Isolation and Oxidative Properties of Intact Mitochondria Isolated from Spinach Leaves

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: Resultsmentioning

confidence: 98%

mentioning

confidence: 99%

Glycine Metabolism and Oxalacetate Transport by Pea Leaf Mitochondria

Day

Wiskich

1981

Section: Introductionmentioning

confidence: 99%

“…Leafmitochondria have not been prepared in a highly purified form, although it is believed that they carry out important functions related to the mechanism of photorespiration (12). It has been demonstrated (4,5,27) that the conversion of glycine to serine occurring in leaf mitochondria is coupled to the synthesis of 3 mol ATP (1,5,10,15,16 plasma membranes and are readily ruptured by gentle mechanical breakage (19)(20)(21).In this communication, we report a method for preparing the highly purified and metabolically active mitochondria from spinach leaf protoplasts, and their oxidative and phosphorylative properties are examined. We also present experimental results concerning the absence of glutamine synthetase in mitochondria in relation to the reassimilation of NH3 in the photorespiratory nitrogen cycle (25,26).…”

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confidence: 99%

Isolation and Characterization of Metabolically Competent Mitochondria from Spinach Leaf Protoplasts

Nishimura¹,

Douce²,

Akazawa³

1982

Self Cite

Intact mitochondria were prepared from spinach (Spinacia oeracea L. The technical advancement of isolating the metabolically competent mitochondria from plant sources, such as etiolated seedlings and storage tissues, has allowed us to study thoroughly the nature of respiratory oxidation and phosphorylation properties (7,22,23). Leafmitochondria have not been prepared in a highly purified form, although it is believed that they carry out important functions related to the mechanism of photorespiration (12). It has been demonstrated (4,5,27) that the conversion of glycine to serine occurring in leaf mitochondria is coupled to the synthesis of 3 mol ATP (1,5,10,15,16 plasma membranes and are readily ruptured by gentle mechanical breakage (19)(20)(21).In this communication, we report a method for preparing the highly purified and metabolically active mitochondria from spinach leaf protoplasts, and their oxidative and phosphorylative properties are examined. We also present experimental results concerning the absence of glutamine synthetase in mitochondria in relation to the reassimilation of NH3 in the photorespiratory nitrogen cycle (25,26).After completion of the manuscript, our attention was drawn to the paper by Bergman et aL (3) describing the isolation of Chlfree spinach leaf mitochondria. MATERIALS AND METHODSProtoplasts. Protoplasts were isolated from freshly harvested spinach leaves (Spinacia oleracea L. var. Kyoho) after digestion of leaf strips (with lower epidermis removed), essentially following the method reported previously (18). The only modification was that the concentration of mannitol was lowered to 0.5 M to prevent osmotic shock, which has an adverse effect during the step of rupturing spinach protoplasts. The final protoplast preparation was shown to have photosynthetic activities of 35 to 70 umol CO2 incorporation/mg Chl.h at 250C.Disruption of Protoplasts and Isolation of Mitochondria by Density Gradient Centrifugation. All operations were carried out at 4°C. Spinach protoplasts (about 4 mg Chl) suspended in 40 ml of buffer-consisting of 0.3 M mannitol, 10 mm Mops3-KOH buffer (pH 7.2), 1 mm EDTA, 0.1% defatted BSA, and 0.6% Polyclar AT-were ruptured in a Teflon homogenizer (3-cm diameter). For the mechanical disruption of protoplasts, about 10 gentle strokes were found to be sufficient to break up plasma membranes from the measurements of the NADH-dependent 02 uptake (experimental details given in "Results").The whole homogenate was then centrifuged at 1,000g for 10 min, the sedimented pellet containing chloroplasts and aggregated organelles (see "Results"). The supernatant fraction was centrifuged at 10,000g for 10 min; the pellet obtained was gently dispersed in 1 ml of suspending buffer containing 0.3 M mannitol, 10 mm Mops-KOH buffer (pH 7.2), and 0.1% defatted BSA and was layered on top of a discontinuous or linear Percoll density gradient. The discontinuous gradient was composed of the following: A, 3 ml 60%o (v/v) Percoll; B, 4 ml 45% (v/v) Percoll; C, 4 ml 28% (v/v) Percoll; a...

“…By differential centrifugation functional mitochondrial preparations can be obtained (3,16). These preparations contain a great deal of contaminating material, mainly thylakoid membrane fragments; the protein to Chl ratio is approximately 15 (3,6). The heavy contamination makes the preparations less suited for studies of molecular composition and enzyme distribution.…”

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confidence: 99%

Method to Obtain a Chlorophyll-free Preparation of Intact Mitochondria from Spinach Leaves

Bergman¹,

Gardeström²,

Ericson³

1980

Mitochondria from green leaves of spinach have been prepared using a three-step procedure involving differential centrifugation, partition in an aqueous dextran polyethylene glycol two-phase system and Percoll gradient centrifugation. The mitochondrial fractions after the different steps of purification were compared. The fmal mitochondrial preparation was totally free from chloroplast material measured as chlorophyll content. The enrichment of mitochondria in relation to peroxisomes and microsomes was approximately 12 and 33 times, respectively, based on NAD:isocitrate dehydrogenase activity, glycolate oxidase activity, and NADPH:cytochrome c oxidoreductase activity. The apparent intactness of the inner and the outer mitochondrial membranes was higher than 90% as measured by latency of enzyme activities. The mitochondria showed high respiratory rates with respiratory control and the ADP/O ratios approached the theoretical limits.