Purification and characterization of the rotenone-insensitive NADH dehydrogenase of mitochondria from Arum maculatum

Cook, Neil D.; Cammack, Richard

doi:10.1111/j.1432-1033.1984.tb08231.x

Cited by 36 publications

(11 citation statements)

References 24 publications

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“…It has been observed in this report and others (4,6,7,15), that neither Ca2+, EDTA, nor EGTA has a marked effect on the solubilized exogenous dehydrogenase activity. These observations are consistent 4a-WAUNNO.…”

Section: Resultssupporting

confidence: 50%

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Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Luethy¹,

Hayes²,

Elthon

1991

Plant Physiol.

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Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak 1, is capable of oxidizing NADPH and the ,B form of NADH. The other two activities, peaks 11 and Ill, oxidize only ,B-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component of peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca2 or Mg2+. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak 11 gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca2+ and Mg2+. As with peak 1, platanetin had no effect on peak 11 activity. Peak Ill was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca2+ and Mg2+, but was inhibited by platanetin. The peak Ill enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak Ill activity is likely the exogenous NADH dehydrogenase.In contrast to animal mitochondria, plant mitochondria are capable of oxidizing cytoplasmic NADH directly. This occurs via an exogenous NADH dehydrogenase located on the cytosolic face of the inner mitochondrial membrane (12,18,21,24

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Section: Resultssupporting

confidence: 50%

“…There have been many attempts to purify the exogenous NADH dehydrogenase with conflicting results (4,5,7,15). This has resulted in part because of the presence of several NAD(P)H dehydrogenases in plant mitochondria.…”

mentioning

confidence: 99%

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Luethy¹,

Hayes²,

Elthon

1991

Plant Physiol.

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“…Their purification to homogeneity should bring progress in this direction and make it possible to develop molecular tools such as antibody production and gene cloning. Recent investigations have been camed out on various plant tissues, but the essential focus of these studies was the externa1 NADH dehydrogenases and not the matrixfacing enzymes of the inner membrane (Cook and Cammack, 1984;Chauveau and Lance, 1991).…”

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confidence: 99%

Purification and Preliminary Characterization of Mitochondrial Complex I (NADH:Ubiquinone Reductase) from Broad Bean (Vicia faba L.)

Leterme

Boutry

1993

Plant Physiol.

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“…Only very recently have some studies been carried out on the isolation of plant mitochondria NADH dehydrogenases from various tissues: cauliflower buds (17), mung bean hypocotyls (6,8), and Arum maculatum spadices (4,5,7,8).…”

mentioning

confidence: 99%

“…Besides, these mitochondria oxidize both NADH and NADPH (at a rate about 40% the rate with NADH) (7). Sonication (5) and various detergents, such as Triton X-100 (6,8,17), deoxycholate (7), and lauryl dimethylamine N-oxide (4), were used to solubilize the NADH dehydrogenases. Due to its high activity in plant mitochondria and to the experimental procedures used, one generally assumes that the isolated enzyme is the rotenoneinsensitive external NADH dehydrogenase, whose molecular properties have been partially characterized.…”

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confidence: 99%

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

Chauveau¹,

Lance²

1991

Plant Physiol.

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Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pi values, sensitivity to Ca2" and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH.By comparison with animal mitochondria, the oxidation of NADH in plant mitochondria is far more complex. Only two oxidation systems have been reported for animal mitochondria: one, localized in the outer membrane, is probably of minor importance; the other one is the well known complex I of the inner membrane, facing the matrix compartment. The latter system is linked to energy transduction, and carries out the reoxidation of the reduced equivalents generated by the many NAD-linked dehydrogenases present in the matrix. It is strongly sensitive to rotenone and piericidin (14).In addition to these two systems, plant mitochondria possess two-and possibly three-other systems carrying out the reoxidation of the NAD(P)H produced in the matrix or in the cytosol (1 1, 24, 29). These additional systems are all localized on the inner membrane. One of them is the external NADH dehydrogenase (10, 22) localized on the outer surface of the inner membrane. It is rotenone-insensitive, but antimycinand cyanide-sensitive (2). It is also stimulated by low Ca2+ concentrations and inhibited by Ca2+ chelators (3,25,27). Two different dehydrogenases could correspond to this system, one specific for NADH and another one specific for NADPH (1,12,18,25). Finally, localized on the inner surface of the inner membrane, another NADH dehydrogenase is also present which may act as a bypass to complex I. This dehydrogenase differs from complex I by its size, its insensitivity to rotenone and piericidin, and its lower affinity for This paper is respectfully dedicated to the memory of Professor J. B. Biale whose soft and warm personality will be long remembered.

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Purification and characterization of the rotenone-insensitive NADH dehydrogenase of mitochondria from Arum maculatum

Cited by 36 publications

References 24 publications

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Purification and Preliminary Characterization of Mitochondrial Complex I (NADH:Ubiquinone Reductase) from Broad Bean (Vicia faba L.)

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

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