ABSTRACT
Ultraviolet (UV) radiation is one of the major risk factors of cataract (loss of eye-lens transparency). The influence of UVB radiation (300 nm; 100 microW cm-2) on the activity and apparent kinetic constants (Km and Vmax) of rat lens hexokinase (HK;EC 2.7.1.1), phosphofructokinase (PFK; EC 2.7.1.11), isocitrate dehydrogenase (ICDH; EC 1.1.1.41) and malate dehydrogenase (MDH; EC 1.1.1.37) of energy metabolism has been investigated by irradiating the lens homogenate of three- and 12-month-old rats. In the three-month-old group specific activities of HK and PFK are reduced by 56 and 43%, respectively, and there is no change in ICDH and MDH activities after a 24 h exposure. On the other hand, in the 12-month-old group the decreases are 72, 71, 24 and 16% for HK, PFK, ICDH and MDH, respectively. UVB irradiation increases the apparent Km of HK and PFK (in both age groups), whereas the Km of ICDH and MDH is not altered. While the decrease in Vmax of these enzymes due to UVB exposure is only marginal in three-month-old rats, it is more pronounced (significant) in 12-month-old rats. A similar decrease in enzyme activities of HK and PFK is also observed upon UVB exposure of the intact rat lens. The photoinduced changes in energy metabolism may in turn have a bearing on lens transparency, particularly at an older age.
Subject(s)
Aging , Lens, Crystalline/enzymology , Lens, Crystalline/radiation effects , Ultraviolet Rays , Animals , Energy Metabolism/radiation effects , Hexokinase/metabolism , Hexokinase/radiation effects , Isocitrate Dehydrogenase/metabolism , Isocitrate Dehydrogenase/radiation effects , Kinetics , Malate Dehydrogenase/metabolism , Malate Dehydrogenase/radiation effects , Male , Phosphofructokinase-1/metabolism , Phosphofructokinase-1/radiation effects , Proteins/metabolism , Rats , Rats, WistarABSTRACT
The effect of total body irradiation (5 Gy) on functional mouse erythroid lineage has been studied. The transferrin binding capacity by bone marrow cells and the activity of glycolytic regulatory enzymes and intracellular levels of 2,3 bisphosphoglycerate in peripheral blood erythrocytes have been determined. Results obtained along one year post-irradiation period suggest a complete recovery in the erythroid cell lineage with respect to the biological endpoints investigated.
Subject(s)
Bone Marrow/radiation effects , Diphosphoglyceric Acids/radiation effects , Erythrocytes/radiation effects , Phosphotransferases/radiation effects , Receptors, Transferrin/radiation effects , Transferrin/metabolism , 2,3-Diphosphoglycerate , Animals , Bone Marrow Cells , Diphosphoglyceric Acids/blood , Erythrocytes/enzymology , Hexokinase/radiation effects , Mice , Mice, Inbred BALB C , Phosphofructokinase-1/radiation effects , Phosphotransferases/blood , Pyruvate Kinase/radiation effects , Whole-Body IrradiationABSTRACT
During irradiation in the presence of decavanadate, the subunits of phosphofructokinase underwent progressive degradation to a fragment of about 78,000 daltons. This cleavage pattern was altered when the photoirradiation was performed in the presence of monomeric vanadate with formation of several smaller peptides. The specificity of the decavanadate induced cleavage was proved by the resistance of other enzymes to the treatment and by the effects of phosphofructokinase ligands. During irradiation, the activity of the enzyme declined. Differences between the rate of inactivation and of cleavage of enzyme subunits suggest the occurrence of multiple processes.
Subject(s)
Muscles/enzymology , Phosphofructokinase-1/radiation effects , Vanadates/pharmacology , Ammonia/pharmacology , Animals , Hydrolysis , Kinetics , Light , Macromolecular Substances , Peptide Fragments/isolation & purification , Phosphofructokinase-1/antagonists & inhibitors , PhotolysisABSTRACT
1. To investigate the mechanism of the reversible inactivation of pig spleen phosphofructokinase by ATP, the effect of order of addition of reactants (substrates, effectors and enzyme solution) was studied by preincubating the enzyme before assay with various combinations of its substrates and effectors. 2. Preincubation of the enzyme with MgATP or ATP at pH7.0 before addition of fructose 6-phosphate caused a rapid and much greater inhibition of activity than that observed when the reaction (carried out at identical substrate concentrations) was initiated with enzyme. 3. The rapid inhibition caused by preincubation with ATP, together with the sigmoidal response to fructose 6-phosphate and activation by AMP, were all blocked by prior photo-oxidation of the enzyme with Methylene Blue, which selectively destroys the inhibitory binding site for ATP [Ahlfors & Mansour (1969) J. Biol. Chem.244, 1247-1251]. 4. Fructose 6-phosphate, but not Mg(2+), protected phosphofructokinase from inhibition during preincubation with ATP in a manner that was sigmoidally dependent on the fructose 6-phosphate concentration. 5. Mg(2+), by protecting the enzyme from the inhibitory effect of preincubation at low pH (7.0) and by preventing its activation during preincubation with fructose 6-phosphate, demonstrated both a weak activating effect in the absence of the other substrates and a stronger inhibitory effect in the presence of fructose 6-phosphate. 6. Positive effectors (K(+), NH(4) (+), AMP and aspartate) protected the enzyme from inhibition during preincubation with MgATP in proportion to their potency as activators, but citrate potentiated the ATP inhibition. P(i) significantly slowed the inactivation process without itself acting as a positive effector. 7. The non-linear dependence of the initial rate of the unmodified enzyme on protein concentration (associated with increased positive homotropic co-operativity to fructose 6-phosphate) was intensified by preincubation with ATP and abolished by photo-oxidation. 8. The results are interpreted in terms of an association-dissociation model which postulates that protonation, at low pH, of a photo-oxidation-sensitive inhibitory site for ATP allows more rapid dissociation of an active tetramer to an inactive dimeric species.
