Spinach glutathione

Glutathione reductase Spinach Increase of activity Guy Carter, 1984... 

Guy, C.L. Carter, J.V. (1984). Characterization of partially purified glutathione reductase from cold-hardened and nonhardened spinach leaf tissue. Cryobiology 21, 454-64. 

The stability of EH2 is very species dependent. All of the above results refer to the pig heart enzyme and, where tested, to other mammalian species. It was initially reported that no long wavelength absorption was observed upon reduction of E. coli enzyme with NADH 109), but reduction by 1 equivalent of NADH or dihydrolipoamide leads to the formation of 25% of the maximal 2-electron-reduced species 108) and similar results are obtained with the Azotobacter enzyme 114)- That this species is the catalytically important one in the E. coli enzyme as well as in the mammalian enzyme has also been demonstrated 50). Reduction with dihydrolipoamide in the rapid reaction spectrophotometer at 2° results in the full formation of EH2 followed by the slow k = 13 min, 1 mAf dihydrolipoamide) four-electron reduction. The spectrum of EHa generated in this way is shown in Fig. 7 and is identical with that of the pig heart enzyme. The 2-electron-reduced form, EHj of lipoamide dehydrogenase of spinach 99) may be somewhat unstable however, spectrally it is difficult to distinguish between instability and formation of the EHa-NADH complex (see above) on the basis of available spectral data. Either phenomenon could lead to inhibition by excess NADH. In glutathione reductase it is possible that the complex can be rapidly reoxidized by glutathione 53). 

In recent years DHA reductase has been purified from several sources and characterized. DHA reductase (EC 1.8.5.1) purified from carp hepatopancreas was specific for glutathione as a reducing agent (72). Km values were 5.7 X M for DHA and 1.5 X 10" M for glutathione. The enzyme was not affected by metal ion chelating agents. DHA reductase from spinach leaves has a MW of about 25,000 daltons... 

This nonenzymatic reaction occurs at a significant rate in crude chloro-plast or plant cell extracts (71, 72). At pH 8, the pH of the stroma in the illuminated chloroplast, dehydroascorbate is reduced to ascorbate by glutathione (GSH), which is present in the stroma of spinach chloroplasts at millimolar concentrations (70). Although nonenzymatic, the reaction is extremely swift at this pH (73). 

Spinach leaves contain a dehydroascorbate reductase enzyme that catalyzes Reaction 13 at acidic and neutral pH values, but the enzyme is not, located within the chloroplast (73-76). GSSG produced by Reaction 13 can be re-converted to GSH by glutathione reductase, an enzyme located in the chloroplast (77). 

After this paper was submitted, further evidence for the operation of an ascorbate-glutathione cycle in spinach (85) and pea (86) chloroplasts was reported. The original failure to detect this cycle in pea... 

Equation (20) is catalyzed by glutathione reductase (E.C. 1.6.4.2), a chloro-plast stromal enzyme (Foyer and Halliwell, 1976 Jablonski and Anderson, 1978) of wide distribution in plants (see Wirth and Latzko, 1978 for review), which has been purified to homogeneity fi-om rice kernels (Ida and Morita, 1971a,b) and spinach (Halliwell and Foyer, 1978). 

Halliwell, B., and C. H. Foyer Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography. Planta 139, 9 (1978). 

Vanoni, M. A., Wong, K. K., Ballon, D. P. Blanchard, J. S. (1990). Glutathione reductase comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach and coli enzymes. Biochemistry, 29,5790-96. 

Under these conditions we are also able to couple the electron transport from ascorbate with spinach Fd-NADP-reductase and thus photoreduce exogenous NADP. Lower rates of NADP photoreduction by the immobilized Nostoc cells occur in the absence of MV. Probably endogenous ferredoxins are able to transfer electrons to the added enzyme. No NADP photoreduction can be observed without added reductase and very low rates are genera y measured under conditions which do not support accumulation of MV in the reaction mixture (e.g., presence of Op, or absence of glutathione whicjji is used as a synergetic reductant to limit the back reaction of MV with photoxidized ascorbate [Krasnovsky et al. 1980]. 

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