1. [Kinetics of dissociation and reactivation of rat liver holotransketolase].
- Author
-
Kubyshin VL, Tomasheva EV, Kulesh IV, and Gorbach ZV
- Subjects
- Animals, Animals, Outbred Strains, Antimetabolites pharmacology, Catalytic Domain, Coenzymes pharmacology, Enzyme Activation, Holoenzymes chemistry, Holoenzymes isolation & purification, Hydrogen-Ion Concentration, Kinetics, Male, Rats, Thiamine Pyrophosphate pharmacology, Transketolase chemistry, Transketolase isolation & purification, Antimetabolites metabolism, Coenzymes metabolism, Holoenzymes metabolism, Liver enzymology, Thiamine Pyrophosphate analogs & derivatives, Thiamine Pyrophosphate metabolism, Transketolase metabolism
- Abstract
The work deals with isolation of transketolase from the rat liver by means of ion-exchange chromatography and substrate elution of enzyme. Experimental data on the regulation of transketolase activity with thiamin pyrophosphate (TPP) and its anticoenzyme analogues are presented. The kinetics of dissociation of holo-TK at pH 4.0 and 5.0 and reactivation of apo-TK at a wide variation of the concentration of TPP and its derivatives with anticoenzyme properties has been studied. The dissociation of holo-TK into apoenzymes and coenzymes at the specified values of pH is characterised by most evident diphasic nature, both fast and slow process being observed. The most part of enzymic activity slowdown falls on the fast phase, while the remaining 20-30% take place within the slow phase. The kinetics research findings illustrate the nonidentity of enzyme active sites with respect to TPP binding with transketolase. The K(m) values for TPP both per the first and second active sites equalled 0.3-4.5 microM and 1.3-19.7 microM, accordingly.
- Published
- 2012