[39] Preparation and properties of succinic—cytochrome c reductase (complex II–III)

Tisdale, H.D.

doi:10.1016/0076-6879(67)10042-6

Cited by 207 publications

(65 citation statements)

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“…There is also a similarity between the bands of the membranes from the denser peak of fraction A and from 6-hr cotyledons. There is an evident difference in that the former contained greater amounts of band numbers 8 and 10 (mol wt 33,800 and 20,000) than did the latter. The membrane from fraction B was quite different from the others in its composition.…”

Section: Preparation and Sucrose Density Gradient Centrifugation Ofmentioning

confidence: 85%

Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition

Sato¹,

Asahi²

1975

Plant Physiol.

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An attempt to isolate intact mitochondria from dry pea seeds (Pisum sativum var. Alaska) ended in failure. Cytochrome oxidase in crude mitochondrial fraction from dry seeds was separated into three fractions by sucrose density gradient centrifugation. Two of the fractions contained malate dehydrogenase, whereas the other did not. Equilibrium centrifugation of mitochondrial membrane on sucrose gradients revealed that the membrane from the fraction without malate dehydrogenase was lighter than that from the others. Differences were observed in relative content of phospholipid to protein and in polypeptide composition analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis among the membranes from three fractions and imbibed cotyledons. Membrane from the fraction without malate dehydrogenase was rich in phospholipid and lacking in polypeptides with relatively high molecular weights as compared with that from others. During imbibition, the fraction without malate dehydrogenase and one of the other two disappeared rapidly after a lag phase lasting for at least 1 hour. Concomitantly, active and stable mitochondria increased in the cotyledons. The results were interpreted to indicate that there were at least three types of mitochondria in dry seeds, the membranes of which differed in their biochemical properties, and that the mitochondria became active and stable through assembly of protein into the membranes during imbibition.Seed germination is accompanied by a marked increase in respiratory activity of the cotyledons or endosperm. Biochemical and electron microscopic studies have revealed that the increase is linked either with the biogenesis of mitochondria or with the development of vesicular mitochondria with a few cristae to crista-rich ones (3). In peas, a rapid development of cotyledon mitochondria, i.e., the formation of their membrane and an increase in their biological function, takes place in the imbibition stage (4, 7). The development does not require de novo synthesis of mitochondrial protein, and seems to result from transport of pre-existing cytoplasmic protein into immature mitochondria (5).The purpose of this investigation was to isolate and characterize immature mitochondria in dry pea seeds and to elucidate how the mitochondria mature to become fully active during 'Present address: Higashi High School, Tosa, Koto, Tokyo 136, Japan.2To whom correspondence should be sent.imbibition. This report describes the existence of at least three types of mitochondrial membranes in dry pea seeds, which differ in their biochemical properties, and a possible mechanism of mitochondrial development in the cotyledons during imbibition. MATERIALS AND METHODSPlant Material. Pea seeds (Piswn sativwn var. Alaska) were purchased from Watanabe Seed Co., Kogota, Miyagi, Japan. The seeds were surface-sterilized with 1% (v/v) NaOCl, washed with deionized H20, and soaked in the dark at 28 C. The cotyledons were harvested at the required time, washed with deionized H20, and used as the source of mitochon...

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Section: Preparation and Sucrose Density Gradient Centrifugation Ofmentioning

confidence: 85%

Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition

Sato¹,

Asahi²

1975

Plant Physiol.

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“…Complex II is clearly substoichiometric to complex III but close to a 1:1 ratio of monomeric complex II and dimeric complex III. A speci c association of the two complexes could not be detected using BN-PAGE, but succinate:cytochrome c reductase has been isolated using cholic acids as detergents (3). The ratio of FAD and cytochrome c 1 was 1:1 in this preparation indicating that two copies of complex II were bound to one complex III dimer that clearly differs from Figure 3.…”

Section: Stoichiometry Of Oxphos Complexes In Bovine Heartmentioning

confidence: 99%

Respiratory Chain Supercomplexes

Schägger¹

2001

IUBMB Life

185

155

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SummaryRespiratory chain supercomplexes have been isolated from mammalian and yeast mitochondria, and bacterial membranes. Functional roles of respiratory chain supercomplexes are catalytic enhancement, substrate channelling, and stabilization of complex I by complex III in mammalian cells. Bacterial supercomplexes are characterized by their relatively high detergent-stability compared to yeast or mammalian supercomplexes that are stable to sonication. The mobility of substrate cytochrome c increases in the order bacterial, yeast, and mammalian respiratory chain. In bacterial supercomplexes, the electron transfer between complexes III and IV involves movement of the mobile head of a tightly bound cytochrome c, whereas the yeast S. cerevisiae seems to use substrate channelling of a mobile cytochrome c, and mammalian respiratory chains have been described to use a cytochrome c pool. Dimeric ATP synthase seems to be speci c for mitochondrial OXPHOS systems. Monomeric complex V was found in Acetobacterium woodii and Paracoccus denitri cans.

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“…The activities of the mitochondrial marker enzyme, succinic-cytochrome c reductase (Tisdale 1967), and of the microsomal marker enzyme, triphosphopyridine nucleotide-cytochrome c reductase (Phillips & Langdon 1962), were measured using the published methods.…”

Section: Preparation Of Subcellular Fractionsmentioning

confidence: 99%

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

Jl¹,

Evans²,

Blanco³

et al. 1999

Journal of Molecular Endocrinology

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Human 3 -hydroxysteroid dehydrogenase/steroid 5 -4 -isomerase (3 -HSD/isomerase) is a bifunctional, single enzyme protein that is membranebound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3 -HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/ mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3 -HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3 -HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3 -HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH 2 -terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/ quaternary structure of this key steroidogenic enzyme.

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[39] Preparation and properties of succinic—cytochrome c reductase (complex II–III)

Cited by 207 publications

References 3 publications

Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition

Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition

Respiratory Chain Supercomplexes

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

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