Four cell types from Vicia faba Linnaeus 'Long Pod' leaflets were assayed for three enzymes unique to the photosynthetic carbon reduction pathway. The enzymes were ribulosebisphosphate carboxylase [3-phospho-p- Gas exchange between a leaf and the atmosphere is almost exclusively through stomata in the epidermis. Stomatal aperture is varied to minimize H20 loss while admitting CO2. The physical basis for opening is swelling of the surrounding guard cell pair in response to the large negative osmotic potential resulting from K+ influx (1). Potassium uptake is electrically balanced by Cl-uptake (2) and the synthesis of organic anions from starch (3-6).An (9) or as by Outlaw and Manchester (6). Analytical enzymes were from Boehringer except RuP2 carboxylase and ribose-P isomerase, which were from Sigma. Agarose was from Miles; most other chemicals were from Sigma. Microscopy Guard cells in epidermal peels were examined by brightfield and fluorescence microscopy. Filters for fluorescence studies were Leitz BG12 and K580. For transmission electron microscopy, tissue was fixed at room temperature for 2 hr under partial vacuum in 100 mM Na cacodylate (pH 7) containing 3% (wt/wt) glutaraldehyde. The tissue was postfixed for 2 hr with 1% OS04 in the above buffer and then dehydrated in a graded ethanol series. The alcohol was replaced by propylene oxide before the tissue was embedded in Spurr's resin (10). Thin sections were stained with uranyl acetate and lead citrate (11) and then were examined in a Hitachi HU-11C electron microscope operated at 75 kV.Enzyme assays Leaflets were illuminated (200 microeinsteins m-2 s-1 of 400-to 700-nm radiation) for at least 15 min before quenching in liquid N2 that had been reduced to its freezing point by evacuation. [Illumination was to activate the enzymes (12)(13)(14).] Leaflets were then freeze-dried at -35°C. Freeze-drying did not change the activities of any of the enzymes reported here.Samples were dissected and weighed (5-15 ng) on a quartz fiber balance in a room with controlled temperature and humidity. The early analytical steps were conducted in a small droplet under oil to prevent evaporation. All enzyme activities were measured at 230C, with the reaction initiated by addition of the tissue sample through the oil. Acid was used to destroy NAD(P)H before enzymatic amplification of the oxidized pyridine nucleotide (NAD, ref. 15; NADP, ref. 16). Tissue blanks and standards were carried through all steps. Except as noted, enzyme specific activities reported are based on dry weight. The concentration of most substrates was determined Abbreviation: RuP2, ribulose bisphosphate.
An apparently defective bacteriophage capable of mediating transduction has been identified in culture filtrates ofDesulfovibrio desulfuricans (American Type Culture Collection 27774). Phage-mediated intraspecies transfer of antibiotic resistance markers occurs with a frequency of 10-5 to 10-6 per recipient cell. The vector contains linear fragments of double-stranded DNA of about 13.5 kilobase pairs, which appear to be random pieces of bacterial DNA. As yet, neither induction nor plaque formation has been observed. To our knowledge, a system of genetic exchange has not been described before for a member of the sulfate-reducing bacteria.The sulfate-reducing bacteria are a heterogeneous group of strictly anaerobic Gram-negative (10), and 0.1% yeast extract (Difco). Resazurin (2.5 ml of a 0.1% stock solution per liter of medium) was used to indicate the reductive state. The medium, in a round-bottom flask in a heating mantle, was continuously flushed with argon while it was brought to a boil. After boiling, the medium was removed from the heat, and 40 ml of reducing agent containing 200 mM NaOH, 160 mM DL-cysteine, and 100 mM Na2S 9H2O was added per liter. The pH of the medium was then adjusted to 6.8-7.0 with 3 M HCl. Before autoclaving, the flasks were sealed with rubber stoppers wired in place. For solidified medium, 16.2 g of Bacto-agar (Difco) and 20 mg of FeCl2' 4H20 were added per liter. The soft agar used for overlayers contained 20 mM Hepes (pH 7.0) and 5 mg of FeCl2 4H2O, 2.5 ml of resazurin solution, 40 ml of reducing agent, and 11 g of Bacto-agar per liter.Anaerobic growth was achieved in liquid culture in Hungate tubes sealed with black rubber stoppers and was measured as an increase in optical density at 660 nm using a Coleman Junior II spectrophotometer. Subculturing, plating, and transduction assays were performed in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI) filled with a nitrogen/hydrogen atmosphere (ca. 95:5 percentage composition) and kept at 30°C.Isolation of Bacteriophages. Bacteriophages were isolated from LS cultures of the antibiotic-resistant strains at mid-to late-exponential phase. For a large phage preparation, a 500-ml culture was centrifuged at 16,000 x g for 15 min at 4°C, and the supernatant was passed through a 0.22-,um pore-sized cellulose acetate filter. The phage were concentrated by a second centrifugation at 108,000 x g for 1 hr at 24°C. The phage pellet was resuspended in a minimal amount (usually 0.5 ml) of fresh LS medium. Smaller amounts of phage were concentrated by using a TLA100.3 rotor at 108,000 x g for 1 hr at 24°C in a TL100 Beckman tabletop ultracentrifuge.Transduction Assay. The wild-type strain 27774, grown to midexponential phase, [-8 x 108 colony-forming units (cfu)/ ml] was used as the recipient. Cell-free filtrates (0.2-,um pore-sized sterile cellulose acetate filters) of the antibiotic resistant strains were the phage source. Typically, 0.5-ml aliquots of the recipient culture were incubated with 0.5-ml volumes of donor filtra...
Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide‐derived urea to NH3 and CO2. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 mM). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent Km (0.8 mM), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross‐reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.
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