ABSTRACT
An additional indication in favour of interaction between sequential glycolytic enzymes is provided by the mutual enhancement of aldolase and glyceraldehyde 3-phosphate dehydrogenase activities. The efficiency of aldolase as the activator is progressively affected by the presence of its substrate, fructose-1,6-diphosphate, and its structural analogue, hexitol-1,6-diphosphate. Such interrelation of two sequential glycolytic enzymes can originate from their conformational interadjustment for the subsequent metabolic channeling between them.
Subject(s)
Fructose-Bisphosphate Aldolase/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Glycolysis/physiology , Muscles/enzymology , Animals , Enzyme Stability , Kinetics , RabbitsABSTRACT
The intrinsic fluorescence (steady-state spectra, anisotropy and nanosecond decay) in combination with phosphorescence at room temperature were used to detect and characterize conformational changes in rabbit muscle aldolase accompanying the NADH-binding process. Ligand binding has entailed a decrease in aldolase fluorescence intensity, a blue shift in its maximum and a polarization increase in a long wavelength part of the emission spectrum. The NADH binding induces the changes in room temperature phosphorescence - higher intensity and longer lifetime. The excited state energy transfer from tryptophans to NADH is not observed, and the character of spectroscopic changes on NADH binding allows us to reveal the spectroscopic heterogeneity among the tryptophan residues. The character, location of protein conformational changes associated with the binding of NADH and their relation to the tryptophans' microenvironment in aldolase are discussed.
Subject(s)
Fructose-Bisphosphate Aldolase/chemistry , Muscles/enzymology , NAD/pharmacology , Animals , Binding Sites , Energy Transfer , Fluorescence Polarization , Luminescent Measurements , Protein Conformation/drug effects , Rabbits , Spectrometry, Fluorescence , Temperature , Tryptophan/chemistryABSTRACT
Fluorescence studies on both the emission of aldolase and NADH bound to the enzyme were carried out. Aldolase was found to bind four molecules of NADH with KD = 6.0 +/- 0.3 microM. KD values for NADPH and NAD+ were 41 +/- 4 microM and 140 +/- 30 microM, respectively. The affinity to NADH was comparable with that of some NAD-dependent dehydrogenases, and was not affected by the substrate or the inhibitor.
Subject(s)
Fructose-Bisphosphate Aldolase/metabolism , Muscles/enzymology , NAD/metabolism , Animals , Kinetics , Protein Binding , Rabbits , Spectrometry, Fluorescence , Substrate SpecificityABSTRACT
A comparative study was performed for rabbit muscle phosphofructokinase (PFK) from normal and atherosclerotic animals. The diagonal electrophoretic technique was used for cysteic acid containing peptides separation. On performic acid oxidation among the tryptic peptides of PFK the disulphide containing peptide was found. Its electrophoretic mobility, compared to that of cysteic acid, gives ground for intrasubunit disulphied bond existence.
