Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain

Abstract

The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed.

Purification of glutathioneS-transferase from Van Lake fish (Chalcalburnus tarichiiPallas) muscle and investigation of some metal ions effect on enzyme activity

“…Our SDS-PAGE result is indicating that the enzyme is a heterodimer, because of exhibiting two protein bands. Similar results have showed other eukaryotic species as follows: rat liver 43 , bovine brain 44 , turkey liver 41 , human liver 45 and kidney 46 . These results support our findings.…”

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfishmacrobrachium vollenhovenii (herklots)

“…While some GSTs have displayed Michaelis-Menten kinetics when GSH was varied [30], a few GSTs have exhibited nonhyperbolic kinetics [28,31]. The data on the initial velocity studies (Figures 2A and 3A) are intersecting patterns that are not parallel and therefore rule out double displacement or pingpong kinetic mechanism [21].…”

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

“…However, there is a lack of agreement as to whether mammalian GSTs exhibit rapid-equilibrium or steady-state kinetics. In some cases, the random, rapid-equilibrium model provided the best fit to the data (Schramm et al, 1984;Ivanetich and Goold, 1989;Young and Briedis, 1989;Phillips and Mantle, 1991), whereas in others the kinetics were best described by the random, steady-state model (Jakobson et al, 1979;Ivanetich et al, 1990). It has been suggested Figure 10.…”

“…Bireactant models have been used to characterize the kinetic mechanisms of mammalian GSTs (Jakobson et al, 1979;Schramm et al, 1984;Ivanetich and Goold, 1989;Young and Briedis, 1989;Ivanetich et al, 1990;Phillips and Mantle, 1991). For mammalian GSTs, analyses of initialvelocity data with bireactant kinetic models have indicated that the kinetic mechanism is random and sequential.…”