Mechanism of Decarboxylation of Glycine and Glycolate by Isolated Soybean Cells

Isolated pea leaf mitochondria oxidatively decarboxylate added glycine. This decarboxylation could be linked to the respiratory chain (in which case it was coupled to three phosphorylations) or (21) with BSA as standard, and Chl was estimated by the method of Arnon (1). Mitochondrial protein was corrected for the contribution by broken thylakoids by assuming a thylakoid protein-to-Chl ratio of 7:1 (13). On this basis, thylakoids contributed 30 to 40% of the total protein in the mitochondrial fraction, and specific activities shown in figures and tables have been corrected for this contribution. 'Whole cell' rates of glycine decarboxylation, expressed as ,umol h-1 mg Chl' were estimated by taking into account the yield of isolated mitochondria and the total Chl extracted from the leaves (19). 02 consumption was measured using a Rank 02 electrode (Rank Bros., Cambridge, U. K.) in 2 to 3 ml of standard reaction medium (0.3 M sorbitol, 10 mm Tes buffer, 10 mm KH2PO4, 2 mm MgCl2, 0.1% BSA [pH 7.21) at 30 C. 02 in air-saturated medium was assumed to be 240 tLM.Enzyme assays were performed spectrophotometrically at room temperature. Mitochondria were disrupted either by incubating for 5 min with 0.5% digitonin (and then treating on a column of Sephadex G-25) (16) or by diluting with reaction medium and sonicating. Malate dehydrogenase was assayed according to Ochoa (26), NAD-malic enzyme according to Chapman and Hatch (6), and fumarase by the method of Huang and Beevers (18).Glycine decarboxylation via a reconstituted OAA-malate shuttle was measured as follows. Mitochondria (approximately 1 mg protein) were added to 0.9 ml standard reaction medium which also contained 5 ,UM antimycin A, 0.1 mm OAA, 4 mm NADP, and 2 to 3 units chicken-liver NADP-malic enzyme. The reaction was initiated by adding 10 mm glycine, and the reduction of NADP followed at 340 nm. NH3 release was measured simultaneously with 02 consumption in a sealed Perspex vessel into which were fitted an Orion ammonia electrode (connected to a Beckman pH meter) and a Clarke-type 02 electrode (Yellow-Springs, OH). The signals from both electrodes were monitored with a twin-channel Honeywell recorder. The capacity of the vessel was 9 ml. Mitochondria (0.5 ml, 3 to 7 mg protein) were added to 8 ml standard reaction 425 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

Section: Discussionmentioning

confidence: 99%

Glycine Metabolism and Oxalacetate Transport by Pea Leaf Mitochondria

Day

Wiskich

1981

“…Chloroplast protein contamination was corrected as described (2). (11,12). Formate dehydrogenase activity was assayed by solubilizing 0.025 to 0.200 ml of the mitochondrial preparation in 2 ml 20 mM Hepes (pH 7.6) with 0.1% Triton X-100 and 1 mm NAD+.…”

Section: Douce Et Al (2) Leaf Tissue Was Homogenized For 4 S In a Wmentioning

confidence: 99%

Formate Oxidation and Oxygen Reduction by Leaf Mitochondria

Oliver¹

1981

Self Cite

Mitochondria isolated from the leaves of several plant species were investigated for the presence of NAD-linked formate dehydrogenase. The NADH produced was oxidized by the electron transport sequence and was coupled to ATP synthesis. The amounts of formate dehydrogenase, and, thereby, the capacity for formate-dependent 02 uptake, varied greatly among species. While no activity was detectable in mitochondria from soybean leaves, the rate of formate oxidation by spinach mitochondria was about one-half the rate of malate oxidation. In spinach, only mitochondria from green tissues oxidized formate. These last two observations raise questions as to the role of this reaction and the possible sources of the formate metabolized.The metabolism of formate in higher plants appears to proceed by three independent mechanisms. Formate is reduced to hydroxymethyl tetrahydrofolate, in which state it can participate in serine synthesis by the serine hydroxymethyl transferase reaction (16). Alternatively, formate can be oxidized to CO2 by two separate reactions. These are the peroxidatic action of catalase (7,8) and the activity of NAD+-dependent formate dehydrogenase (7,9). Both the serine hydroxymethyl transferase and the NAD-dependent formate dehydrogenase activities are mitochondrial, whereas the catalase reaction is peroxisomal (7,15 MATERIALS AND METHODS Tobacco (Nicotinia tabacum), lentils (Lens culinaris), and soybeans (Glycine max) were grown in a greenhouse. Spinach (Spinacia oleracea) was either greenhouse-grown or purchased locally.Peas (Pisum sativum), bush beans (Phaseolus vulgaris), chard (Beta vulgaris cicla), beets (Beta vulgaris), and lettuce (Lactuca sativa) were grown in a garden plot.Mitochondria were isolated by a modification of the method of Douce et al. (2). Leaf tissue was homogenized for 4 s in a WaringBlendor in 250 ml partially frozen slurry containing 0.3 M mannitol, 2 mm EDTA, 30 mm Hepes (pH 7.5), 0.2% (w/v) BSA, 0.6% (w/v) polyvinylpolypyrolidone, and 0.05% (v/v) 2-mercaptoethanol. Better coupling ratios were obtained if the BSA level was increased to 1% for bush beans and beets. The homogenate was rapidly filtered through four layers of cheesecloth and two layers of Miracloth (Chicopee Mills, Milltown, NJ) and centrifuged at 1,000g for 10 min. The supernatant was then centrifuged at l0,OOOg for 10 min. The pellet was resuspended in about 30 ml reaction mix, which was comprised of 0.3 M mannitol, 5 mm MgCl2, 10 mm KCI, 10 mm K-phosphate (pH 7.2), and 0.1% BSA (2). Resuspension was greatly facilitated by use of a No. 5 red sable artist brush. The mitochondrial preparation was centrifuged again at l,000g for 10 min, and the mitochondria were collected from the supernatant by centrifuging at 6,000g for 10 min (1). This pellet was either resuspended in the reaction mix and used directly or further purified by phase partitioning (4). Chloroplast protein contamination was corrected as described (2). (11,12). Formate dehydrogenase activity was assayed by solubilizing 0.025 to 0.200 ml of the mi...

“…These measurements also are underestimates in that they assume, for example, that the time between addition of the inhibitor and complete blockage of glycolate oxidation is negligible. Uncertainties in the mechanism of glycolate metabolism prevent precise analysis of the magnitude of photorespiration from estimates of glycolate synthesis, but 25% to 50%Yo of the carbon in glycolate is probably lost under most conditions (12). Photorespiratory CO2 release, therefore, could range from a minimum of 50%1o to over 100o of net photosynthesis.…”

Section: Methodsmentioning

confidence: 99%

“…As this glycolate is metabolized through the glycolate pathway, 25-100%o of the carbon contained in the glycolate is lost as CO2 by the process of photorespiration (12). Could this carbon be conserved, large increases in dry matter accumulation should be possible (25).…”

mentioning

confidence: 99%

The Effect of Glyoxylate on Photosynthesis and Photorespiration by Isolated Soybean Mesophyll Cells

Oliver

1980

Self Cite

Incubating isolated soybean leaf mesophyO ceOs with glyoxylate increased the rates of CO2 fixation by as much as 150%. In order to cause this stimulation, the glyoxylate must be presented to the cels before the NaHCO. (13,20). Photosynthesis was assayed in stoppered 10-ml Fernbach flasks in a reaction mixture that contained 0.3 M sorbitol, 50 mm Tris-HCl (pH 7.8), 2 mm NaNO3, 2 mm EDTA, 1 mm MnCl2, 1 mM MgCl2, 0.5 mm K2HPO4, and 2 mm DTT (added fresh daily).The isolated soybean leaf cells, containing 5-30 jig Chl and suspended in the same solution, were generally added to previously equilibrated flasks in a 25 C waterbath and illuminated for 15 s to 20 min in the absence of added NaHCO3. Next the NaH'4CO3 was added and the illumination continued for an additional 5 min before the reaction was stopped by adding 0.1 ml 3 N HCI. The amount of acid-stable radioactivity was determined by liquid scintillation counting of 0.1-or 0.2-ml aliquots of the acidified reaction mixture.All reactions were in equilibrium with air unless otherwise indicated. In some experiments the reaction vessels were equilibrated with C02-free gases by blowing the gas vigorously against the liquid surface for 10 min before the cells were added. The activity of the RuBP carboxylase in the cells was assayed by first making the membrane more permeable with toluene (7) and then adding the carboxylation substrates RuBP and NaH14CO3. The cells were preilluminated and assayed in the same solution that was used in the photosynthetic studies except that the MgC12 concentration was increased to 25 mm (1). After the cells were illuminated at 25 C for 5 min, 25-,lI samples of the cell mixture were transferred to the reaction flasks. Thirty s later the reaction was terminated by adding 0.1 ml 3 N HCI. The reaction flasks contained 50 pl of a 4:1 (v/v) ethanol to toluene solution, 0.35 mM RuBP, the indicated amount of NaH'4CO3, (2)(3)(4)uCi/,umol)