunits, were recognized by an antibody raised against a unique amino acid sequence predicted from Goα‐1 cDNA. These results suggest that αo 1,2 subunits and αo 3,4 subunits are encoded by G.oα‐2 cDNA and Goα‐1 cDNA, respectively.

Heterogeneity of three electrophoretically distinct Go α‐subunits in mammalian brain

“…All membranes obtained from whole brain tissue but not membranes from distinct cell lines tested show a third Goe,-protein with a pl value of about 5.45 which is detected by 0~o~-and ~o.~,,~o,-specific antibodies but not by ~o2-specific antibodies. The most acidic ~to-forrn on 2D-SDS-PAGE and the slowest migrating a,, protein on urea-containing SDS-PAGE is identical with neither the two :to2 nor two ~oj isoforms reported previously [10,11]. Therefore, it is not Gag* described by Goldsmith etal.…”

“…Purification of the corresponding proteins revealed the existence of two isoforms of each :¢oj and :to,. [8][9][10][11]. Additionally, a third ~o form was detected in brain membranes, using specific antibodies [12][13][14][15].…”

“…All three ~o proteins appear as 2 isoforms in two different gel systems. Since myristoylation slightly changes the mobility of cto proteins in urea-containing SDS-PAGE [19], it is feasible that the two isoforms of the proteins found in this study, by Katada's group [10,11], and Goldsmith et al [8] differ in their co-or posttranslational modifications. Experiments are un.…”

“…So far three splice variants of the ~,,-gene coding for two Cto proteins have been identified by molecular cloning [4][5][6][7]. Interestingly, purification of the two splice variants revealed the existence of two isoforms of each ~Xot and ~o2 [8][9][10][11]. Recently, several groups reported on a third form of ao in brain membranes, using urea-containing SDS-PAGE gels [12][13][14][15].…”

Characterization of the Major Bovine Brain Go α Isoforms

“…Three of the isoforms, ␣ oA , ␣ oB , and ␣ oC , are purified associated with ␤␥ dimers as G oA , G oB , and G oC , while ␣ oD , an inconsistently observed ␣ o isoform, is not. Immunological evidence and peptide mapping suggest that ␣ oB and ␣ oD are protein products of the ␣ o2 mRNA, whereas ␣ oA and ␣ oC are translated from the ␣ o1 mRNA (23)(24)(25)(26). The functional significance of these different ␣ o isoforms is not yet clear, but the G oA , G oB , and G oC heterotrimers each contain a different assortment of ␤␥ dimers (23).…”