Purification and immunological studies of glutathione reductase from rat liver. Evidence for an antigenic determinant at the nucleotide-binding domain of the enzyme

Glutathione reductase has been purified to homogeneity by a method which is an improvement of an earlier procedure (Carlberg, I. and Mannervik, B. (1975) J. Biol. Chem. 250, 5475-5480). The new steps in the purification scheme include affinity chromatography on 2',5' ADP-Sepharose 4B. Antibodies to glutathione reductase from rat liver were raised in rabbits and used for analysis of the enzyme by quantitative 'rocket' immunoelectrophoresis. Glutathione reductase from human erythrocytes, porcine erythrocytes, and calf-liver gave precipitin lines showing partial identity with the rat liver enzyme in Ouchterlony double diffusion experiments. Enzyme from spinach, yeast (Saccharomyces cerevisiae), and the photosynthetic bacterium Rhodospirillum rubrum did not give precipitates with the antibodies to the enzyme from rat liver. Titration of glutathione reductase from the different sources with antibodies confirmed the cross-reactivity of the mammalian enzymes; the human enzyme giving the strongest heterologous reaction. No reaction was observed with the enzyme from spinach, yeast, and Rhodospirillum rubrum. NADPH, NADP+, and 2',5' ADP were found to inhibit the interaction between antibodies and glutathione reductase from rat liver and human erythrocytes. NADH, glutathione, or glutathione disulfide did not protect the enzyme from reacting with the antibodies. It is concluded that glutathione reductase has an antigenic binding site for the antibodies at the pyridine nucleotide-binding site of the enzyme molecule.     

Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.

We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.     

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.     

Isolation and Partial Purification of Prophenoloxidase from Daucus carota L. Cell Cultures

The enzyme, phenoloxidase, was isolated and partially purified as an inactive enzyme, a proenzyme, from plant cell cultures of Daucus carota, Nicotiana tabacum, and Haplopappus gracilis. The prophenoloxidase was found to be specifically activated by Ca²⁺ or Mn²⁺ ions in concentrations above 1 millimolar. Calmodulin was not involved in this activation. Concentrations of Ca²⁺ or Mn²⁺ below 1 millimolar could not induce activation of the prophenoloxidase, but if trypsin was added simultaneously with Ca²⁺ or Mn²⁺ at a concentration of 1 millimolar or below, the proenzyme was converted to its active form. The inactive form of phenoloxidase was found to be a soluble enzyme, whereas after activation the enzyme aggregated, and a significant amount of the enzyme activity could become pelleted.     

Disruption of estrous cycles in exercise-trained rats

The female Sprague-Dawley rat was evaluated as an animal model for the menstrual irregularities that are common in women athletes. Daily vaginal smears revealed that estrous cycles were markedly disrupted in rats during a 10-week exercise training program, while cycles remained normal in sedentary rats. Compared to 9 sedentary rats, the 10 exercise-trained rats had longer mean cycle lengths and fewer estrus smears. Six of the exercise-trained rats, but none of the sedentary rats, had an "anestrus period" with more than twice the normal interval between estrus smears; one exercise-trained rat became essentially acyclic. Weight gain during the 10-week training program was lower in exercise-trained rats than in sedentary rats. Colonic temperatures, monitored at rest and during 30 min of exercise, were slightly lower in exercise-trained rats with irregular estrous cycles than in exercise-trained rats with regular cycles, indicating that unusually elevated body temperatures during exercise are not responsible for exercise-related reproductive acyclicity. It is concluded that the female Sprague-Dawley rat may be a useful animal model for the study of menstrual irregularities associated with exercise training.     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.