Oxidation and phosphorylation associated with the conversion of glycine to serine

121

A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracca L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, a-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the prepartions. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors.Cyanide completely inhibited the oxidation of NADH. The oxidation of succinate, malate, and glycine was only partially inhibited.The development of techniques for the isolation of tightly coupled mitochondria from etiolated and storage tissues (12,17,18) has resulted in considerable progress toward understanding the physiological properties of the plant mitochondria (11,12,17,18). In marked contrast, relatively few methods are available in the literature for the isolation of intact mitochondria from the leaves of higher plants (8,12,17,18). Therefore, little information is available about the oxidative and phosphorylation properties of mitochondria from green leaves (10,12,17,18).The object of this paper is to describe the preparation, oxidative capacities, and response to various inhibitors of spinach leaf mitochondria. MATERIALS AND METHODSPreparation of Mitochondria. Fresh spinach (Spinacia oleracea L.) leaves were obtained from a local market and used immediately. Young spinach leaves (0.8 kg after deribbing) were cut into 3 liters of chilled medium containing 0.3 M mannitol, 4 mM cysteine, 1 mM EDTA, 30 mm MOPS3 buffer (pH 7.5), 0.2% defatted BSA, and 0.6% insoluble PVP. The leaves were disrupted at low speed for 2 sec in a 1-gallon Waring Blendor. The homogenate was squeezed through six layers of cheesecloth and mitochondria were isolated as fast as possible by differential centrifugation according to the method of Bonner (4). We found that very short grinding times in a Waring Blendor were the most successful. Longer grinding times had a deleterious effect on the mitochondrial oxidative and phosphorylative capacities.Mitochondrial Assays. Mitochondrial respiration was mea- sured polarographically using a Clark 02 electrode in a closed cell at 25 C. Oxygen consumption measurements were performed in a reaction medium (3.3 ml) containing 0.3 M mannitol, 5 mM MgCl2, 10 mm KCI, 10 mm Na-phosphate buffer (pH 7.2), and 0.1% defatted BSA. The 02 concentration in airsaturated medium was taken as 240 UM (9).Rotenone (Sigma), FCCP (Boehringer), oligomycin (Sigma), and antimycin A (Sigma) were dissolved in absolute ethanol. Salicyl-hydroxamic acid dissolved in formamide was a gift of Dr. Vignais.Mitochondrial Protein Determination. Total protein was determined by the Folin-Ciocalteau phenol reagent (16). Chl was extracted from the mitochondrial pellet in 80% acetone and measured according to Arnon (1). If we assume a protein to Chl ratio of 7, in broken thylakoids (15), the amount of mito...

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Isolation and Oxidative Properties of Intact Mitochondria Isolated from Spinach Leaves

Douce¹,

Moore²,

Neuburger³

1977

121

“…We followed NH3 release using an ammonia electrode in a vessel which also allowed the use of an 02 electrode; such simultaneous measurements allow a more dynamic picture to be obtained than is possible with the "4CO2 measurements. State three-state four transitions can be observed with both 02 uptake and NH3 release ( The ratio of 02 consumed to NH3 or CO2 released was 1:2 (Tables II and III), in agreement with the conversion of 2 mol of glycine to 1 mol each of serine, NH3, CO2, and NADH2, which reduces 0.5 mol of 02 (3,4).…”

Section: Resultssupporting

confidence: 73%

Glycine Metabolism and Oxalacetate Transport by Pea Leaf Mitochondria

Day

Wiskich

1981

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

“…This glycine decarboxylation is coupled to serine synthesis through the activity of serine hydroxymethyltransferase (2,13).…”

mentioning

confidence: 99%

Properties and Intramitochondrial Localization of Serine Hydroxymethyltransferase in Leaves of Higher Plants

Woo¹

1979

from spinach leaves was absolutely dependent on tetrahydrofolate; pyridoxal phosphate has no effect on the activity. The stability of this activity in the isolated mitochondria was dependent on the presence of sulfhydryl compounds. It was apparently more stable at pH 7.0 to 7.5 than at higher pH even though the pH optimum of serine hydroxymethyltransferase was 8.5 for both the mitochondrial and cytoplasmic fractions. Distribution studies have indicated that serine hydroxymethyltransferase was predominantly located in the mitochondria. The activity of serine hydroxymethyltransferase was observed to be co-compartmented with glycine decarboxylation and malate dehydrogenase behind the mitochondrial inner membrane. This activity could be solubilized by KCI from osmotically ruptured mitochondrial membrane fractions but substantial activity (35 to 40%) was still retained with the membrane fractions at 0.3 M KCI. This suggests that the glycine decarboxylation-serine hydroxymethyltransferase complex may be closely bound to the internal surface of the mitochondrial inner membrane.The relationship of this integrated enzyme complex to CO2 evolution and serine synthesis during photorespiration and the physiological role of the dicarboxylate shuttle were discussed.The CO2 released during photorespiration is derived principally from the decarboxylation of glycine in leaf mitochondria (4,32,33). This glycine decarboxylation is coupled to serine synthesis through the activity of serine hydroxymethyltransferase (2, 13).The stoichiometry of the reaction for glycine decarboxylation and serine synthesis in isolated leaf mitochondria suggests that the activity of serine hydroxymethyltransferase is equivalent to the rate of glycine decarboxylation. We have reported rates of serine hydroxymethyltransferase in isolated leaf mitochondria of C3 plants comparable to those of glycine decarboxylation (15).The activity of serine hydroxymethyltransferase in nonphotosynthetic (29) and photosynthetic (6, 21) tissues of higher plants has been reported but the intracellular localization of this enzyme in these tissues is not known, although this enzyme is reported to be present in pea chloroplasts (26). Recent studies with rat liver (20) and mutants of Saccharomyces cerevisiae (34) have revealed the presence of cytoplasmic and mitochondrial alloenzymes of serine hydroxymethyltransferase. In rat liver mitochondria the enzyme is located within the matrix (7). Earlier studies have indicated localization of glycine decarboxylation activity behind the inner membrane of the mitochondria (33). This would also imply the localization of serine hydroxymethyltransferase in this compartment.We have examined the properties of this mitochondrial serine hydroxymethyltransferase from spinach leaves and provided evidence for its co-compartmentation with glycine decarboxylation activity behind the inner membrane of the mitochondria. MATERIALS AND METHODSPreparation of Mitochondria from Spinach Leaves. Spinach leaves (20 g) were deribbed and blended for 2 x...