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

Abstract

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.

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

“…In contrast to NADH oxidation, NADPH oxidation was not induced by cold storage, providing support to the hypothesis that cytosolic NADH and NADPH are o+&zed by separate enzymes (Mdler and Lin, 1986;Chauveau and Lance, 1991;Fredlund et al, 1991). Whereas the specific induction of an exogenous NADH dehydrogenase in cold-stored and aged beetroot tissues is a well-recognized phenomenon (Day et al, 1976;Arron and Edwards, 1979;Rayner and Wiskich, 1983;Fredlund et al, 1991), the physiological role of the extemal NAD(P)H dehydrogenase in fresh tissues remains to be established (Arron and Edwards, 1979;Rayner and Wiskick, 1983;Soole et al, 1990aSoole et al, , 1990bFredlund et al, 1991;Luethy et al, 1991).…”

“…This conclusion, however, is essentially based on measurements of electron transport activities and does not imply a reliable molecular model taking into account the Caz+ response of the NAD(P)H dehydrogenase(s). The Ca2+ dependence of the externa1 NAD(P)H Cehydrogenase, which varies among species (Moore and Akerman, 1982;Nash and Wiskich, 1983;Rugolo and Zannoni, 1992) and tissues of the same species (this work) both in vivo (mitochondria) and in vitro (solubilized enzymes) (Cottingham and Moore, 1984;Cammack, 1984,1985;Chauveau and Lance, 1991;Luethy et al, 1991Luethy et al, , 1992, has tentatively been explained in several ways, such as (a) conformational change of the enzyme; (b) presence of a membrane-bound Ca2'-regulatory protein closely associated with the enzyme; and/or (c) a differential Ca2+-induced interaction between the enzyme and the ubiquinone pool (Mdler and Lin, 1986;Soole et al, 1990b).…”

Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

“…Chauveau and Lance (1991) recently isolated a similar (as judged by the Mono Q elution profile and overall protein composition) activity from Arum maculatum. The Avum activity oxidized only NADH, was readily solubilized from mitochondria by freeze-thawing, and contained flavoprotein.…”

“…Although these characteristics can distinguish exogenous NAD(P)H DH activity in situ, most of these characteristics are lost when the enzymes are released from the mitochondrial membrane (Moller and Lin, 1986). There have been many efforts to punfy exogenous NAD(P)H DHs Cammack, 1984, 1985; Moore, 1984, 1988;Klein and Burke, 1984;Cottingham et al, 1986;Chauveau and Lance, 1991; Luethy et al, 19911, which have led us to propose that exogenous NAD(P)H DH activity can result from up to three different enzymes depending on the species (Luethy et al., 1992). We had previously purified a 32-kD NADH DH and a 42-kD NAD(P)H DH from red beet root (Betu vulguris L.) mitochondria .…”

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

“…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).…”