ABSTRACT
In the experiments with enzyme preparations of Na,K-ATPase from normal brain tissue (NBT) and tumorous brain tissue (TBT) the following data were established: 1) the cooperativity of Na,K-ATPase with Na+ from NBT is temperature-dependent, the Hill coefficient (nH) at 37, 27.0-30.5 and 20-22 degrees C being 1.80 +/- 0.07, 1.30 +/- 0.09 and 1.10 +/- 0.08, respectively; the cooperativity of Na+ with Na,K-ATPase from TBT was absent; 2) the cooperativity for ouabain (nH-1.30 +/- 0.05) was revealed only in the case of Na-pump from TBT; 3) the protective effect of ATP against the inhibitory action of pCMB is temperature-dependent and differs significantly in enzyme preparations from NBT and TBT; 4) the parameters of the temperature inactivation of enzyme preparations at 45-52 degrees C, especially the change of entropy (delta S*) were different in the case of NBT and TBT; 5) a peptide fraction isolated from sheep brain differently inhibited the Na,K-ATPase from NBT and TBT. In conclusion, these data demonstrate that there are significant differences in functioning of Na,K-ATPase from NBT and TBT, and that besides lipid-protein interactions the local domenic conformational changes in the enzyme molecule may play a definite role in these differences.
Subject(s)
Brain/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Adenosine Triphosphate/pharmacology , Animals , Brain/drug effects , Brain Neoplasms/enzymology , Carbodiimides/pharmacology , Chloromercuribenzoates/pharmacology , Humans , Ouabain/pharmacology , Protein Conformation/drug effects , Rats , Reference Values , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/drug effects , Structure-Activity Relationship , TemperatureABSTRACT
It has been shown that the desensibilization of the enzymic preparations of Na+, K+-ATPase by urea, DS-Na, digitonin and CHAPS reduces differently the amount of alpha beta-protomer in the enzymic preparations and the Hill coefficients of Na+ and K+. The factors (urea, DS-Na) which cause a more pronounced decrease in the amount of beta-protomer reduce the nH of Na+ for Na+, K+-ATPase and nH of K+ for Na+, K+-ATPase and K+-pNPPase to unit. The analysis of the effects of ATP and pNPP indicates that ATP has a protective effect only in the case of urea and DS-Na, but this effect is not exerted by pNPP (nonallosteric substrate). A conclusion is drawn that cooperative interactions of Na+, K+-ATPase from the brain with Na+ require more higher level of the oligomeric structure of enzyme than cooperative interactions with K+. At the same time these cooperative interactions in the both cases need subunits interactions in the protomer and interactions between cation sites with relatively high affinity.
Subject(s)
Potassium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Animals , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Kinetics , Macromolecular Substances , Protein Conformation , RatsABSTRACT
Curves of inhibition of rat brain Na, K-ATPase and K-pNPPase by prostaglandin E2 (PGE2) showed a sigmoidal shape with nH for PGE2 of 1.4 +/- 0.1 and 1.3 +/- 0.1, respectively. The desensitization of the enzymes with 0.25 M urea (4 degrees, 15 min) caused a loss of their cooperative interaction with PGE2. 2.0 mM PGE2 shifts the temperature break in the Arrhenius plots for the ATPase from 19.8 degrees to 23 degrees and simultaneously increased the Ea below the break by 9.5 kcal/mol. After treatment of the ATPase with phospholipase A2 PGE2 showed no cooperative interaction with the enzyme. Modulation of membrane enzymes by means of the surrounding lipid phasic state appears to be the general mechanism of their indirect allosteric regulation.
