Tag Archives: pentose phosphate pathway

Kinetics of dissociation and reactivation of rat liver holotransketolase

V. L. Kubyshin, A. V. Tomashova, I. V. Kulesh, Z. V. Gorbach

Educational Establishment Grodno State Agrarian University, Belarus;
e-mail: lena7843041mal@rambler. ru

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 рН 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 Km values for TPP both per the first and second active sites equalled 0.3-4.5 μМ and 1.3-19.7 μМ, accordingly.

Expression of phosphoribosyl pyrophosphate synthetase genes in U87 glioma cells with ERN1 knockdown: effect of hypoxia and endoplasmic reticulum stress

O. H. Minchenko, I. A. Garmash, O. V. Kovalevska,
D. O. Tsymbal, D. O. Minchenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: ominchenko@yahoo.com

Activation of pentose phosphate pathway is an important factor of enhanced cell proliferation and tumor growth. Phosphoribosyl pyrophosphate synthetase (PRPS) is a key enzyme of this pathway and plays a central role in the synthesis of purines and pyrimidines. Hypoxia as well as ERN1 (from endoplasmic reticulum to nuclei-1) mediated endoplasmic reticulum stress response-signalling pathway is linked to the proliferation because the blockade of ERN1 suppresses tumor growth, including glioma. We studied the expression of different PRPS genes in glioma cells with ERN1 knockdown under hypoxic condition. It was shown that hypoxia decreases the expression of PRPS1 and PRPS2 genes in both types of glioma cells, being more pronounced in cells without ERN1 function, but PRPSAP1 and PRPSAP2 gene expressions are suppressed by hypoxia only in glioma cells with blockade of ERN1. Moreover, the blockade of endoribonuclease activity of ERN1 does not affect the expression of PRPS1 and PRPS2 as well as PPRS-associated protein genes in U87 glioma cells. At the same time, the induction of endoplasmic reticulum stress by tunicamycin in glioma cells with suppressed activity of ERN1 endoribonuclease decreases the expression level of PRPS1 and PRPS2 genes only. Results of this investigation clearly demonstrated that the expression of different genes encoding subunits of PRPS enzyme is affected by hypoxia in U87 glioma cells, but the effect of hypoxia is modified by suppression of endoplasmic reticulum stress signaling enzyme ERN1.