|Budget Amount *help
¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥5,000,000 (Direct Cost: ¥5,000,000)
Previous studies from this laboratory have proposed that membrane-associated nucleoside diphosphate kinase(m-NDP kinase) may play a role in regulation of adenylate cyclase by channeling GTP, an essential cofactor of adenylate cyclase regulation, into GTP binding protein(Gs) in a hormone dependent manner. To understand the true role of m-NDP kinase, in the present study, the m-NDP kinase was solubilized and purified to apparent homogenerity from rat liver purified plasma membranes, and characterized in comparison with the cytosolic enzyme purified from the same tissue(s-NDP kinase). Some physical properties determined were; molecular weight(monomer), 18,300; sedimentation coefficient(s_<20,w>), 6.2s; isoelectric point(pI), 6.0. These values and kinetic parameters of the m-NDP kinase were almost identical to those of the s-NDP_<125>kinase whose characteristics were more externsively studied. A peptide mapping study of the I-labelled m- and s-NDP kinases gave essentially identical pattern
s. Polycolonal antibodies against the s-NDP kinase, which also cross-reacted with the m-NDP kinase, were prepared. Immunoblotting studies with the affinity-purified antibodies revealed that the monomer molecular weight of the purified m- and s-NDP kinases was identical to the values of unpurified enzymes present in membranes and crude extract. These results demonstrate that the purified m-NDP kinase underwent no remarkable modification during solubilization and purification, and that the m- and s-NDP kinases are quite similar in protein structure, if at all different.
Next, whether the m-NDP kinase has a direct interaction with the component(GTP-binding protein(Gs)) of the glucagon- and <beta>-adrenergic agonist-sensitive adenylate cyclase systems in rat liver membranes was examined by extraction with octylglucoside, followed by immunoprecipitation by affinity-purified monospecific anti-NDP kinase antibodies. The results demonstrated that the m-NDP kinase and the Gs were extractable as a complexed form and that the complex formation was reversibly regulated, through cell surface receptors, by hormones which had an ability to cause activation of the rat liver adenylate cyclase. Also, it was suggested that guanine nucleotides rather than hormones were primary regulators of the m-NDP kinase-Gs interaction.
These results suggest that the interaction between m-NDP kinase and Gs may play a role as a GTP channeling mechanism in membrane signal transduction. Less