Publication: Sıçan beyin G proteinlerinin karakterizasyonu
Abstract
Heterotrimerik guanin nükleotit bağlayan proteinler (G proteinler) hücre sinyal iletiminde önemli rol oynamaktadır. Hücre zarının sitoplazmaya bakan yüzeyinde yerleşik G proteinleri $alpha$, β, γ alt birimlerinden oluşmaktadır. $alpha$ alt birimi guanin nükleotit (GTP ve GDP) bağlama ve GTP'yi hidrolizleme (GTPaz) etkinliğine sahiptir. G proteinleri özgünlüklerini belirleyen $alpha$ alt birimine göre Gs,Gi,Gq ve G12 olmak üzere 4 büyük alt gruba ayrılmıştır. Bu çalışmada sıçan beyninde GTP bağlama etkinliğini ölçmek için uygun koşullar belirlendi. Sıçan beyninden hazırlanan ham zar (P30) ve zar özüt (S142) kesimlerinde GTP bağlama etkinliği GTP'nin hidrolizlenmeyen analogu [35 S] GTPγS ile ölçüldü. GTPγS bağlama etkinliğinin zamana ve protein derişimine bağlı olarak arttığı ve 60mM MgCl2 varlığında maksimum olduğu saptanmıştır. Western emdirim yöntemi kullanılarak beyinde G$alpha$ ve β γ altbirimleri, beyin korteksinde G$alpha$o ve G$alpha$i proteinlerinin ekspresyonları gösterilmiştir.
Background and Design.- Heterotrimeric guanine nucleotide binding protein (G proteins) play a central role in regulation of signal transmission in the cell. G proteins which are localized in the inner surface of the cell membrane consist of $alpha$, β- and γ- subunits. The $alpha$-subunit which binds guanine nucleotides (GTP and GDP) contains intrinsic GTPase activity. G proteins are divided into four families based on their $alpha$-subunits which confer their specifity: G$alpha$s, G$alpha$i, G$alpha$q and G$alpha$12. This study was designed to measure GTP binding activity and G protein expression in rat brain. Membrane extracts were prepared from whole brain and brain cortex. GTP binding activity in crude membrane fractions (P30) and membrane extracts (S142) was measured using [35S] GTPγS, the non-hydrolyzable analogue of GTP. Conclusion.- We observed that [35S] GTPγS binding increased with time and with increasing amounts of membrane proteins. We also demonstrated that GTPγS binding was strongly magnesium dependent and was maximum at 60mM MgCl2 concentration. The presence of G protein $alpha$ and βγ subunits in whole brain and of G$alpha$o and G$alpha$i in brain cortex was shown by Western blot analysis.
Background and Design.- Heterotrimeric guanine nucleotide binding protein (G proteins) play a central role in regulation of signal transmission in the cell. G proteins which are localized in the inner surface of the cell membrane consist of $alpha$, β- and γ- subunits. The $alpha$-subunit which binds guanine nucleotides (GTP and GDP) contains intrinsic GTPase activity. G proteins are divided into four families based on their $alpha$-subunits which confer their specifity: G$alpha$s, G$alpha$i, G$alpha$q and G$alpha$12. This study was designed to measure GTP binding activity and G protein expression in rat brain. Membrane extracts were prepared from whole brain and brain cortex. GTP binding activity in crude membrane fractions (P30) and membrane extracts (S142) was measured using [35S] GTPγS, the non-hydrolyzable analogue of GTP. Conclusion.- We observed that [35S] GTPγS binding increased with time and with increasing amounts of membrane proteins. We also demonstrated that GTPγS binding was strongly magnesium dependent and was maximum at 60mM MgCl2 concentration. The presence of G protein $alpha$ and βγ subunits in whole brain and of G$alpha$o and G$alpha$i in brain cortex was shown by Western blot analysis.