Responses of superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) activities were evaluated in leaf tissue from intact cotton plants (Cotton Branch 1697) H202 and O2(3, 20) and concludes with the ultimate reduction of H202 (4, 13, 16). This cycling of 02 leads to no net evolution or uptake but results in a transport of electrons or 02 intermediates to locations removed from the '

Antioxidant metabolites in eastem white pine (Pinus strobus L.) needles increased two-to fourfold from the summer to the winter season. Antioxidant enzymes in needle tissue increased between 2-and 122-fold during this same period. These seasonal changes were determined by monitoring ascorbate and glutathione concentrations and the activity of ascorbate peroxidase, glutathione reductase (GR), and superoxide dismutase. Levels of antioxidant metabolites and enzymes were observed always to be lowest during the summer, or active growing season, and highest during the winter, or dormant season. These data correlated well with the thermal kinetic window for purified GR obtained from summer needles. The minimum, apparent KmDm for two isoforms of GR (GRA and GRo) occurred at 5 and 10°C, respectively. The upper limit of the thermal kinetic window (200% of the minimum K.) for GRA and GRB was 20 and 250C, respectively, indicating that needle temperatures exceeding 250C may result in impairment of antioxidant metabolism. The needle content and kinetic properties of GR, the increased activities of other enzymes, and the high substrate concentrations observed during the winter are consistent with the protective function this pathway may provide against photooxidative, winter injury.

Glutathione reductase was purified from pea seedlings using a procedure that included 2',5'-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chro.natofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The Km for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 gm for plastidic and extraplastidic glutathione reductase, respectively, at 25°C. The Km for NADPH was 4.8 and 4.0 gm for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L1 ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozonefumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.

Platelets activated with thrombin release bactericidal factors. We studied the role of the susceptibility of viridans streptococci to these bactericidal factors in the development of infective endocarditis (IE). By using the experimental endocarditis rabbit model, the initial adherence and the development of IE were assessed for 10 viridans streptococcal strains differing in their susceptibilities to releasate (material released) from thrombin-activated platelets. Six strains were susceptible and four strains were resistant to these releasates. The numbers of vegetations (VGs) colonized at 5 min and 48 h after intravenous challenge with 10 4 CFU were determined. At 5 min after challenge, significantly more VGs were colonized with bacteria of the six platelet releasate-susceptible strains than with bacteria of the four releasate-resistant strains (P < 0.005). In the releasate-susceptible group of strains, the number of colonized VGs decreased significantly between 5 min and 48 h after intravenous inoculation (P < 0.001). Such a decrease was not observed with the releasate-resistant strains. As a result, the final developments of IE due to releasate-susceptible and-resistant strains were not significantly different. The releasate-susceptible strain 1 and the releasate-resistant strain 2 were selected for more detailed experiments. Rabbits were killed at 5 and 30 min and 2, 4, and 48 h after inoculation. The number of culture-positive VGs as well as the number of adherent bacteria on the individual VGs were determined. The 90% infective dose for each strain was 10 5 CFU. At low inoculum concentrations (10 3 and 10 4 CFU) a larger proportion of the inoculated bacteria of both strains was found to be adherent on VGs than at higher challenge doses. The number of culture-positive VGs as well as the number of adherent bacteria per VG decreased rapidly in the first 30 min after challenge with strain 1 but not after challenge with strain 2. Additional experiments with the platelet releasate-susceptible strain S224 and the platelet releasate-resistant strain S182 confirmed the data obtained with strains 1 and 2 and indicated that releasate-susceptible strains disappeared from the VGs with time, whereas releasate-resistant strains persisted. In vitro studies with VGs excised 5 min after challenge with strain 1 or 2 showed that clearance of the releasate-susceptible strain 1 was not caused by complement bactericidal activity or surface phagocytosis by polymorphonuclear cells. Bacterial cells of strain 1 adherent on excised VGs were rapidly cleared by exposure to fresh clotting blood or to releasates from thrombin-stimulated platelet suspensions. In contrast, strain 2 bacteria adherent on VGs were hardly affected by these treatments. These data strongly indicate that bactericidal factors released from platelets upon thrombin stimulation are involved in the clearance of bacteria early after their adherence to VGs. Therefore, development of IE is the combined result of the abilities of viridans streptococci to adhere to VGs and ...

Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2', 5'-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLCgel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GRA and GRB). Purified GRA had a specific activity of 1.81 microkatals per milligram of protein and GRB had a specific activity of 6.08 microkatals per milligram of protein. GRA accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GRB accounted for 83%. The native molecular mass for GRA was 103 to 104 kilodaltons and for GRB was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GRA and 57 kilodaltons for GR.. The pH optimum for GRA was 7.25 to 7.75 and for GR. was 7.25. At 250C the Km for GSSG was 15.3 and 39.8 micromolar for GRA and GRB, respectively. For NADPH, the Km was 3.7 and 8.8 micromolar for GRA and GRB, respectively. Antibody produced from purified GR. was reactive with both native and denatured GR., but was cross-reactive with only native GRA.Plants have demonstrated altered antioxidant levels when exposed to air pollutants, low temperature, drought, or xenobiotics such as herbicides (1, 28). These plant-stress interactions, at one time or another, have been associated with increased cellular concentrations of superoxide anion radicals (02-s-) and hydrogen peroxide (H202). Superoxide radicals and H202 can directly attack lipid membranes and certain sulfhydryl-containing enzymes of the reductive pentose cycle (24), or they can further interact to generate hydroxyl radicals (OH) which also can react with membrane lipids and proteins (2,28). Such alterations to cell metabolism and membrane biochemistry would limit normal growth and vigor. Mechanisms for the scavenging of toxic free radicals have previously been reviewed in the plant literature (1-3, 17, 28).