Phospholipid Metabolism in Plant Mitochondria

Self Cite

Cytidine 5'-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn2" being preferred and giving maximum activity at 2.5 millimolar. The estimated K,. for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 miUimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K,. for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K,. estimated for C1TP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold.

mentioning

confidence: 94%

Biosynthesis of Cytidine 5′-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm

Kleppinger-Sparace¹,

Moore²

1985

Self Cite

“…To determine the intramitochondrial localization of enzymes involved in proline oxidation, mitochondria were isolated from 120 g of tissue and suspended in 2.0 ml of 10 mm phosphate buffer (pH 7.4). Fractionation was achieved with a modification of the procedure of Sparace and Moore (15). The entire procedure was conducted at 4 C. The mitochondrial suspension was homogenized for 5 min (10 rotating strokes/per min) in a Duall 25 glass homogenizer, diluted to 120 ml with buffer and allowed to swell for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Submitochondrial Location and Electron Transport Characteristics of Enzymes Involved in Proline Oxidation

Elthon¹,

Stewart²

1981

Isolated corn mitochondria (Zea mays cv. B73 x Mo17) were fractionated and the fragments were separated on a 20-45% (weight/weight) continuous sucrose gradient. Soluble enzymes remained at the top of the gradient overlapping with the outer membranes, while inner membrane vesicles and intact inner membranes were distributed farther down the gradient. Proline oxidase and A'-pyrroline-5-carboxylic acid dehydrogenase activities were associated only with the inner mitochondrial membrane. Glutamate dehydrogenase was confirmed as a matrix enzyme.Both proline and A'-pyrroline-5-carboxylic acid supported oxygen uptake in isolated mitochondria. Proline dependent oxygen uptake was relatively independent of pH with a maximum rate at pH 7.2. In contrast, A'1-pyrroline-5-carboxylic acid-dependent oxygen uptake was sensitive to pH with an optimum at pH 6.1. The oxidation of proline and A'-pyrroline- Proline oxidation is a mitochondrial process (2, 10) that can be inhibited by water stress (16). Although extensive research has been conducted on proline accumulation and its significance during water stress, little has been directed toward elucidating the inhibition of proline oxidation. Inhibition of proline oxidation contributes to proline accumulation. To determine the mechanism of this process, it is necessary to establish the intramitochondrial location and properties of the enzymes involved. The first enzyme involved in the oxidation of proline is proline oxidase, which catalyzes the conversion of proline to P5C'. P5C is then oxidized to glutamate by P5C dehydrogenase. Glutamate can be converted to the Krebs cycle intermediate a-KG by glutamate dehydrogenase, or it can be metabolized by other pathways.The location of these enzymes in animal mitochondria is fairly well established. Proline oxidase is associated with the outer phase of the inner mitochondrial membrane, PSC dehydrogenase and glutamate dehydrogenase are considered matrix enzymes (1, 5). In plants, their submitochondrial location is not well defined. The location of proline oxidase and PSC dehydrogenase is not known, while glutamate dehydrogenase is considered a matrix enzyme (4).The purpose of this paper is to further characterize these enzymes in isolated corn mitochondria.' Abbreviations: P5C, A'-pyrroline-5-carboxylic acid; a-KG, a-ketoglutarate; SRM, standard reaction medium; RCR, respiratory control ratio; OAA, oxaloacetic acid; DCIP, 2,6-dichloroindophenol. MATERIALS AND METHODSCorn seedlings (Zea mays cv. B73 x Mo17) were grown in the dark at 30 ± 2 C in moist vermiculite. Mitochondria were isolated from the shoots of 3-to 4-day-old seedlings according to Day and Hanson (6). Protein was estimated by the method of Lowry et al. (12) using BSA (Fraction V) as the standard. Assays were conducted at 26 ± 2 C in 3.0 ml of SRM (6) was measured by the P5C dependent reduction of NAD+ in SRM containing 0.05% Triton X-100, 2 mm NAD+, and 0.19 mM DL-P5C. Changes in A at 340 nm were used to measure these reactions. P5C was synthesized according to Williams ...

“…Cyt b5 in the outer mitochondrial membranes or ER may serve as the acceptor for the glyoxysomal e-. Antimycin insensitive NADH:CCR activity, indicating the presence of Cyt b5, has been detected in the outer membranes of mitochondria from castor bean endosperm (15).…”

Section: Resultsmentioning

confidence: 99%

β-Oxidation and Glyoxylate Cycle Coupled to NADH: Cytochrome c and Ferricyanide Reductases in Glyoxysomes

Donaldson¹,

Fang²

1987

ABSTRACIGlyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contmination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by 8-oxidation and the glyoxylate cycle.