Membrane-associated nucleoside diphosphate kinase from rat liver. Purification, characterization, and comparison with cytosolic enzyme.
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 homogeneity 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 (s20,w), 6.2 S; isoelectric point (pI), 6.0. These values and kinetic parameters of the m-NDP kinase were almost identical to those of the s-NDP kinase whose characteristics were more extensively studied. A peptide mapping study of the 125I-labeled m- and s-NDP kinases gave essentially identical patterns. Polyclonal 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. The physiological relevance of the m-NDP kinase in relation to the adenylate cyclase system is discussed.
Kimura
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Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Japan.
Shimada
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N
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002462:Cell Membrane / Q000201:enzymology; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification*; D051381:Rats
D000596:Amino Acids; D000906:Antibodies; D000936:Antigen-Antibody Complex; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase