ABSTRACT
The binding of nucleotides to nucleoside-diphosphate kinase from pig heart was studied using the dye rose Bengal as an optical probe. By difference absorption spectroscopy and by equilibrium dialysis it was shown that one dye molecule strongly bound per enzyme subunit. By competition with nucleotides it was shown that two nucleotide-binding sites exist on each subunit of either unphosphorylated or phosphorylated enzyme: one of them binds ATP or ADP tightly, the other one binds rose Bengal tightly and ADP loosely. As detected by different affinities for rose Bengal the enzyme exists in two conformations: a 'relaxed' conformation induced by the binding of ADP, and a 'tense' conformation induced by the binding of ATP or by phosphorylation.
Subject(s)
Adenine Nucleotides/metabolism , Nucleoside-Diphosphate Kinase/metabolism , Phosphotransferases/metabolism , Rose Bengal , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Binding Sites , Kinetics , Magnesium/pharmacology , Phosphorylation , Protein Conformation , SwineABSTRACT
The binding of nucleotides to pig heart nucleoside-diphosphate kinase was studied using Rose Bengal as an optical probe. ATP, in the absence of Mg2+, binds slowly to the enzyme, with a second order rate constant of about 3000 M-1 . s-1, whereas in its presence the binding is much faster. This finding suggests the regulation of the nucleoside-diphosphate kinase activity by uncomplexed ATP, and that ATP binds normally to the enzyme via a metal ion bridge.
Subject(s)
Adenosine Triphosphate/metabolism , Nucleoside-Diphosphate Kinase/metabolism , Phosphotransferases/metabolism , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Animals , Chemical Phenomena , Chemistry, Physical , Kinetics , Magnesium/metabolism , Myocardium/enzymology , Protein Conformation , Rose Bengal , SwineABSTRACT
The affinity for Blue Sepharose of several proteins of known structure showed a pH dependence governed by their isoelectric points; Blue Sepharose behaved like a strong cationic ion exchanger because of the negative charges of its dye ligand, Cibacron Blue. A study of the protein-Cibacron Blue interactions by phase partition and equilibrium dialysis revealed the presence of high-affinity binding sites both in the case of the (di)nucleotide-dependent enzymes that possess the structural domain known as "dinucleotide fold", and in the case of other proteins consisting almost entirely of alpha-helix (human haemoglobin, cytochrome c) or beta-sheet (human immunoglobulin G). The presence of additional sites of low affinity, probably situated at the protein surface, was also inferred from the equilibrium dialysis data. In some instances, in contrast with the Sepharose-immobilized dye, the interaction of free Cibacron Blue with proteins was not pH dependent. Steric factors could be responsible for such a differential behaviour. It is suggested that certain nucleotide-dependent enzymes might also bind to Blue Sepharose by ion exchange. Preparative applications of these findings are illustrated and discussed in terms of the optimization of affinity chromatography experiments.
Subject(s)
Proteins/isolation & purification , Sepharose/analogs & derivatives , Triazines , Animals , Cattle , Chromatography, Ion Exchange , Hemoglobinometry , Hydrogen-Ion Concentration , Immunoglobulin G/analysis , L-Lactate Dehydrogenase/analysis , Mitochondria, Heart/enzymology , Muscles/enzymology , Ovalbumin/analysis , Pyruvate Kinase/isolation & purification , SwineABSTRACT
The nucleosidediphosphate kinase phosphorylation reaction led to the incorporation of 0.95 +/- 0.1 phosphate groups per enzyme subunit. The equilibrium constant of the phosphorylation reaction was 0.26. The inhibition of the nucleosidediphosphate kinase activity by Cibacron blue 3GA was competitive with respect to ATP, the donor nucleotide (apparent Ki = 0.28 microM) and uncompetitive with respect to 8-bromoinosine 5'-diphosphate, the acceptor nucleotide (apparent Ki = 0.31 microM). By difference spectroscopy it was shown that each enzyme subunit bound one Cibacron blue 3GA molecule, whereas the phosphorylated enzyme had no affinity for the dye. ATP was an effective competitor, being able to displace the dye from its bound state. The complex behaviour noted was taken as evidence for cooperative interaction between the enzyme subunits. The data obtained using polarographic techniques agreed with these results.
Subject(s)
Myocardium/enzymology , Nucleoside-Diphosphate Kinase/metabolism , Phosphotransferases/metabolism , Triazines/metabolism , Adenosine Triphosphate/pharmacology , Animals , Binding Sites/drug effects , Catalysis , Phosphorylation , Protein Binding , Spectrophotometry , SwineABSTRACT
Various analogues of adenosine 5'-diphosphate with modifications in the heterocyclic base residue were tested as substrates of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC. 2.7.1.40) and guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). The significance of different structural elements for the enzyme-substrate interaction is discussed. While pyruvate kinase shows a rather broad specificity for these analogues, phosphoenolpyruvate carboxykinase has a more stringent requirement for nucleotides, the intact keto and NH groups at C6 and N1 of the pyrimidine ring representing essential sites for the phosphoenolpyruvate carboxykinase substrate interaction. The biological significance of the different substrate specificities of pyruvate kinase and phosphoenolpyruvate carboxykinase is discussed as a possible metabolic control factor.