ABSTRACT
We expressed the fusion proteins of the muscarinic acetylcholine receptor M2 subtype (M2 receptor) with a maltose-binding protein (MBP) and various G protein α subunits (Gα(i1-i3/o)) at its N- and C-terminals, respectively (MBP-M2-Gα(i/o)), in Escherichia coli, and examined the effect of G protein ßγ subunits (Gßγ) on the receptor-Gα interaction as assessed by agonist- and GDP-dependent [(35)S]GTPγS binding of the fusion proteins. We found that (i) Gßγ promoted both the agonist-dependent and -independent [(35)S]GTPγS binding with little effect on the guanine nucleotide-sensitive high-affinity agonist binding, (ii) the specific [(35)S]GTPγS binding activity was much greater for MBP-M2-Gα(oA) than for MBP-M2-Gα(i1-i3) in the absence of Gßγ, whereas Gßγ preferentially promoted the agonist-dependent decrease in the affinity for GDP of MBP-M2-Gα(i1-i3) rather than of MBP-M2-Gα(oA), and (iii) the proportion of agonist-dependent [(35)S]GTPγS binding was roughly 50% irrespective of species of Gα and the presence or absence of Gßγ. These results demonstrate that receptor-Gα fusion proteins expressed in E. coli could be useful for studies of receptor-G interaction.
Subject(s)
Escherichia coli/metabolism , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Maltose-Binding Proteins/metabolism , Receptor, Muscarinic M2/metabolism , Recombinant Fusion Proteins/metabolism , Escherichia coli/genetics , GTP-Binding Protein alpha Subunits/chemistry , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein gamma Subunits/chemistry , Humans , Maltose-Binding Proteins/chemistry , Receptor, Muscarinic M2/chemistry , Receptor, Muscarinic M2/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
AIM: The purpose of the present study was to elucidate the change of D-dimer and the possibility of deep vein thrombosis screening by D-dimer during pregnancy. METHODS: One thousand, one hundred and thirty-one pregnant women were enrolled in the study from April 2006 to March 2007. D-dimer was measured by latex immunoassay at 6 to 14 and 30 to 36 weeks of gestation, respectively, and the veins of the lower extremities were examined by ultrasound at 30 to 36 weeks of gestation. RESULTS: The mean and standard error of D-dimer was 1.1 +/- 1.0 microg/mL in the first trimester and 2.2 +/- 1.1 microg/mL in the third trimester, and both values were significantly higher than adult values. In addition, D-dimer significantly increased during pregnancy. D-dimer was not significantly different between singleton and twin pregnancies in the first trimester, but in the third trimester, the values of twin pregnancies were higher than singleton pregnancies (2.2 +/- 1.6 vs 3.7 +/- 2.5 microg/mL). The mean value of D-dimer of ultrasonographically positive women was 2.6 +/- 2.0 microg/mL, which was significantly higher than the value for negative woman during the third trimester (2.2 +/- 1.6 microg/mL). The positive predictive value was 7.4% and negative predictive value was 95.5% for ultrasonographically positive women when D-dimer was set at 3.2 microg/mL. CONCLUSION: We clearly found a change of D-dimer during pregnancy. When D-dimer was higher than 3.2 microg/mL, the percentage of ultrasonographically positive women was high. We propose that women with D-dimer higher than 3.2 microg/mL are closely monitored for prevention of pulmonary thromboembolism.
